OA19469A - Compositions and methods of inhibiting MASP-3 for the treatment of various deseases and disorders. - Google Patents

Abstract

The present invention relates to MASP-3 inhibitory antibodies and compositions comprising such antibodies for use in inhibiting the adverse effects of MASP-3 dependent complement activation.

Description

Uniess specifically defined herein, ail terms used herein hâve the same meaning as would be understood by those of ordinary skill in the art of the présent invention. The following définitions are provided in order to provide clarity with respect to the terms as they are used in the spécification and claims to describe the présent invention.

As used herein, the lectin pathway effector arm 1 (“LEA-1”) refers to lectindependent activation of factor B and factor D by MASP-3.

As used herein, the lectin pathway effector arm 2 (“LEA-2”) refers to MASP-2dependent complément activation.

As used herein, the term “MASP-3-dependent complément activation” comprises two components: (i) lectin MASP-3-dependent activation of factor B and factor D, encompassed in LEA-1-mediated complément activation, occurs in the presence of Ca*⁴, commonly leading to the conversion of C3bB to C3bBb and of pro-factor D to factor D; and (ii) lectinindependent conversion of factor B and factor D, which can occur in the absence of Ca^, commonly leading to the conversion of C3bB to C3bBb and of pro-factor D to factor D. LEA-1-mediated complément activation and lectin-independent conversion of factor B and factor D hâve been determined to cause opsonization and/or lysis. While not wishing to be bound by any particular theory, it is believed that only when multiple C3b molécules associate and bind in close proximity, the C3bBb C3 convertase changes its substrate specificity and cleaves C5 as the alternative pathway C5 convertase termed C3bBb(C3b)n.

As used herein, the term “MASP-2-dependent complément activation”, also referred to herein as LEA-2-mediated complément activation, comprises MASP-2 lectin-dependent activation, which occurs in the presence of Ca*⁴, leading to the formation of the lectin pathway C3 convertase C4b2a and upon accumulation of the C3 cleavage product C3b subsequently to the C5 convertase C4b2a(C3b)n, which has been determined to cause opsonization and/or lysis.

As used herein, the term traditional understanding of the alternative pathway also referred to as the “traditional alternative pathway” refers to the alternative pathway prior to the instant discovery described herein, i.e., complément activation that is triggered, for example, by zymosan from fungal and yeast cell walls, lipopolysaccharide (LPS) from Gram négative outer membranes, and rabbit érythrocytes, as well as from many pure polysaccharides, viruses, bacteria, animal tumor cells, parasites and damaged cells, and which has traditionally been thought to arise from spontaneous proteolytic génération of C3b from complément factor C3. As used herein, activation of the traditional alternative pathway, also referred to herein as the “alternative pathway”, is measured in Mg⁺⁺/EGTA buffer (i.e., in the absence of Ça**).

As used herein, the term lectin pathway refers to complément activation that occurs via the spécifie binding of sérum and non-serum carbohydrate-binding proteins including mannan-binding lectin (MBL), CL-11 and the ficolins (H-fïcolin, M-ficolin, or L-ficolin). As described herein, the inventors hâve discovered that the lectin pathway is driven by the two effector arms, lectin pathway effector arm I (LEA-I), which is now known to be MASP-3dependent, and lectin pathway effector arm 2 (LEA-2), which is MASP-2-dependent. As used herein, activation of the lectin pathways are assessed using Ca⁺⁺ containing buffers.

As used herein, the term classical pathway refers to complément activation that is triggered by antibody bound to a foreign particle and requires binding of the récognition molécule Clq.

As used herein, the term “HTRA-l” refers to the serine peptidase High-temperature requirement serine protease AI.

As used herein, the term “MASP-3 inhibitory agent” refers to any agent that directly inhibits MASP-3-dependent complément activation, including agents that bind to or directly interact with MASP-3, including MASP-3 antibodies and MASP-3 binding fragments thereof, natural and synthetic peptides, compétitive substrates, small-molecules, expression inhibitors and isolated natural inhibitors, and also encompasses peptides that compete with MASP-3 for binding to another récognition molécule (e.g., MBL, CL-II, H-fîcolin, Mficolin, or L-ficolin) in the lectin pathway. In one embodiment, the MASP-3 inhibitory agent is spécifie to MASP-3, and does not bind to MASP-1 or MASP-2. An inhibitory agent that directly inhibits MASP-3 can be referred to as a direct MASP-3 inhibitory agent (e.g., a MASP-3 antibody), while an inhibitory agent that indirectly inhibits MASP-3 can be referred to as an indirect MASP-3 inhibitory agent (e.g., a MASP-I antibody that inhibits MASP-3 activation). An example of a direct MASP-3 inhibitory agent is a MASP-3 spécifie inhibitory agent, such as a MASP-3 inhibitory agent that specifically binds to a portion of human MASP-3 (SEQ ID NO:2) with a binding affinity of at least 10 times greater than to other components in the complément system. Another example of a direct MASP-3 inhibitory agent is a high affinity MASP-3 antibody that specifically binds to the serine protease domain of human MASP-3 (SEQ ID NO:2), with an affinity of less than 500 pM and does not bind to human MASP-I (SEQ ID NO:8). In one embodiment, a MASP-3 inhibitory agent indirectly inhibits MASP-3 activity, such as, for example, an inhibitor of MASP-3 activation, including an inhibitor of MASP-1-mediated MASP-3 activation (e.g., a MASP-1 antibody or MASP-1 binding fragments thereof, natural and synthetic peptides, small-molecules, expression inhibitors and isolated natural inhibitors, and also encompasses peptides that compete with MASP-I for binding to MASP-3). In a preferred embodiment, a MASP-3 inhibitory agent, such as an antibody or antigen-binding fragment thereof or antigen binding peptide inhibits MASP-3-mediated maturation of factor D. In another embodiment, a MASP-3 inhibitory agent inhibits MASP-3-mediated activation of factor B. MASP-3 inhibitory agents useful in the method of the invention may reduce MASP-3-dependent complément activation by greater than 10%, such as greater than 20%, greater than 50%, or greater than 90%. In one embodiment, the MASP-3 inhibitory agent reduces MASP-3-dependent complément activation by greater than 90% (i.e., resulting in MASP-3 complément activation of only 10% or less). It is expected that MASP-3 inhibition will block, in full or in part, both LEA-1related lysis and opsonization and lectin-independent conversion of factor B and factor Drelated lysis and opsonization.

In one embodiment, a high affinity MASP-3 inhibitory antibody binds to the serine protease domain of MASP-3 (amino acid residues 450 to 728 of SEQ ID NO:2) with an affinity of less than 500 pM (e.g., less than 250 pM, less than 100 pM, less than 50 pM, or less than 10 pM) and inhibit the alternative pathway of complément activation in the blood of a mammalian subject by at least 50% (e.g., at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% or greater).

An “antibody” is an immunoglobulin molécule capable of spécifie binding to a target, such as a polypeptide, through at least one epitope récognition site located in the variable région (also referred to herein as the variable domain) of the immunoglobulin molécule.

As used herein, the term antibody encompasses antibodies and antibody fragments thereof, derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate including human), or from a hybridoma, phage sélection, recombinant expression or transgenic animais (or other methods of producing antibodies or antibody fragments”), that specifically bind to a target polypeptide, such as, for example, MASP-1, MASP-2 or MASP-3 polypeptides or portions thereof. It is not intended that the term “antibody” is limited as regards to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage sélection, recombinant expression, transgenic animal, peptide synthesis, etc). Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; panspecific, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies); humanized antibodies; murine antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies; and anti-idiotype antibodies, and may be any intact antibody or fragment thereof. As used herein, the term “antibody” encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof,such as a single variable région antibody (dAb), or other known antibody fragments such as Fab, Fab', F(ab')2, Fv and the like, single chain (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen-binding fragment of the required specificity, humanized antibodies, chimeric antibodies, bi-specific antibodies, and any other modified configuration of the immunoglobulin molécule that comprises an antigenbinding site or fragment (epitope récognition site) of the required specificity.

A “monoclonal antibody refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non-naturally occurring) that are involved in the sélective binding of an epitope. Monoclonal antibodies are highly spécifie for the target antigen. The term monoclonal antibody encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv), variants thereof, fusion proteins comprising an antigen-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molécule that comprises an antigen-binding fragment (epitope récognition site) of the required specificity and the ability to bind to an epitope. It is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage sélection, recombinant expression, transgenic animais, etc.). The term includes whole immunoglobulins as well as the fragments etc. described above under the définition of antibody.

As used herein, the term antibody fragment refers to a portion derived from or related to a full-length antibody, such as, for example, a MASP-1, MASP-2 or MASP-3 antibody, generally including the antigen binding or variable région thereof. Illustrative examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molécules and multispecific antibodies formed from antibody fragments.

In certain embodiments, antibodies and antigen-binding fragments thereof as described herein include a heavy chain (VH) and a light chain (VL) complementaritydetermining région (“CDR”) set, respectively interposed between a heavy chain and a light chain Framework région (FR) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. As used herein, the term “CDR set” refers to the three hypervariable régions of a heavy or light chain V région. Proceeding from the Nterminus of a heavy or light chain, these régions are denoted as “CDR1,” “CDR2,” and “CDR3” respectively. An antigen-binidng site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V région.

As used herein, the term “FR set” refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V région. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V région into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs. Within FRs, certain amino acid residues and certain structural features are very highly conserved. In this regard, ail V région sequences contain an internai disulfide loop of around 90 amino acid residues. With the V régions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen-binding surface. It is generally recognized that there are conserved structural régions of FRs which influence the folded shape of the CDR loops into certain “canonical” structures- regardless of the précisé CDR amino acid sequence.

The structures and locations of immunoglobuiin variable régions may be determined by reference to Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 4^th Edition, US Department of Health and Human Services, 1987, and updates thereof, now available on the Internet (immuno.bme.nwu.edu.).

As used herein, a single-chain Fv or scFv antibody fragment comprises the Vpj and Vl domains of an antibody, wherein these domains are présent in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the Vpj and Vl domains, which enables the scFv to form the desired structure for antigen binding.

As used herein, a chimeric antibody is a recombinant protein that contains the variable domains and complementarity-determining régions derived from a non-human species (e.g., rodent) antibody, while the remainder of the antibody molécule is derived from a human antibody. in some embodiments, a chimeric antibody is comprised of an antigenbinding fragment of a MASP-3 inhibitory antibody operably linked or otherwise fused to a heterologous Fc portion of a different antibody. In some embodiments, the heterologous Fc domain may be from a different Ig class from the parent antibody, including IgA (including subclasses IgAl and IgA2), IgD, IgE, IgG (including subclasses IgGl, IgG2, IgG3 and IgG4) and IgM.

As used herein, a “humanized antibody” is a chimeric molécule, generally prepared using recombinant techniques, having an antigen-binding site derived from an 5 immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molécule based upon the structure and/or sequence of a human immunoglobulin. The antigen-binding site may comprise either complété variable régions fused onto constant domains or only the CDRs grafted onto appropriate framework régions in the variable domains. Epitope binding sites may be wild type or may be modified by one or more amino 10 acid substitutions. Another approach focuses not only on providing human-derived constant régions, but also on modifying the variable régions as well so as to reshape them as closely as possible to human form. In some embodiments, humanized antibodies preserve ail CDR sequences (for example, a humanized mouse antibody which contains ail six CDRs from the mouse antibodies). In other embodiments, humanized antibodies hâve one or more CDRs 15 (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody.

An antibody “specifically binds” to a target if it binds with greater affinity and/or avidity that it binds to other substances. In one embodiment, the antibody, or antigen-binding 20 fragment thereof, specifically binds to the serine protease domain of human MASP-3 (amino acid residues 450 to 728 of SEQ ID NO:2). In one embodiment, the antibody, or antigenbinding fragment thereof, specifically binds to one or more of the epitopes described in TABLE 4, TABLE 28 or shown in FIGURE 62.

As used herein, the term mannan-binding lectin (MBL) is équivalent to 25 mannan-binding protein (MBP).

As used herein, the membrane attack complex (MAC) refers to a complex of the terminal five complément components (C5b combined with C6, C7, C8 and C9) that inserts into and disrupts membranes (also referred to as C5b-9).

As used herein, a subject includes ail mammals, including without limitation 30 humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.

As used herein, the amino acid residues are abbreviated as follows: alanine (Ala;A), asparagine (Asn;N), aspartic acid (Asp;D), arginine (Arg;R), cysteine (Cys;C), glutamic acid (Glu;E), glutamine (Gln;Q), glycine (Gly;G), histidine (His;H), isoleucine (Ile;I), leucine (Leu;L), lysine (Lys;K), méthionine (Met;M), phenylalanine (Phe;F), proline (Pro;P), serine 5 (Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine (Tyr;Y), and valine (Val;V).

In the broadest sense, the naturally occurring amino acids can be divided into groups based upon the Chemical characteristic of the side chain of the respective amino acids. By hydrophobie amino acid is meant either Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. By hydrophilic amino acid is meant either Gly, Asn, Gin, Ser, Thr, Asp, Glu, Lys, Arg or 10 His. This grouping of amino acids can be further subclassed as follows. By uncharged hydrophilic amino acid is meant either Ser, Thr, Asn or Gin. By acidic amino acid is meant either Glu or Asp. By basic amino acid is meant either Lys, Arg or His.

As used herein the term conservative amino acid substitution is illustrated by a substitution among amino acids within each of the following groups: (I) glycine, alanine, 15 valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.

The term oligonucleotide as used herein refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term also 20 covers those oligonucleobases composed of naturally-occurring nucléotides, sugars and covalent intemucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring modifications.

As used herein, an epitope refers to the site on a protein (e.g., a human MASP-3 protein) that is bound by an antibody. Overlapping epitopes include at least one (e.g., two, 25 three, four, five, or six) common amino acid residue(s), including linear and non-linear epitopes.

As used herein, the terms polypeptide, peptide, and protein are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification. The MASP-3 proteins described herein can contain or be 30 wild-type proteins or can be variants that hâve not more than 50 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) conservative amino acid substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

In some embodiments, the human MASP-3 protein can hâve an amino acid sequence that is, or is greater than, 70 (e.g., 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100) % identical to the human MASP-3 protein having the amino acid sequence set forth in SEQ ID NO: 2.

In some embodiments, peptide fragments can be at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or 600 or more) amino acid residues in length (e.g., at least 6 contiguous amino acid residues in SEQ ID NO:2). In some embodiments, an antigenic peptide fragment of a human MASP-3 protein is fewer than 500 (e.g., fewer than 450, 400, 350, 325, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6) amino acid residues in length (e.g., fewer than 500 contiguous amino acid residues in SEQ ID NO:2.

In some embodiments, in the context of generating an antibody that binds MASP-3, the peptide fragments are antigenic and retain at least 10% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% or more) ofthe ability ofthe full-length protein to induce an antigenic response in a mammal (see below under Methods for Producing an Antibody).

Percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

In représentative embodiments, the human MASP-3 protein (SEQ ID NO:2) is encoded by the cDNA sequence set forth as SEQ ID NO:1. Those skilled in the art will 5 recognize that the cDNA sequence disclosed in SEQ ID NO:1 represents a single allele of human MASP-3, and that allelic variation and alternative splicing are expected to occur. Allelic variants of the nucléotide sequences shown in SEQ ID NO:1, including those containing silent mutations and those in which mutations resuit in amino acid sequence changes, are within the scope of the présent invention. Allelic variants of the MASP-3 10 sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures, or may be identified by homology comparison search (e.g., BLAST searching) of databases containing such information.

As used herein, an isolated nucleic acid molécule is a nucleic acid molécule (e.g., a polynucleotide) that is not integrated in the genomic DNA of an organism. For example, a 15 DNA molécule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molécule. Another example of an isolated nucleic acid molécule is a Chemically-synthesized nucleic acid molécule that is not integrated in the genome of an organism. A nucleic acid molécule that has been isolated from a particular species is smaller than the complété DNA molécule of a chromosome from that species.

As used herein, a nucleic acid molécule construct is a nucleic acid molécule, either single- or double-stranded, that has been modified through human intervention to contain segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature.

As used herein, an expression vector is a nucleic acid molécule encoding a gene that is expressed in a host cell. Typically, an expression vector comprises a transcription 25 promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be operably linked to the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulâtes the activity of the core promoter.

As used herein, the term “about” as used herein is meant to specify that the spécifie 30 value provided may vary to a certain extent, such as a variation in the range of ±10%, preferably ±5%, most preferably ±2% are included in the given value. Where ranges are stated, the endpoints

Where ranges are stated, the endpoints are included within the range unless otherwise stated or otherwise évident from the context.

As used herein the singular forms a, an and the include plural aspects unless the context clearly dictâtes otherwise. Thus, for example, reference to an excipient includes a plurality of such excipients and équivalents thereof known to those skilled in the art, reference to an agent includes one agent, as well as two or more agents; reference to an antibody includes a plurality of such antibodies and reference to a framework région includes reference to one or more framework régions and équivalents thereof known to those skilled in the art, and so forth.

Each embodiment in this spécification is to be applied mutatis nuitandis to every other embodiment unless expressly stated otherwise. It is contemplated that any embodiment discussed in this spécification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

II. THE LECTIN PATHWAY: A NEW UNDERSTANDING

i. OverView: the Lectin pathway has been redefined

As described herein, the inventors hâve made the surprising discovery that the lectin pathway of complément has two effector arms to activate complément, both driven bj lectin pathway activation complexes formed of carbohydrate récognition components (MBL, CL-11 and ficolins): i) the effector arm formed by the lectin pathway-associated serine proteases MASP-1 and MASP-3, referred to herein as “lectin pathway effector arm 1” or “LEA-1”; and (ii) the MASP-2 driven activation effector arm, referred to herein as “lectin pathway effector arm 2”, or “LEA-2”. Both LEA-1 and LEA-2 can effect lysis and/or opsonization.

It has also been determined that lectin-independent conversion of factor B by MASP3 and lectin-independent conversion of factor D by HTRA-1, MASP-1 and MASP-3, which both can occur in the absence of Ca⁺⁺, commonly lead to the conversion of C3bB to C3bBb and of pro-factor D to factor D. Therefore, inhibiting MASP-3 can inhibit both LEA-1 and the lectin-independent activation of factor B and/or factor D, which can resuit in the inhibition of lysis and/or opsonization.

FIGURE 1 illustrâtes this new understanding of the pathways of complément activation. As shown in FIGURE 1, LEA-1 is driven by lectin-bound MASP-3, which can activate the zymogen of factor D to its active form and/or cleave the C3b- or C3b(H20)-bound factor B, leading to conversion of the C3bB zymogen complex into its enzymatically active form C3bBb. Activated factor D, generated by MASP-3, can also convert the C3bB or C3b(H2Û) zymogen complexes into their enzymatically active form. MASP-1 is capable of rapid self-activation, whereas MASP-3 is not. In many cases, MASP-1 is the activator of MASP-3.

While in many examples lectins (i.e., MBL, CL-11 or ficolins) can direct activity to cellular surfaces, FIGURE 1 also outlines the lectin-independent functions of MASP-3, MASP-1, and HTRA-1 in factor B activation and/or factor D maturation. As with the lectinassociated form of MASP-3 in LEA-1, the lectin-independent form of MASP-3 is capable of medîating conversion of C3bB or C3b(H2Û) to C3bBb (see also FIGURES 29 and 30) and pro-factor D to factor D (see FIGURE 32). MASP-1 (see also FIGURE 32) and the nonMASP-reiated protein HTRA-1 can also activate factor D (Stanton et al., Evidence That the TITRAI Interactome Influences Susceptibility to Age-Related Macidar Degeneration, presented at The Association for Research in Vision and Ophthalmoiogy 2011 conférence on May 4, 2011) in a manner in which no lectin component is required.

Thus, MASP-1 (via LEA-I and lectin-independent forms), MASP-3 (via LEA-1 and lectin-independent forms), and HTRA-1 (lectin-independent only) are capable of either direct or indirect activation at one or more points along a MASP-3 - factor D - factor B axis. In doing so, they generate C3bBb, the C3 convertase of the alternative pathway, and they stimulate the production and déposition of C3b on microbial surfaces. C3b déposition piays a critical rôle in opsonization, labeiing the surfaces of microbes for destruction by host phagocytic cells, such as macrophages. As an exampie herein (FIGURES 28A and 28B), MASP-3 is critical for opsonization of S. aureus. C3b déposition occurs rapidiy on 5. aureus exposed to human sérum in a MASP-3-dependent fashion (FIGURES 28A and 28B).

The contributions of LEA-1 and the lectin-independent functions of MASP-3, MASP1, or HTRA-1 are not limited to opsonization, however. As diagrammed in FIGURE 1, these three components can also cause cell iysis by indirect or direct activation of factor B, and the production of C3b. These components form complexes that generate the alternative pathway C5 convertase, C3bBb(C3b)_n. As described further herein, the requirement for MASP-3 and MBL, but not MASP-2 (and, therefore, not LEA-2 in this exampie), in the lysis of N. meningitidis (see FIGURES 11, 12 and 13) demonstrates a rôle for LEA-1 in lysis. In summary, the opsonization results obtained from the 5. aureiis studies and the lysis results observed in the N. meningitidis studies support the rôle of LEA-1 in both processes (as diagrammed in FIGURE 1). Furthermore, these studies demonstrate that both opsonization and lysis can resuit from the conversion of C3bB or C3b(H20) and/or of pro-factor D to factor 5 D; therefore, both processes can be outcomes of the lectin-independent rôles of MASP-3,

MASP-1, or HTRA-1. Thus, the model developed by the inventors in FIGURE 1 supports the use of inhibitors of principally MASP-3, but also MASP-1 and/or HTRA-1, to block opsonization and/or lysis and to treat pathologies caused by dysrégulation of these processes.

1. Lectin Pathway Effector Arm (LEA-1)

The first effector arm of the lectin pathway, LEA-1, is formed by the lectin pathwayassociated serine proteases MASP-1 and MASP-3. As described herein, the inventors hâve now shown that, in the absence of MASP-3 and in the presence of MASP-1, the alternative pathway is not effectively activated on surface structures. These results demonstrate that MASP-3 plays a previously undisclosed rôle in initiating the alternative pathway, and this is confirmed using the MASP-3-deficient 3MC sérum obtained from patients with the rare 3MC autosomal récessive disorder (Rooryck C, et al., Nat Genet. 43(3):197-203 (2011)) with mutations that render the serine protease domain of MASP-3 dysfunctional. Based on these novel fmdings, it is expected that complément activation involving the alternative pathway, as conventionally defined, is MASP-3-dependent. In fact, MASP-3, and its activation of 20 LEA-1, may represent the hitherto elusive initiator of the alternative pathway.

As further described in Examples 1-4 herein, in MASP-2-deficient sera, the inventors observed a higher activity of lectin-dependent alternative pathway activation resulting in a higher bactericidal activity (i.e., lytic activity) against N. meningitidis. While not wishing to be bound by any particular theory, it is believed that in absence of MASP-2, MASP-1-bearing 25 carbohydrate récognition complexes are more likely to bind close to MASP-3-bearing carbohydrate récognition complexes to activate MASP-3. It is known that, in many instances, activation of MASP-3 is dépendent on MASP-1 activity, as MASP-3 is not an auto-activating enzyme and very often requires the activity of MASP-1 to be converted from its zymogen form into its enzymatically active form. MASP-1 (like MASP-2) is an auto30 activaiing enzyme, while MASP-3 does not auto-activate and, in many instances, needs the enzymatic activity of MASP-1 to be converted into its enzymatically active form. See, Zundel S, et al., J Immunol., 172(7):4342-50 (2004). In absence of MASP-2, ail lectin pathway récognition complexes are either loaded with MASP-1 or MASP-3. Therefore, the absence of MASP-2 facilitâtes the MASP-1-mediated conversion of MASP-3 into its enzymatically active form. Once MASP-3 is activated, activated MASP-3 initiâtes alternative pathway activation, now referred to as “LEA-1” activation, through a MASP-3mediated conversion of C3bB to C3bBb and/or conversion of pro-factor D to factor D. C3bBb, also referred to as the alternative pathway C3 convertase, cleaves additional C3 molécules yielding déposition of opsonic C3b molécules. If several C3b fragments bind in close proximity to the C3bBb convertase complex, this results in the formation of the alternative pathway C5 convertase C3bBb(C3b)n, which promûtes formation of MAC. Additionally, C3b molécules deposited on the surface form new sites for factor B binding, which can now be cleaved by factor D and/or MASP-3 to form additional sites where alternative pathway C3 and C5 convertase complexes can be formed. This latter process is needed for effective lysis and does not require lectins once the initial C3b déposition has occurred. A recent publication (Iwaki D. et al., J Immunol 187(7):3751-8 (2011)) as well as data generated from the inventors (FIGURE 30) demonstrate that the alternative pathway C3 convertase zymogen complex C3bB is converted into its enzymatically active form by activated MASP-3. The inventors now hâve discovered that the MASP-3-mediated cleavage of factor B represents a subcomponent of the newly described LEA-1, which promûtes lectindependent formation ofthe alternative pathway C3 convertase C3bBb.

2. Lectin Pathway Effector Arm (LEA-2)

The second effector arm of the lectin pathway, LEA-2, is formed by the lectin pathwayassociated serine protease MASP-2. MASP-2 is activated upon binding of the récognition components to their respective pattern, and may also be activated by MASP-1, and subsequently cleaves the complément component C4 into C4a and C4b. After the binding of the cleavage product C4b to plasma C2, C4b-bound C2 becomes substrate of a second MASP-2-mediated cleavage step which converts C4b-bound C2 into the enzymatically active complex C4bC2a and a small C2b cleavage fragment. C4b2a is the C3-converting C3 convertase of the lectin pathway, converting the abundant plasma component C3 into C3a and C3b. C3b binds to any surface in close proximity via a thioester bond. If several C3b fragments bind in close proximity to the C3 convertase complex C4b2a, this convertase alters its specificity to convert C5 into C5b and C5a, forming the C5 convertase complex C4b2a(C3b)n. While this C5 convertase can initiate formation of MAC, this process is thought to be insufficiently effective to promote lysis on its own. Rather, the initial C3b opsonins produced by LEA-2 form the nucléus for the formation of new alternative pathway C3 convertase and C5 convertase sites, which ultimately lead to abundant MAC formation and lysis. This latter event is mediated by factor D activation of factor B associated with . LEA-2-formed C3b, and hence is dépendent on LEA-1 by virtue of the essential rôle for MASP-1 in the maturation of factor D. There is also a MASP-2-dependent C4-bypass activation route to activate C3 in the absence of C4, which plays an important rôle in the pathophysiology of ischemia-reperfusion injury, since C4-deficient mice are not protected from ischemia-reperfusion injury while MASP-2-deficient mice are (Schwaeble et al., PNAS, 2011 supra). LEA-2 is also tied to the coagulation pathway, involving the cleavage of prothrombin to thrombin (common pathway) and also the cleavage of factor XII (Hageman factor) to convert into its enzymatically active form Xlla. Factor Xlla in tum cleaves factor XI to Xla (intrinsic pathway). The intrinsic pathway activation of the clotting cascade leads to fibrin formation, which is ofcritical importance for thrombus formation.

FIGURE 1 illustrâtes the new understanding of the lectin pathway and alternative pathway based on the results provided herein. FIGURE 1 delineates the rôle of LEA-2 in both opsonization and lysis. While MASP-2 is the initiator of “downstream” C3b déposition (and résultant opsonization) in multiple lectin-dependent settings physiologically (FIGURES 18A, 18B, 18C), it also plays a rôle in lysis of serum-sensitive bacteria. As illustrated in FIGURE 1, the proposed molecular mechanism responsible for the increased bactericidal activity of MASP-2-deficient or MASP-2-depIeted serum/plasma for serum-sensitive pathogens such as N. meningitidis is that, for the lysis of bacteria, lectin pathway récognition complexes associated with MASP-1 and MASP-3 hâve to bind in close proximity to each other on the bacterial surface, thereby allowing MASP-l to cleave MASP-3. In contrast to MASP-1 and MASP-2, MASP-3 is not an auto-activating enzyme, but, in many instances, requires activatîon/cleavage by MASP-l to be converted into its enzymatically active form.

As further shown in FIGURE 1, activated MASP-3 can then cleave C3b-bound factor B on the pathogen surface to initiale the alternative activation cascade by formation of the enzymatically active alternative pathway C3 and C5 convertases C3bBb and C3bBb(C3b)n, respectively. MASP-2-bearing lectin-pathway activation complexes hâve no part in the activation of MASP-3 and, in the absence of or after déplétion of MASP-2, all-lectin pathway activation complexes will either be loaded with MASP-l or MASP-3. Therefore, in the absence of MASP-2, the likelihood is markedly increased that on the microbial surface MASP-1- and MASP-3-bearing lectin-pathway activation complexes will corne to sit in close proximity to each other, leading to more MASP-3 being activated and thereby leading to a higher rate of MASP-3-mediated cleavage of C3b-bound factor B to form the alternative pathway C3 and C5 convertases C3bBb and C3bBb(C3b)n on the microbial surface. This leads to the activation of the terminal activation cascades C5b-C9 that forms the Membrane Attack Complex, composed of surface-bound C5b associated with C6, C5bC6 associated with C7, C5bC6C7 associated with C8, and C5bC6C7C8, leading to the polymerization of C9 that inserts into the bacterial surface structure and forms a pore in the bacterial wall, which 10 will lead to osmolytic killing of the complement-targeted bacterium.

The core of this novel concept is that the data provided herein clearly show that the lectin pathway activation complexes drive the following two distinct activation routes, as illustrated in FIGURE 1:

i) LEA-1: A MASP-3-dependent activation route that initiâtes and drives activation of 15 complément by generating the alternative pathway convertase C3bBb through initial cleavage and activation of factor B on activator surfaces, which will then catalyze C3b déposition and formation of the alternative pathway convertase C3bBb. The MASP-3-driven activation route plays an essential rôle in the opsonization and lysis of microbes and drives the alternative pathway on the surface of bacteria, leading to optimal rates of activation to 20 generate membrane attack complexes; and ii) LEA-2: A MASP-2-dependent activation route leading to the formation of the lectin pathway C3 convertase C4b2a and, upon accumulation of the C3 cleavage product C3b, subsequently to the C5 convertase C4b2a(C3b)n. In the absence of complément C4, MASP-2 can form an alternative C3 convertase complex which involves C2 and clotting factor XI.

In addition to its rôle in lysis, the MASP-2-driven activation route plays an important rôle in bacterial opsonization leading to microbes being coated with covalently bound C3b and cleavage products thereof (i.e., iC3b and C3dg), which will be targeted for the uptake and killing by C3 receptor-bearing phagocytes, such as granulocytes, macrophages, monocytes, 30 microglia cells and the réticuloendothélial system. This is the most effective route of clearance of bacteria and microorganisms that are résistant to complément lysis. These include most of the gram-positive bacteria.

In addition to LEA-1 and LEA-2, there is the potential for lectin-independent activation of factor D by MASP-3, MASP-1 and/or HTRA-1, and there is also the potential for lectin-independent activation of factor B by MASP-3.

While not wishing to be bound by any particular theory, it is believed that each of (i) LEA-1, (ii) LEA-2 and (iii) lectin-independent activation of factor B and/or factor D lead to opsonization and/or the formation ofMAC with résultant lysis.

ii. Background of MASP-1, MASP-2 and MASP-3

Three mannan-binding lectin-associated serine proteases (MASP-1, MASP-2 and MASP-3) are presently known to be associated in human sérum with the mannan-binding lectin (MBL). Mannan-binding lectin is also called ‘mannose-binding protein’ or ‘mannosebinding lectin’ in the recent literature. The MBL-MASP complex plays an important rôle in innate immunity by virtue of the binding of MBL to carbohydrate structures présent on a wide variety of microorganisms. The interaction of MBL with spécifie arrays of carbohydrate structures brings about the activation of the MASP proenzymes which, in tum, activate complément by cleaving the complément components C4 and C2 to form the C3 convertase C4b2b (Kawasaki et al., J. Biochem 106:483-489 (1989); Matsushita & Fujita, J. ExpMed. 176:1497-1502 (1992); Ji et al., J. Immunol 150:571-578 (1993)).

The MBL-MASP proenzyme complex was, until recently, considered to contain only one type of protease (MASP-1), but it is now clear that there are two other distinct proteases (i.e., MASP-2 and MASP-3) associated with MBL (Thiel et al., Nature 386:506-510 (1997); Dahl et al., Immunity 15:127-135 (2001)), as well as an additional sérum protein of 19 kDa, referred to as “MApl9” or “sMAP” (Stover et al., J. Immunol 162:3481-3490 (1999); Stover et al., J. Immunol 163:6848-6859 (1999); Takahashi étal., Int. Immunol 11:859-63 (1999)).

MApl9 is an alternatively spliced gene product of the structural gene for MASP-2 and lacks the four C-terminal domains of MASP-2, including the serine endopeptidase domain. The abundantly expressed truncated mRNA transcript encoding MApl9 is generated by an alternative splicing/polyadenylation event of the MASP-2 gene. By a similar mechanism, the MASP-1/3 gene gives rise to three major gene products, the two serine proteases MASP-1 and MASP-3 and a truncated gene product of 44 kDa referred to as “MAp44” (Degn et al., J. Immunol 183(11):7371-8 (2009); Skjoedt et al., J Biol Chem 285:8234-43 (2010)).

MASP-1 was first described as the P-100 protease component of the sérum Rareactive factor, which is now recognized as being a complex composed of MBL plus MASP (Matsushita et al., Collectins andInnate Immunity, (1996); Ji et al., JImmunol 150:571-578 (1993). The ability of an MBL-associated endopeptidase within the MBL-MASPs complex to act on the complément components C4 and C2 in a manner apparently identical to that of the Cls enzyme within the Clq-(Clr)2-(Cls)2 complex of the classical pathway of complément suggests that there is a MBL-MASPs complex which is functionally analogous to the Clq-(Clr)2-(Cls)2 complex. The Clq-(Clr)2-(Cls)2 complex is activated by the interaction of Clq with the Fc régions of antibody IgG or IgM présent in immune complexes. This brings about the autoactivation of the Clr proenzyme which, in tum, activâtes the Cls proenzyme which then acts on complément components C4 and C2.

The stoichiometry of the MBL-MASPs complex differs from the one found for the C1 q-(C 1 r)2-(C 1 s)2 complex in that different MBL oligomers appear to associate with different proportions of MASP-l/MApl9 or MASP-2/MASP-3 (Dahl et al., Immunity 15:127-135 (2001). The majority of MASPs and MApl9 found in sérum are not complexed with MBL (Thiel et al., J Immunol 165:878-887 (2000)) and may associate in part with ficolins, a recently described group of lectins having a fibrinogen-like domain able to bind to N-acetylglucosamine residues on microbial surfaces (Le et al., FEBS Lett 425:367 (1998); Sugimoto et al., J. Biol Chem 273:20721 (1998)). Among these, human L-ficolin, H-ficolin and M-ficolin associate with MASPs as well as with MApl9 and may activate the lectin pathway upon binding to the spécifie carbohydrate structures recognized by ficolins (Matsushita et al., JImmunol 164:2281-2284 (2000); Matsushita et al., JImmunol 168:35023506 (2002)). In addition to the ficolins and MBL, an MBL-like lectin collectin, called CL11, has been identified as a lectin pathway récognition molécule (Hansen et al. J Immunol 185:6096-6104 (2010); Schwaeble et al. PNAS 108:7523-7528 (2011)). There is overwhelming evidence underlining the physiological importance of these alternative carbohydrate récognition molécules and it is therefore important to understand that MBL is not the only récognition component ofthe lectin activation pathway and that MBL deficiency is not to be mistaken for lectin-pathway deficiency. The existence of possibly an array of alternative carbohydrate-recognition complexes structurally related to MBL may broaden the spectrum of microbial structures that initiate a direct response of the innate immune System via activation of complément.

Ail lectin pathway récognition molécules are characterized by a spécifie MASPsbinding motif within their collagen-homologous stalk région (Wallis et al. J. Biol Chem

279:14065-14073 (2004)). The MASP-binding site in MBLs, CL-11 and ficolins is characterized by a distinct motif within this domain: Hyp-Gly-Lys-Xaa-Gly-Pro. where Hyp is hydroxyproline and Xaa is generally an aliphatic residue. Point mutations in this sequence disrupt MASP binding.

1. Respective structures, sequences, chromosomal localization and splice variants of MASP-1 and MASP-3

FIGURE 2 is a schematic diagram îllustrating the domain structure of the human MASP-1 polypeptide (SEQ ID NO:8), human MASP-3 polypeptide (SEQ ID NO:2) and human MAp44 polypeptide and the exons encoding the same. As shown in FIGURE 2, the serine proteases MASP-1 and MASP-3 consist of six distinct domains arranged as found in Clr and Cl s; i.e., (I) an N-terminal Clr/Cls/sea urchin VEGF/bone morphogenic protein (or CUBI) domain; (II) an epidermal growth factor (EGF)-like domain; (III) a second CUB domain (CUBII); (IV and V) two complément control protein (CCP1 and CCP2) domains; and (VI) a serine protease (SP) domain.

The cDNA-derived amino acid sequences of human and mouse MASP-1 (Sato et al., Int Immunol 6:665-669 (1994); Takada et al., Biochetn Biophys Res Commun 196:1003-1009 (1993); Takayama et al., J. Immunol 152:2308-2316 (1994)), human, mouse, and rat MASP-2 (Thiel et al., Nature 386:506-510 (1997); Endo et al., J Immunol 161:4924-30 (1998); Stover et al., J. Immunol 162:3481-3490 (1999); Stover et al., J. Immunol 163:6848-6859 (1999)), as well as human MASP-3 (Dahl et al., Immunity 15:127-135 (2001)) indicate that these proteases are serine peptidases having the characteristic triad of His, Asp and Ser residues within their putative catalytic domains (Genbank Accession numbers: human MASP-1: BAA04477.1 (SEQ ID NO:8); mouse MASP-1: BAA03944; rat MASP-1: AJ457084; Human MASP-3:AAK84071 (SEQ ID NO:2); mouse MASP-3: AB049755, as accessed on Genbank on 2/15/2012 (SEQ ID NO:3); rat MASP-3 (SEQ ID NO:4); chicken MASP-3 (SEQ ID NO:5); rabbit MASP-3 (SEQ ID NO:6); and Cynomolgus monkey (SEQ ID NO:7).

As further shown in FIGURE 2, upon conversion of the zymogen to the active form, the heavy chain (alpha, or A chain) and light chain (beta, or B chain) are split to yield a disulphide-linked A-chain and a smaller B-chaîn representing the serine protease domain. The single-chain proenzyme MASP-1 is activated (like proenzyme Clr and Cls) by cleavage of an Arg-lle bond located between the second CCP domain (domain V) and the serine protease domain (domain VI). Proenzymes MASP-2 and MASP-3 are considered to be . 51 activated in a similar fashion to that of MASP-1. Each MASP protein forms homodimers and is individually associated with MBL and the ficolins in a Ca^-dependent manner.

The human MASP-1 polypeptide (SEQ ID NO:8) and MASP-3 polypeptide (SEQ ID NO:2) arise from one structural gene (Dahl et al., Immunity 15:127-135 (2001), which has been mapped to the 3q27-28 région of the long arm of chromosome 3 (Takada et al., Genomics 25:757-759 (1995)). The MASP-3 and MASP-1 mRNA transcripts are generated from the primary transcript by an alternative splicing/polyadenylation process. The MASP-3 translation product is composed of an alpha chain, which is common to both MASP-1 and MASP-3, and a beta chain (the serine protease domain), which is unique to MASP-3. As shown in FIGURE 2, the human MASP-1 gene encompasses 18 exons. The human MASP-1 cDNA is encoded by exons 2, 3, 4, 5, 6, 7, 8, 10, 11, 13, 14, 15, 16, 17 and 18. As further shown in FIGURE 2, the human MASP 3 gene encompasses twelve exons. The human MASP-3 cDNA (set forth as SEQ ID NO:1) is encoded by exons 2, 3, 4, 5, 6, 7, 8, 10, 11 and 12. An alternative splice results in a protein termed MBL-associated protein 44 (MAp44), arising from exons 2, 3, 4, 5, 6, 7, 8 and 9. .

The human MASP-1 polypeptide (SEQ ID NO: 8 from Genbank BAA04477.1) has 699 amino acid residues, which includes a leader peptide of 19 residues. When the leader peptide is omitted, the calculated molecular mass of MASP-1 is 76,976 Da. As shown in FIGURE 2, the MASP-1 amino acid sequence contains four N-linked glycosylation sites. The domains of the human MASP-1 protein (with reference to SEQ ID NO:8) are shown in FIGURE 2 and include an N-terminal Clr/Cls/sea urchin VEFG/bone morphogenic protein (CUBI) domain (aa 25-137 of SEQ ID NO:8), an epidermal growth factor-like domain (aa 139-181 of SEQ ID NO:8), a second CUB domain (CUBII) (aa 185-296 of SEQ ID NO:8), as well as a tandem of complément control protein (CCP1 aa 301-363 and CCP2 aa 367-432 of SEQ ID NO:8) domains and a serine protease domain (aa 449-694 of SEQ ID NO:8).

The human MASP-3 polypeptide (SEQ ID NO:2, from Genbank AAK84071) has 728 amino acid residues (as shown in FIGURE 3), which includes a leader peptide of 19 residues (shown as the underlined amino acid residues in FIGURE 3).

When the leader peptides are omitted, the calculated molecular mass of MASP-3 is 81,873 Da. As shown in FIGURE 2, there are seven N -linked glycosylation sites in MASP3. The domains ofthe human MASP-3 protein (with reference to SEQ ID NO:2) are shown in

FIGURE 2 and include an N-terminal Clr/Cls/sea urchin VEGF/bone morphogenic protein (CUBI) domain (aa 25-137 of SEQ ID NO:2), an epidermal growth factor-like domain (aa 139-181 of SEQ ID NO:2), a second CUB domain (CUBII) (aa 185-296 of SEQ ID NO:2), as vvell as a tandem of complément control protein (CCP1 aa 299-363 and CCP2 aa 5 367-432 of SEQ ID NO:2) domains and a serine protease domain (aa 450-728 of SEQ ID

NO:2).

The MASP-3 translation product is composed of an alpha chain (heavy chain), containing the CUB-1-EGF-CUB-2-CCP-1-CCP-2 domains (alpha chain: aa 1-448 of SEQ ID NO:2) which is common to both MASP-1 and MASP-3, and a light chain (beta chain: aa 10 449-728 of SEQ ID NO:2), containing the serine protease domain, which is unique to MASP3.

2. Comparison of MASP-3 amino acid sequences from various species

FIGURE 4 provides a multi-species alignment of MASP-3 showing a comparison of 15 full-length MASP-3 protein from human (SEQ ID NO:2), cynomolgus monkey (SEQ ID NO:7), rat (SEQ ID NO:4), murine (SEQ ID NO:3), chicken (SEQ ID NO:5) and rabbit (SEQ ID NO:6). FIGURE 5 provides a multi-species alignment of the serine protease (SP) domain from human (aa 450-728 of SEQ ID NO:2); rabbit (aa 450-728 of SEQ ID NO:6); murine (aa aa455-733 of SEQ ID NO:3); rat (aa 455-733 of SEQ ID NO:4) and chicken (aa aa448-730 of 20 SEQ ID NO:5).

As shown in FIGURE 4, there is a high level of amino acid sequence conservation of MASP-3 polypeptide amongst different species, particularly in the SP domain (FIGURE 5). As further shown in FIGURE 5, the catalytic triad (H at residue 497; D at residue 553 and S at residue 664 with reference to full length human MASP-3 (SEQ ID NO:2) is conserved 25 across species. TABLE 1 summarizes the percent identity of the MASP-3 SP domain across species.

TABLE 1: Percent Identity of the MASP-3 SP domain Across Species

	Cyno	Rabbit	Rat	Mouse	chicken
Human	95%	94%	92%	91%	79%
Cyno		94%	90%	90%	79%
Rabbit			92%	92%	81%

Rat				97%	78%
mouse					78%

MASP-3 has no proteolytic activity towards C4, C2 or C3 substrates. Conversely, MASP-3 was initially reported to act as an inhibitor of the lectin pathway (Dahl et al., Immunity 15:127-135 (2001)). This conclusion may hâve corne about because in contrast to MASP-1 and MASP-2, MASP-3 is not an autoactivating enzyme (Zundel S. et al., J Immunol 172:4342-4350 (2004); Megyeri et al., J. Biol. Chem. 288:8922-8934 (2013).

Recently, evidence for possible physiological fonctions of MASP-1 and MASP-3 emerged from transgenic mouse studies using a mouse strain with a combined MASP-1 and MASP-3 deficiency. While MASP-1/3-knockout mice hâve a functional lectin pathway (Schwaeble et al., PNAS 108:7523-7528 (2011)), they appear to lack alternative pathway activity (Takahashi et al., JEM 207(1):29-37 (2010)). Lack of alternative pathway activity appears to be due to a processing defect of complément factor D, which is necessary for alternative pathway activity. In MASP-1/3 knockout mice, ail factor D is circulating as a proteolytically inactive pro-form, whereas in the sérum of normal mice, substantially ail of factor D is in the active form. Biochemical analysis suggested that MASP-1 may be able to convert complément factor D from its zymogen form into its enzymatically active form (FIGURE 32; Takahashi et al., JEM 207(1 ):29-37 (2010)). MASP-3 also cleaves pro-factor D zymogen and produce active factor D in vitro (FIGURE 32; Takahashi et al., JEM 207(1):29-37 (2010)). Factor D is présent as an active enzyme in circulation in normal individuals, and MASP-1 and MASP-3, as well as HTRA-1, may be responsible for this activation. Furthermore, mice with combined MBL and ficolin deficiencies still produce normal levels of factor D and hâve a fully functional alternative pathway. Thus, these physiological functions of MASP-1 and MASP-3 do not necessarily involve lectins, and are thus unrelated to the lectin pathway. Recombinant mouse and human MASP-3 also appear to cleave factor B and support C3 déposition on S¹, aureus in vitro (FIGURE 29; Iwaki D. et al., JImmunol 187(7):3751-8 (2011)).

An unexpected physiological rôle for MASP-3 has emerged from recent studies of patients with 3MC syndrome (previously designated the Camevale, Mingarelli, Maipuech, and Michels syndrome; OMIM # 257920). These patients display severe developmental abnormalities, including cleft palate, cleft lip, cranial malformations and mental retardation.

Genetic analysis identified 3MC patients that were homozygous for a dysfunctional MASP-3 gene (Rooryck et al., Nat Genet. 43(3):197-203 (2011)). Another group of 3MC patients was found to be homozygous for a mutation in the MASP-1 gene that leads to the absence of functional MASP-1 and MASP-3 proteins. Yet another group of 3MC patients lacked a 5 functional CL-11 gene. (Rooryck et al., Nat Genet. 43(3):197-203 (2011)). Thus, the CL-11 MASP-3 axis appears to play a rôle during embryonic development. The molecular mechanisms of this developmental pathway are unclear. It is unlikely, however, to be mediated by a conventional complement-driven process since individuals with deficiencies of common complément components C3 do not develop this syndrome. Thus, prior to the 10 discovery of the instant inventors, as described herein, a functional rôle for MASP-3 in lectindependent complément activation was previously not established.

The structures of the catalytic fragment of MASP-1 and MASP-2 hâve been determined by X-ray crystallography. Structural comparison of MASP-1 protease domain with those of other complément proteases revealed the basis of its relaxed substrate 15 specificity (Dobô et al., J. Immunol 183:1207-1214 (2009)). While the accessibility ofthe substrate binding groove of MASP-2 is restricted by surface loops (Harmat et al., J Mol Biol 342:1533-1546 (2004)), MASP-1 has an open substrate binding pocket which resembles that of trypsin rather than other complément proteases. A thrombin-like property of the MASP-1 structure is the unusually large 60 amino acid loop (loop B) which may interact with 20 substrates. Another interesting feature of the MASP-1 structure is the internai sait bridge between the SI Asp 189 and Arg224. A similar sait bridge can be found in the substrate binding pocket of factor D, which can regulate its protease activity. Cl s and MASP-2 hâve almost identical substrate specificities. Surprisingly, some of the eight surface loops of MASP-2, which détermine the substrate specificities, hâve quite different conformations 25 compared to those of Cl s. This means that the two functionally related enzymes interact with the same substrates in a different manner. The structure of zymogen MASP-2 shows an inactive protease domain with disrupted oxyanion hole and substrate binding pocket (Gâl et al., J Biol Chem 280:33435-33444 (2005)). Surprisingly, zymogen MASP-2 shows considérable activity on a large protein substrate, C4. It is likely that the structure of 30 zymogen MASP-2 is quite flexible, enabling the transition between the inactive and the active forms. This flexibility, which is reflected in the structure, may play a rôle in the autoactivation process.

Northern blot analysis indicates that liver is the major source of MASP-l and MASP2 mRNA. Using a 5' spécifie cDNA probe for MASP-1, major MASP-1 transcript was seen at 4.8 kb and a minor one at approximately 3.4 kb, both présent in human and mouse liver (Stover et al., Genes Immunity 4:374-84 (2003)). MASP-2 mRNA (2.6 kb) and MApl9 mRNA (1.0 kb) are abundantly expressed in liver tissue. MASP-3 is expressed in the liver, and also in many other tissues, including neuronal tissue (Lynch N. J. et al., J Immunol 174:4998-5006 (2005)).

A patient with a history of infections and chronic inflammatory disease was found to hâve a mutated form of MASP-2 that fails to form an active MBL-MASP complex (Stengaard-Pedersen et al., N Engl J Med 349:554-560 (2003)). Some investigators hâve determined that deficiency of MBL leads to a tendency to frequent infections in childhood (Super et al., Lancet 2:1236-1239 (1989); Garred et al., Lancet 346:941-943 (1995) and a decreased résistance to HIV infection (Nielsen et al., Clin Exp Immunol 100:219-222 (1995); Garred et al., Mol Immunol 33 (suppl 1):8 (1996)). However, other studies hâve not demonstrated a significant corrélation of low MBL levels with increased infections (Egli et al., PLoS One. 8(l):e5l983 (2013); Ruskamp et al., J Infect Dis. 198(11):1707-13 (2008); Israëls et al., Arch Dis Child Fêtai Néonatal Ed. 95(6):F452-6l (2010)). While the literature is mixed, deficiency, or non-utilization, of MASP may hâve an adverse effect on an individual’s ability to mount immédiate, non-antibody-dependent defense against certain pathogens.

Supporting data for the new understanding, underscoring traditional assay conditions that are devoid of Ca** and results obtained using a more physiological set of conditions that include Ca**.

Several independent lines of strong experimental evidence are provided herein pointing to the conclusion that the lectin pathway activation route of complément activâtes complément via two independent effector mechanisms: i) LEA-2: a MASP-2-driven path that médiates complement-driven opsonisation, chemotaxis (Schwaeble et al., PNAS 108:75237528 (2011)), and cell lysis, and ii) LEA-1: a novel MASP-3-dependent activation route that initiâtes complément activation by generating the alternative pathway convertase C3bBb through cleavage and activation of factor B on activator surfaces, which will then catalyze C3b déposition and formation of the alternative pathway convertase C3bBb, which can resuit in cell lysis as well as microbial opsonization. In addition, as described herein, separate ·

lectin-independent activation of factor B and/or factor D by MASP-1, MASP-3, or HTRA-1, or a combination of any the three, can also lead to complément activation via the alternative pathway.

A lectin pathway-dependent MASP-3-driven activation of the alternative pathway appears to contribute to the well-established factor D-mediated cleavage of C3b-bound factor B to achieve optimal activation rates for complement-dependent lysis through the terminal activation cascade to lyse bacterial cells through the formation of C5b-9 membrane attack complexes (MAC) on the cellular surface (FIGURES 12-13). This rate-limited event appears to require optimal coordination as it is defective in the absence of MASP-3 functional activity as well as in the absence of factor D functional activity. As described in Examples 14 herein, the inventors discovered this MASP-3-dependent lectin pathway function when studying the phenotype of MASP-2 deficiency and MASP-2 inhibition in experimental mouse models of N. menigitidis infection. Gene-targeted, MASP-2-deficient mice and wildtype mice treated with antibody-based MASP-2 inhibitors were highly résistant to experimental N. meningitidis infection (see FIGURES 6-10). When the infectious dose was adjusted to give approximately 60% mortality in the wild-type littermates, ail of the MASP-2deficient or MASP-2-depleted mice cleared the infection and survived (see FIGURE 6 and FIGURE 10). This extremely high degree of résistance was reflected in a significant increase of sérum bactericidal activity in MASP-2-deficient or MASP-2-depleted mouse sérum. Further experiments showed that this bactericidal activity was dépendent on alternative pathway-driven bacterial lysis. Mouse sera déficient of factor B, or factor D, or C3 showed no bactericidal activity towards N. meningitidis, indicating that the alternative pathway is essential for driving the terminal activation cascade. A surprising resuit was that mouse sera déficient of MBL-A and MBL-C (both being the lectin-pathway récognition molécules that recognize N. meningitidis') as well as mouse sera déficient of the lectin pathway-associated serine proteases MASP-1 and MASP-3 had lost ail bacteriolytic activity towards N. meningitidis (FIGURE 13). A recent paper (Takahashi M. et al., JEM207: 29-37 (2010)) and work presented herein (FIGURE 32) demonstrate that MASP-1 can convert the zymogen form of factor D into its enzymatically active form and may in part explain the loss of lytic activity through the absence of enzymatically active factor D in these sera. This does not explain the lack of bactericidal activity in MBL-deficient mice since these mice hâve normal enzymatically active factor D (Banda et al., Mol Imunol 49(1-2):281-9 (2011)).

Remarkably, when testing human sera from patients with the rare 3MC autosomal récessive disorder (Rooryck C, et al., Nat Genet. 43(3):197-203) with mutations that render the serine protease domain of MASP-3 dysfunctional, no bactericidal activity against N. meningilidis was détectable (n.b.: these sera hâve MASP-1 and factor D, but no MASP-3).

The hypothesis that human sérum requires lectin pathway-mediated MASP-3dependent activity to develop bactericidal activity is further supported by the observation that MBL-deficient human sera also fail to lyse N. meningilidis (FIGURES 11-12). MBL is the only human lectin-pathway récognition molécule that binds to this pathogen. Since MASP-3 does not auto-activate, the inventors hypothesize that the higher bacteriolytic activity in MASP-2-deficient sera could be explained by a favored activation of MASP-3 through MASP-1 since, in the absence of MASP-2, ail lectin-pathway activation complexes that bind to the bacterial surface will be loaded with either MASP-1 or MASP-3. Since activated MASP-3 cleaves both factor D (FIGURE 32) and factor B to generate their respective enzymatically active forms in vitro (FIGURE 30 and Iwaki D., et al., J. Immunol.187(7):3751-3758 (2011)), the most likely function of MASP-3 is to facilitate the formation of the alternative pathway C3 convertase (i.e., C3bBb).

While the data for the lectin-dependent rôle are compelling, multiple experiments suggest that MASP-3 and MASP-1 are not necessarily obligated to function in a complex with lectin molécules. Experiments such as that shown in FIGURE 28B demonstrate the ability of MASP-3 to activate the alternative pathway (as demonstrated by C3b déposition on 5. aureus) under conditions (i.e., the presence of EGTA) in which complexes with lectin would not be présent. FIGURE 28A demonstrates that déposition under these conditions is dépendent upon factor B, factor D, and factor P, ail critical components of the alternative pathway. Addtionally, factor D activation by MASP-3 and MASP-1 (FIGURE 32), and factor B activation by MASP-3 (FIGURE 30) can occur in vitro in the absence of lectin. Finally, hemolysis studies of mouse érythrocytes in the presence of human sérum demonstrate a clear rôle for both MBL and MASP-3 for cell lysis. However, the deficiency of MBL does not completely reproduce the severity of the deficiency of MASP-3, in contrast to what would be expected if ail functional MASP-3 were complexed with MBL. Thus, the inventors do not wish to be constrained by the notion that ali of the rôles for MASP-3 (and MASP-1) demonstrated herein can be attributed solely to function associated with lectin.

The identification of the two effector arms of the lectin pathway, as well as the possible lectin-independent functions of MASP-l, MASP-3, and HTRA-1, represent novel opportunities for therapeutic interventions to effectively treat defined human pathologies caused by excessive complément activation in the presence of microbial pathogens or altered 5 host cells or metabolic deposits. As described herein, the inventors hâve now discovered that in the absence of MASP-3 and in the presence of MASP-1, the alternative pathway is not activated on surface structures (see FIGURES 15-16, 28B, 34-35A,B, 38-39). Since the alternative pathway is important in driving the rate-limiting events leading to bacterial lysis as well as cell lysis (Mathieson PW, et al., J Exp Med 177(6):1827-3 (1993)), our results demonstrate that activated MASP-3 plays an important rôle in the lytic activity of complément. As shown in FIGURES 12-13, 19-21, 36-37, and 39-40, in sérum of 3MC patients lacking MASP-3 but not MASP-1, the lytic terminal activation cascade of complément is defective. The data shown in FIGURES 12 and 13 demonstrate a loss of bacteriolytic activity in absence of MASP-3 and/or MASP-1/MASP-3 functional activity.

Likewise, the loss of hemolytic activity in MASP-3-deficient human sérum (FIGURES 1921, 36-37 and 39-40), coupled with the ability to reconstitute hemolysis by adding recombinant MASP-3 (FIGURES 39-40), strongly supports the conclusion that activation of the alternative pathway on target surfaces (which is essential to drive complement-mediated lysis) dépends on the presence of activated MASP-3. Based on the new understanding of the 20 lectin pathway detailed above, alternative pathway activation of target surfaces is thus dépendent upon LEA-1, and/or lectin-independent activation of factor B and/or factor D, which is also mediated by MASP-3, and therefore, agents that block MASP-3-dependent complément activation will prevent alternative pathway activation on target surfaces.

The disclosure of the essential rôle of MASP-3-dependent initiation of alternative 25 pathway activation implies that the alternative pathway is not an independent, stand-alone pathway of complément activation as described in essentially ail current medical textbooks and recent review articles on complément. The current and widely held scientific view is that the alternative pathway is activated on the surface of certain particulate targets (microbes, zymosan, and rabbit érythrocytes) through the amplification of spontaneous tick-over C3 30 activation. However, the absence of any alternative pathway activation in sera of MASP-1 and MASP-3 double-deficient mice and human 3MC patient sérum on both zymosan-coated plates and two different bacteria (N. meningitidis and S. auretts), and the réduction of hemolysis of érythrocytes in MASP-3-deficient sera from human and mouse indicate that initiation of alternative pathway activation on these surfaces requires functional MASP-3. The required rôle for MASP-3 may be either lectin-dependent or-independent, and leads to formation ofthe alternative pathway C3 convertase and C5 convertase complexes, i.e. C3bBb and C3bBb(C3b)n, respectively. Thus, the inventors here disclose the existence of a previously elusive initiation routes for the alternative pathway. This initiation route is dépendent upon (i) LEA-1, a newly discovered activation arm of the lectin pathway, and/or (ii) lectin-independent rôles ofthe proteins MASP-3, MASP-1, and HTRA-1.

3. The use of MASP-3 inhibitory agents for the Treatment of Alternative Pathwayrelated Diseases and Conditions.

As described herein, high affinity MASP-3 inhibitory antibodies (e.g., with a binding affinity of less than 500 pM) which hâve been shown to completely inhibit the alternative pathway in mammalian subjects such as rodents and non-primates at molar concentrations less than the concentration of the MASP-3 target (e.g., at a molar ratio of from about 1:1 to about 2.5:1 (MASP-3 target to mAb) (see in Examples 11-21). As described in Example 11, a single dose administration of a high affinity MASP-3 inhibitory antibody, mAb 13B1, to mice led to near-complete ablation of systemic alternative pathway complément activity for at least 14 days. As further described in Example 12, in a study conducted in a wellestablished animal model associated with PNH it was demonstrated that mAb 13B1 significantly improved the survival of PNH-like red blood cells and protected PNH-like red blood cells significantly better than did C5 inhibition. As described in Example 13, it was further demonstrated that mAb 13B1 reduced the incidence and severity of disease in a mouse model of arthritis. The results in this example demonstrate that représentative high affinity MASP-3 inhibitory mAbs 13B1, 10D12 and 4D5 are highly effective at blocking the alternative pathway in primates. Single dose administration of mAb I3B1, 10D12 or 4D5 to cynomolgus monkeys resulted in sustained ablation of systemic alternative pathway activity lasting for approximately 16 days. The extent of alternative pathway ablation in cynomolgus monkeys treated with high affinity MASP-3 inhibitory antibodies was comparable to that achieved by factor D blockade in vitro and in vivo, indicating complété blockade of factor D conversion by the MASP-3 inhibitory antibodies. Therefore, high affinity MASP-3 inhibitory mAbs hâve therapeutic utility in the treatment of patients suffering from diseases related to alternative pathway hyperactivity

Accordingly, in one aspect the invention provides methods of inhibiting the alternative pathway in a mammalian subject in need thereof comprising administering to the subject a composition comprising an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to the serine protease domain of human MASP-3 (amino acid residues 450 to 728 of SEQ ID NO:2) with high affinity (having a Kd of less than 500 pM), in an amount effective to inhibits alternative pathway complément activation in the subject. In some embodiments, the subject is suffering from an alternative pathwayrelated disease or disorder, (i.e., a disease or disorder related to alternative pathway hyperactivity), such as for example, paroxysmal noctumal hemoglobinuria (PNH), agerelated macular degeneration (AMD, including wet and dry AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS),thrombotic thrombocytopénie purpura (TTP) or transplant-associated TMA), asthma, dense deposit disease, pauci-immune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behcet’s disease, multiple sclerosis, Guillain Barre Syndrome, Alzheimer’s disease, Amylotrophic latéral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy, Uveitis, Chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD), hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis, Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy and Myasthenia Gravis, as further described below.

A. THE ROLE OF MASP-3 IN PAROXYSMAL NOCTURNAL HEMOGLOBINURIA AND THERAPEUTIC METHODS USING MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION with MASP-2 INHIBITORY AGENTS

OverView of PNH

Paroxysmal nocturnal hemoglobinuria (PNH), sometimes also referred to as Marchiafava-Micheli syndrome, is an acquired, potentially life-threatening disease of the blood. PNH may develop on its own, referred to as primary PNH or in the context of other bone marrow disorders such as aplastic anémia, referred to as secondary PNH. The majority of cases are primary PNH. PNH is characterized by complement-induced destruction of red blood cells (hemolysis), low red blood cell counts (anémia), thrombosis and bone marrow failure. Laboratory findings in PNH show changes consistent with 5 intravascular hemolytic anémia: low hemoglobin, raised lactate dehydrogenase, raised réticulocyte counts (immature red cells released by the bone marrow to replace the destroyed cells), raised bilirubin (a breakdown product of hemoglobin), in the absence of autoreactive RBC-binding antibodies as a possible cause.

The hallmark of PNH is the chronic complement-mediated hemolysis caused by the 10 unregulated activation of terminal complément components, including the membrane attack complex, on the surface of circulating RBCs. PNH RBCs are subject to uncontrolled complément activation and hemolysis due to the absence of the complément regulators CD55 and CD59 on their surface (Lindorfer, M.A., et al., Blood 115(11):2283-91 (2010), Risitano, et al., Mini-Reviews in Médicinal Chemistry, 11:528-535 (2011)). CD55 and CD59 are 15 abundantly expressed on normal RBCs and control complément activation. CD55 acts as a négative regulator of the alternative pathway, inhibiting the assembly of the alternative pathway C3 convertase (C3bBb) complex and accelerating the decay of preformed convertase, thus blocking the formation of the membrane attack complex (MAC). CD59 inhibits the complément membrane attack complex directly by binding the C5b678 complex 20 and preventing C9 from binding and polymerizing.

While hemolysis and anémia are the dominant clinical features of PNH, the disease is a complex hématologie disorder that further includes thrombosis and bone marrow failure as part ofthe clinical findings (Risitano et al, Mini Reviews in Med Chem, 11:528-535 (2011)). At the molecular level, PNH is caused by the abnormal clonal expansion of hematopoietic 25 stem cells lacking a functional PIG A gene. PIG A is an X-linked gene encoding a glycosylphosphatidyl inositol transferase required for the stable surface expression of GPI-anchored class A glycoproteins, including CD55 and CD59. For reasons that are presently under investigation, hematopoietic stem cells with a dysfunctional PIG A gene that arise as the resuit of spontaneous somatic mutations can undergo clonal expansion to the point where 30 their progeny constitute a significant portion of the peripheral hematopoietic cell pool. While both érythrocyte and lymphocyte progeny of the mutant stem cell clone lack CD55 and CD59, only the RBCs undergo overt lysis after they enter the circulation.

Current treatment for PNH includes blood transfusion for anémia, anticoagulation for thrombosis and the use of the monoclonal antibody eculizumab (Soliris®), which protects blood cells against immune destruction by inhibiting the complément System (Hillmen P. et al., N. Engl. J. Med. 350(6):552-559 (2004)). Eculizumab (Soliris®) is a humanized monoclonal antibody that targets the complément component C5, blocking its cleavage by C5 convertases, thereby preventing the production of C5a and the assembly of MAC. Treatment of PNH patients with eculizumab has resulted in a réduction of intravascular hemolysis, as measured by lactate dehydrogenase (LDH), leading to hemoglobin stabilization and transfusion independence in about half of the patients (Risitano et al, Mini-Reviews in Médicinal Chemistry, 11(6) (2011)). While nearly ail patients undergoing therapy with eculizumab achieve normal or almost normal LDH levels (due to control of intravascular hemolysis), only about one third ofthe patients reach a hemoglobin value about 1 Igr/dL, and the remaining patients on eculizumab continue to exhibit moderate to severe (i.e., transfusion-dependent) anémia, in about equal proportions (Risitano A.M. et al., Blood 113:4094-100 (2009)). As described in Risitano et al., Mini-Reviews in Médicinal Chemistry 11:528-535 (2011), it was demonstrated that PNH patients on eculizumab contained large amounts of C3 fragments bound to their PNH érythrocytes (while untreated patients did not). This finding lead to the récognition that in Soliris treated PNH patients, PNH RBCs that are no longer hemolyzed due to C5 blockade now can accumulate copious amounts of membrane-bound C3 fragments, which operate as opsonins, resulting in their entrapment in the réticuloendothélial cells through spécifie C3 receptors and subséquent extravascular hemolysis. Thus, while preventing intravascular hemolysis and the resulting sequelae, eculizumab therapy simply diverts the disposition of these RBCs from intravascular to extravascular hemolysis, resulting in substantial residual untreated anémia in many patients (Risitano A.M. et al., Blood 113:4094-100 (2009)). Therefore, therapeutic strategies in addition to the use of eculizumab are needed for those patients developing C3-fragmentmediated extravascular hemolysis, because they continue to require red cell transfusions. Such C3 fragment targeting approaches hâve demonstrated utility in experimental Systems (Lindorfer et al., Blood 115:2283-91, 2010).

Complement-initiating mechanisms in PNH

The causal link between defective surface expression of the négative complément regulators CD55 and CD59 in PNH, combined with the effectiveness of eculizumab in preventing intravascular hemolysis, clearly define PNH as a condition mediated by the complément System. While this paradigm is widely accepted, the nature of the events initiating complément activation, and the complément activation pathway(s) involved remain unresolved. Because CD55 and CD59 negatively regulate the terminal amplification steps in 5 the complément cascade common to ail complément initiation pathways, deficiency of these molécules will lead to exaggerated formation and membrane intégration of membrane attack complexes, regardless of whether complément activation is initiated by the lectin pathway, by the classical pathway or by spontaneous turnover of the alternative pathway. Thus, in PNH patients, any complément activation events that lead to C3b déposition on the RBC surface 10 can trigger subséquent amplification and pathological hemolysis (intravascular and/or extravascular) and precipitate a hemolytic crisis. A clear mechanistic understanding of the molecular events triggering hemolytic crisis in PNH patients has remained elusive. Because no complément initiating event is overtly évident in PNH patients undergoing a hemolytic crisis, the prevailing view is that complément activation in PNH may occur spontaneously 15 owing to low level “tick-over” activation of the alternative pathway, which is subsequently magnified by inappropriate control of terminal complément activation due to lack of CD55 and CD59.

However, it is important to note that in its natural history, PNH usually develops or is exacerbated after certain events, such as an infection or an injury (Risitano, Biologics 2:20520 222 (2008)), which hâve been shown to trigger complément activation. This complément activation response is not dépendent on prior immunity of the host towards the inciting pathogen, and hence likely does not involve the classical pathway. Rather, it appears that this complément activation response is initiated by lectin binding to foreign or “altered self’ carbohydrate patterns expressed on the surface of microbial agents or damaged host tissue.

Thus, the events precipitating hemolytic crisis in PNH are tightly linked to complément activation initiated via lectins. This makes it very likely that lectin activation pathways provide the initiating trigger that ultimately leads to hemolysis in PNH patients.

Using well-defined pathogens that activate complément via lectins as experimental models to dissect the activation cascades at the molecular level, we demonstrate that, 30 depending on the inciting microbe, complément activation can be initiated by either LEA-2 or

LEA-1, leading to opsonization and/or lysis. This same principle of dual responses (i.e., opsonization and/or lysis) to lectin initiation events will likely also apply to other types of infectious agents, or to complément activation by lectins following tissue injury to the host, or other lectin-driven complément activation events that may precipitate PNH. On the basis of this duality in the lectin pathway, we infer that LEA-2- and/or LEA-1-initiated complément activation in PNH patients promûtes opsonization and/or lysis of RBCs with C3b and subséquent extravascular and intravascular hemolysis. Therefore, in the setting of PNH, inhibition of both LEA-1 and LEA-2 would be expected to address both intravascular and extravascular hemolysis, providing a significant advantage over the C5 inhibitor eculizumab.

It has been determined that exposure to S. pneumoniae preferentially triggers lectindependent activation of LEA-2, which leads to opsonization of this microbe with C3b. Since S. pneumonia is résistant to MAC-mediated lysis, its clearance from circulation occurs through opsonisation with C3b. This opsonization and subséquent removal from circulation is LEA-2-dependent, as indicated by compromised bacterial control in MASP-2-deficient mice and in mice treated with MASP-2 monoclonal antibodies (PLOS Pathog., 8: e 1002793. (2012)).

In exploring the rôle of LEA-2 in the innate host responses to microbial agents, we tested additional pathogens. A dramatically different outcome was observed when Neisseria meningitidis was studied as a model organism. N. meningitidis also activâtes complément via lectins, and complément activation is required for contaînment of N. meningitidis infections in the naïve host. However, LEA-2 plays no host protective functional rôle in this response: As shown in FIGURES 6 and 7, blockade of LEA-2 through genetic ablation of MASP-2 does not reduce survival following infection with N. meningitidis. To the contrary, LEA-2 blockade by MASP-2 ablation significantly improved survival (FIGURES 6 and 7) as well as illness scores (FIGURE 9) in these studies. LEA-2 blockade by administration of MASP-2 antibody yielded the same resuit (FIGURE 10), eliminating secondary or compensatory effects in the knockout-mouse strain as a possible cause. These favorable outcomes in LEA-2-ablated animais were associated with a more rapid élimination of N. meningitidis from the blood (FIGURE 8). Also, as described herein, incubation of N. meningitidis with normal human sérum killed N. meningitidis (FIGURE 11). Addition of a functional monoclonal antibody spécifie for human MASP-2 that blocks LEA-2, but not administration of an isotype control monoclonal antibody, may enhance this killing response. Yet, this process dépends on lectins and at least a partially functional complément System, as

MBL-deficient human sérum or heat-inactivated human sérum was unable to kill N. meningitidis (FIGURE 11). Collectively, these novel findings suggest that N. meningitidis infections in the presence of a functional complément system are controlled by a lectindependent but LEA-2-independent pathway of complément activation.

The hypothesis that LEA-1 may be the complément pathway responsible for lectindependent killing of N. meningitidis was tested using a sérum specimen from a 3MC patient. This patient was homozygous for a nonsense mutation in exon 12 of the MASP-1/3 gene. As a resuit, this patient lacked a functional MASP-3 protein, but was otherwise complément sufficient (exon 12 is spécifie for the MASP-3 transcript; the mutation has no effect on 10 MASP-1 function or expression levels) (see Nat Genet 43(3):197-203 (2011)). Normal human sérum eflficiently kills N. meningitidis, but heat-inactivated sérum déficient in MBL (one ofthe récognition molécules for the Lectin pathway) and MASP-3-deficient sérum were unable to kill N. meningitidis (FIGURE 12). Thus, LEA-1 appears to médiate N. meningitidis killing. This finding was confirmed using sérum samples from knockout mouse 15 strains. While complément containing normal mouse sérum readily killed N. meningitidis, MBL-deficient or MASP-l/3-deficient mouse sérum was as ineffective as heat-inactivated sérum that lacks functional complément (FIGURE 13). Conversely, MASP-2-déficient sérum exhibited efficient killing of N. meningitidis.

These findings provide evidence for a hitherto unknown duality in the lectin pathway 20 by revealing the existence of separate LEA-2 and LEA-1 pathways of lectin-dependent complément activation. In the examples detailed above, LEA-2 and LEA-1 are nonredundant and médiate distinct, functional outcomes. The data suggest that certain types of lectin pathway activators (including, but not limited to S. pneumonia) preferentially initiale complément activation via LEA-2 leading to opsonization, while others (exemplified by N.

meningitidis) preferentially initiale complément activation via LEA-1 and promote cytolytic processes. The data do not, however, necessarily limit LEA-2 to opsonization and LEA-1 to cytolytic processes, as both pathways in other settings can médiate opsonization and/or lysis.

In the context of lectin-dependent complément activation by N. meningitidis, LEA-2 and LEA-1 arms appear to compete with each other, as blockade of LEA-2 enhanced LEA-1 30 dépendent lytic destruction of the organism in vitro (FIGURE 13). As detailed above, this finding can be explained by the increased likelihood of lectin MASP-1 complexes residing in close proximity to lectin MASP-3 complexes in the absence of MASP-2, which will enhance

LEA-1 activation and thus promote more effective lysis of N. meningitides. Because lysis of N. meningitidis is the main protective mechanism in the naïve host, blockade of LEA-2 in vivo increases N. meningitidis clearance and leads to enhanced killing.

While the examples discussed above illustrate opposing effects of LEA-2 and LEA-I with respect to outcomes following infection with N. meningitidis, there may be other settings where both LEA-2 and LEA-1 may synergize to produce a certain outcome. As detailed below, in other situations of pathological complément activation via lectins such as those présent in PNH, LEA-2- and LEA-I-driven complément activation may cooperate in a synergistic manner to contribute to the overall pathology of PNH. In addition, as described herein, MASP-3 also contributes to the lectin-independent conversion of factor B and factor D, which can occur in the absence of Ca++, commonly leading to the conversion of C3bB to C3bBb and of pro-factor D to factor D, which may further contribute to the pathology of PNH.

Biology and expected functional activity in PNH ’

This section describes the inhibitory effects of LEA-2 and LEA-1 blockade on hemolysis in an in vitro model of PNH. The findings support the utility of LEA-2-blocking agents (including, but not limited to, antibodies that bind to and block the function of MASP2) and LEA-1-blocking agents (including, but not limited to, antibodies that bind to and block the function of MASP-1-mediated activation of MASP-3, MASP-3, or both) to treat subjects suffering from one or more aspects of PNH, and also the use of inhibitors of LEA-2 and/or LEA-I, and/or MASP-3-dependent, lectin-independent complément activation (including MASP-2 inhibitors, MASP-3 inhibitors, and dual- or bispecific MASP-2/MASP-3 or MASPl/MASP-2 inhibitors, and pan-specific MASP-1/MASP-2/MASP-3 inhibitors) to ameliorate the effects of C3-fragment-mediated extravascular hemolysis in PNH patients undergoing therapy with a C5-inhibitor such as eculizumab.

MASP-2 inhibitors to block opsonization and extravascular hemolysis of PNH RBCs through the réticuloendothélial System

As detailed above, PNH patients become anémie owing to two distinct mechanisms of RBC clearance from circulation: intravascular hemolysis via activation of the membrane attack complex (MAC), and extravascular hemolysis following opsonization with C3b and subséquent clearance following complément receptor binding and uptake by the réticuloendothélial System. The intravascular hemolysis is largely prevented when a patient is treated with eculizumab. Because eculizumab blocks the terminal lytic effector mechanism that occurs downstream of both the complement-initiating activation event as well as the ensuing opsonization, eculizumab does not block extravascular hemolysis (Risitano A.M. et al., Blood 113:4094-100 (2009)). Instead, RBCs that would hâve undergone hemolysis in untreated PNH patients now can accumulate activated C3b proteins on their surface, which augments uptake by the réticuloendothélial system and enhances their extravascular hemolysis. Thus, eculizumab treatment effectively diverts RBC disposition from intravascular hemolysis to potential extravascular hemolysis. As a resuit, some eculizumabtreated PNH patients remain anémie. It follows that agents that block complément activation upstream and prevent the opsonization of PNH RBCs may be particularly suitable to block the extravascular hemolysis occasionally seen with eculizumab.

The microbial data presented here suggest that LEA-2 is often the dominant route for lectin-dependent opsonization. Furthermore, when lectin-dependent opsonization (measured as C3b déposition) was assessed on three prototypic lectin activation surfaces (mannan, FIGURE 17A; zymosan, FIGURE 17B, and S. pneumonia; FIGURE 17C), LEA-2 appears to be the dominant route for lectin-dependent opsonization under physiologie conditions (i.e., in the presence of Ca⁺⁺ wherein ail complément pathways are operational). Under these experimental conditions, MASP-2-deficient sérum (which lacks LEA-2) is substantially less effective in opsonizing the test surfaces than WT sérum. MASP-l/3-deficient sérum (which lacks LEA-1) is also compromised, though this effect is much less pronounced as compared to sérum lacking LEA-2. The relative magnitude of the contributions of LEA-2 and LEA-1 to lectin-driven opsonization is further illustrated in FIGURES 18A-18C. While the alternative pathway of complément has been reported to support opsonization of lectin activating surfaces in the absence of lectin pathway or classical pathway (Selander et al., J Clin Invest 116(5):1425-1434 (2006)), the alternative pathway in isolation (measured under Ca^-free assay conditions) appears substantially less effective than the LEA-2- and LEA-1 initiated processes described herein. By extrapolation, these data suggest that opsonization of PNH RBCs may also be preferentially initiated by LEA-2 and, to a lesser extent, by LEA-1 (possibly amplified by the alternative pathway amplification loop), rather than the resuit of lectin-independent alternative pathway activation. Therefore, LEA-2 inhibitors may be expected to be most effective at limiting opsonization and preventing extravascular hemolysis in PNH. However, récognition of the fact that lectins other than MBL, such as ficolins, bind to non-carbohydrate structures such as acetylated proteins, and that MASP-3 preferentially associâtes with H-ficolin (Skjoedt et al., Immunobiol. 215:921-931, 2010), leaves open the possibility of a significant rôle for LEA-1 in PNH-associated RBC opsonization as well. Therefore, LEA-1 inhibitors are expected to hâve additional anti-opsonization effects, and the combination of LEA-1 and LEA-2 inhibitors is expected to be optimal and médiate the most robust treatment benefit in limiting opsonization and extravascular hemolysis in PNH patients. Thus, LEA-2 and LEA-1 act additively or synergistically to promote opsonization, and a crossreactive or bispecific LEA-1/LEA-2 inhibitor is expected to be most effective at blocking opsonization and extravascular hemolysis in PNH.

Rôle of MASP-3 inhibitors in PNH

Using an in vitro model of PNH, we demonstrated that complément activation and the resulting hemolysis in PNH are indeed initiated by LEA-2 and/or LEA-1 activation, and that it is not an independent function of the alternative pathway. These studies used mannansensitized RBCs of various mouse stains, including RBCs from Crry-deficient mice (an important négative regulator of the terminal complément pathway in mice) as well as RBCs from CD55/CD59-deficient mice, which lack the same complément regulators that are absent in PNH patients). When mannan-sensitized Crry-deficient RBCs were exposed to complement-sufficient human sérum, the RBCs effectively hemolysed at a sérum concentration of 3% (FIGURE 19 and 20) while complement-deficient sérum (HI: heatinactivated) was not hemolytic. Remarkably, complement-sufficient sérum where LEA-2 was blocked by addition of MASP-2 antibody had reduced hemolytic activity, and 6% sérum was needed for effective hemolysis. Similar observations were made when CD55/CD59deficient RBCs were tested (FIGURE 22). Complement-sufficient human sérum supplemented with MASP-2 monoclonal antibody (i.e., sérum where LEA-2 is suppressed) was about two-fold less effective than untreated sérum in supporting hemolysis. Furthermore, higher concentrations of LEA-2-blocked sérum (i.e., treated with antiMASP-2 monoclonal antibody) were needed to promote effective hemolysis of untreated WT RBCs compared to untreated sérum (FIGURE 21).

Even more surprisingly, sérum from a 3MC patient homozygous for a dysfunctional MASP-3 protein (and hence lacking LEA-I) was completely unable to hemolyze mannansensitized Crry-deficient RBCs (FIGURE 20 and FIGURE 21). A similar outcome was observed when unsensitized normal RBCs were used: As shown in FIGURE 21, LEA-I defective sérum isolated from a 3MC patient was completely ineffective at mediating hemolysis. Collectively, these data indicate that whereas LEA-2 contributes significantly to the intravascular hemolysis response, LEA-1 is the prédominant complement-initiating pathway leading to hemolysis. Thus, while LEA-2 blocking agents are expected to significantly reduce intravascular hemolysis of RBCs in PNH patients, LEA-1 blocking agents are expected to hâve a more profound effect and largely eliminate complement-driven hemolysis. It should be noted that the sérum of the LEA-1-déficient 3MC patient used in this study possessed a diminished but functional alternative pathway when tested under conventional alternative pathway assay conditions (FIGURE 15). This finding suggests that LEA-1 makes a greater contribution to hemolysis than alternative pathway activity as conventionally defined in this experimental setting of PNH. By inference, it is implied that LEA-1-blocking agents will be at least as effective as agents blocking other aspects of the alternative pathway in preventing or treating intravascular hemolysis in PNH patients.

Rôle of MASP-2 inhibitors in PNH

The data presented herein suggest the following pathogenic mechanisms for anémia in PNH: intravascular hemolysis due to unregulated activation of terminal complément components and lysis of RBC by formation of MAC, which is initiated predominantly, though not exclusively, by LEA-1, and extravascular hemolysis caused by opsonization of RBCs by C3b, which appears to be initiated predominately by LEA-2. While a discemible rôle for LEA-2 in initiating complément activation and promoting MAC formation and hemolysis is apparent, this process appears substantially less effective than LEA-1-initiated complément activation leading to hemolysis. Thus, LEA-2-blocking agents are expected to significantly reduce intravascular hemolysis in PNH patients, though this therapeutic activity is expected to be only partial. By comparison, a more substantial réduction in intravascular hemolysis in PNH patients is expected for LEA-1-blocking agents.

Extravascular hemolysis, a less dramatic, yet equally important mechanism of RBC destruction that leads to anémia in PNH, is primarily the resuit of opsonization by C3b, which appears to be predominantly mediated by LEA-2. Thus, LEA-2-blocking agents may be expected to preferentially block RBC opsonization and the ensuing extravascular hemolysis in PNH. This unique therapeutic activity of LEA-2-blocking agents is expected to provide a significant treatment benefit to ail PNH patients as no treatment currently exists for those PNH patients who expérience this pathogenic process.

LEA-2 inhibitors as adjunct treatment to LEA-1 inhibitors or terminal complément blocking agents

The data presented herein detail two pathogenic mechanisms for RBC clearance and anémia in PNH which can be targeted, separately or in combination, by distinct classes of therapeutic agents: the intravascular hemolysis initiated predominantly, though not exclusively, by LEA-1 and thus expected to be effectively prevented by a LEA-1-blocking agent, and extravascular hemolysis due to C3b opsonization driven predominantly by LEA-2, 10 and thus effectively prevented by a LEA-2-blocking agent.

It is well documented that both intravascular and extravascular mechanisms of hemolysis lead to anémia in PNH patients (Risitano et al., Blood 113:4094-4100 (2009)). Therefore, it is expected that a LEA-1-blocking agent that prevents intravascular hemolysis in combination with a LEA-2 blocking agent that primarily prevents extravascular hemolysis 15 will be more effective than either agent alone in preventing the anémia that develops in PNH patients. In fact, the combination of LEA-1- and LEA-2-blocking agents is expected to prevent ail relevant mechanisms of complément initiation in PNH and thus block ail symptoms of anémia in PNH.

It is also known that C5-blocking agents (such as eculizumab) effectively block 20 intravascular hemolysis but do not interfère with opsonization. This leaves some anti-C5treated PNH patients with substantial residual anémia due to extravascular hemolysis mediated by LEA-2 that remains untreated. Therefore, it is expected that a C5-bIocking agent (such as eculizumab) that prevents intravascular hemolysis in combination with a LEA2 blocking agent that reduces extravascular hemolysis will be more effective than either agent 25 alone in preventing the anémia that develops in PNH patients.

Other agents that block the terminal amplification loop of the complément system leading to C5 activation and MAC déposition (including, but not limited to agents that block properdin, factor B or factor D or enhance the inhibitory activity of factor I, factor H or other complément inhibitory factors) are also expected to inhibit intravascular hemolysis. However, 30 these agents are not expected to interfère with LEA-2-mediated opsonization in PNH patients. This leaves some PNH patients treated with such agents with substantial residual anémia due to extravascular hemolysis mediated by LEA-2 that remains untreated. Therefore, it is expected that treatment with such agents that prevent intravascular hemolysis in combination with a LEA-2-blocking agent that minimizes extravascular hemolysis will be more effective than either agent alone in preventing the anémia that develops in PNH patients. In fact, the combination of such agents and a LEA-2 blocking agent is expected to 5 prevent ail relevant mechanisms of RBC destruction in PNH and thus block ail symptoms of anémia in PNH.

Use of LEA-1 and LEA-2 multiple, bispecific or pan-specific antibodies to treat PNH

As detailed above, the use of a combination of pharmacologie agents that individually block LEA-1 and LEA-2, and thus in combination block ail complément activation events 10 that médiate the intravascular as well as the extravascular hemolysis, is expected to provide the best clinical outcome for PNH patients. This outcome can be achieved for example, by co-administration of an antibody that has LEA-1-blocking activity together with an antibody that has LEA-2-blocking activity. In some embodiments, LEA-1- and LEA-2-blocking activities are combined into a single molecular entity, and that such entity with combined 15 LEA-1- and LEA-2-blocking activity will effectively block intravascular as well as the extravascular hemolysis and prevent anémia in PNH. Such an entity may comprise or consist of a bispecific antibody where one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and diminishes LEA-2 and the second antigen-combining site specifically recognizes MASP-2 and further blocks LEA-2. Alternatively, such an entity may consist of a 20 bispecific monoclonal antibody where one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may optimally consist of a bispecific monoclonal antibody where one antigen-combining site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 and diminishes LEA-2 while the second antigen25 combining site specifically recognized MASP-2 and further blocks LEA-2. Based on the similarities in the overall protein sequence and architecture, it can also be envisioned that a conventional antibody with two identical binding sites can be developed that specifically binds to MASP-1 and to MASP-2 and to MASP-3 in a functional manner, thus achieving functional blockade of LEA-1 and LEA-2. Such an antibody with pan-MASP inhibitory 30 activity is expected to block both the intravascular as well as the extravascular hemolysis and thus effectively treat the anémia in PNH patients.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies hâve been generated which hâve therapeutic utility for inhibition ofthe alternative pathway in AP-related diseases or conditions, such as PNH.

Accordingly, in one embodiment, the présent invention provides a method for treating a subject suffering from, or at risk for developing PNH comprising an effective amount of a high affinity monoclonal antibody or antigen binding fragment thereof as disclosed herein that binds to human MASP-3 and inhibits alternative pathway complément activation to treat or reduce the risk of PNH in the subject.

In one embodiment, the présent invention provides a method for treating a subject suffering from, or at risk for developing paroxysmal nocturnal hemoglobinuria (PNH), comprising administering to the subject a pharmaceutical composition comprising an effective amount of a monoclonal antibody or antigen binding fragment thereof as disclosed herein that binds to human MASP-3 and inhibits alternative pathway complément activation to treat or reduce the risk of PNH in the subject, such as, wherein said antibody or antigen binding fragment thereof comprises (a) a heavy chain variable région comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable région comprising (i) VLCDRI comprising SEQ ID NO: 142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259, (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161. In some embodiments, the pharmaceutical composition increases the survival of red blood cells in the subject suffering from PNH. In some embodiments, wherein the subject suffering from or at risk for developing PNH exhibits one or more symptoms selected from the group consisting of (i) below normal levels of hemoglobin, (ii) below normal levels of platelets; (iii) above normal levels of réticulocytes, and (iv) above normal levels of bilirubin. In some embodiments, the pharmaceutical composition is administered systemically (e.g., subcutaneously, intra-muscularly, intravenously, intraarterially or as an inhalant) to a subject suffering from, or at risk for developing PNH. In some embodiments, the subject suffering from or at risk for PNH has previously undergone, or is currently undergoing treatment with a terminal complément inhibitor that inhibits cleavage of complément protein C5. In some embodiments, the method further comprises administering to the subject a terminal complément inhibitor that inhibits cleavage of complément protein C5. In some embodiments, the terminal complément inhibitor is a humanized anti-C5 antibody or antigen-binding fragment thereof. In some embodiments, the terminal complément inhibitor is eculizumab.

B. THE ROLE OF MASP-3 IN AGE-RELATED MACULAR DEGENERATION AND THERAPEUTIC METHODS USING MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION WITH AND MASP-2 INHIBITORY AGENTS

Age related macular degeneration (AMD) is the leading cause of visual impairment and blindness in the elderly and accounts for up to 50% of cases of blindness in developed countries. The prevalence of AMD is around 3% in adults and increases with âge such that almost two-thirds of the population over 80 years of âge will hâve some signs. It is estimated that over 1.75 million individuals in the United States hâve advanced AMD and the prevalence is increasing as the population âges and is expected to reach almost 3 million by 2020 (Friedman, D.S., et al., Arch. Ophthalmol. 122:564-572, 2004). AMD is an abnormality of the retinal pigment epithelium (RPE) that results in degeneration of the photoreceptors of the overlying central retina, or macula, and loss of central vision. Early and intermediate forms of AMD are characterized by progressive deposits of drusen, a yellowish material containing lipid, protein, lipoprotein, and cellular débris, in the subretinal space adjacent to the RPE, along with pigmentary irregularities in the retina. Advanced AMD consists of two clinical subtypes: non-neovascular géographie atrophie (‘dry’) AMD and neovascular exudative (‘wet’) AMD. Although dry AMD accounts for 80-90% of advanced AMD, the majority of sudden and severe vision loss occurs in patients with wet AMD. It is not known whether the two types of AMD represent differing phenotypes arising from similar pathologies or two distinct conditions. Currently no therapy has been approved by the United States Food and Drug Administration (FDA) to treat dry AMD. FDAapproved treatment options for wet AMD include intravitreal injections of anti-angiogenic drugs (ranibizumab, pegaptanib sodium, aflibercept), laser therapy, photodynamic laser therapy, and implantable telescope.

The etiology and pathophysiology of AMD are complex and incompletely understood. Several lines of evidence support the rôle of dysrégulation of the complément System in the pathogenesis of AMD. Gene association studies hâve identified multiple genetic loci associated with AMD, including genes coding for a range of complément proteins, factors, and regulators. The strongest association is with polymorphisms in the complément factor H (CFH) gene, with the Y402H variant homozygotes having approximately 6-fold and hétérozygotes approximately 2.5-fold increased risk for developing AMD compared to the non-risk génotype (Khandhadia, S., et al., Immunobiol. 217:127-146, 2012). Mutations in other complément pathway encoding genes hâve also been associated with increased or decreased risk of AMD, including complément factor B (CFB), C2, C3, factor I, and CFH-related proteins 1 and 3 (Khandhadia et al.). Immunohistochemical and proteomic studies in donor eyes from AMD patients showed that proteins of the complément cascade to be increased and localized in drusen (Issa, P.C., et al., Graefes. Arch. Clin. Exp. Ophthalmol. 249:163-174, 2011). Furthermore, AMD patients hâve increased systemic complément activation as measured in peripheral blood (Issa et al., 2011, supra).

The alternative pathway of complément appears to be more relevant than the classical pathway in the pathogenesis of AMD. Clq, the essential récognition component for activation of the classical pathway, was not detected in drusen by immunohistochemical analyses (Mullins et al., FASEB J. 14:835 846, 2000; Johnson et al., Exp. Eye Res. 70:441 449, 2000). Genetic association studies hâve implicated CFH and CFB genes. These proteins are involved in the alternative pathway amplification loop, with CFH being a fluid phase inhibitor and CFB being an activating protease component of the alternative pathway. The Y402H variant of CFH affects interaction with ligand binding, including binding with Creactive protein, heparin, M protein, and glycosaminoglycans. This altered binding to ligands may reduce binding to cell surfaces, which in tum may lead to reduced factor I mediated dégradation of C3b activation fragment and impaired régulation of the alternative C3 convertase, resulting in over activation ofthe alternative pathway (Khandhadia et al., 2012, supra). Variations in the CFB gene are associated with a protective effect for the development of AMD. A functional variant ÎB32Q had 4 times less binding affinity to C3b than the risk variant ÎB32R, resulting in a réduction in C3 convertase formation (Montes, T. et al., Proc. Natl. Acad. Sci. U.S.A. 106:4366-4371,2009).

Complement-initiating mechanisms in AMD

The human genetic linkage studies discussed above suggest an important rôle for the complément system in AMD pathogenesis. Furthermore, complément activation products are abundantly présent in drusen (Issa, P.C., et al., Graefes. Arch. Clin. Exp. Ophthalmol.

249:163-174, 2011), a hallmark pathologie lésion in both wet and dry AMD. However, the nature of the events initiating complément activation, and the complément activation pathway(s) involved remain incompletely understood. '

It is important to note that drusen deposits are composed of cellular débris and oxidative waste products originating from the retina that accumulate beneath the RPE as the eye âges. In addition, oxidative stress appears to play an important rôle (Cai et al; Front Biosci., 17:1976-95, 2012), and has been shown to cause complément activation in RPE (J Biol Chem., 284(25):16939-47, 2009). It is widely appreciated that both oxidative stress and cellular or tissue injury activate the complément System lectins. For example, Collard et al. hâve demonstrated that endothélial cells exposed to oxidative stress trigger abundant complément déposition mediated by lectins (Collard CD et al., Mol Immunol., 36(13-14):9418, 1999; Collard C.D. et al., Am J Pathol., 156(5):1549-56, 2000), and that blockade of lectin binding and lectin-dependent complément activation improves outcomes in experimental models of oxidative stress injury (Collard C.D. et al., Am J Pathol., 156(5): 1549-56, 2000). Thus, it appears likely that oxidative waste products présent in drusen also activate complément via the lectins. By inference, lectin-dependent complément activation may play a pivotai rôle in AMD pathogenesis.

The rôle of the complément System has been evaluated in mouse models of AMD. In the light-damage mouse model, an experimental model for oxidative stress-mediated photoreceptor degeneration, knockout mice with an élimination of the classical pathway (Clqa-/- on a C57BL/6 background) had the same sensitivity to light damage compared to wild-type littermates, whereas élimination of complément factor D ofthe alternative pathway (CFD-/-) resulted in protection from light damage (Rohrer, B. et al., Invest. Ophthalmol. Vis. Sci. 48:5282-5289, 2007). In a mouse model of choroidal neovascularization (CNV) induced by laser photocoagulation of the Bruch membrane, knockout mice without complément Factor B (CFB-/-) were protected against CNV compared with wild-type mice (Rohrer, B. et al., Invest. Ophthalmol. Vis. Sci. 50:3056-3064, 2009). In the same model, intravenous administration of a recombinant form of complément Factor H targeted to sites of complément activation (CR2-fH) reduced the extent of CNV. This protective effect was observed whether CR2-fH was administered at the time of laser injury or therapeutically (after laser injury). A human therapeutic version of CR2-fH (TT30) was also efTicacious in the murine CNV model (Rohrer, B. et al. J. Ocul. Pharmacol. Ther.,28:402-409, 2012).

Because fB is activated by LEA-1, and because MASP-1 and MASP-3 contribute to the maturation of factor D, these findings imply that LEA-1 inhibitors may hâve therapeutic benefit in AMD patients. In addition, recent results reported from a Phase 2 study hâve shown that monthly intravitreal injection with Lampalizumab (previously referred to as FCFD4514S and anti-factor D, which is an antigen-binding fragment of a humanized monoclonal antibody directed against Factor D) reduced géographie atrophy area progression in patients with géographie atrophy secondary to AMD (Yaspan B.L. et al., Sci Transi. Med. 9, Issue 395, June 21, 2017).

Initial experimental studies in a rodent model of AMD using MBL-deficient mice did not support a critical rôle for the lectin pathway in pathogenic complément activation (Rohrer et al., Mol Immunol. 48:el-8, 2011). However, MBL is only one of several lectins, and lectins other than MBL may trigger complément activation in AMD. Indeed, our previous work has shown that MASP-2, the rate-limiting serine protease that is critically required for lectin pathway function, plays a critical rôle in AMD. As described in US Patent No. 7,919,094 (assigned to Omeros Corporation), incorporated herein by reference, MASP-2deficient mice and mice treated with MASP-2 antibody were protected in a mouse model of laser-induced CNV, a validated preclinical model of wet AMD (Ryan et al., Tr Am Opth Soc LXXVII:707-745, 1979). Thus, inhibitors of LEA-2 are expected to effectively prevent CNV and improve outcomes in AMD patients.

Thus, in view of the above, LEA-1 and LEA-2 inhibitors are expected to hâve independent therapeutic benefit in AMD. In addition, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone, or may provide effective treatment for a wider spectrum of patient subsets. Combined LEA-1 and LEA-2 inhibition may be accomplished by co-administration of a LEA-1-blocking agent and a LEA-2-blocking agent. Optimally, LEA-1 and LEA-2 inhibitory function may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP1/3 and a MASP-2-specific binding site, or a dual specificity antibody where each binding site can bind to and block MASP-1/3 or MASP-2.

In accordance with the foregoing, an aspect of the invention thus provides a method for inhibiting LEA-1 dépendent complément activation to treat age-related macular degeneration (wet and dry forms) by administering a composition comprising a therapeutically effective amount of a MASP 1 inhibitory agent, a MASP 3 inhibitory agent, or a combination of a MASP 1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from such a condition. The MASP 1, MASP 3, or MASP 1/3 inhibitory composition may be administered locally to the eye, such as by irrigation, intravitreal administration, or application of the composition in the form of a gel, salve or drops.

Altemately, the MASP I, MASP 3, or MASP 1/3 inhibitory agent may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

In one embodiment, the method according to this aspect of the invention further comprises inhibiting LEA-2-dependent complément activation in a subject suffering from age-related macular degeneration, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3 or MASP1/3 inhibitory agent to the subject in need thereof. As detailed above, the use of a combination of pharmacologie agents that individually block LEA-1 and LEA-2 is expected to provide an improved therapeutic outcome in AMD patients as compared to the inhibition of LEA-1 alone. This outcome can be achieved for example, by co-administration of an antibody that has LEA-1 blocking activity together with an antibody that has LEA-2-blocking activity. In some embodiments, LEA-1- and LEA-2-blocking activities are combined into a single molecular entity, and that such entity with combined LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody where one antigen-combining site specifîcally recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifîcally recognizes MASP-2 and blocks LEA-2. Altematively, such an entity may consist of a bispecific monoclonal antibody where one antigen-combining site specifîcally recognizes

MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifîcally recognizes MASP-2 and blocks LEA-2. Such an entity may optimally consist of a bispecific monoclonal antibody where one antigen-combining site specifîcally recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifîcally recognized MASP-2 and blocks LEA-2.

The MASP 2 inhibitory composition may be administered locally to the eye, such as by irrigation, intravitreal injection or topical application of the composition in the form of a gel, salve or drops. Altemately, the MASP 2 inhibitory agent may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and optional MASP 2 inhibitory compositions of the présent invention may be carried out by a single administration of the composition (e.g., a single composition comprising MASP-2 and MASP-3 inhibitory agents, or bispecific or dual inhibitory agents, or co-administration of separate compositions), or a limited sequence of administrations, for treatment of AMD. Alternatively, the composition 10 may be administered at periodic intervals such as daily, biweekly, weekly, every other week, monthly or bimonthly over an extended period of time for treatment of AMD.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies hâve been generated which hâve therapeutic utility for inhibition of the alternative pathway in AP-related diseases or conditions, such as AMD.

Accordingly, in one embodiment, the présent invention provides a method for treating a subject suffering from, or at risk for developing AMD comprising an effective amount of a high affinity monoclonal antibody or antigen binding fragment thereof as disclosed herein that binds to human MASP-3 and inhibits alternative pathway complément activation to treat or reduce the risk of AMD in the subject. In one embodiment, the présent invention provides a method for treating a subject suffering from, or at risk for developing AMD comprising administering to the subject a pharmaceutical composition comprising an effective amount of a monoclonal antibody or antigen binding fragment thereof as disclosed herein that binds to human MASP-3 and inhibits alternative pathway complément activation to treat or reduce the risk of AMD in the subject, such as, for example, wherein said antibody or antigen binding fragment thereof comprises (a) a heavy chain variable région comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable région comprising (i) VLCDRI comprising SEQ ID NO: 142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259, (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising

SEQIDNO:161.

.

C. THE ROLE OF MASP-3 IN ISCHEMIA REPERFUSION INJURY AND THERAPEUTIC METHODS USING MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITORY AGENTS

Tissue ischemia is the basis for a wide spectrum of clinical disorders. Although timely restoration of blood flow is essential to préservation of ischémie tissue, it has long been recognized that reperfusion, which can occur either spontaneously or through therapeutic intervention, may lead to additional tissue injury, a phenomenon that has been termed ischemia reperfusion (I/R) injury (Eltzschig, H.K. and Tobias, E., Nat. Med. 17:1391-

1401, 2011). I/R injury may affect single organs, such as the heart (acute coronary syndrome), kidney (acute kidney injury), intestine (intestinal I/R), and brain (stroke). I/R injury may also affect multiple organs, such as following major trauma and resuscitation (multiple organ failure), circulatory arrest (hypoxie brain injury, acute kidney injury), peripheral vascular disease, and sickle cell disease (acute chest syndrome, acute kidney injury). Major surgery may be associated with I/R injury, including cardiac surgery (acute heart failure after cardiopulmonary bypass), thoracic surgery (acute lung injury), peripheral vascular surgery (compartment syndrome), vascular surgery (acute kidney injury), and solid organ transplantation (acute graft failure). Currently there are no spécifie thérapies that target I/R injury and there is a need for effective treatments in order to maximize the salvage of tissue in the ischémie zone and improve functional outcome in these common settings.

The pathophysiology of I/R injury is complex and characterized by a robust inflammatory response following reperfusion. Activation of the complément System has been implicated as an important component of I/R injury and inhibition of complément activity has been efficacious in a variety of animal models (Diepenhorst, G.M.P. et al., Ann. Surg.

249:889-899, 2009). The relative importance of the classical, lectin, and alternative pathways in I/R injury is largely unsettled and may dififer depending on the organs affected. Recently the availability of knockout mice déficient in spécifie complément proteins and pathwayspecific inhibitors has generated data that implicate the lectin and alternative pathways in I/R injury.

The rôle of the alternative pathway in gastrointestinal I/R injury was investigated using factor D-deficient (-/-) and heterozygotus (+/-) mice (Stahl, G.L., et al. Am. J. Pathol. 162:449-455, 2003). Following transient gastrointestinal ischemia, intestinal and pulmonary injury were reduced but not prevented in factor D-deficient mice compared with heterozygotus mice, and addition of human factor D to Factor D (-/-) mice restored I/R injury. The same model was evaluated in Clq-deficient and MBL-A/C-deficient mice and the results showed that gastrointestinal I/R injury was independent of Clq and classical 5 pathway activation, but that MBL and lectin pathway activation was required for intestinal injury (Hart, M.L., et al. J. Immunol. 174:6373-6380, 2005). Conversely, the Clq récognition molécule of the classical pathway was responsible for pulmonary injury after intestinal I/R (Hart, M.L., et al. J. Immunol. 174:6373-6380, 2005). One hypothesis is that activation of complément during I/R injury occurs through natural IgM binding to self10 antigens présent on the surface of ischémie (but not normal) tissue, for example non-muscle myosin heavy chains type II. In a mouse gastrointestinal I/R injury model, immunocomplexes from gut tissue were evaluated for the presence of initiating factors in the classical (Clq), lectin (MBL), or alternative (Factor B) pathways (Lee, H., et al., Mol. Immunol. 47:972-981, 2010). The results showed that Clq and MBL were detected whereas Factor B was not 15 detected in these immunocomplexes, indicating involvement of the classical and lectin pathways but not the alternative pathway. In the same model, Factor B-deficient mice were not protected from local tissue injury, providing additional support for the lack of involvement of the alternative pathway. The rôle of the lectin pathway in gastrointestinal I/R injury was directly evaluated in MASP-2-deficient mice and the results showed that gastrointestinal injury was reduced in these mice compared with wide-type Controls; treatment with MASP-2 monoclonal antibody was similarly protective (Schwaeble, W.J., et al., Proc. Natl. Acad. Sci. 108:7523-7528, 2011). Taken together, these results provide support for the involvement of the lectin pathway in gastrointestinal I/R injury, with conflicting data regarding involvement ofthe alternative pathway.

In a mouse myocardial I/R injury model, a pathogenic rôle was demonstrated for the lectin pathway as MBL-deficient mice were protected from myocardial injury whereas Clqdeficient and C2/fB-deficient mice were not (Walsh, M.C. et al., J. Immunol. 175:541-546, 2005). Protection from myocardial I/R injury was also observed in MASP-2-deficient mice (Schwaeble, W.J., et al., Proc. Natl. Acad. Sci. 108:7523-7528, 2011). Treatment of rats in a 30 myocardial I/R model with monoclonal antibodies against rat MBL resulted in reduced postischemic reperfusion injury (Jordan, J.E., et al., Circulation 104:1413 18, 2001). In a study of myocardial infarction patients treated with angioplasty, MBL deficiency was associated with reduced 90-day mortality compared to MBL-sufficient counterparts (M Trendelenburg étal., Eur Heart J. 31:1181, 2010). Furthermore, myocardial infarction patients that develop cardiac dysfunction after angioplasty hâve approximately ~ three-fold higher MBL levels compared to patients with functional recovery (Haahr-Pedersen S., et al., J Inv Cardiology, 21:13, 2009). MBL antibodies also reduced complément déposition on endothélial cells in vitro after oxidative stress indicating a rôle for the lectin pathway in myocardial I/R injury (Collard, C.D., et al., Am. J. Pathol. 156:1549 56, 2000). In a mouse hétérotopie isograft heart transplant model of I/R injury, the rôle of the alternative pathway was investigated using the pathway-specific fusion protein CR2-IH (Atkinson, C., et al., J. Immunol. 185:7007-7013, 2010). Systemic administration of CR2-fH immediately posttransplantation resulted in a réduction in myocardial I/R injury to an extent comparable to treatment with CR2-Crry, which inhibits ail complément pathways, indicating that the alternative pathway is of key importance in this model.

In a mouse model of rénal I/R injury, the alternative pathway was implicated as factor B-deficient mice were protected from a décliné in rénal function and tubular injury, compared with wild-type mice (Thurman, J.M., et al., J. Immunol. 170:1517-1523, 2003). Treatment with an inhibitory monoclonal antibody to factor B prevented complément activation and reduced murine rénal I/R injury (Thurman, J.M., et al., J. Am. Soc. Nephrol. 17:707-715, 2006). In a bilateral rénal I/R injury model, MBL-A/C-deficient mice were protected from kidney damage compared with wild-type mice and recombinant human MBL reversed the protective effect in MBL-A/C-deficient mice, implicating a rôle for MBL in this model (Moller-Kristensen, M., et al., Scand. J. Immunol. 61:426-434, 2005). In a rat unilatéral rénal I/R injury model, inhibition of MBL with a monoclonal antibody to MBL-A preserved rénal function after I/R (van der Pol, P., et al., Am. J. Transplant. 12:877-887, 2010). Interestingly, the rôle of MBL in this model did not appear to involve activation of the terminal complément components, as treatment with a C5 antibody was ineffective in preventing rénal injury. Rather, MBL appeared to hâve a direct toxic effect on tubular cells, as human proximal tubular cells incubated with MBL in vitro intemalized MBL with subséquent cellular apoptosis. In a swine model of rénal I/R, Castellano G. et al., (Am J Pathol, 176(4):1648-59, 2010), tested a Cl inhibitor, which irreversibly inactivâtes Clr and Cl s proteases in the classical pathway and also MASP-1 and MASP-2 proteases in MBL complexes ofthe lectin pathway, and found that Cl inhibitor reduced complément déposition in peritubular capillaries and glomerulus and reduced tubular damage.

The alternative pathway appears to be involved in experimental traumatic brain injury as factor B-deficient mice had reduced systemic complément activation as measured by 5 sérum C5a levels and reduced posttraumatic neuronal cell death compared with wide-type mice (Leinhase, I., et al., BMC Neurosci. 7:55-67, 2006). In human stroke, complément components Clq, C3c, and C4d were detected by immunohistochemical staining in ischémie lésions, suggesting activation via the classical pathway (Pedersen, E.D., et al., Scand. J. Immunol. 69:555-562, 2009). Targeting of the classical pathway in animal models of 10 cérébral ischemia has yielded mixed results, with some studies demonstrating protection while others showing no benefit (Arumugam, T.V., et al., Neuroscience 158:1074-1089, 2009). Experimental and clinical studies hâve provided strong evidence for lectin pathway involvement. In experimental stroke models, deficiency of either MBL or MASP-2 results in reduced infarct sizes compared to wild-type mice (Cervera A, et al.; PLoS One 3;5(2):e8433, 15 2010; Osthoff M. et al., PLoS One, 6(6):e21338, 2011). Furthermore, stroke patients with low levels of MBL hâve a better prognosis compared to their MBL-sufficient counterpart (OsthoffM. et al., PLoS One, 6(6):e21338, 2011).

In a baboon model of cardiopulmonary bypass, treatment with a factor D monoclonal antibody inhibited systemic inflammation as measured by plasma levels of C3a, sC5b-9, and 20 IL-6, and reduced myocardial tissue injury, indicating involvement of the alternative pathway in this model (Undar, A., et al., Ann. Thorac. Surg. 74:355-362, 2002).

Thus, depending on the organ affected by I/R, ail three pathways of complément can contribute to pathogenesis and adverse outcomes. Based on the experimental and clinical findings detailed above, LEA-2 inhibitors are expected to be protective in most settings of 25 1/R. Lectin-dependent activation of LEA-1 may cause complément activation via the alternative pathway at least in some settings. In addition, LEA-2-initiated complément activation may be further amplified by the alternative pathway amplification loop and thus exacerbate I/R-related tissue injury. Thus, LEA-1 inhibitors are expected to provide additional or complementary treatment benefits in patients suffering from an ischemia-related 30 condition.

In view of the above, LEA-1 and LEA-2 inhibitors are expected to hâve independent therapeutic benefits in treating, preventing or reducing the severity of ischemia-reperfusion related conditions. In addition, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefits compared to either agent alone. An optimally effective treatment for an I/R-related condition therefore comprises active pharmaceutical ingrédients that, alone or in combination, block both LEA-1 and LEA-2. Combined LEA-1 and LEA-2 inhibition may be accomplished by co-administration of a LEA-1 blocking agent and a LEA2 blocking agent. Preferentially, LEA-1 and LEA-2 inhibitory function may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a MASP-2-specific binding site, or a dual specificity antibody where each binding site can bind to and block MASP-1/3 or MASP-2.

In accordance with the foregoing, an aspect of the invention thus provides a method for inhibiting LEA-1 dépendent complément activation for treating, preventing or reducing the severity of ischemia reperfusion injuries by administering a composition comprising a therapeutically effective amount of a LEA-1 inhibitory agent comprising a MASP 1 inhibitory agent, a MASP 3 inhibitory agent, or a combination of a MASP 1/3 inhibitory agent, in a pharmaceutical carrier to a subject experiencing ischémie reperfusion. The MASP 1, MASP 3, or MASP 1/3 inhibitory composition may be administered to the subject by intra arterial, intravenous, intracranial, intramuscular, subeutaneous, or other parentéral administration, and potentially orally for non peptidergic inhibitors, and most suitably by intra arterial or intravenous administration. Administration of the LEA-1 inhibitory compositions of the présent invention suitably commences immediately after or as soon as possible after an ischemia reperfusion event. In instances where reperfusion occurs in a controlled environment (e.g., following an aortic aneurism repair, organ transplant or reattachment of severed or traumatized limbs or digits), the LEA-1 inhibitory agent may be administered prior to and/or during and/or after reperfusion. Administration may be repeated periodically as determined by a physician for optimal therapeutic effect.

In some embodiments, the methods are used to treat or prevent an ischemiareperfusion injury associated with at least one of aortic aneurysm repair, cardiopulmonary bypass, vascular reanastomosis in connection with organ transplants and/or extremity/digit replantation, stroke, myocardial infarction, and hémodynamie resuscitation following shock 30 and/or surgical procedures.

In some embodiments, the methods are used to treat or prevent an ischemiareperfusion injury in a subject that is about to undergo, is undergoing, or has undergone an organ transplant. In some embodiments, the methods are used to treat or prevent an ischemica-reperfusion injury in a subject that is about to undergo, is undergoing, or has undergone an organ transplant, provided that the organ transplant is not a kidney transplant.

In one embodiment, the method according to this aspect of the invention further 5 comprises inhibiting LEA-2-dependent complément activation in a subject experiencing ischémie reperfusion, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to the subject. As detailed above, the use of a combination of pharmacologie agents that individually block LEA-1 and LEA-2, is expected to provide an improved therapeutic 10 outcome in treating, preventing, or reducing the severity of ischemia reperfusion injuries as compared to the inhibition of LEA-1 alone. This outcome can be achieved for example, by co-administration of an antibody that has LEA-1-blocking activity together with an antibody that has LEA-2-blocking activity. In some embodiments, LEA-1- and LEA-2-blocking activities are combined into a single molecular entity, and that such entity with combined

LEA-1- and LEA-2-bIocking activity. Such an entity may comprise or consist of a bispecific antibody where one antigen-combining site specifically recognizes MASP-1 and blocks LEA1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Altematively, such an entity may consist of a bispecific monoclonal antibody where one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the 20 second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may optimally consist of a bispecific monoclonal antibody where one antigencombining site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically recognized MASP-2 and blocks LEA-2.

The MASP 2 inhibitory composition may be administered to a subject in need thereof 25 by intra arterial, intravenous, intracranial, intramuscular, subeutaneous, or other parentéral administration, and potentially orally for non peptidergic inhibitors, and most suitably by intra arterial or intravenous administration. Administration of the MASP-2 inhibitory compositions of the présent invention suitably commences immediately after or as soon as possible after an ischemia reperfusion event. In instances where reperfusion occurs in a 30 controlled environment (e.g., following an aortic aneurism repair, organ transplant or reattachment of severed or traumatized limbs or digits), the MASP-2 inhibitory agent may be administered prior to and/or during and/or after reperfusion. Administration may be repeated periodically as determined by a physician for optimal therapeutic effect.

Application of the MASP-3 inhibitory compositions and optional MASP 2 inhibitory compositions of the présent invention may be carried out by a single administration of the composition (e.g., a single composition comprising MASP-2 and MASP-3 inhibitory agents, or bispecific or dual inhibitory agents, or co-administration of separate compositions), or a limited sequence of administrations, for treatment or prévention of ischemia reperfusion injuries. Alternatively, the composition may be administered at periodic intervals such as daily, biweekly, weekly, every other week, monthly or bimonthly over an extended period of time for treatment of a subject experiencing ischémie reperfusion.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies hâve been generated which hâve therapeutic utility for inhibition of the alternative pathway in AP-related diseases or conditions, such in a subject experiencing ischémie reperfusion.

Accordingly, in one embodiment, the présent invention provides a method for treating a subject suffering from, or at risk for developing ischemia-reperfusion comprising an effective amount of a high affinity monoclonal antibody or antigen binding fragment thereof as disclosed herein that binds to human MASP-3 and inhibits alternative pathway complément activation to treat or reduce the risk of tissue injury associated with ischemiareperfusion in the subject.

D. THE ROLE OF MASP-3 IN INLAMMATORY AND NONINFLAMMATORY ARTHRITIDES AND THERAPEUTIC METHODS USING MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION WITH AND MASP-2 INHIBITORY AGENTS

Rheumatoid arthritis (RA) is a chronic inflammatory disease of synovial joints that may also hâve systemic manifestations. RA affects approximately 1% of the world population, with women being two to three times more likely to be afflicted. Joint inflammation manifests in swelling, pain, and stiffness. As the disease progresses there may be joint érosion and destruction, resulting in impaired range of motion and deformities. Treatment goals in RA include prévention or control of joint damage, prévention of loss of joint function and disease progression, relief of symptoms and improvement in quality of life, and achievement of drug-free remission. Pharmacological treatment of RA includes diseasemodifying anti-rheumatic drugs (DMARDs), analgésies, and anti-inflammatory agents (glucocorticoids and non-steroidal anti-inflammatory drugs). DMARDs are the most important treatment because they can induce durable remissions and delay or hait the progression of joint destruction, which is irréversible. Traditional DMARDs include small molécules such as methotrexate, sulfasalazine, hydroxychloroquine, gold salts, leflunomide, D-penicillamine, cyclosporine, and azathioprine. If traditional DMARDs are inadéquate to control the disease then several biologie agents targeting inflammatory cells or mediators are available treatment options, such as tumor necrosis factor inhibitors (etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab), cytokine antagonists (anakinra and tocilizumab), rituximab, and abatacept.

Although adaptive immunity is clearly central to RA pathogenesis as evidenced by genetic association with T-cell activation genes and the presence of autoantibodies, innate immune mechanisms hâve also been implicated (Mclnnes, I.B. and Schett, G. New Engl. J. Med. 365:2205-2219, 2011). In human RA, synovial fluid levels of the alternative pathway cleavage fragment Bb were several fold higher than samples from patients with crystalinduced arthritis or degenerative joint disease, implicating preferential activation of the alternative pathway in RA patients (Brodeur, J.P., et al., Arthritis Rheum. 34:1531-1537, 1991). In the experimental anti-type II collagen antibody-passive transfer model of arthritis, factor B-deficient mice had decreased inflammation and joint damage compared with wildtype mice, whereas C4-deficient mice had similar disease activity as wild-type mice, indicating the requirement for the alternative pathway and not the classical pathway in this model (Banda, N.K. et al., J. Immunol. 177:1904-1912, 2006). In the same experimental model of collagen antibody-induced arthritis (CAIA), mice with only classical pathway active or only lectin pathway active were not capable of developing arthritis (Banda, N.K. et al., Clin. Exp. Immunol. 159:100-108, 2010). Data from this study suggested that either the classical or lectin pathways were capable of activating low levels of C3 in vitro. However, in the absence of the alternative pathway amplification loop, the level of joint déposition of C3 was inadéquate to produce clinical disease. A key step in the activation of the alternative pathway is conversion of the zymogen of factor D (pro-factor D) to mature factor D, which is mediated by MASP-1 and/or MASP-3 (Takahashi, M., et al., J. Exp. Med. 207:29-37, 2010) and/or HTRA1 (Stanton et al., Evidence That the HTRA1 Interactome Influences

Susceptibility to Age-Related Macular Degeneration, presented at The Association for Research in Vision and Ophthalmology 2011 conférence on May 4, 2011). The rôle of MASP-1/3 was evaluated in murine CALA and the results showed that MASP-1/3 déficient mice were protected from arthritis compared with wild-type mice (Banda, N.K., et al., J.

Immunol. 185:5598-5606, 2010). In MASP-1/3-deficient mice, pro-factor D but not mature factor D was detected in sérum during the évolution of CAIA, and the addition of human factor D in vitro reconstituted C3 activation and C5a génération using sera from these mice. In contrast, in a murine model of the effector phase of arthritis, C3-deficient mice developed very mild arthritis compared to WT mice while factor B-deficient mice still developed 10 arthritis, indicating independent contribution of both the classical/lectin and alternative pathways (Hietala, M.A. et al., Eur. J. Immunol. 34:1208-1216, 2004). In the K/BxN T cell receptor transgenic mouse model of inflammatory arthritis, mice lacking C4 or Clq developed arthritis similar to wild-type mice whereas mice lacking factor B either did not develop arthritis or had mild arthritis, demonstrating the requirement for the alternative 15 pathway and not the classical pathway in this model (Ji H. et aL, Immunity 16:157-168, 2002). In the K/BxN model, mice lacking MBL-A were not protected from serum-induced arthritis, but as the rôle of MBL-C was not investigated, a potential rôle for the lectin pathway could not be eliminated (Ji et aL, 2002, supra).

Two research groups hâve independently proposed that lectin-dependent complément 20 activation promûtes inflammation in RA patients via interaction of MBL with spécifie IgG glycoforms (Malhotra et al., Nat. Med. 1:237 243, 1995; Cuchacovich et al., J. Rheumatol. 23:44 51, 1996). It is noted that rheumatoid conditions are associated with a marked increase in IgG glycoforms that lack galactose (referred to as IgGO glycoforms) in the Fc région ofthe molécule (Rudd et al., Trends Biotechnology 22:524 30, 2004). The percentage of IgGO 25 glycoforms increases with disease progression of rheumatoid conditions, and returns to normal when patients go into remission. In vivo, IgGO is deposited on synovial tissue and MBL is présent at increased levels in synovial fluid in individuals with RA. Aggregated agalactosyl IgG (IgGO) associated with RA can bind MBL and therefore can initiate lectindependent complément activation via LEA-1 and/or LEA-2. Furthermore, results from a 30 clinical study looking at allelic variants of MBL in RA patients suggest that MBL may hâve an inflammatory enhancing rôle in the disease (Garred et al., J. Rheumatol. 27:26 34, 2000).

Therefore, the lectin-dependent complément activation via LEA-1 and/or LEA-2 may play an important rôle in the pathogenesis of RA.

Complément activation also plays in important rôle in juvénile rheumatoid arthritis (Mollnes, T.E., et al., Arthritis Rheum. 29:1359 64, 1986). Similar to adult RA, in juvénile rheumatoid arthritis, elevated sérum and synovial fluid levels of alternative pathway complément activation product Bb compared to C4d (a marker for classical or LEA-2 activation), indicate that complément activation is mediated predominantly by LEA-1 (El Ghobarey, A.F. et al., J. Rheumatology 7:453 460, 1980; Agarwal, A., et al., Rheumatology 39:189 192, 2000).

Similarly, complément activation plays an important rôle in psoriatic arthritis. Patients with this condition hâve increased complément activation products in their circulation, and their red blood cells appear to hâve lower levels ofthe complément regulator CD59 (Triolo,. Clin Exp Rheumatol., 21(2):225-8, 2003). Complément levels are associated with disease activity, and hâve a high prédictive value to détermine treatment outcomes (Chimenti at al., Clin Exp Rheuntatol., 30(1):23-30, 2012). In fact, recent studies suggest that the effect of anti-TNF therapy for this condition is attributable to complément modulation (Ballanti et al., Autoimmun Rev., 10(10):617-23, 2011). While the précisé rôle of complément in psoriatic arthritis has not been determined, the presence of C4d and Bb complément activation products in the circulation of these patients suggests an important rôle in pathogenesis. On the basis of the products observed, it is believed that LEA-1, and possibly also LEA-2 are responsible for pathologie complément activation in these patients.

Osteoarthritis (OA) is the most common form of arthritis, affecting over 25 million people in the United States. OA is characterized by breakdown and eventual loss of joint cartilage, accompanied by new bone formation and synovial prolifération, leading to pain, stiffness, loss of joint fùnction, and disability. Joints that are frequently affected by OA are hands, neck, lower back, knees and hips. The disease is progressive and current treatments are for symptomatic pain relief and do not alter the natural history of disease. The pathogenesis of OA is unclear, but a rôle for complément has been implicated. In a proteomic and transcriptomic analyses of synovial fluid from patients with OA, several components of complément were aberrantly expressed compared to samples from healthy individuals, including classical (Cls and C4A) and alternative (factor B) pathways, and also C3, C5, C7, and C9 (Wang, Q., et al., Nat. Med. 17:1674-1679, 2011). Moreover, in a mouse model of OA induced by médial meniscectomy, C5-deficient mice had less cartilage loss, osteophyte formation and synovitis than C5-positive mice, and treatment of wild-type mice with CR2-IH, a fusion protein that inhibits the alternative pathway, attenuated the development of OA (Wang et al., 2011 supra).

Ross River virus (RRV) and chikungunya virus (CHIKV) belong to a group of mosquito-bome viruses that can cause acute and persistent arthritis and myositis in humans. In addition to causing endemic disease, these viruses can cause épidémies that involve millions of infected individuals. The arthritis is believed to be initiated by viral réplication and induction of host inflammatory response in the joint and the complément System has been 10 invoked as a key component in this process. Synovial fluid from humans with RRV-induced polyarthritis contains higher levels of C3a than synovial fluid from humans with OA (Morrison, T.E., et al., J. Virol. 81:5132-5143, 2007). In a mouse model of RRV infection, C3-deficient mice developed less severe arthritis compared with wild-type mice, implicating the rôle of complément (Morrison et al., 2007, supra). The spécifie complément pathway 15 involved was investigated and mice with inactivated lectin pathway (MBL-A-/- and MBL-C/-) had attenuated arthritis compared with wide-type mice. In contrast, mice with inactivated classical pathway (Clq-/-) or alternative pathway (factor B-/-) developed severe arthritis, indicating that the lectin pathway initiated by MBL had an essential rôle in this model (Gunn, B.M., et al., PLoS Pathog. 8:el002586, 2012). Because arthritides involve damage to the 20 joints, the initial joint damage caused by various étiologies may trigger a secondary wave of complément activation via LEA-2. In support of this concept, our previous work has demonstrated that MASP-2 KO mice hâve reduced joint injury compared to WT mice in the collagen-induced model of RA.

In view ofthe body ofevidence detailed above, LEA-1 and LEA-2 inhibitors, alone or 25 in combination, are expected to be therapeutically useful for the treatment of arthritides. An optimaiiy effective treatment for arthritides may therefore comprise active pharmaceutical ingrédients that, alone or in combination, can block both LEA-1 and LEA-2. Combined LEA1 and LEA-2 inhibition may be accomplished by co-administration of an LEA-1 blocking agent and a LEA2 blocking agent. Preferentially, LEA-1 and LEA-2 inhibitory function may 30 be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a MASP-2-specific binding site, or a dual specificity antibody where each binding site can bind to and block MASP-1/3 or MASP-2.1n accordance with the foregoing, an aspect of the invention thus provides a method for inhibiting LEA-1 dépendent complément activation for treating, preventing, or reducing the severity of inflammatory or non-inflammatory arthritides, including osteoarthritis, rheumatoid arthritis, juvénile rheumatoid arthritis and psoriatic arthritis, by administering a composition comprising a therapeutically effective amount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of a MASP 1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from, or at risk for developing, inflammatory or non-inflammatory arthritides. The MASP-1, MASP-3, or MASP 1/3 inhibitory composition may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, subcutaneous, or other parentéral administration, or by oral administration. Altematively, administration may be by local delivery, such as by intraarticular injection. The LEA-1 inhibitory agent may be administered periodically over an extended period of time for treatment or control of a chronic condition, or may be by single or repeated administration in the period before, during and/or following acute trauma or injury, including surgical procedures performed on the joint.

In one embodiment, the method according to this aspect of the invention further comprises inhibiting LEA-2-dependent complément activation in a subject suffering from, or at risk for developing, inflammatory or non-inflammatory arthritides (including osteoarthritis, rheumatoid arthritis, juvénile rheumatoid arthritis and psoriatic arthritis), by administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP1/3 inhibitory agent to the subject. As detailed above, the use of a combination of pharmacologie agents that individually block LEA-1 and LEA-2, is expected to provide an improved therapeutic outcome in treating or preventing arthritides as compared to the inhibition of LEA-1 alone. This outcome can be achieved for example, by co-administration of an antibody that has LEA-1-blocking activity together with an antibody that has LEA-2blocking activity. In some embodiments, LEA-1- and LEA-2-blocking activities are combined into a single molecular entity, and that such entity with combined LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody where one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Altematively, such an entity may consist of a bispecific monoclonal antibody where one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may optimally consist of a bispecific monoclonal antibody where one antigencombining site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory composition may be administered to the subject in need thereof systemically, such as by intra arterial, intravenous, intramuscular, subcutaneous, or other parentéral administration, or potentially by oral administration for non peptidergic inhibitors. Alternatively, administration may be by local delivery, such as by intra-articular injection. The MASP-2 inhibitory agent may be administered periodically over an extended period of time for treatment or control of a chronic condition, or may be by single or repeated administration in the period before, during and/or following acute trauma or injury, including surgical procedures performed on the joint.

Application of the MASP-3 inhibitory compositions and optional MASP 2 inhibitory compositions of the présent invention may be carried out by a single administration of the composition (e.g., a single composition comprising MASP-2 and MASP-3 inhibitory agents, or bispecific or dual-inhibitory agents, or co-administration of separate compositions), or a limited sequence of administrations, for treating, preventing or reducing the severity of inflammatory or non-inflammatory arthritides. Alternatively, the composition may be administered at periodic intervals such as daily, biweekly, weekly, every other week, monthly or bimonthly over an extended period of time for treatment of a subject suffering from inflammatory or non-inflammatory arthritides.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies hâve been generated which hâve therapeutic utility for inhibition ofthe alternative pathway in AP-related diseases or conditions, such as arthritis.

Accordingly, in one embodiment, the présent invention provides a method for treating a subject suffering from, or at risk for developing arthritis (inflammatory and noninflammatory arthritides) comprising administering to the subject a pharmaceutical composition comprising an effective amount of a high affinity monoclonal antibody or antigen binding fragment thereof as disclosed herein that binds to human MASP-3 and inhibits alternative pathway complément activation to treat or reduce the risk of arthritis in the subject, such as, for example, wherein said antibody or antigen binding fragment thereof comprises (a) a heavy chain variable région comprising (i) VHCDRl comprising SEQ ID

NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable région comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161. In 5 some embodidments, the subject is suffering from arthritis selected fronm the group consisting of osteoarthritis, rheumatoid arthritis, juvénile rheumatoid arthritis, ankylosing spondylitis, Behcet’s disease, infection-related arthritis and psoriatic arthritis. In some embodiments, the pharmaceutical composition is administered systemically (i.e., subcutaneously, intra-muscularly, intravenously, intra-arterially or as an inhalant). In some 10 embodiments, the pharmaceutical composition is administered locally to a joint.

E. . THE ROLE OF MASP-3 IN DISSEMINATED INTRAVASCULAR COAGULATION (DIC) AND THERAPEUTIC METHODS USING MASP-3 15 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION WITH AND MASP-2 INHIBITORY AGENTS

Disseminated intravascular coagulation (DIC) is a syndrome of pathologie overstimulation of the coagulation system that can manifest clinically as hemorrhage and/or 20 thrombosis. DIC does not occur as a primary condition but rather in association with a variety of disease processes, including tissue damage (trauma, bums, heat stroke, transfusion reaction, acute transplant rejection), neoplasia, infections, obstetric conditions (placenta previa, amniotic fluid embolism, toxemia of pregnancy), and miscellaneous conditions such as cardiogenic shock, near drowning, fat embolism, aortic aneurysm. Thrombocytopenia is a 25 frequent abnormality in patients in the intensive care unit, with an incidence of 35% to 44%, and DIC is the etiology in about 25% of these cases, i.e., DIC occurs in approximately 10% of critically ill patients (Levi, M. and Opal, S.M. Crit. Care 10:222-231, 2006). The pathophysiology of DIC is that the underlying disease process initiâtes a physiological coagulation response. However, the prothrombotic substances overwhelm the normal 30 counterbalancing mechanisms such that there is the inappropriate déposition of fibrin and platelets in the microcirculation, leading to organ ischemia, hypofibrinogenemia, and thrombocytopenia. The diagnosis of DIC is based on the clinical présentation in the appropriate underlying illness or process, along with abnormalities in laboratory parameters (prothrombin time, partial thromboplastin time, fibrin dégradation products, D-dimer, or platelet count). The primary treatment of D1C is to address the underlying condition that is the responsible trigger. Blood product support in the form of red blood cells, platelets, fresh 5 frozen plasma, and cryoprecipitate may be necessary to treat or prevent clinical complications.

The rôle of the complément pathways in DIC has been investigated in several studies. Complément activation was evaluated in pédiatrie patients with meningococcal infection comparing the clinical course in relation to MBL génotype (Sprong, T. et al., Clin. Infect. 10 Dis. 49:1380-1386, 2009). At admission to the hospital, patients with MBL deficiency had lower circulating levels of C3bc, terminal complément complex, C4bc, and C3bBbP than MBL-sufficient patients, indicating lower extent of common complément, terminal complément, and alternative pathway activation. Furthermore, extent of systemic complément activation correlated with disease severity and parameters of DIC and the MBL15 déficient patients had a milder clinical course than MBL-sufficient patients. Therefore, although MBL deficiency is a risk factor for susceptibility to infections, MBL deficiency during septic shock may be associated with lower disease severity.

As demonstrated in Examples 1-4 herein, experimental studies hâve highlighted the important contribution of MBL and MASP-1/3 in innate immune response to Neisseria 20 menigitidis, the etiological agent of meningococcal infection. MBL-deficient sera from mice or humans, MASP-3 déficient human sera, or the MASP-1/3 knockout mouse are less effective at activating complément and lysing meningococci in vitro compared to wild-type sera. Similarly, naïve MASP-1/3 knockout mice are more susceptible to neisserial infection than their wild-type counterparts. Thus, in the absence of adaptive immunity, the LEA-1 25 pathway contributes to innate-host résistance to neisserial infection. Conversely, LEA-1 augments pathologie complément activation triggering a harmful host response, including DIC.

In a murine model of arterial thrombosis, MBL-null and MASP-1/-3 knockout mice had decreased FeC13-induced thrombogenesis compared with wild-type or C2/factor B-null 30 mice, and the defect was reconstituted with recombinant human MBL (La Bonte, L.R., et al., J. Immunol. 188:885-891,2012). In vitro, MBL-null or MASP-1/-3 knockout mouse sera had decreased thrombin substrate cleavage compared with wild-type or C2/factor B-null mouse sera; addition of recombinant human MASP-1 restored thrombin substrate cleavage in MASP-1/-3 knockout mouse sera (La Bonte et al., 2012, supra). These results indicate that MBL/MASP complexes, in particular MASP-1, play a key rôle in thrombus formation. Thus, LEA-1 may play an important rôle in pathologie thrombosis, including DIC.

Experimental studies hâve established an equally important rôle for LEA-2 in pathologie thrombosis. In vitro studies further demonstrate that LEA-2 provides a molecular link between the complément System and the coagulation System. MASP-2 has factor Xa-like activity and activâtes prothrombin through cleavage to form thrombin, which can subsequently clear fibrinogen and promote fibrin clôt formation (see also Krarup et al., PLoS 10 One, 18:2(7):e623, 2007).

Separate studies hâve shown that lectin-MASP complexes can promote clôt formation, fibrin déposition and fibrinopeptide release in a MASP-2 dépendent process (Gulla et al., Immunoïogy, 129(4):482-95, 2010). Thus, LEA-2 promûtes simultaneous lectin-dependent activation of complément and the coagulation system.

In vitro studies hâve further shown that MASP-1 has thrombin-like activity (Presanis

J.S., et al., Mol Immunol, 40(13):921-9, 2004), and cleaves fibrinogen and factor XIII (Gulla K. C. et la., Immunoïogy, 129(4):482-95, 2010), suggesting that LEA-1 may activate coagulation pathways independently or in concert with LEA-2.

The data detailed above suggest that LEA-1 and LEA-2 provide independent links between lectin-dependent complément activation and coagulation. Thus, in view of the above, LEA-1 and LEA-2 inhibitors are expected to hâve independent therapeutic benefits in treating a subject suffering from disseminated intravascular coagulation. In some embodiments, the subject is suffering from disseminated intravascular coagulation secondary to sepsis, trauma, infection (bacterial, viral, fungal, parasitic), malignancy, transplant 25 rejection, transfusion reaction, obstetric complication, vascular aneurysm, hepatic failure, heat stroke, bum, radiation exposure, shock, or severe toxic reaction (e.g., snake bite, insect bite, transfusion reaction). In some embodiments, the trauma is a neurological trauma. In some embodiments, the infection is a bacterial infection, such as a Neisseria meningitidis infection.

In addition, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefits compared to either agent alone. As both LEA-1 and LEA-2 are known to be activated by conditions that lead to DIC (for example infection or trauma), LEA-1- and

LEA-2-blocking agents, either separately or in combination, are expected to hâve therapeutic utility in the treatment of DIC. LEA-1 and LEA-2 blocking agents may prevent different cross-talk mechanisms between complément and coagulation. LEA-1- and LEA-2-blocking agents may thus hâve complementary, additive or synergistic effects in preventing DIC and other thrombotic disorders.

In addition, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone, or may provide effective treatment for a wider spectrum of patient subsets. Combined LEA-1 and LEA-2 inhibition may be accomplished by co-administration of a LEA-1-blocking agent and a LEA-2-blocking agent. Optimally, LEA-1 and LEA-2 inhibitory function may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a MASP-2-specifîc binding site, or a dual specificity antibody where each binding site and bind to and block MASP-1/3 or MASP-2.

In accordance with the foregoing, an aspect of the invention thus provides a method for inhibiting LEA-1 dépendent complément activation for treating, preventing, or reducing the severity of disseminated intravascular coagulation in a subject in need thereof comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent, a MASP 3 inhibitory agent, or a combination of a MASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subject experiencing, or at risk for developing, disseminated intravascular coagulation. The MASP1, MASP-3, or MASP-1/3 inhibitory composition may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled. For treatment or prévention of DIC secondary to trauma or other acute event, the LEA-1 inhibitory composition may be administered immediately following the traumatic injury or prophylactically prior to, during, immediately following, or within one to seven days or longer, such as within 24 hours to 72 hours, after trauma-inducing injury or situations such as surgery in patients deemed at risk of DIC. In some embodiments, the LEA-1 inhibitory composition may suitably be administered in a fast acting dosage form, such as by intravenous or intra arterial delivery of a bolus of a solution containing the LEA-1 inhibitory agent composition.

In one embodiment, the method according to this aspect of the invention further comprises inhibiting LEA-2-dependent complément activation for treating, preventing, or reducing the severity of disseminated intravascular coagulation in a subject in need thereof, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent 5 and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to the subject. As detailed above, the use of a combination of pharmacologie agents that individually block LEA-I and LEA-2 is expected to provide an improved therapeutic outcome in treating or preventing disseminated intravascular coagulation as compared to the inhibition of LEA-I alone. This outcome can be achieved for example, by co-administration of an antibody that has LEA-1 10 blocking activity together with an antibody that has LEA-2-blocking activity. In some embodiments, LEA-1- and LEA-2-blocking activities are combined into a single molecular entity, and that such entity with combined LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody where one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site 15 specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody where one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may optimally consist of a bispecific monoclonal antibody where one antigen-combining site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the subject in need thereof systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non 25 peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled. For DIC secondary to trauma or other acute event, the MASP-2 inhibitory composition may be administered immediately following the traumatic injury or prophylactically prior to, during, immediately following, or within one to seven days or longer, such as within 24 hours to 72 hours, after trauma-inducing injury or 30 situations such as surgery in patients deemed at risk of DIC. In some embodiments, the MASP-2 inhibitory composition may suitably be administered in a fast acting dosage form, such as by intravenous or intra arterial delivery of a bolus of a solution containing the MASP2 inhibitory agent composition.

Application of the MASP-3 inhibitory compositions and optional MASP-2 inhibitory compositions of the présent invention may be carried out by a single administration of the 5 composition (e.g., a single composition comprising MASP-2 and MASP-3 inhibitory agents, or bispecific or dual-inhibitory agents, or co-administration of separate compositions), or a limited sequence of administrations, for treating, preventing, or reducing the severity of disseminated intravascular coagulation in subject in need thereof. Alternatively, the composition may be administered at periodic intervals such as daily, biweekly, weekly, every 10 other week, monthly or bimonthly over an extended period of time for treatment of a subject experiencing, or at risk for developing disseminated intravascular coagulation.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies hâve been generated which hâve therapeutic utility for inhibition ofthe alternative pathway in AP-related diseases or conditions, such as disseminated intravascular coagulation.

Accordingly, in one embodiment, the présent invention provides a method for treating a subject suffering from, or at risk for developing disseminated intravascular coagulation comprising an effective amount of a high affinity monoclonal antibody or antigen binding fragment thereof as disclosed herein that binds to human MASP-3 and inhibits alternative pathway complément activation to treat or reduce the risk of developing disseminated 20 intravascular coagulation, such as, for example, wherein said antibody or antigen binding fragment thereof comprises (a) a heavy chain variable région comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable région comprising (i) VLCDR1 comprising SEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258 or 25 SEQ ID NO:259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO:161.

F. THE ROLE OF MASP-3 IN THROMBOTIC MICROANGIOPATHY (TMA), INCLUDING HEMOLYTIC UREM1C SYNDROME (HUS), ATYPICAL 30 HEMOLYTIC UREMIC SYNDROME (AHUS) AND THROMBOTIC THROMBOCYTOPENIC PURPURA (TTP) AND THERAPEUTIC METHODS USING

MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP2 INHIBITORY AGENTS

Thrombotic microangiopathy (TMA) refers to a group of disorders characterized 5 clinically by thrombocytopenia, microangiopathic hemolytic anémia, and variable organ ischemia. The characteristic pathological features of TMA are platelet activation and the formation of microthrombi in the small artérioles and venules. The classic TMAs are hemolytic urémie syndrome (HUS) and thrombotic thrombocytopénie purpura (TTP). HUS is distinguished from TTP by the presence of acute rénal failure. HUS occurs in two forms: 10 diarrhea-associated (D+) or typical HUS, and diarrhea négative (D-) or atypical HUS (aHUS).

HUS

D+HUS is associated with a prodromal diarrheal illness usually caused by Escherichia coli 0157 or another Shiga-toxin-producing strain of bacteria, accounts for over 90% of the 15 HUS cases in children, and is the most common cause of acute rénal failure in children. Although human infection with Escherichia coli 0157 is relatively frequent, the percentages of bloody diarrhea that progresses to D+HUS ranged from 3% to 7% in sporadic cases and 20% to 30% in some outbreaks (Zheng, X.L. and Sadler, J.E., Annu. Rev. Pathol. 3:249-277, 2008). HUS usually occurs 4 to 6 days after the onset of diarrhea and approximately two20 third of children require dialysis in the acute phase of the disease. Treatment of D+HUS is supportive as no spécifie treatments hâve been shown to be effective. The prognosis of D+HUS is favorable, with the majority of patients regaining rénal function.

The pathogenesis of D+HUS involves bacteria-produced Shiga toxins that bind to membranes on microvascular endothélial cells, monocytes, and platelets. The 25 microvasculature of the kidney is most often affected. Following binding, the toxin is intemalized, leading to release of proinflammatory mediators and eventual cell death. It is thought that endothélial cell damage triggers rénal microvascular thrombosis by promoting the activation of the coagulation cascade. There is evidence for activation of the complément system in D+HUS. In children with D+HUS, plasma levels of Bb and SC5b-9 were 30 increased at the time of hospitalization compared to normal Controls and, at day 28 after hospital discharge, the plasma levels had normalized (Thurman, J.M. et al., Clin. J. Am. Soc. Nephrol. 4:1920-1924, 2009). Shiga toxin 2 (Stx2) was found to activate human complément in the fluid phase in vitro, predominantly via the alternative pathway as activation proceeded in the presence of ethylene glycol tetraacetic acid which blocks the classical pathway (Orth, D. et al., J. Immunol. 182:6394-6400, 2009). Furthermore, Stx2 bound factor H and not factor I, and delayed the cofactor activity of factor H on cell surfaces (Orth et al, 2009, supra). These results suggest that Shiga toxin may cause rénal damage through multiple potential mechanisms, including a direct toxic effect, and indirectly through activation of complément or inhibition of complément regulators. Toxic effects on the vascular endothélium are expected to activate complément via LEA-2, as ' evidenced by the effectiveness of MASP-2 blockade in preventing complement-mediated reperfusion injury in various vascular beds as described in Schwaeble, W.J., et al., Proc. Natl. Acad. Sci. 108:7523-7528, 2011.

In a murine model of HUS induced by co-injection of Shiga toxin and lipopolysaccharide, factor B-deficient mice had less thrombocytopenia and were protected from rénal impairment compared with wild-type mice, implicating LEA-1-dépendent activation of the alternative pathway in microvascular thrombosis (Morigi, M. et al., J. Immunol. 187:172-180, 2011). As described herein, in the same model, administration of MASP-2 antibody was also effective and increased survival following STX challenge, implicating LEA-2-dependent complément pathway in microvascular thrombosis.

Based on the foregoing, LEA-1 and LEA-2 inhibitors are expected to hâve independent therapeutic benefit in the treatment or prévention of HUS. In addition, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone, or may provide effective treatment for a wider spectrum of patient subsets. Combined LEA-1 and LEA-2 inhibition may be accomplished by co-administration of a LEA-1-blocking agent and a LEA-2-blocking agent. Optimally, LEA-1 and LEA-2 inhibitory function may be encompassed in a single molecular entity, such as a bispecifïc antibody composed of MASP-1/3 and a MASP-2-specific binding site, or a dual-specificity antibody where each binding site can bind to and block MASP-1/3 or MASP-2.

aHUS

Atypical HUS is a rare disease, with an estimated incidence of 2 per million in the United States (Loirat, C. and Fremeaux-Bacchi, V. Orphanet J. Rare Dis. 6:60-90, 2011). Atypical HUS can develop at any âge, although the majority of patients hâve an onset during childhood. Atypical HUS is heterogeneous: some cases are familial, some are recurring, and

100 some are triggered by an infectious illness, typically upper respiratory tract or gastroenteritis. The onset of aHUS is usually sudden and most patients require dialysis at admission. Extra rénal manifestations are présent in about 20% of patients and may involve the central nervous system, myocardial infarction, distal ischémie gangrené, or multiorgan failure. Treatment of 5 aHUS includes supportive care for organ dysfunction, plasma infusion or plasma exchange, and eculizumab, a humanized monoclonal antibody that targets C5 that was recently approved for use in the United States and European Union. The prognosis in aHUS is not as good as in D+HUS, with approximately 25% mortality during the acute stage and most survivors develop end-stage rénal disease.

Atypical HUS has been characterized as a disease of complément dysrégulation in that approximately 50% of patients hâve mutations in genes encoding complément regulatory proteins (Zheng and Sadler, 2008 supra). Most mutations are seen in factor H (FH); other mutations include membrane cofactor protein (MCP), factor I (FI), factor B, and C3. Functional studies showed that the mutations in FH, MCP, and FI lead to loss of function and 15 therefore more complément activation, whereas mutations in factor B are gain of function.

The effects of these mutations predominantly affect the alternative pathway. These genetic abnormalities are risk factors rather than the only cause of disease as approximately 50% of family members who carry the mutation do not présent with the disease by âge 45 (Loirat and Fremeaux-Bacchi, 2011 supra).

Factor H is a complément control protein that protects host tissue from alternative pathway complément attack. FH régulâtes the alternative pathway amplification loop in three ways: it is a cofactor for FI, which cleaves C3b, it inhibits the formation of the alternative pathway C3 convertase, C3bBb, and it binds to polyanions on cell surfaces and tissue matrices and blocks déposition of C3b (Atkinson, J.P. and Goodship, T.H.J., J. Exp. Med.

6:1245-1248, 2007). The majority of FH mutations in aHUS patients occur in the C-terminal short consensus repeat domains of the protein, which resuit in defective binding of FH to heparin, C3b, and endothélium, but do not alter plasma C3 régulation which résides among N-terminal domains (Pickering, M.C. et al., J. Exp. Med. 204:1249-1256, 2007). FH-deficient mice hâve uncontrolled plasma C3 activation and spontaneously develop 30 membranoproliferative glomerulonephritis type II, but not aHUS. However, FH-deficient mice that transgenically expressed a mouse FH protein functionally équivalent to aHUSassociated human FH mutants spontaneously develop a HUS but not membranoproliferative

101 glomerulonephritis type II, providing in vivo evidence that defective control of alternative pathway activation in rénal endothélium is a key event in the pathogenesis of FH-associated aHUS (Pickering et al., 2007 supra). Another form of FH-associated aHUS occurs in patients who hâve anti-FH autoantibodies resulting in a loss of FH functional activity; most of these patients hâve délétions in genes encoding five FH-related proteins (Loirat and Fremeaux-Bacchi, 2011, supra).

Similar to FH, MCP inhibits complément activation by regulating C3b déposition on target cells. MCP mutations resuit in proteins with low C3b-binding and cofactor activity, thus allowing for dysregulated alternative pathway activation. FI is a serine protease that cleaves C3b and C4b in the presence of cofactors, such as FH and MCP, and thereby prevents the formation of C3 and C5 convertases and inhibits both the alternative and the classical complément pathways. Most of the Fl-associated aHUS mutations resuit in reduced FI activity for the dégradation of C3b and C4b (Zheng and Stadler, 2008, supra). FB is a zymogen that carries the catalytic sites of the alternative pathway convertase C3bBb. Functional analysis showed that the aHUS associated FB mutations resuit in increased alternative pathway activation (Loirat and Fremeaux-Bacchi, 2011, supra). Heterozygous mutations in C3 are associated with aHUS. Most C3 mutations induce a defect of C3 to bind MCP, leading to an increased capacity of FB to bind C3b and increased formation of C3 convertase (Loirat and Fremeaux-Bacchi, 2011, supra). Thus, aHUS is a disease closely associated with mutations in the complément genes that lead to inadéquate control of the alternative pathway amplification loop. Since the alternative pathway amplification loop is dépendent on factor B proteolytic activity, and since LEA-1 is required for factor B activation (either by MASP-3 dépendent cleavage or by factor D-mediated cleavage wherein the MASP-I contributes to the maturation of factor D), LEA-1-blocking agents are expected to prevent uncontrolled complément activation in susceptible individuals. As a resuit, it is expected that LEA-1 blocking agents will effectively treat aHUS.

While the central rôle of a deregulated alternative pathway amplification loop in aHUS is widely accepted, the triggers initiating complément activation and the molecular pathways involved are unresolved. Not ail individuals carrying the above-described mutations develop aHUS. In fact, familial studies hâve suggested that the penetrance of aHUS is only ~50% (Sullivan M. et al., Ann Hum Genet 74:17-26 2010). The natural history of the disease suggests that aHUS most often develops after an initiating event such as an

102 infectious épisode or an injury. Infectious agents are well known to activate the complément System. In the absence of pre-existing adaptive immunity, complément activation by infectious agents may be primarily initiated via LEA-I or LEA-2. Thus, lectin-dependent complément activation triggered by an infection may represent the initiating trigger for 5 subséquent pathological amplification of complément activation in aHUS-predisposed individuals, which may ultimately lead to disease progression. Accordingly, another aspect of the présent invention comprises treating a patient suffering with aHUS secondary to an infection by administering an effective amount of a LEA-1- or a LEA-2-inhibitory agent.

Other forms of injury to host tissue will activate complément via LEA-2, in particular 10 injury to the vascular endothélium. Human vascular endothélial cells subject to oxidative stress, for example, respond by expressing surface moieties that bind lectins and activate the LEA-2 pathway of complément (Collard et al., Am J. Pathol 156(5):1549-56, 2000). Vascular injury following ischemia/reperfusion also activâtes complément via LEA-2 in vivo (Moller-Kristensen et al., Scand J Immunol 61(5):426-34, 2005). Lectin pathway activation 15 in this setting has pathological conséquences for the host, and as shown in Examples 22 and 23, inhibition of LEA-2 by blocking MASP-2 prevents further host tissue injury and adverse outcomes (see also Schwaeble PNAS, 2011, supra).

Thus, other processes that precipitate aHUS are also known to activate LEA-1 or LEA-2. It is therefore likely that the LEA-1 and/or LEA-2 pathway may represent the initial 20 complément activating mechanism that is inappropriately amplified in a deregulated fashion in individuals genetically predisposed to aHUS, thus initiating aHUS pathogenesis. By inference, agents that block activation of complément via LEA-1 and/or LEA-2 are expected to prevent disease progression or reduce exacerbations in aHUS susceptible individuals.

In further support of this concept, recent studies hâve identified Streptococcus25 pneumoniae as an important etiological agent in pédiatrie cases of aHUS. (Lee, C.S. et al, Nephrology, 17(1):48-52 (2012); Banerjee R. et al., Pediatr Infect Dis J., 30(9):736-9 (2011)). This particular etiology appears to hâve an unfavorable prognosis, with significant mortality and long-term morbidity. Notably, these cases involved non-enteric infections leading to manifestations of microangiopathy, uremia and hemolysis without evidence of 30 concurrent mutations in complément genes known to prédisposé to aHUS. It is important to note that S. pneumoniae is particularly effective at activating complément, and does so predominantly through LEA-2. Thus, in cases of non-enteric HUS associated with

103 pneumococca! infection, manifestations of microangiopathy, uremia and hemolysis are expected to be driven predominantly by activation of LEA-2, and agents that block LEA-2, including MASP-2 antibodies, are expected to prevent progression of aHUS or reduce disease severity in these patients. Accordingly, another aspect of the présent invention comprises 5 treating a patient suffering with non-enteric aHUS that is associated with S. pneumoniae infection by administering an effective amount of a MASP-2 inhibitory agent.

TTP

Thrombotic thrombocytopénie purpura (TTP) is a life-threatening disorder of the blood-coagulation System caused by autoimmune or hereditary dysfunctions that activate the 10 coagulation cascade or the complément System (George, JN, N Engl J Med·, 354:1927-35, 2006). This results in numerous microscopie clots, or thomboses, in small blood vessels throughout the body, which is a characteristic feature of TMAs. Red blood cells are subjected to shear stress, which damages their membranes, leading to intravascular hemolysis. The resulting reduced blood flow and endothélial injury results in organ damage, 15 including brain, heart, and kidneys. TTP is clinically characterized by thrombocytopenia, microangiopathic hemolytic anémia, neurological changes, rénal failure and fever. In the era before plasma exchange, the fatality rate was 90% during acute épisodes. Even with plasma exchange, survival at six months is about 80%.

TTP may arise from genetic or acquired inhibition of the enzyme ADAMTS-13, a 20 metalloprotease responsible for cleaving large multimers of von Willebrand factor (vWF) into smaller units. ADAMTS-13 inhibition or deficiency ultimately results in increased coagulation (Tsai, H. J Am Soc Nephrol 14: 1072-1081, 2003). ADAMTS-13 régulâtes the activity of vWF; in the absence of ADAMTS-13, vWF forms large multimers that are more likely to bind platelets and prédisposés patients to platelet aggregation and thrombosis in the 25 microvasculature.

Numerous mutations in ADAMTS13 hâve been identified in individuals with TTP. The disease can also develop due to autoantibodies against ADAMTS-13. In addition, TTP can develop during breast, gastrointestinal tract, or prostate cancer (George JN., Oncology (Williston Park). 25:908-14, 2011), pregnancy (second trimester or postpartum), (George JN., 30 Curr Opin Hematol 10:339-344, 2003), or is associated with diseases, such as HIV or autoimmune diseases like systemic lupus erythematosis (Hamasaki K, et al., Clin Rheumatol. 22:355-8, 2003). TTP can also be caused by certain drug thérapies, including heparin, • 104 quinine, immune mediated ingrédient, cancer chemotherapeutic agents (bleomycin, cisplatin, cytosine arabinoside, daunomycin gemcitabine, mitomycin C, and tamoxifen), cyclosporine A, oral contraceptives, penicillin, rifampin and anti-platelet drugs including ticlopidine and clopidogrel (Azarm, T. et al., J Res Med Soi., 16: 353-357, 2011). Other factors or 5 conditions associated with TTP are toxins such as bee venoms, sepsis, splenic séquestration, transplantation, vasculitis, vascular surgery, and infections like Streptococcus pneumoniae and cytomégalovirus (Moake JL., N Engl J Med., 347:589-600, 2002). TTP due to transient functional ADAMTS-13 deficiency can occur as a conséquence of endothélial cell injury associated with S. pneumoniae infection (Pediatr Nephrol, 26:631-5, 2011).

Plasma exchange is the standard treatment for TTP (Rock GA, et al., N Engl J Med

325:393-397, 1991). Plasma exchange replaces ADAMTS-13 activity in patients with genetic defects and removes ADAMTS-13 autoantibodies in those patients with acquired autoimmune TTP (Tsai, H-M, Hematol Oncol Clin North Am., 21(4): 609-v, 2007). Additional agents such as immunosuppressive drugs are routinely added to therapy (George, 15 JN, N Engl J Med, 354:1927-35, 2006). However, plasma exchange is not successful for about 20% of patients, relapse occurs in more than a third of patients, and plasmapheresis is costly and technically demanding. Furthermore, many patients are unable to tolerate plasma exchange. Consequently, there remains a critical need for additional and better treatments for TTP.

Because TTP is a disorder of the blood coagulation cascade, treatment with antagonists of the complément system may aid in stabilizing and correcting the disease. While pathological activation of the alternative complément pathway is linked to aHUS, the rôle of complément activation in TTP is less clear. The functional deficiency of ADAMTS13 is important for the susceptibility to TTP, however it is not suffïcient to cause acute épisodes.

Environmental factors and/or other genetic variations may contribute to the manifestation of TTP. For example, genes encoding proteins involved in the régulation of the coagulation cascade, vWF, platelet function, components of the endothélial vessel surface, or the complément system may be implicated in the development of acute thrombotic microangiopathy (Galbusera, M. et al., Haematologica, 94: 166-170, 2009). In particular, 30 complément activation has been shown to play a critical rôle; sérum from thrombotic microangiopathy associated with ADAMTS-13 deficiency has been shown to cause C3 and MAC déposition and subséquent neutrophil activation which could be abrogated by

105 complément inactivation (Ruiz-Torres MP, et al., Thromb Haemost, 93:443-52, 2005). In addition, it has recently been shown that during acute épisodes of TTP there are increased levels of C4d, C3bBbP, and C3a (M. Réti et al., J Thromb Haemost. 10(5):791-798, 2012), consistent with activation of the classical, lectin and alternative pathways. This increased amount of complément activation in acute épisodes may initiate the terminal pathway activation and be responsible for further exacerbation of TTP.

The rôle of ADAMTS-13 and vWF in TTP clearly is responsible for activation and aggregation of platelets and their subséquent rôle in shear stress and déposition in microangiopathies. Activated platelets internet with and trigger both the classical and alternative pathways of complément. Platelet-mediated complément activation increases the inflammatory mediators C3a and C5a (Peerschke E. et al., Mol Immunol, 47:2170-5 (2010)). Platelets may thus serve as targets of classical complément activation in inherited or autoimmune TTP.

As described above, the lectin-dependent activation of complément, by virtue of the thrombin-like activity of MASP-1 and the LEA-2-mediated prothombin activation, is the dominant molecular pathway linking endothélial injury to the coagulation and microvascular thrombosis that occurs in HUS. Similarly, activation of LEA-1 and LEA-2 may directly drive the coagulation System in TTP. LEA-1 and LEA-2 pathway activation may be initiated in response to the initial endothélium injury caused by ADAMTS-13 deficiency in TTP. It is therefore expected that LEA-1 and LEA-2 inhibitors, including but not limited to antibodies that block MASP-2 function, MASP-1 function, MASP-3 function, or MASP-1 and MASP-3 function will mitigate the microangiopathies associated with microvascular coagulation, thrombosis, and hemolysis in patients suffering from TTP.

Patients suffering from TTP typically présent in the emergency room with one or more of the following: purpura, rénal failure, low platelets, anémia and/or thrombosis, including stroke. The current standard of care for TTP involves intra-catheter delivery (e.g., intravenous or other form of cathéter) of replacement plasmapheresis for a period of two weeks or longer, typically three times a week, but up to daily. If the subject tests positive for the presence of an inhibitor of ADAMTS13 (i.e., an endogenous antibody against ADAMTS13), then the plasmapheresis may be carried out in combination with immunosuppressive therapy (e.g., corticosteroids, rituxan, or cyclosporine). Subjects with

106 refractory TTP (approximately 20% of TTP patients) do not respond to at least two weeks of plasmapheresis therapy.

In accordance with the foregoing, in one embodiment, in the setting of an initial diagnosis of TTP, or in a subject exhibiting one or more symptoms consistent with a 5 diagnosis of TTP (e.g., central nervous system involvement, severe thrombocytopenia (a platelet count of less than or equal to 5000/pL if off aspirin, less than or equal to 20,000/pL if on aspirin), severe cardiac involvement, severe pulmonary involvement, gastro-intestinal infarction or gangrené), a method is provided for treating the subject with an effective amount of a LEA-2 inhibitory agent (e.g., a MASP-2 antibody) or a LEA-1 inhibitory agent 10 (e.g., a MASP-1 or MASP-3 antibody) as a first line therapy in the absence of plasmapheresis, or in combination with plasmapheresis. As a first-line therapy, the LEA-1 and/or LEA-2 inhibitory agent may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration. In some embodiments, the LEA-1 and/or LEA-2 inhibitory agent is 15 administered to a subject as a first-line therapy in the absence of plasmapheresis to avoid the potential complications of plasmapheresis, such as hemorrhage, infection, and exposure to disorders and/or allergies inhérent in the plasma donor, or in a subject otherwise averse to plasmapheresis, or in a setting where plasmapheresis is unavailable. In some embodiments, the LEA-1 and/or LEA-2 inhibitory agent is administered to the subject suffering from TTP 20 in combination (including co-administration) with an immunosuppressive agent (e.g., corticosteroids, rituxan or cyclosporine) and/or in combination with concentrated ADAMTS13.

In some embodiments, the method comprises administering a LEA-1 and/or LEA-2 inhibitory agent to a subject suffering from TTP via a cathéter (e.g., intravenously) for a first 25 time period (e.g., an acute phase lasting at least one day to a week or two weeks) followed by administering a LEA-1 and/or LEA-2 inhibitory agent to the subject subcutaneously for a second time period (e.g., a chronic phase of at least two weeks or longer). In some embodiments, the administration in the first and/or second time period occurs in the absence of plasmapheresis. In some embodiments, the method is used to maintain the subject to 30 prevent the subject from suffering one or more symptoms associated with TTP.

In another embodiment, a method is provided for treating a subject suffering from refractory TTP (i.e., a subject that has not responded to at least two weeks of plasmaphoresis

107 therapy), by administering an amount ofa LEA-1 and/or LEA-2 inhibitor effective to reduce one or more symptoms of TTP. In one embodiment, the LEA-1 and/or LEA-2 inhibitor is administered to a subject with refractory TTP on a chronic basis, over a time period of at least two weeks or longer via subcutaneous or other parentéral administration. Administration 5 may be repeated as determined by a physician until the condition has been resolved or is controlled.

In some embodiments, the method further comprises determining the level of at least one complément factor (e.g., C3, C5) in the subject prior to treatment, and optionally during treatment, wherein the détermination of a reduced level of the at least one complément factor 10 in comparison to a standard value or healthy control subject is indicative of the need for continued treatment with the LEA-1 and/or LEA-2 inhibitory agent.

In some embodiments, the method comprises administering, either subcutaneousiy or intravenously, a LEA-1 and/or LEA-2 inhibitory agent to a subject suffering from, or at risk for developing, TTP. Treatment is preferably daily, but can be as infrequent as monthiy. 15 Treatment is continued until the subject’s platelet count is greater than 150,000/mi for at least two consecutive days.

In summary, LEA-1 and LEA-2 inhibitors are expected to hâve independent therapeutic benefit in the treatment of TMAs, including HUS, aHUS and TTP. In addition, LEA-1 and LEA-2 inhibitors used together are expected to achieve additional treatment 20 benefit compared to either agent aione, or may provide effective treatment for a wider spectrum of patient subsets suffering from variant forms of TMA. Combined LEA-1 and LEA-2 inhibition may be accomplished by co-administration ofa LEA-1 blocking agent and a LEA2 blocking agent. Optimaily, LEA-1 and LEA-2 inhibitory function may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP25 1/3 and a MASP-2-specifïc binding site, or a dual specificity antibody where each binding site can bind to and block MASP-1/3 or MASP-2.

In accordance with the foregoing, an aspect of the invention thus provides a method for inhibiting LEA-1 dépendent complément activation for treating, preventing, or reducing the severity of a thrombotic microangiopathy, such as hemoiytic urémie syndrome (HUS), 30 atypicai hemoiytic urémie syndrome (aHUS) or thrombotic thrombocytopénie purpura (TTP) comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibitory agent comprising a MASP 1 inhibitory agent, a MASP 3 inhibitory agent,

108 or a combination of a MASP 1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from, or at risk for developing a thrombotic microangiopathy. The MASP 1, MASP 3, or MASP 1/3 inhibitory composition may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other 5 parentéral administration, or potentially by oral administration for non peptidergic agents.

Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

In one embodiment, the method according to this aspect of the invention further comprises inhibiting LEA-2-dependent complément activation for treating, preventing, or 10 reducing the severity of a thrombotic microangiopathy, such as hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS) or thrombotic thrombocytopénie purpura (TTP) comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering from, or at risk for developing a thrombotic microangiopathy. As detailed above, 15 the use of a combination of pharmacologie agents that individually block LEA-1 and LEA-2, is expected to provide an improved therapeutic outcome in treating or preventing or reducing the severity of a thrombotic microangiopathy as compared to the inhibition of LEA-1 alone. This outcome can be achieved for example, by co-administration of an antibody that has LEA-1-blocking activity together with an antibody that has LEA-2-blocking activity. In 20 some embodiments, LEA-1- and LEA-2-blocking activities are combined into a single molecular entity, and that such entity with combined LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody where one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may 25 consist of a bispecific monoclonal antibody where one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may optimally consist of a bispecific monoclonal antibody where one antigen-combining site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site 30 specifically recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other

109 parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled. .

Application of the MASP-3 inhibitory compositions and optional MASP-2 inhibitory compositions of the présent invention may be carried out by a single administration of the composition (e.g., a single composition comprising MASP-2 and MASP-3 inhibitory agents, or bispecific or dual inhibitory agents, or co-administration of separate compositions), or a limited sequence of administrations, for treating, preventing or reducing the severity of a thrombotic microangiopathy in a subject suffering from, or at risk for developing, a thrombotic microangiopathy. Alternatively, the composition may be administered at periodic intervals such as daily, biweekly, weekly, every other week, monthly or bimonthly over an extended period of time for treatment of a subject in need thereof.

As described in Examples 11-21 herein, high affïnity MASP-3 inhibitory antibodies hâve been generated which hâve therapeutic utility for inhibition ofthe alternative pathway in AP-related diseases or conditions, such as a thrombotic microangiopathy (e.g., hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS), or thrombotic thrombocytopénie purpura (TTP).

Accordingly, in one embodiment, the présent invention provides a method for treating a subject suffering from, or at risk for developing a thrombotic microangiopathy (e.g.,hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS), or thrombotic thrombocytopénie purpura (TTP), comprising an effective amount of a high affïnity monoclonal antibody or antigen binding fragment thereof as disclosed herein that binds to human MASP-3 and inhibits alternative pathway complément activation to treat or reduce the risk of developing a thrombotic microangiopathy (e.g., hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS), thrombotic thrombocytopénie purpura (TTP), or transplant-related TMA (TA-TMA), such as, for example, wherein said antibody or antigen binding fragment thereof comprises (a) a heavy chain variable région comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable région comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161.

110

G. THE ROLE OF MASP-3 IN ASTHMA AND THERAPEUTIC METHODS USING MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITORY AGENTS

Asthma is a common chronic inflammatory disease of the airways. Approximately 25 million people in the United States hâve asthma, including seven million children under the âge of 18, with more than half experiencing at least one asthma attack each year, leading to more than 1.7 million emergency department visits and 450,000 hospitalizations annually (world-wide-web at gov/health/prof/lung/asthma/naci/asthma-info/index.htm., accessed on May 4, 2012). The disease is heterogeneous with multiple clinical phenotypes. The most common phenotype is allergie asthma. Other phenotypes include nonallergic asthma, aspirinexacerbated respiratory disease, post-infectious asthma, occupational asthma, airbome irritant-induced asthma, and exercise-induced asthma. The cardinal features of allergie asthma include airway hyperresponsiveness (AHR) to a variety of spécifie and nonspecific stimuli, excessive airway mucus production, pulmonary eosinophilia, and elevated concentration of sérum IgE. The symptoms of asthma include coughing, wheezing, chest tightness, and shortness of breath. The goal of asthma treatment is to control the disease and minimize exacerbations, daily symptoms, and allow patients to be physically active. Current treatment guidelines recommend stepwise treatments until asthma control is attained. The first treatment step is as needed rapid-acting inhaled p2-agonist, followed by addition of controller médications such as inhaled corticosteroids, long-acting inhaled p2-agonists, leukotriene modifier drugs, theophylline, oral glucocorticosteroids, and anti-IgE monoclonal antibody.

Although asthma is multifactorial in origin, it is generally accepted that it arises as a resuit of inappropriate immunological responses to common environmental antigens in genetically susceptible individuals. Asthma is associated with complément activation and the anaphylatoxins (AT) C3a and C5a hâve proinflammatory and immunoregulatory properties that are relevant to the development and modulation of the allergie response (Zhang, X. and Kohl, J. Expert. Rev. Clin. Immunol., 6:269-277, 2010). However, the relative involvement of the classical, alternative, and lectin pathways of complément in asthma is not well understood. The alternative pathway may be activated on the surface of allergens and the

111 lectin pathway may be activated through récognition of allergen polysaccharide structures, both processes leading to the génération of AT. Complément may be activated by different pathways depending on the causative allergen involved. Highly allergie grass pollen of the Parietaria family for example is very effective at promoting MBL-dependent activation of C4, implicating LEA-2. Conversely, house dust mite allergen does not require MBL for complément activation (Varga et al. Mol Immunol., 39(14):839-46, 2003).

Environmental triggers of asthma may activate complément by the alternative pathway. For example, in vitro exposure of human sérum to cigarette smoke or diesel exhaust particles resulted in activation of complément and the effect was unaffected by the presence of EDTA, suggesting activation was via the alternative rather than classical pathway (Robbins, R.A. et al, Am. J. Physiol. 260: L254-L259, 1991; Kanemitsu, H., et al., Biol. Pharm. Bull. 21:129-132, 1998). The rôle of complément pathways in allergie airway inflammation was evaluated in a mouse ovalbumin sensitization and challenge model. Wildtype mice developed AHR and airway inflammation in response to aeroallergen challenge. A Crry-Ig fusion protein which inhibits ail pathways of complément activation, was effective in preventing AHR and lung inflammation when administered systemically or locally by inhalation in the mouse ovalbumine model of allergie lung inflammation (Taube et al., Am J Respir Crit Care Med., 168( 11):1333-41, 2003).

In comparison to wild-type mice, factor B-deficient mice demonstrated less AHR and airway inflammation whereas C4-deficient mice had similar effects as wild-type mice (Taube, C., et al., Proc. Natl. Acad. Sci. USA 103:8084-8089, 2006). These results support a rôle for alternative pathway and not classical pathway involvement in the murine aeroallergen challenge model. Further evidence for the importance of the alternative pathway was provided in a study of factor H (FH) using the same mouse model (Takeda, K., et al., J. Immunol. 188:661-667, 2012). FH is a négative regulator of the alternative pathway and acts to prevent autologous injury of self tissues. Endogenous FH was found to be présent in airways during allergen challenge and inhibition of FH with a recombinant compétitive antagonist increased the extent of AHR and airway inflammation (Takeda et al., 2012, supra). Therapeutic delivery of CR2-fH, a chimeric protein that links the iC3b/C3d binding région of CR2 to the complement-regulatory région of FH which targets the complément regulatory activity of fH to sites of existing complément activation, protected the development of AHR and eosinophil infiltration into the airways after allergen challenge

112 (Takeda et al., 2012, supra). The protective effect was demonstrated with ovalbumin as well as ragweed allergen, which is a relevant allergen in humans.

The rôle of lectin-dependent complément activation in asthma was evaluated in a mouse model of fungal asthma (Hogaboam et al., J. Leukocyte Biol. 75:805 814, 2004). These studies used mice genetically déficient in mannan binding lectin A (MBL-A), a carbohydrate binding protein that functions as the récognition component for activation of the lectin complément pathways. MBL-A(+/+) and MBL-A(-/-) Aspergillus. fumigatus sensitized mice were examined at days 4 and 28 after an i.t. challenge with A. fumigatus conidia. AHR in sensitized MBL-A(-/-) mice was significantly attenuated at both times after conidia challenge compared with the sensitized MBL-A (+/+) group. Lung TH2 cytokine levels (IL-4, IL-5 and IL-13) were significantly lower in A. fumigatus-sensxùzeà MBL-A(-/-) mice compared to the wild-type group at day 4 after conidia. These results indicate that MBL-A and the lectin pathway hâve a major rôle in the development and maintenance of AHR during chronic fungal asthma.

The findings detailed above suggest the involvement of lectin-dependent complément activation in the pathogenesis of asthma. Experimental data suggest that factor B activation plays a pivotai rôle. In light of the fondamental rôle for LEA-1 in the lectin-dependent activation of factor B and subséquent activation of the alternative pathway, it is expected that LEA-1 blocking agents will be bénéficiai for the treatment of certain forms of asthma mediated by the alternative pathway. Such a treatment may thus be particularly useful in house dust mite-induced asthma, or asthma caused by environmental triggers such as cigarette smoke or diesel exhaust. Asthmatic responses triggered by grass pollen on the other hand are likely to invoke LEA-2-dependent complément activation. Therefore, LEA-2blocking agents are expected to be particularly useful in treating the asthmatic conditions in this subset of patients.

In view of the data detailed above, the inventors believe that LEA-1 and LEA-2 médiate pathologie complément activation in asthma. Depending on the inciting allergie agent, LEA-1 or LEA-2 may be preferentially involved. Thus, a LEA-1-blocking agent combined with a LEA-2-blocking agent may hâve utility in the treatment of multiple forms of asthma regardless of the underlying etiology. LEA-1 and LEA-2-blocking agents may hâve complementary, additivé or synergistic effects in preventing, treating or reversing pulmonary inflammation and symptoms of asthma.

113

Combined LEA-1 and LEA-2 inhibition may be accomplished by co-administration of a LEA-1-blocking agent and a LEA2-blocking agent. Optimally, LEA-1 and LEA-2 inhibitory function may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a MASP-2-specific binding site, or a dual specificity antibody where each binding site can bind to and block MASP-1/3 or MASP-2.

In accordance with the foregoing, an aspect of the invention thus provides a method for inhibiting LEA-1 dépendent complément activation for treating, preventing, or reducing the severity of asthma, comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of a MASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from, or at risk for developing asthma. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

In one embodiment, the method according to this aspect of the invention further comprises inhibiting LEA-2-dependent complément activation for treating, preventing, or reducing the severity of asthma, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering from, or at risk for developing asthma. As detailed above, the use of a combination of pharmacologie agents that individually block LEA-1 and LEA-2, is expected to provide an improved therapeutic outcome in treating or preventing or reducing the severity of asthma as compared to the inhibition of LEA-1 alone. This outcome can be achieved for example, by co-administration of an antibody that has LEA-1-blocking activity together with an antibody that has LEA-2-blocking activity. In some embodiments, LEA-1- and LEA-2blocking activities are combined into a single molecular entity, and that such entity with combined LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody where one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody where one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1

114 and the second antigen-combining site specifîcally recognizes MASP-2 and blocks LEA-2. Such an entity may optimally consist of a bispecific monoclonal antibody where one antigencombining site specifîcally recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifîcally recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and optional MASP-2 inhibitory compositions of the présent invention may be carried out by a single administration of the composition (e.g., a single composition comprising MASP-2 and MASP-3 inhibitory agents, or bispecific or dual inhibitory agents, or co-administration of separate compositions), or a limited sequence of administrations, for treating, preventing or reducing the severity of a asthma in a subject suffering from, or at risk for developing asthma. Altematively, the composition may be administered at periodic intervals such as daily, biweekly, weekly, every other week, monthly or bimonthly over an extended period of time for treatment of a subject in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies hâve been generated which hâve therapeutic utility for inhibition ofthe alternative pathway in AP-related diseases or conditions, such as asthma.

Accordingly, in one embodiment, the présent invention provides a method for treating a subject suffering from, or at risk for developing asthma comprising an effective amount of a high affinity monoclonal antibody or antigen binding fragment thereof as disclosed herein that binds to human MASP-3 and inhibits alternative pathway complément activation to treat or reduce the risk of developing asthma, such as, for example, wherein said antibody or antigen binding fragment thereof comprises (a) a heavy chain variable région comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable région comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ IDNO:161.

115

H. THE ROLE OF MASP-3 IN DENSE DEPOSIT DISEASE, AND THERAPEUTIC METHODS USING MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITORY AGENTS

Membranoproliferative glomerulonephritis (MPGN) is a kidney disorder characterized morphologically by mesangial cell prolifération and thickening of the glomerular capillary wall due to subendothelial extension of the mesangium. MPGN is classified as primary (also referred to as idiopathic) or secondary, with underlying diseases such as infectious diseases, systemic immune complex diseases, neoplasms, chronic liver disease, and others. Idiopathic MPGN includes three morphologie types. Type I, or classical MPGN, is characterized by subendothelial deposits of immune complexes and activation of the classical complément pathway. Type II, or dense deposit disease (DDD), is characterized by additional intra-membraneous dense deposits. Type III is characterized by additional subepithelial deposits. Idiopathic MPGN is rare, accounting for approximately 4 to 7% of primary rénal causes of nephrotic syndrome (Alchi, B. and Jayne, D. Pediatr. Nephrol. 25:1409-1418, 2010). MPGN primarily affects children and young adults and may présent as nephrotic syndrome, acute nephritic syndrome, asymptomatic proteinuria and hematuria, or récurrent gross hematuria. Rénal dysfunction occurs in the majority of patients and the disease has a slowly progressive course, with approximately 40% of patients developing endstage rénal disease within 10 years of diagnosis (Alchi and Jayne, 2010, supra). Current treatment options include corticosteroids, immunosuppressives, antiplatelet regimens, and plasma exchange. .

DDD is diagnosed by the absence of immunoglobulin and presence of C3 by immunofluorescence staining of rénal biopsies, and électron microscopy shows characteristic dense osmiophilic deposits along the glomerular basement membranes. DDD is caused by dysrégulation of the alternative pathway of complément (Sethi et al, Clin J Am Soc Nephrol. 6(5):1009-17, 2011), which can arise from a number of different mechanisms. The most common complément System abnormality in DDD is the presence of C3 nephritic factors which are autoantibodies to the alternative pathway C3 convertase (C3bBb) that increases its half-life and therefore activation of the pathway (Smith, R.J.H. et al., Mol. Immunol. 48:1604-1610, 2011). Other alternative pathway abnormalitîes include factor H autoantibody

116 that blocks the function of factor H, gain of function C3 mutations, and genetic deficiency of factor H (Smith et al., 2011, supra). Recent case reports show that eclizumab (anti-C5 monoclonal antibody) treatment was associated with improvements in rénal function in two patients with DDD (Daina, E. et al., New Engl. J. Med. 366:1161-1163, 2012; Vivarelli, M. et 5 al., New Engl. J. Med. 366:1163-1165, 2012), suggesting a causative rôle for complément activation in rénal outcomes.

Given the above genetic, functional and immunohistochemical and anecdotal clinical data, the central rôle for complément in the pathogenesis of DDD is well established. Thus, interventions that block the disease-causing mechanisms of complément activation, or the 10 subséquent complément activation products, are expected to be therapeutically useful to treat this condition.

While the human genetic data suggest that inappropriate control or excessive activation of the alternative pathways amplification loop plays a key rôle, complementinitiating events hâve not been identified. Immunohistochemical studies in rénal biopsies 15 show evidence of MBL déposition in diseased tissue, suggesting involvement of the lectin pathways in the initiation of pathological complément activation in DDD (Lhotta et al, Nephrol Dial Transplant., 14(4):881-6, 1999). The importance of the alternative pathway has been further corroborated in experimental models. Factor H-deficient mice develop progressive proteinuria and the rénal pathological lésions characteristic of the human 20 condition (Pickering et al., Nat Genet., 31(4):424, 2002). Pickering et al. further demonstrated that ablation of factor B, which médiates LEA-1-dépendent activation of the alternative pathway, fully protects factor H-deficient mice from DDD (Pickering et al., Nat Genet., 31(4):424, 2002).

Thus it is expected that agents that block LEA-1 will effectively block lectin25 dépendent activation of the alternative pathway, and will thus provide an effective treatment for DDD. Given that the alternative pathway amplification loop is dysregulated in DDD patients, it can further be expected that agents that block the amplification loop will be effective. Since LEA-1-targeting agents that block MASP-1 or MASP-1 and MASP-3 inhibit the maturation of factor D, such agents are predicted to effectively block the alternative 30 pathway amplification loop.

As detailed above, pronounced MBL déposition has been found in diseased rénal specimens, highlighting the probable involvement of lectin-driven activation events in DDD

117 pathogenesis. Once an initial tissue injury to the glomerular capillaries is established, it is likely that additional MBL binding to injured glomerular endothélium and underlying mesangial structures occurs. Such tissue injuries are well known to lead to activation of LEA-2, which can thus cause further complément activation. Therefore, LEA-2-blocking agents are also expected to hâve utility in preventing further complément activation on injured glomerular structures, and thus forestall further disease progression towards end stage rénal failure.

The data detailed above suggest that LEA-1 and LEA-2 promote separate pathologie complément activation processes in DDD. Thus, a LEA-1-blocking agent and a LEA-2 blocking agent, either alone or in combination are expected to be useful for treating DDD.

When used in combination, LEA-1- and LEA-2-blocking agents are expected to be more efficacious than either agent alone, or useful for treating different stages of the disease. LEA-1- and LEA-2-blocking agents may thus hâve complementary, additive or synergistic effects in preventing, treating or reversing DDD-associated rénal dysfunction.

Combined LEA-1 and LEA-2 inhibition may be accomplished by co-administration of a LEA-1 blocking agent and a LEA2 blocking agent. Optimally, LEA-1 and LEA-2 blocking agents with inhibitory function may be encompassed in a single molecular entity, such as a bispecific antibody composed of MASP-1/3 and a MASP-2-specific binding site, or a dualspecificity antibody where each binding site can bind to and block MASP-1/3 or MASP-2.

In accordance with the foregoing, an aspect of the invention thus provides a method for inhibiting LEA-1 dépendent complément activation for treating, preventing, or reducing the severity of dense deposit disease, comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibitory agent comprising a MASP 1 inhibitory agent, a MASP 3 inhibitory agent, or a combination of a MASP 1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from, or at risk for developing dense deposit disease. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subeutaneous or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

In another aspect, a method is provided for inhibiting LEA-2-dependent complément activation for treating, preventing, or reducing the severity of dense deposit disease,

118 comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent to a subject suffering from, or at risk for developing dense deposit disease. In another aspect, a method is provided comprising inhibiting both LEA-1 and LEA-2-dependent complément activation for treating, preventing, or reducing the severity of dense deposit disease, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-l/3-inhibitory agent to a subject suffering from, or at risk for developing dense deposit disease. .

In some embodiments, the method comprises inhibiting both LEA-1-dépendent complément activation and LEA-2-dependent complément activation. As detailed above, the use of a combination of pharmacologie agents that individually block LEA-1 and LEA-2, is expected to provide an improved therapeutic outcome in treating, preventing or reducing the severity of dense deposit disease as compared to the inhibition of LEA-1 alone. This outcome can be achieved for example, by co-administration of an antibody that has LEA-1blocking activity together with an antibody that has LEA-2-blocking activity. In some embodiments, LEA-1- and LEA-2-blocking activities are combined into a single molecular entity, and that such entity with combined LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody where one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody where one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may optimally consist of a bispecific monoclonal antibody where one antigen-combining site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically recognized MASP-2 and blocks LEA-2.

The LEA-1 and/or LEA-2 inhibitory agents may be administered to the subject systemicaiiy, such as by intra arterial, intravenous, intramuscuiar, inhaiational, nasal, subeutaneous or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resoived or is controiled.

Application of the MASP-3 inhibitory compositions and/or the MASP-2 inhibitory compositions of the présent invention may be carried out by a single administration of the

119 composition (e.g., a single composition comprising MASP-2 and/or MASP-3 inhibitory agents, or bispecific or dual inhibitory agents, or co-administration of separate compositions), or a limited sequence of administrations, for treating, preventing or reducing the severity of dense deposit disease in a subject in need thereof. Alternatively, the composition may be administered at periodic intervals such as daily, biweekly, weekly, every other week, monthly or bimonthly over an extended period of time for treatment of a subject in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies hâve been generated which hâve therapeutic utility for inhibition of the alternative pathway in AP-related diseases or conditions, such as dense deposit disease.

Accordingly, in one embodiment, the présent invention provides a method for treating a subject suffering from, or at risk for developing dense deposit disease comprising an effective amount of a high affinity monoclonal antibody or antigen binding fragment thereof as disclosed herein that binds to human MASP-3 and inhibits alternative pathway complément activation to treat or reduce the risk of developing dense deposit disease, such as, for example, wherein said antibody or antigen binding fragment thereof comprises (a) a heavy chain variable région comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable région comprising (i) VLCDR1 comprising SEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ IDNO:161.

I. THE ROLE OF MASP-3 IN PAUCI-IMMUNE NECROTIZING CRESCENTIC GLOMERULONEPHRITIS, AND THERAPEUTIC METHODS USING MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION WITH AND MASP-2 INHIBITORY AGENTS

Pauci-immune necrotizing crescentic glomerulonephritis (NCGN) is a form of rapidly progressive glomerulonephritis in which glomerular capillary walls show signs of inflammation yet hâve a paucity of détectable immunocomplex déposition or antibodies against the glomerular basement membrane. The condition is associated with a rapid décliné in rénal function. Most patients with NCGN are found to hâve antineutrophil cytoplasmic autoantibodies (ANCA) and thus belong to a group of diseases termed ANCA-associated

120 vasculitis. Vasculitis is a disorder of blood vessels characterized by inflammation and fibrinoid necrosis of the vessel wall. Systemic vasculitides are classified based on vessel size: large, medium, and small. Several forms of small-vessel vasculitis are associated with the presence of ANCA, namely Wegener granulomatosis, microscopie polyangiitis, ChurgStrauss syndrome, and renal-limited vasculitis (NCGN). They can also be a manifestation of underlying conditions such as systemic lupus erythematosus. The target antigens for ANCA include proteinase-3 (PR3) and myeloperoxidase (MPO). Pauci-immune NCGN is rare, with a reported incidence of approximately 4 per million in Wessex, United Kingdom (Hedger, N. et al., Nephrol. Dial. Transplant. 15:1593-1599, 2000). In the Wessex sériés of 128 patients with pauci-immune NCGN, 73% were ANCA-positive and initial dialysis was required by 59% of patients and 36% needed long-term dialysis. Treatments for pauci-immune NCGN include corticosteroids and immunsuppressive agents such as cyclophosphamide and azathioprine. Additional treatment options for ANCA-associated vasculitides include rituximab and plasma exchange (Chen, M. and Kallenberg, C.G.M. Nat. Rev. Rheumatol. 6:653-664, 2010).

Although NCGN is characterized by a paucity of complément déposition, the alternative pathway of complément has been implicated in its pathogenesis. A rénal biopsy évaluation of 7 patients with MPO-ANCA-associated pauci-immune NCGN detected the presence of membrane attack complex, C3d, factor B, and factor P (which were not detected in biopsies from normal Controls or patients with minimal change disease), whereas C4d and mannose binding lectin were not detected, suggesting sélective activation of the alternative pathway (Xing, G.Q. et al. J. Clin. Immunol. 29:282-291, 2009). Experimental NCGN can be induced by transfer of anti-MPO IgG into wild-type mice or anti-MPO splénocytes into immune-deficient mice (Xiao, H. et al. J. Clin. Invest. 110:955-963, 2002). In this mouse model of NCGN, the rôle of spécifie complément activation pathways was investigated using knockout mice. After injection of anti-MPO IgG, C4-/- mice developed rénal disease comparable to wild-type mice whereas C5-/- and factor B-/- mice did not develop rénal disease, indicating that the alternative pathway was involved in this model and the classical and lectin pathways were not (Xiao, H. et al. Am. J. Pathol. 170:52-64, 2007). Moreover, incubation of MPO-ANCA or PR3-ANCA IgG from patients with TNF-primed human neutrophils caused release of factors that resulted in complément activation in normal human sérum as detected by génération of C3a; this effect was not observed with IgG from healthy

121 subjects, suggesting the potential pathogenic rôle of ANCA in neutrophil and complément activation (Xiao et al., 2007, supra).

Based on the rôle outlined above for the alternative pathway in this condition, it is expected that blocking the activation of the alternative pathway will hâve utility in the treatment of ANCA positive NCGN. Given the requirement for fB activation for pathogenesis, it is expected that inhibitors of LEA-1 will be particularly useful in treating this condition, and in preventing the further décliné in rénal function in these patients.

Yet another subset of patients develops progressive rénal vasulitis with crescent formation accompanied by a rapid décliné in rénal function in the absence of ANCA. This form of the condition is termed ANCA-negative NCGN and constitutes about one third of ali patients with pauci immune NCGN (Chen et al, JASN 18(2): 599-605, 2007). These patients tend to be younger, and rénal outcomes tend to be particularly severe. (Chen et al., Nat Rev Nephrol., 5(6):313-8, 2009). A discriminating pathological feature of these patients is the déposition of MBL and C4d in rénal lésions (Xing et al., J Clin Immunol. 30(1):144-56, 2010). MBL and C4d staining intensity in rénal biopsies correlated negatively with rénal function (Xing et al., 2010, supra). These findings suggest an important rôle for lectindependent complément activation in pathogenesis. The fact that C4d, but not factor B is commonly found in diseased tissue specimens indicates LEA-2 involvement.

Based on the rôle of lectin-dependent complément activation in ANCA négative NCGN described above, it is expected that blocking the activation of the LEA-2 pathway will hâve utility in the treatment of ANCA négative NCGN.

The data detailed above suggest that LEA-1 and LEA-2 médiate pathologie complément activation in ANCA-positive and ANCA-negative NCGN, respectively. Thus, a LEA-1-blocking agent combined with a LEA-2-bIocking agent is expected to hâve utility in the treatment of ail forms of pauci-immune NCGN, regardless of the underlying etiology. LEA-I- and LEA-2-blocking agents may thus hâve complementary, additive or synergistic effects in preventing, treating or reversing NCGN-associated rénal dysfunction.

LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone, or may provide effective treatment for a wider spectrum of patient subsets. Combined LEA-1 and LEA-2 inhibition may be accomplished by coadministration of a LEA-1 blocking agent and a LEA-2 blocking agent. Optimally, LEA-1 and LEA-2 inhibitory function may be encompassed in a single molecular entity, such as a

122 bispecific antibody composed of MASP-1/3 and a MASP-2-specific binding site, or a dualspecificity antibody where each binding site can bind to and block MASP-1/3 or MASP-2.

In accordance with the foregoing, an aspect of the invention thus provides a method for inhibiting LEA-1 dépendent complément activation for treating, preventing, or reducing the severity of pauci-immune necrotizing crescentic glomerulonephritis, comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of a MASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from, or at risk for developing pauci-immune necrotizing crescentic glomerulonephritis. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

In another aspect, a method is provided for inhibiting LEA-2-dependent complément activation for treating, preventing, or reducing the severity of pauci-immune necrotizing crescentic glomerulonephritis, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent to a subject suffering from, or at risk for developing pauciimmune necrotizing crescentic glomerulonephritis. In another aspect, a method is provided comprising inhibiting both LEA-1 and LEA-2-dependent complément activation for treating, preventing, or reducing the severity of pauci-immune necrotizing crescentic glomerulonephritis, comprising administering a therapeutically effective amount of a MASP2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject in need thereof.

In some embodiments, the method comprises inhibiting both LEA-1-dépendent complément activation and LEA-2-dependent complément activation. As detailed above, the use of a combination of pharmacologie agents that individually block LEA-1 and LEA-2, is expected to provide an improved therapeutic outcome in treating or preventing or reducing the severity of pauci-immune necrotizing crescentic glomerulonephritis as compared to the inhibition of LEA-1 alone. This outcome can be achieved for example, by co-administration of an antibody that has LEA-1-blocking activity together with an antibody that has LEA-2blocking activity. In some embodiments, LEA-1- and LEA-2-blocking activities are

123 combined into a single molecular entity, and that such entity with combined LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody where one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody where one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may optimally consist of a bispecific monoclonal antibody where one antigencombining site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and/or the MASP-2 inhibitory compositions of the présent invention may be carried out by a single administration of the composition (e.g., a single composition comprising MASP-2 and/or MASP-3 inhibitory agents, or bispecific or dual inhibitory agents, or co-administration of separate compositions), or a limited sequence of administrations, for treating, preventing or reducing the severity of pauci-immune necrotizing crescentic glomerulonephritis. Alternatively, the composition may be administered at periodic intervals such as daily, biweekly, weekly, every other week, monthly or bimonthly over an extended period of time for treatment of a subject in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies hâve been generated which hâve therapeutic utility for inhibition ofthe alternative pathway in AP-related diseases or conditions, such as Pauci-immune necrotizing crescentic glomerulonephritis (NCGN).

Accordingly, in one embodiment, the présent invention provides a method for treating a subject suffering from, or at risk for developing Pauci-immune necrotizing crescentic glomerulonephritis (NCGN) comprising an effective amount of a high affinity monoclonal antibody or antigen binding fragment thereof as disclosed herein that binds to human MASP19469

124 and inhibits alternative pathway complément activation to treat or reduce the risk of developing Pauci-immune necrotizing crescentic glomerulonephritis (NCGN), such as, for example, wherein said antibody or antigen binding fragment thereof comprises (a) a heavy chain variable région comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable région comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161.

J. THE ROLE OF MASP-3 IN TRAUMATIC BRAIN INJURY, AND THERAPEUTIC METHODS USING MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION WITH AND MASP-2 INHIBITORY AGENTS

Traumatic brain injury (TBI) is a major global health problem that leads to at least 10 million deaths or hospitalizations annually (Langlois, J.A. et al., J. Head Trauma Rehabil. 21:375-378, 2006). In 2003 there were an estimated 1.6 million TBIs in the United States, including 1.2 million emergency department visits, 290,000 hospitalizations, and 51,000 deaths (Rutland-Brown, W. et al., J. Head Trauma Rehabil. 21:544-548, 2006). The majority of TBIs in the United States are caused by faits and motor vehicle traffic. TBI can resuit in long-term or lifelong physical, cognitive, behavioral, and emotional conséquences. Over 5 million Americans are living with long-term or lifelong disability associated with a TBI (Langlois et al., 2006, supra).

TBI may involve pénétration of the brain substance (“penetrating” injuries) or injuries that do not penetrate the brain (“closed” injuries). The injury profiles and associated neurobehavioral sequelae can be quite different between penetrating and closed TBI. Although each injury is unique, certain brain régions are particularly vulnérable to traumainduced damage, including the frontal cortex and subfrontal white matter, the basal ganglia and diencephalon, the rostral brain stem, and the temporal lobes including the hippocampi (McAlIister, T.W. Dialogues Clin. Neurosci. 13:287-300, 2011). TBI can lead to changes in several neurotransmitter Systems, including release of glutamate and other excitatory amino acids during the acute phase and chronic alterations in the catecholaminergic and cholinergic Systems, which may be associated with neurobehavioral disability (McAlIister, 2011, supra).

125

Survivors of significant TBI often suffer from cognitive defects, personality changes, and increased psychiatrie disorders, particularly dépréssion, anxiety, and post-traumatic stress disorder. Despite intense research, no clinically effective treatment for TBI that can reduce mortality and morbidity and improve functional outcome has yet to be found.

Complément factors and TBI

Numerous studies hâve identified a relationship of complément proteins and neurological disorders, including Alzheimer’s disease, multiple sclerosis, myasthenia gravis, Guillain-Barré syndrome, cérébral lupus, and stroke (reviewed in Wagner, E., et al., Nature Rev Drug Disc. 9: 43-56, 2010). Recently a rôle for Clq and C3 in synapse élimination has been demonstrated, thus complément factors are likely involved in both normal CNS function and neurodegenerative disease (Stevens, B. et al., Cell 131:1164-1178, 2007). The gene for MASP-1 and MASP-3 is extensively expressed in the brain and also in a glioma cell line, T98G (Kuraya, M. et al., Int Immunol., 15:109-17, 2003), consistent with a rôle ofthe lectin pathway in the CNS.

MASP-1 and MASP-3 are key to immédiate defense against pathogens and altered self-cells, but the lectin pathway also is responsible for severe tissue damage after stroke, heart attack, and other ischemia reperfusion injuries. Similarly, MASP-1 and MASP-3 are likely mediators in the tissue damage caused by TBI. Inhibition of Factor B in the alternative pathway has been shown to attenuate TBI in two mouse models. Factor B knockout mice are protected from complement-mediated neuroinflammation and neuropathology after TBI (Leinhase I, et al., BMC Neurosci. 7:55, 2006). In addition, anti-factor B antibody attenuated cérébral tissue damage and neuronal cell death in TBI induced mice (Leinhase I, et al., J Neuroinflammation 4:13, 2007). MASP-3 directly activâtes Factor B (Iwaki, D. et al., J Immunol. 187:3751-8, 2011) and therefore is also a likely mediator in TBI. Similar to inhibition of Factor B, LEA-1 inhibitors, such as antibodies against MASP-3 are expected to provide a promising strategy for treating tissue damage and subséquent sequelae in TBI.

Thus, LEA-1 and LEA-2 inhibitors may hâve independent therapeutic benefit in TBI. In addition, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone, or may provide effective treatment for a wider spectrum of patient subsets. Combined LEA-1 and LEA-2 inhibition may be accomplished by co-administration of a LEA-1-blocking agent and a LEA2-blocking agent. Optimally, LEA-1 and LEA-2 inhibitory function may be encompassed in a single molecular entity, such as a

126 bispecific antibody composed of MASP-1/3 and a MASP-2-specific binding site, or a dualspecificity antibody where each binding site can bind to and block MASP-1/3 or MASP-2.

In accordance with the foregoing, an aspect of the invention thus provides a method for inhibiting LEA-1 dépendent complément activation for treating, or reducing the severity of traumatic brain injury, comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of a MASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from a traumatic brain injury. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, intracranial, subcutaneous or other parentéral administration, or potentielly by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

In another aspect, a method is provided for inhibiting LEA-2-dependent complément activation for treating, or reducing the severity of traumatic brain injury, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent to a subject suffering from a traumatic brain injury. In another aspect, a method is provided comprising inhibiting both LEA-1 and LEA-2-dependent complément activation for treating, or reducing the severity of traumatic brain injury, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering from a traumatic brain injury.

In some embodiments, the method comprises inhibiting both LEA-1-dépendent complément activation and LEA-2-dependent complément activation. As detailed above, the use of a combination of pharmacologie agents that individually block LEA-1 and LEA-2 is expected to provide an improved therapeutic outcome in treating or reducing the severity of traumatic brain injury as compared to the inhibition of LEA-1 alone. This outcome can be achieved for example, by co-administration of an antibody that has LEA-1-blocking activity together with an antibody that has LEA-2-blocking activity. In some embodiments, LEA-1 and LEA-2-blocking activities are combined into a single molecular entity, and that such entity with combined LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody where one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes

127

MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody where one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may optimally consist of a bispecific monoclonal antibody 5 where one antigen-combining site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous, intracranial, 10 or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and/or the MASP-2 inhibitory compositions of the présent invention may be carried out by a single administration of the 15 composition (e.g., a single composition comprising MASP-2 and/or MASP-3 inhibitory agents, or bispecific or dual inhibitory agents, or co-administration of separate compositions), or a limited sequence of administrations, for treating or reducing the severity of traumatic brain injury. Alternatively, the composition may be administered at periodic intervals such as daily, biweekly, weekly, every other week, monthly or bimonthly over an extended period of 20 time for treatment of a subject in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies hâve been generated which hâve therapeutic utility for inhibition of the alternative pathway in AP-related diseases or conditions, such as traumatic brain injury.

Accordingly, in one embodiment, the présent invention provides a method for treating 25 a subject suffering from, or at risk for developing traumatic brain injury comprising an effective amount of a high affinity monoclonal antibody or antigen binding fragment thereof as disclosed herein that binds to human MASP-3 and inhibits alternative pathway complément activation to treat or reduce the risk of developing traumatic brain injury, such as, for example, wherein said antibody or antigen binding fragment thereof comprises (a) a 30 heavy chain variable région comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable région comprising (i) VLCDR1 comprising

128

SEQ ID NO: 142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ IDNO:144 and (iii) VLCDR3 comprising SEQ IDN0:16I.

K. THE ROLE OF MASP-3 IN ASPIRATION PNEUMONIA, AND

THERAPEUTIC METHODS USING MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITORY AGENTS

Aspiration is defined as the inhalation of either oropharyngeal or gastric contents into the lower airways. Aspiration may resuit in complications of aspiration (chemical) pneumonitis, primary bacterial aspiration pneumonia, or secondary bacterial infection of chemical pneumonitis. Risk factors for aspiration include decreased levels of consciousness (e.g., head trauma, alcohol or drug-induced alterations in sensorium, stroke), various gastrointestinal and esophageal abnormalities, and neuromuscular diseases. It is estimated that 5-15% of the 4.5 million cases of community-acquired pneumonia are due to aspiration pneumonia (Marik, P.E. New Engl. J. Med. 344:665-671, 2001). Treatment of chemical pneumonitis is mainly supportive and the use of empiric antibiotics is controversial. Treatment of bacterial aspiration pneumonia is with appropriate antibiotics, which is based on whether the aspiration occurred in the community or in the hospital as the likely causative organisms differ between these settings. Measures should be taken to prevent aspiration in high-risk patients, for example elderly patients in nursing homes who hâve impaired gag reflexes. Measures that hâve been shown to be effective prophylaxis include élévation of the head of the bed while feeding, dental prophylaxis, and good oral hygiene. Prophylactic antibiotics hâve not been shown to be effective and are discouraged as they may lead to the emergence of résistant organisms.

Modulation of complément components has been proposed for numerous clinical indications, including infectious disease—sepsis, viral, bacterial, and fungal infections—and pulmonary conditions—respiratory distress syndrome, chronic obstructive pulmonary disease, and cystic fibrosis (reviewed in Wagner, E., et al., Nature Rev Drug Disc. 9: 43-56, 2010). Support for this proposai is provided by numerous clinical and genetic studies. For example, there is a significantly decreased frequency of patients with low MBL levels with clinical tuberculosis (Soborg et al., Journal of Infectious Diseases 188:777-82, 2003), suggesting that low levels of MBL are associated with protection from disease.

129

In a murine model of acid aspiration injury, Weiser MR et al., J. Appl. Physiol. 83(4): 1090-1095, 1997, demonstrated that C3-knockout mice were protected from serious injury; whereas C4-knockout mice were not protected, indicating that complément activation is mediated by the alternative pathway. Consequently, blocking the alternative pathway with LEA-1 inhibitors is expected to provide a therapeutic benefit in aspiration pneumonia.

Thus, LEA-1 and LEA-2 inhibitors may hâve independent therapeutic benefit in aspiration pneumonia. In addition, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone, or may provide effective treatment for a wider spectrum of patient subsets. Combined LEA-1 and LEA-2 inhibition may be accomplished by co-administration of a LEA-1-blocking agent and a LEA-2-blocking agent. Optimally, LEA-1 and LEA-2 inhibitory function may be encompassed in a single molecular entity, such as a bi-specific antibody composed of MASP-1/3 and a MASP-2specific binding site, or a dual-specificity antibody where each binding site binds to and blocks MASP-1/3 or MASP-2.

An aspect of the invention thus provides a method for inhibiting LEA-1 dépendent complément activation to treat aspiration pneumonia by administering a composition comprising a therapeutically effective amount of a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of a MASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from such a condition or other complément mediated pneumonia. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered locally to the lung, as by an inhaler. Altemately, the MASP-1, MASP-3, or MASP-1/3 inhibitory agent may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

In accordance with the foregoing, an aspect of the invention thus provides a method for inhibiting LEA-1 dépendent complément activation for treating, preventing or reducing the severity of aspiration pneumonia, comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of a MASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from, or at risk for developing aspiration pneumonia. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be

130 administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

In another aspect, a method is provided for inhibiting LEA-2-dependent complément activation for treating, preventing or reducing the severity of aspiration pneumonia, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent to a subject suffering from, or at risk for developing aspiration pneumonia. In another aspect, a method is provided comprising inhibiting both LEA-1 and LEA-2-dependent complément activation for treating, or reducing the severity of aspiration pneumonia, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering from aspiration pneumonia.In some embodiments, the method comprises inhibiting both LEA-1-dépendent complément activation and LEA-2-dependent complément activation. As detailed above, the use of a combination of pharmacologie agents that individually block LEA-1 and LEA-2, is expected to provide an improved therapeutic outcome in treating or reducing the severity of aspiration pneumonia as compared to the inhibition of LEA-1 alone. This outcome can be achieved for example, by co-administration of an antibody that has LEA-1-blocking activity together with an antibody that has LEA-2-blocking activity. In some embodiments, LEA-1 and LEA-2-blocking activities are combined into a single molecular entity, and that such entity with combined LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody where one antigen-combining site specifïcally recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifïcally recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody where one antigen-combining site specifïcally recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifïcally recognizes MASP-2 and blocks LEA-2. Such an entity may optimally consist of a bispecific monoclonal antibody where one antigen-combining site specifïcally recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifïcally recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous, or other

131 parentéral administration, or potentially by oral administration for non-peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and/or the MASP-2 inhibitory 5 compositions of the présent invention may be carried out by a single administration of the composition (e.g., a single composition comprising MASP-2 and/or MASP-3 inhibitory agents, or bispecific or dual-inhibitory agents, or co-administration of separate compositions), or a limited sequence of administrations, for treating, preventing or reducing the severity of aspiration pneumonîa in a subject in need thereof. Alternatively, the composition may be 10 administered at periodic intervals such as daily, biweekly, weekly, every other week, monthly or bimonthly over an extended period of time for treatment of a subject in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies hâve been generated which hâve therapeutic utility for inhibition of the alternative pathway in AP-related diseases or conditions, such as aspiration pneumonîa.

Accordingly, in one embodiment, the présent invention provides a method for treating a subject suffering from, or at risk for developing aspiration pneumonîa comprising an effective amount of a high affinity monoclonal antibody or antigen binding fragment thereof as disclosed herein that binds to human MASP-3 and inhibits alternative pathway complément activation to treat or reduce the risk of developing aspiration pneumonîa, such 20 as, for example, wherein said antibody or antigen binding fragment thereof comprises (a) a heavy chain variable région comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable région comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ IDNO:144 and (iii) VLCDR3 comprising SEQ IDNO:161.

L. THE ROLE OF MASP-3 IN ENDOPHTHALMITIS, AND THERAPEUTIC METHODS USING MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION WITH AND MASP-2 INHIBITORY AGENTS

Endophthalmitis is an inflammatory condition of the intraocular cavities and is usually caused by infection. Endophthalmitis may be endogeneous, resulting from

132 hematogenous spread of organisms from a distant source of infection (e.g., endocarditis), or exogeneous, from direct inoculation of an organism from the outside as a complication of ocular surgery, foreign bodies, and/or blunt or penetrating ocular trauma. Exogeneous endophthalmitis is much more common than endogenous and most cases of exogeneous endophthalmitis occur following ocular surgery. In the United States, cataract surgery is the leading cause of endophthalmitis and occurs in 0.1-0.3% of this procedure, with an apparent increase in the incidence over the last decade (Taban, M. et al., Arch. Ophthalmol. 123:613620, 2005). Post-surgical endophthalmitis may présent either acutely, within 2 weeks of surgery, or delayed, months after surgery. Acute endophthalmitis typically présents with pain, redness, lid swelling, and decreased visual acuity. Delayed-onset endophthalmitis is less common than the acute form and patients may report only mild pain and photosensitivity. Treatment of endophthalmitis dépends on the underlying cause and may include systemic and/or intravitreal antibiotics. Endophthalmitis may resuit in decreased or loss of vision.

As previously described for AMD, multiple complément pathway genes hâve been associated with ophthalmologic disorders, and these specifically include genes of the lectin pathway. For example, MBL2 has been identified with subtypes of AMD (Dinu V, et al., Genet Epidemiol 31: 224-37, 2007). The LEA-1 and LEA-2 pathways are likely to be involved in ocular inflammatory conditions such as endophthalmitis (Chow SP et al., Clin Experiment Ophthalmol. 39:871-7, 2011). Chow et al. examined MBL levels of patients with endophthalmitis and demonstrated that both MBL levels and functional lectin pathway activity are significantly elevated in inflamed human eyes but virtually undetectable in noninflamed control eyes. This suggests a rôle for MBL and the lectin pathway in sightthreatening ocular inflammatory conditions, particularly endophthalmitis. Furthermore, in a murine model of comeal fungal keratitis, the MBL-A gene was one of five upregulated inflammatory pathway genes (Wang Y., et al., Mol Vis 13: 1226-33, 2007).

Thus, LEA-1 and LEA-2 inhibitors are expected to hâve independent therapeutic benefit in treating endophthalmitis. In addition, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent alone, or may provide effective treatment for a wider spectrum of patient subsets. Combined LEA-1 and LEA-2 inhibition may be accomplished by co-administration of a LEA-1-blocking agent and a LEA2-blocking agent. Optimally, LEA-1 and LEA-2 inhibitory function may be encompassed in a single molecular entity, such as a bi-specific antibody composed of MASP-1/3 and a

133

MASP-2-specific binding site, or a dual-specificity antibody where each binding site binds to and blocks MASP-1/3 or MASP-2

In accordance with the foregoing, an aspect of the invention thus provides a method for inhibiting LEA-1 dépendent complément activation for treating, preventing, or reducing the severity of endophthalmitis, comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of a MASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from, or at risk for developing endophthalmitis. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered locally to the eye, such as by irrigation or application of the composition in the form of a topical gel, salve or drops, or by intravitreal administration. Altemately, the MASP-1, MASP-3, or MASP-1/3 inhibitory agent may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

In another aspect, a method is provided for inhibiting LEA-2-dependent complément activation for treating, preventing, or reducing the severity of endophthalmitis, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent to a subject suffering from, or at risk for developing endophthalmitis. In another aspect, a method is provided comprising inhibiting both LEA-1 and LEA-2-dependent complément activation for treating, or reducing the severity of endophthalmitis, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering from endophthalmitis.

In some embodiments, the method comprises inhibiting both LEA-1-dépendent complément activation and LEA-2-dependent complément activation. As detailed above, the use of a combination of pharmacologie agents that individually block LEA-1 and LEA-2 is expected to provide an improved therapeutic outcome in treating or preventing or reducing the severity of endophthalmitis, as compared to the inhibition of LEA-1 alone. This outcome can be achieved for example, by co-administration of an antibody that has LEA-l-blocking activity together with an antibody that has LEA-2-blocking activity. In some embodiments, LEA-1- and LEA-2-blocking activities are combined into a single molecular entity, and that

134 such entity with combined LEA-1- and LEA-2-bIocking activity. Such an entity may comprise or consist of a bispecific antibody where one antigen-combining site specifically recognizes MASP-I and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a 5 bispecific monoclonal antibody where one antigen-combining site specifically recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may optimally consist of a bispecific monoclonal antibody where one antigen-combining site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically 10 recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered locally to the eye, such as by irrigation or application of the composition in the form of a topical gel, salve or drops, or by intravitreal injection. Altemately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, 15 subcutaneous or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and/or the MASP-2 inhibitory compositions of the présent invention may be carried out by a single administration of the 20 composition (e.g., a single composition comprising MASP-2 and/or MASP-3 inhibitory agents, or bispecific or dual inhibitory agents, or co-administration of separate compositions), or a limited sequence of administrations, for treating, preventing or reducing the severity of endophthalmitis in a subject in need thereof. Alternatively, the composition may be administered at periodic intervals such as daily, biweekly, weekly, every other week, monthly 25 or bimonthly over an extended period of time for treatment of a subject in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies hâve been generated which hâve therapeutic utility for inhibition ofthe alternative pathway in AP-related diseases or conditions, such as endophthalmitis.

Accordingly, in one embodiment, the présent invention provides a method for treating 30 a subject suffering from, or at risk for developing endophthalmitis comprising an effective amount of a high affinity monoclonal antibody or antigen binding fragment thereof as disclosed herein that binds to human MASP-3 and inhibits alternative pathway complément

135 activation to treat or reduce the risk of developing endophthalmitis, such as, for example, wherein said antibody or antigen binding fragment thereof comprises (a) a heavy chain variable région comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID 5 NO:88; and (b) a light chain variable région comprising (i) VLCDR1 comprising SEQ ID

NO: 142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising

SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161.

M. THE ROLE OF MASP-3 IN NEUROMYELITIS OPTICA, AND 10 THERAPEUTIC METHODS USING MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITORY AGENTS

Neuromyelitis optica (NMO) is an autoimmune disease that targets the optic nerves and spinal cord. This results in inflammation of the optic nerve, known as optic neuritis, and the spinal cord, known as myelitis. Spinal cord lésions in NMO may lead to weakness or 15 paralysis in the legs or arms, blindness, bladder and bowel dysfunction, and sensory dysfunction.

NMO shares several similarities to multiple sclerosis (MS), since both are due to immune attack of CNS targets and both resuit in demyelination (Papadopoulos and Verkman, Lancet Neurol., 11 (6):535-44, 2013). However, the molecular targets, treatments, and lésions 20 for NMO are distinct from those of MS. While MS is largely mediated by T cells, NMO patients typically hâve antibodies that target the water channel protein aquaporin 4 (AQP4), a protein found in astrocytes that surround the blood-brain barrier. Interferon beta is the most commonly used therapy for MS, but it is generally acknowledged to be harmful in NMO. The inflammatory lésions of NMO are found in the spinal cord and optic nerve and may 25 progress to the brain, including white and gray matter. The demyelination that occurs in NMO lésions is mediated by complément (Papadopoulos and Verkman, Lancet Neurol., 11(6):535-44, 2013).

Complement-dependent cytotoxicity appears to be the major mechanism causing development of NMO. Over 90% of NMO patients hâve IgG antibodies against AQP4 30 (Jarius and Wildemann, Jarius S, Wildemann B., Nat Rev Neurol. 2010 Jul;6(7):383-92). These antibodies initiate formation of a lésion at the blood brain barrier. The initial antigenantibody complex—AQP4/AQP4-IgG—on the surface of astrocytes activâtes the classical

136 pathway of complément. This results in formation of the membrane attack complex on the astrocyte surface, leading to granulocyte infiltration, demyelination, and ultimately necrosis of astrocytes, oligodendrocytes and neurons (Misu et al., Acta Neuropathol 125(6):815-27, 2013). These cellular events are reflected in tissue destruction and formation of cystic, necrotic lésions.

The classical pathway of complément clearly is critical for NMO pathogenesis. NMO lésions show a vasculocentric déposition of immunoglobulin and activated complément components (Jarius et al., Nat Clin Pract Neurol. 4(4):202-14, 2008). In addition, complément proteins such as C5a hâve been isolated from cerebrospinal fluid of NMO patients (Kuroda et al., J Neuro immunol.,254(\-2)'Λ78-82, 2013). Furthermore, sérum IgG obtained from NMO patients can cause complement-dependent cytotoxicity in a mouse NMO model (Saadoun et al., Brain, 133(Pt 2):349-61, 2010). A monoclonal antibody against Clq prevents the complément mediated destruction of astrocytes and lésions in a mouse model of NMO (Phuan et al., Acta Neuropathol, 125(6):829-40, 2013).

The alternative pathway of complément serves to amplify overall complément activity. Harboe and colleagues (2004) demonstrated that sélective blockade of the alternative pathway inhibited more than 80% of membrane attack complex formation induced by the classical pathway (Harboe et al., Clin Exp Immunol 138(3):439-46, 2004). Tüzün and colleagues (2013) examined both classical and alternative pathway products in NMO patients (Tüzün E, et al., JNeuroimmunol. 233(1-2): 211-5, 2011). C4d, the breakdown product of C4, was measured to evaluate classical pathway activity and was increased in NMO patient sera compared to Controls (an élévation of 2.14-fold). In addition, an increase of Factor Bb, the breakdown product of the alternative pathway Factor B, was observed in NMO patients compared to MS patients or normal control individuals (an élévation of 1.33-fold). This suggests that alternative pathway function is also increased in NMO. This activation would be expected to increase overall complément activation, and in fact sC5b-9, the final product ofthe complément cascade, was significantly increased (a 4.14-fold élévation).

Spécifie inhibitors of MASP-3 are expected to provide benefit in treating patients suffering from NMO. As demonstrated herein, sérum lacking MASP-3 is unable to activate Factor B, an essential component of C5 convertase, or Factor D, the central activator of the alternative pathway. Therefore, blocking MASP-3 activity with an inhibitory agent such as an antibody or small molécule would also be expected to inhibit activation of Factor B and

137

Factor D. Inhibition of these two factors will arrest the amplification of the alternative pathway, resulting in diminished overaii complément activity. MASP-3 inhibition should thus significantly improve therapeutic outcomes in NMO.

Thus, LEA-1 and/or LEA-2 inhibitors are expected to hâve independent therapeutic 5 benefit in treating NMO. In addition, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit compared to either agent aione, or may provide effective treatment for a wider spectrum of patient subsets. Combined LEA-I and LEA-2 inhibition may be accomplished by co-administration of a LEA-1-biocking agent and a LEA-2-blocking agent. Optimaiiy, LEA-1 and LEA-2 inhibitory function may be encompassed in a single 10 molecular entity, such as a bi-specific antibody composed of MASP-1/3 and a MASP-2specific binding site, or a dual-specificity antibody where each binding site binds to and blocks MASP-1/3 or MASP-2

In accordance with the foregoing, an aspect of the invention thus provides a method for inhibiting LEA-1 dépendent complément activation for treating, preventing, or reducing 15 the severity of NMO, comprising administering a composition comprising a therapeutically effective amount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of a MASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from, or at risk for developing NMO. The MASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administered locaily to the 20 eye, such as by irrigation or application of the composition in the form of a topicai gel, salve or drops, or by intravitreal administration. Aitemately, the MASP-1, MASP-3, or MASP-1/3 inhibitory agent may be administered to the subject systemicaiiy, such as by intra arterial, intravenous, intramuscuiar, inhalationai, nasal, subcutaneous or other parenterai administration, or potentialiy by oral administration for non peptidergic agents. 25 Administration may be repeated as determined by a physician until the condition has been resolved or is controiied.

In another aspect, a method is provided for inhibiting LEA-2-dependent complément activation for treating, preventing, or reducing the severity of NMO, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent to a subject 30 suffering from, or at risk for developing NMO. In another aspect, a method is provided comprising inhibiting both LEA-1 and LEA-2-dependent complément activation for treating, or reducing the severity of NMO, comprising administering a therapeutically effective

138 amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering from NMO.

In some embodiments, the method comprises inhibiting both LEA-I-dépendent complément activation and LEA-2-dependent complément activation. As detailed above, the use of a combination of pharmacologie agents that individually block LEA-I and LEA-2 is expected to provide an improved therapeutic outcome in treating or preventing or reducing the severity ofNMO, as compared to the inhibition of LEA-1 alone. This outcome can be achieved for example, by co-administration of an antibody that has LEA-1-blocking activity together with an antibody that has LEA-2-blocking activity. In some embodiments, LEA-Iand LEA-2-blocking activities are combined into a single molecular entity, and that such entity with combined LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody where one antigen-combining site specifïcally recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifïcally recognizes MASP-2 and blocks LEA-2. Alternatively, such an entity may consist of a bispecific monoclonal antibody where one antigen-combining site specifïcally recognizes MASP-3 and thus blocks LEA-1 and the second antigen-combining site specifïcally recognizes MASP-2 and blocks LEA-2. Such an entity may optimally consist of a bispecific monoclonal antibody where one antigen-combining site specifïcally recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifïcally recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered locally to the eye, such as by irrigation or application of the composition in the form of a topical gel, salve or drops, or by intravitreal injection. Altemately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and/or the MASP 2 inhibitory compositions of the présent invention may be carried out by a single administration of the composition (e.g., a single composition comprising MASP-2 and/or MASP-3 inhibitory agents, or bispecific or dual inhibitory agents, or co-administration of separate compositions), or a limited sequence of administrations, for treating, preventing or reducing the severity of

139

NMO in a subject in need thereof. Altematively, the composition may be administered at periodic intervals such as daily, biweekly, weekly, every other week, monthly or bimonthly over an extended period of time for treatment of a subject in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies 5 hâve been generated which hâve therapeutic utility for inhibition of the alternative pathway in AP-related diseases or conditions, such as neuromyelitis optica (NMO).

Accordingly, in one embodiment, the présent invention provides a method for treating a subject suffering from, or at risk for developing neuromyelitis optica (NMO). comprising an effective amount of a high affinity monoclonal antibody or antigen binding fragment 10 thereof as disclosed herein that binds to human MASP-3 and inhibits alternative pathway complément activation to treat or reduce the risk of developing neuromyelitis optica (NMO), such as, for example, wherein said antibody or antigen binding fragment thereof comprises (a) a heavy chain variable région comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising 15 SEQ ID NO:88; and (b) a light chain variable région comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161.

N. THE ROLE OF MASP-3 IN BEHCET’S DISEASE, AND THERAPEUTIC 20 METHODS USING MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY IN COMBINATION WITH MASP-2 INHIBITORY AGENTS

Behçet's disease, or Behçet's syndrome, is a rare, immune-mediated small-vessel systemic vasculitis that often présents with mucous membrane ulcération and ocular problems. Behçet's disease (BD) was named in 1937 after the Turkish dermatologist Hulusi 25 Behçet, who first described the triple-symptom complex of récurrent oral ulcers, génital ulcers, and uveitis. BD is a systemic, relapsing inflammatory disorder of unknown cause. The inflammatory perivasculitis of BD may involve the gastrointestinal tract, pulmonary, musculoskeletal, cardiovascular, and neurological Systems. BD can be fatal due to ruptured vascular aneurysms or severe neurological complications. Optic neuropathy and atrophy may 30 resuit from vasculitis and occlusion of the vessels supplying the optic nerve. See AI-Araji A, et al., Lancet Neurol., 8(2):192-204, 2009.

140

The highest incidence of BD is in the Middle East and Far East régions, but it is rare in Europe and North America. BD is often initially controlled with corticosteroids and immunosuppressants, but many cases are refractory with serious morbidity and mortality. Biologie agents, including interferon-alpha, IVIG, anti-TNF, anti-IL-6, and anti-CD20, hâve 5 shown benefit in some cases, but there is no consensus on best treatment.

While BD is clearly an inflammatory disorder, its pathobiology is not clear. There are genetic associations with HLA antigens, and genome wide association studies hâve implicated numerous cytokine genes (Kirino et al., Nat Genet, 45(2):202-7, 2013). The hyperactivity of the immune system appears to be regulated by the complément system. 10 Increased levels of C3 hâve been observed in BD patient sera (Bardak and Aridogan, Ocid Immunol Inflamm 12(1):53-8, 2004), and elevated C3 and C4 in the cerebrospinal fluid correlates with disease (Jongen et al., Arch Neurol, 49(10):1075-8, 1992).

Tüzün and colleagues (2013) examined both classical and alternative pathway products in sera of BD patients (Tüzün E, et al., J Neuroimmunol, 233(1-2):211-5, 2011). 4d, 15 the breakdown product of C4, is generated upstream of the alternative pathway and was measured to evaluate initial classical pathway activity. C4d was increased in BD patient sera compared to Controls (an élévation of 2.18-fold). Factor Bb is the breakdown product of Factor B, and was measured to détermine activity of the alternative pathway. BD patients had an increase of factor Bb compared to normal control individuals (an élévation of 2.1920 fold) consistent with an increase in BD alternative pathway function. Because the alternative pathway of complément serves to amplify overall complément activity, this activation would be expected to increase overall complément activation. Harboe and colleagues (2004) demonstrated that sélective blockade of the alternative pathway inhibited more than 80% of membrane attack complex formation induced by the classical pathway (Harboe M, et al., Clin 25 Exp Immunol, 138(3):439-46, 2004). In fact, sC5b-9, the final product of the complément cascade, was significantly increased in BD patients (a 5.46-foId élévation). Spécifie inhibitors of MASP-3 should provide benefit in BD. Blocking MASP-3 should inhibit activation of Factor B and Factor D. This will stop the amplification of the alternative pathway, resulting in a diminished response of overall complément activity. MASP-3 30 inhibition should thus significantly improve therapeutic outcomes in BD. Thus, LEA-1 and/or LEA-2 inhibitors are expected to hâve independent therapeutic benefit in treating BD. In addition, LEA-1 and LEA-2 inhibitors used together may achieve additional treatment benefit

141 compared to either agent alone, or may provide effective treatment for a wider spectrum of patient subsets. Combined LEA-1 and LEA-2 inhibition may be accomplished by coadministration of a LEA-l-blocking agent and a LEA-2-blocking agent. Optimally, LEA-1 and LEA-2 inhibitory fonction may be encompassed in a single molecular entity, such as a bi5 spécifie antibody composed of MASP-1/3 and a MASP-2-specific binding site, or a dualspecificity antibody where each binding site binds to and blocks MASP-1/3 or MASP-2.

In accordance with the foregoing, an aspect of the invention thus provides a method for inhibiting LEA-1 dépendent complément activation for treating, preventing, or reducing the severity of BD, comprising administering a composition comprising a therapeutically 10 effective amount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combination of a MASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subject suffering from, or at risk for developing BD. The MASPI, MASP-3, or MASP-I/3 inhibitory composition may be administered locally to the eye, such as by irrigation or application of the composition in the form of a topical gel, salve or 15 drops, or by intravitreal administration. Altemately, the MASP-1, MASP-3, or MASP-1/3 inhibitory agent may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subeutaneous or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been 20 resolved or is controlled.

In another aspect, a method is provided for inhibiting LEA-2-dependent complément activation for treating, preventing, or reducing the severity of BD, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent to a subject suffering from, or at risk for developing BD. In another aspect, a method is provided comprising inhibiting 25 both LEA-1 and LEA-2-dependent complément activation for treating, or reducing the severity of BD, comprising administering a therapeutically effective amount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering from BD.

In some embodiments, the method comprises inhibiting both LEA-I-dépendent 30 complément activation and LEA-2-dependent complément activation. As detailed above, the use of a combination of pharmacologie agents that individually block LEA-1 and LEA-2 is expected to provide an improved therapeutic outcome in treating or preventing or reducing

142 the severity of BD, as compared to the inhibition of LEA-1 alone. This outcome can be achieved for example, by co-administration of an antibody that has LEA-1-blocking activity together with an antibody that has LEA-2-blocking activity. In some embodiments, LEA-1 and LEA-2-blocking activities are combined into a single molecular entity, and that such 5 entity with combined LEA-1- and LEA-2-blocking activity. Such an entity may comprise or consist of a bispecific antibody where one antigen-combining site specifically recognizes MASP-1 and blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Altematively, such an entity may consist of a bispecific monoclonal antibody where one antigen-combining site specifically recognizes MASP-3 and 10 thus blocks LEA-1 and the second antigen-combining site specifically recognizes MASP-2 and blocks LEA-2. Such an entity may optimally consist of a bispecific monoclonal antibody where one antigen-combining site specifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 while the second antigen-combining site specifically recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered locally to the eye, such as by irrigation or application of the composition in the form of a topical gel, salve or drops, or by intravitreal injection. Altemately, the MASP-2 inhibitory agent may be administered to the subject systemically, such as by intra arterial, intravenous, intramuscular, inhalational, nasal, subcutaneous or other parentéral administration, or potentially by oral administration for non peptidergic agents. Administration may be repeated as determined by a physician until the condition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and/or the MASP-2 inhibitory compositions of the présent invention may be carried out by a single administration of the composition (e.g., a single composition comprising MASP-2 and/or MASP-3 inhibitory 25 agents, or bispecific or dual inhibitory agents, or co-administration of separate compositions), or a limited sequence of administrations, for treating, preventing or reducing the severity of BD in a subject in need thereof. Altematively, the composition may be administered at periodic intervals such as daily, biweekly, weekly, every other week, monthly or bimonthly over an extended period of time for treatment of a subject in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitory antibodies hâve been generated which hâve therapeutic utility for inhibition ofthe alternative pathway in AP-related diseases or conditions, such as Behçet's disease (BD).

143

Accordingly, in one embodiment, the présent invention provides a method for treating a subject suffering from, or at risk for developing Behçet's disease (BD) comprising an effective amount of a high affinity monoclonal antibody or antigen binding fragment thereof as disclosed herein that binds to human MASP-3 and inhibits alternative pathway complément activation to treat or reduce the risk of developing Behçet's disease (BD), such as, for example, wherein said antibody or antigen binding fragment thereof comprises (a) a heavy chain variable région comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable région comprising (i) VLCDR1 comprising SEQ ID NO: 142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ IDNO:144 and (iii) VLCDR3 comprising SEQ IDNO:161.

MASP-3 Inhibitory Agents

With the récognition that the lectin pathway of complément is composed oftwo major complément activation arms, LEA-1 and LEA-2, and that there also is a lectin-independent complément activation arm, cornes the realization that it would be highly désirable to specifically inhibit one or more of these effector arms that cause a pathology associated withaltemative pathway complément activation, such as at least one of paroxysmal noctumal hemoglobinuria (PNH), age-related macular degeneration (AMD, including wet and dry AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS), thrombotic thrombocytopénie purpura (TTP) or transplant-associated TMA), asthma, dense deposit disease, pauci-immune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet’s disease, multiple sclerosis (MS), Guillain Barre Syndrome, Alzheimer’s disease, Amylotrophic latéral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy, Uveitis, Chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD), hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), ANCÀ vasculitis, Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy and Myasthenia Gravis, without completely shutting down the immune defense capabilities of complément (i.e., leaving the classical pathway intact). This would

144 leave the Clq-dependent complément activation System intact to handle immune complex processing and to aid in host defense against infection.

Compositions for inhibiting LEA-1-mediated complément activation

As described herein, the inventors hâve unexpectedly discovered that activation of LEA-1, leading to lysis, is MASP-3-dependent. As further described herein, under physiological conditions, MASP-3-dependent LEA-1 activation also contributes to opsonization, thereby providing an additive effect with LEA-2-mediated complément activation. As demonstrated herein, in the presence of Ca**, factor D is not required, as MASP-3 can drive activation of LEA-1 in factor D'⁷’ sera. MASP-3, MASP-1, and HTRA-1 are able to convert pro-factor D to active factor D. Likewise, MASP-3 activation appears, in many instances, to be dépendent on MASP-1, since MASP-3 (in contrast to MASP-1 and MASP-2) is not an auto-activating enzyme and is incapable of converting into its active form without the help of MASP-1 (Zundel, S. et al., J.Immunol. 172: 4342-4350 (2004); Megyeri et al., J. Biol. Chem. 288:8922-8934 (2013). As MASP-3 does not autoactivate and, in many instances, requîtes the activity of MASP-1 to be converted into its enzymatically active form, the MASP-3-mediated activation of the alternative pathway C3 convertase C3Bb can either be inhibited by targeting the MASP-3 zymogen or already-activated MASP-3, or by targeting MASP-1-mediated activation of MASP-3, or both, since, in many instances, in the absence of MASP-1 functional activity, MASP-3 remains in its zymogen form and is not capable of driving LEA-1 through direct formation ofthe alternative pathway C3 convertase (C3bBb).

Therefore, in one aspect of the invention, the preferred protein component to target in the development of therapeutic agents to specifically inhibit LEA-1 is an inhibitor of MASP3 (including inhibitors of MASP-1-mediated MASP-3 activation (e.g., a MASP-1 inhibitor that inhibits MASP-3 activation)).

In accordance with the foregoing, in one aspect, the invention provides methods of inhibiting the adverse effects of LEA-1 (i.e., hemolysis and opsonization) by administering a MASP-3 inhibitory agent, such as a MASP-3 inhibitory antibody in a subject suffering from, or at risk for developing, a disease or disorder selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS) and thrombotic thrombocytopénie purpura (TTP), asthma, dense deposit

145 disease, pauci-immune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica Behcet’s disease, multiple sclerosis, Guillain Barre Syndrome, Alzheimer’s disease, Amylotrophic latéral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy, Uveitis, Chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD), hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis, Antiphospholipid syndrome, Atherosclerosis, IgA Nephropathy and Myasthenia Gravis, comprising administering to the subject a pharmaceutical composition comprising an amount of a MASP-3 inhibitory agent effective to inhibit MASP-3-dependent complément activation and a pharmaceutically acceptable carrier.

MASP-3 inhibitory agents are administered in an amount effective to inhibit MASP-3-dependent complément activation in a living subject suffering from, or at risk for developing, paroxysmal noctumal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS) or thrombotic thrombocytopénie purpura (TTP)), asthma, dense deposit disease, pauci-immune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behcet’s disease, multiple sclerosis, Guillain Barre Syndrome, Alzheimer’s disease, Amylotrophic latéral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy, Uveitis, Chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD), hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis, Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy and Myasthenia Gravis. In the practice of this aspect of the invention, représentative MASP-3 inhibitory agents include: molécules that inhibit the biological activity of MASP-3, including molécules that inhibit at least one or more of the following: lectin MASP-3-dependent activation of factor B, lectin MASP-3-dependent activation of pro-factor D, MASP-3dependent, lectin-independent activation of factor B, and MASP-3-dependent, lectinindependent activation of pro-factor D (such as small-molecule inhibitors, MASP-3 antibodies and fragments thereof, or blocking peptides which interact with MASP-3 or

146 interfère with a protein-protein interaction), and molécules that decrease the expression of MASP-3 (such as MASP-3 antisense nucleic acid,molécules, MASP-3 spécifie RNAi molécules and MASP-3 ribozymes). A MASP-3 inhibitory agent may effectively block MASP-3 protein-to-protein interactions, interfère with MASP-3 dimerization or assembly, 5 block Ca^44- binding, interfère with the MASP-3 serine protease active site, or reduce MASP-3 protein expression, thereby preventing MASP-3 from activating LEA-1-mediated, or lectinindependent, complément activation. The MASP-3 inhibitory agents can be used alone as a primary therapy or in combination with other therapeutics as an adjuvant therapy to enhance the therapeutic benefits of other medical treatments, as further described herein.

High Affinity monoclonal MASP-3 inhibitory antibodies

As described in Examples 11-21 herein, and summarized in TABLES 2A, 2B and TABLE 3 below, the inventors hâve generated surprisingly high affinity (i.e. <500 pM) MASP-3 inhibitory antibodies that bind to an epitope in the serine protease domain of human 15 MASP-3. As described herein, the inventors hâve demonstrated that these high affinity MASP-3 antibodies are capable of inhibiting alternative pathway complément activation in human sérum, rodents and non-human primates. The variable light and heavy chain régions of these antibodies hâve been sequenced, isolated and analyzed in both a Fab format and in a full-length IgG format. As described in Example 15 and shown in dendrograms depicted in 20 FIGURES 50A and 50B, the antibodies can be grouped according to sequence similarity. A summary of the heavy chain variable régions and the light chain variable régions of these antibodies is shown in FIGURES 49A and 49B and provided in TABLES 2A and 2B below. Humanized versions of représentative high affinity MASP-3 inhibitory antibodies were generated as described in Example 19 and are summarized in TABLE 3.

TABLE 2A: MASP-3 high affinity inhibitory Antibody Sequences:mouse parental

MASP-3 Antibody Reference No	Group	Heavy Chain Variable Région (amino acid)	Light Chain Variable Région (amino acid)	Heavy chain variable région (DNA)	Light chain variable région (DNA)
4D5	IA	SIN:24	SIN:40	SIN:217	SIN:233
1F3	IA	SIN:25	SIN:41	SIN:218	SIN:234
4B6	IA	SIN:26	SIN:42	SIN:219	SIN:235
IA10	IA	SIN:27	SIN:42	SIN:220	SIN:235
10D12	IB	SIN:28	SIN:43	SIN:22l	SIN:236

147

35C1	IB	SIN:29	SIN:44	SIN:222	SIN:237
13B1	IC	SIN:30	SIN:45	SIN:223	SIN:238
1G4	II	SIN:31	SIN:46	SIN:224	SIN:239
1E7	IIIA	SIN:32	SIN:47	SIN:225	SIN:240
2D7	IIIA	SIN:33	SIN:48	SIN:226	SIN:241
49CU	IIIA	SIN:34	SIN:49	SIN:227	SIN:242
I5D9	IIIB	SIN:35	S1N:5O	SIN:228	SIN:243
2F5	IIIB	SIN:36	SIN:51	SIN:229	SIN:244
1B11	IIIC	SIN:37	SIN:52	SIN:230	SIN:245
2F2	IIID	SIN:38	SIN:53	SIN:231	SIN:246
11B6	IIID	SIN:39	SIN:54	S1N:232	SIN:247

Note: “SIN” refers to “SEQ ID NO:”

TABLE 2B: MASP-3 high affinity inhibitory antibodies:CDRs

MASP-3 Antibody Reference No.	Heavy Chain Variable Région (amino acid)	Light Chain Variable Région (amino acid)	Heavy Chain: CDR1; CDR2; CDR3 (SEQ IDNOs)	Light Chain: CDR1; CDR2; CDR3 (SEQ IDNOs)
4D5	SIN:24	SIN:40	56;58;60	142; 144; 146
IF3	S1N:25	SIN:41	62;63;65	149; 144; 146
4B6	SIN:26	SIN:42	62;67;65	149; 144; 146
1A10	SIN:27	SIN:42	62;69;65	149; 144; 146
I0D12	SIN:28	SIN:43	72;74;76	153;155;157
35C1	SIN:29	SIN:44	79;74;82	159;155;160
13B1	SIN:30	SIN:45	84;86;88	142;144;161
1G4	SIN:31	SIN:46	91;93;95	163;165; 167
1E7	SIN:32	SIN:47	109;l 10;l 12	182; 184; 186
2D7	SIN:33	SIN:48	125; 127; 129	196;198;200
49C11	SIN:34	S1N:49	132;133;135	203;165;204
15D9	SIN:35	SIN:50	137;138;140	206;207;208
2F5	SIN:36	SIN:51	98;99;101	169;171;173
1B11	SIN:37	SIN:52	103;105;107	176; 178; 180
2F2	SIN:38	SIN:53	114;116;118	188;178;190
11B6	SIN:39	SIN:54	114;121;123	191;178;193

TABLE 3: Représentative high affinity MASP-3 inhibitory antibodies: humanized and modified to remove post-translational modification sites _________________

MASP-3 Antibody Reference No.	Heavy Chain Variable Région aa (SEQIDNO)	Light Chain Variable Région aa (SEQ ID NO)	Heavy Chain: CDR1; CDR2; CDR3 (SEQ IDNOs)	Light Chain: CDR1;CDR2; CDR3 (SEQ ID NOs)
4D5 parent	24	40	56;58;60	142:144:146
h4D5-14-l	248	250	56;58;60	142;144;146
h4D5-19-l	249	250	56;58;60	142;144;146
h4D5-14-l-NA	248	278	56;58:60	258;144;146
h4D5-19-l-NA	249	278	56;58;60	258; 144; 146

148

10D12 parent	28	43	72;74;76	153;155;157
h 10D12-45-21	251	253	72;74;76	153:155:157
h 10D12-49-21	252	253	72;74;76	153;155;157
h 10D12-45-21-GA	251	279	72;74;76	263;155;157
h!0D12-49-21-GA	252	279	72:74:76	263:155:157
13B1 parent	30	45	84;86;88	142;144;161
hl3Bl-9-l	254	256	84;275;88	142;144;161
h!3Bl-10-l	255	256	84;86;88	142:144:161
hl3Bl-9-l-NA	254	280	84;275;88	258:144:161
hl3Bl-10-l-NA	255	280	84;86;88	258;144;161

Accordingly, in one aspect, the présent invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifîcally binds to the serine protease domain of human MASP-3 (amino acid residues 450 to 728 of SEQ ID NO:2) with high 5 affinity (having a Kd of Iess than 500 pM), wherein the antibody or antigen-binding fragment thereof inhibits alternative pathway complément activation. In some embodiments, the high affinity MASP-3 inhibitory antibody, or antigen-binding fragment thereof inhibits the alternative pathway at a molar ratio of from about 1:1 to about 2.5:1 target MASP-3 to mAb in a mammalian subject.

The inhibition of alternative pathway complément activation is characterized by at least one or more of the following changes in a component of the complément System that occurs as a resuit of administration of a high affinity MASP-3 inhibitory antibody in accordance with various embodiments of the invention: inhibition of hemolysis and/or opsonization; inhibition of lectin-independent conversion of factor B; inhibition of lectin15 independent conversion of factor D, inhibition of MASP-3 serine protease substrate-specific cleavage; the réduction of hemolysis or the réduction of C3 cleavage and C3b surface déposition; the réduction of Factor B and Bb déposition on an activating surface; the réduction of resting levels (in circulation, and without the experimental addition of an activating surface) of active Factor D relative to pro-Factor D; the réduction of levels of 20 active Factor D relative to pro-Factor D in response to an activating surface; and/or the production of resting and surface-induced levels of fluid-phase Ba, Bb, C3b, or C3a.

For example, as described herein the high affinity MASP-3 inhibitory antibodies, are antibodies or antigen-binding fragments thereof capable of inhibiting factor D maturation (i.e., cleavage of pro-factor D to factor D) in a mammalian subject. In some embodiments, the 25 high affinity MASP-3 inhibitory antibodies are capable of inhibiting factor D maturation in

149 full sérum to a level less than 50% than that found in untreated control sérum (such as less than 40%, for example less than 30%, such as less than 25%, for example less than 20%, such as less than 15%, for example less than 10%, such as less than 5% untreated control sérum not contacted with a MASP-3 inhibitory antibody).

In preferred embodiments, the high affinity MASP-3 inhibitory antibodies selectively inhibit the alternative pathway, leaving the Clq-dependent complément activation System functionally intact.

In another aspect, the présent disclosure features a nucleic acid molécule that encodes one or both of the heavy and light chain polypeptides of any of the MASP-3 inhibitory antibodies or antigen-binding fragments disclosed herein. Also featured is a vector (e.g., a cloning or expression vector) comprising the nucleic acid and a cell (e.g., an insect cell, bacterial cell, fungal cell, or mammalian cell) comprising the vector. The disclosure futher provides a method for producing any of the MASP-3 inhibitory antibodies or antigen-binding fragments disclosed herein. The methods include, providing a cell containing an expression vector which contains a nucleic acid that encodes one or both of the heavy and light chain polypeptides of any of the antibodies or antigen-binding fragments disclosed herein. The cell or culture of cells is cultured under conditions and for a time sufficient to allow expression by the cell (or culture of cells) of the antibody or antigen-binding fragment thereof encoded by the nucleic acid. The method can also include isolating the antibody or antigen binding fragment thereof from the cell (or culture of cells) or from the media in which the cell or cells were cultured.

MASP-3 epitopes and peptides

As described in Example 18, illustrated in FIGURE 62 and summarized in TABLE 4 below, the high affinity MASP-3 inhibitory antibodies and antigen-binding fragments thereof according to the présent invention were found to specifically recognize one or more epitopes within the serine protease domain of human MASP-3 (amino acid residues 450 to 728 of SEQ ID NO:2). “Specifically recognises” means that the antibody binds to said epitope with significantly higher affinity than to any other molécule or part thereof.

TABLE 4: Représentative High Affinity MASP-3 inhibitory antibodies: Epitope Binding Régions of MASP-3 (see also FIGURE 62)

150

Peptide Binding Fragments (Epitopes) with reference to human MASP-3 (w/leader)	MASP-3 mAb Ref No.
wsVLRSQRRDTTVIsoo (SIN:9)	IF3,4B6,4D5, IA10, I0DI2,
494TAAHVLRSQRRDTTV508 (SIN:10)	I3BI
544DFNIQNYNHDIALVQ558 (SIN:11)	1F3,4B6,4D5, IA10
626PHAECKTSYESRS638 (SIN: 12)	I3BI
639GNYSVTENMFC649 (SIN:13)	IF3,4B6,4D5, ΙΑΙ0
7O4VSNYVDWVWE7I3 (SIN:14)	IF3,4B6,4D5, 1AI0
498VLRSQRRDTTV503 (SIN: 15) Core sequence of Group I	1F3, 4B6, 4D5, 1A10, 10D12, 13B1
435ECGQPSRSLPSLV447 (SIN: 16)	1B11
454RNAEPGLFPWQ464 (SIN: 17) Core sequence ofGroups II and III	1G4, 1E7, 2D7, I5D9,2F5, 1B11
479KWFGSGALLSASWIL493 (SIN 18)	15D9, 2F5
514EHVTVYLGLH523 (SIN: 19)	IE7, 2D7, 1G4
562PVPLGPHVMP571 (SIN:20)	15D9.2F5
583APHMLGL589 (SIN:21)	IB11
614SDVLQYVKLP623 (SIN:22)	1B11
667AFV1FDDLSQRW678 (SIN:23)	1G4, 1E7,2D7, 15D9.2F5

151

Accordingly, in some embodiments, the high affinity MASP-3 inhibitory antibody or antigen-binding fragment thereof specifically binds to an epitope located within the serine protease domain of human MASP-3, wherein said epitope is located within at least one or more of: VLRSQRRDTTVI (SEQ ID NO:9), TAAHVLRSQRRDTTV (SEQ ID NO: 10), DFNIQNYNHDIALVQ (SEQ ID NO:11), PHAECKTSYESRS (SEQ ID NO:12), GNYSVTENMFC (SEQ ID NO: 13), VSNYVDWVWE (SEQ ID NO: 14) and/or VLRSQRRDTTV (SEQ ID NO: 15). In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within SEQ ID NO: 15. In some embodiments, the antibody or antigen-binding fragment binds to an epitope within SEQ ID NO:9. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within SEQ ID NO: 12. In some embodidments, the antibody or antigenbinding fragment thereof binds to an epitope within SEQ ID NO: 10 and SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within at least one of SEQ ID NO:11, SEQ ID NO: 13 and/or SEQ ID NO:14.

In other embodiments, the high affinity MASP-3 inhibitory antibody or antigenbinding fragment thereof specifically binds to an epitope located within the serine protease domain of human MASP-3, wherein said epitope is located within at least one or more of: ECGQPSRSLPSLV (SEQ ID NO: 16), RNAEPGLFPWQ (SEQ ID NO: 17); KWFGSGALLSASWIL(SEQ ID NO: 18); EHVTVYLGLH (SEQ ID NO: 19); PVPLGPHVMP (SEQ ID NO:20); APHMLGL (SEQ ID NO:21); SDVLQYVKLP (SEQ ID NO:22); and/or AFVIFDDLSQRW (SEQ ID NO:23). In one embodiment, the antibody or antigen-binding fragment binds to an epitope within SEQ ID NO: 17. In one embodiment, the antibody or antigen binding fragment binds to an epitope within EHVTVYLGLH (SEQ ID NO: 19) and/or AFVIFDDLSQRW (SEQ ID NO:23). In one embodiment, the antibody or antigen-binding fragment binds to an epitope within SEQ ID NO: 18, SEQ ID NO:20 and/or SEQ ID NO:23. In one embodiment, the antibody or antigen-binding fragment binds to an epitope within at least one of SEQ ID NO: 16, SEQ ID NO: 21 and/or SEQ ID NO:22.

CDR Régions:

In one aspect of the présent invention the antibody or functional équivalent thereof comprises spécifie hypervariable régions, designated CDRs. Preferably, the CDRs are CDRs

152 according to the Kabat CDR définition. CDRs or hypervariable régions may for example be identified by sequence alignment to other antibodies. The CDR régions of the high affinity MASP-3 inhibitory antibodies are shown in TABLES 18-23.

Group IA mAbs

In one aspect, the invention provides an isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 comprising: (a) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:209 (XXDIN, wherein X at position 1 is S or T and wherein X at position 2 is N or D); a HC-CDR2 set forth as SEQ ID NO:210 (WIYPRDXXXKYNXXFXD, wherein X at position 7 is G or D; X at position 8 is S, T or R; X at position 9 is I or T; X at position 13 is E or D; X at position 14 is K or E; and X at position 16 is T or K); and a HC-CDR3 set forth as SEQ ID NO:211 (XEDXY, wherein X at position 1 is L or V, and wherein X at position 4 is T or S); and (b) a light chain variable région comprising a LC-CDR1 set forth as SEQ ID NO:212 (KSSQSLLXXRTRKNYLX, wherein X at position 8 is N, I, Q or A; wherein X at position 9 is S or T; and wherein X at position 17 is A or S); a LC-CDR2 set forth as SEQ ID NO: 144 (WASTRES) and a LCCDR3 set forth as SEQ ID NO: 146 (KQSYNLYT). In one embodiment, the HC-CDR1 of the heavy chain variable région according to (a) comprises SEQ ID NO:56 (TDDIN). In one embodiment, the HC-CDR1 of the heavy chain variable région according to (a) comprises SEQ ID NO:62 (SNDIN). In one embodiment, the HC-CDR2 of the heavy chain variable région according to (a) comprises SEQ ID NO:58 (WIYPRDDRTKYNDKFKD). In one embodiment, the HC-CDR2 of the heavy chain variable région according to (a) comprises SEQ ID NO:63 (WIYPRDGSIKYNEKFTD). In one embodiment, the HC-CDR2 of the heavy chain variable région according to (a) comprises SEQ ID NO:67 (WIYPRDGTTKYNEEFTD). In one embodiment, the HC-CDR2 of the heavy chain variable région according to (a) comprises SEQ ID NO:69 (WIYPRDGTTKYNEKFTD). In one embodiment, the HC-CDR3 of the heavy chain variable région according to (a) comprises SEQ ID NO:60 (LEDTY). In one embodiment, the HC-CDR3 of the heavy chain variable région according to (a) comprises SEQ ID NO:65 (VEDSY). In one embodiment, the LC-CDRI of the light chain variable région comprises SEQ ID NO: 142 (KSSQSLLNSRTRKNYLA); SEQ ID NO:257 (KSSQSLLQSRTRKNYLA), SEQ ID NO:258 (KSSQSLLASRTRKNYLA); or SEQ ID NO:259 (KSSQSLLNTRTRKNYLA). In one embodiment, the LC-CDRI comprises SEQ ID

153

NO:258 (KSSQSLLASRTRKNYLA). In one embodiment, the LC-CDR1 comprises SEQ ID NO: 149 (KSSQSLLISRTRKNYLS).

In one embodiment, the HC-CDR1 comprises SEQ ID NO:56, the HC-CDR2 comprises SEQ ID NO:58, the HC-CDR3 comprises SEQ ID NO:60 and the LC-CDR1 5 comprises SEQ ID NO: 142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259; the LCCDR2 comprises SEQ ID NO: 144 and the LC-CDR3 comprises SEQ ID NO: 146.

In one embodiment, the HC-CDR1 comprises SEQ ID NO:62, the HC-CDR2 comprises SEQ ID NO:63, SEQ ID NO:67 or SEQ ID NO:69, the HC-CDR3 comprises SEQ ID NO:65 and the LC-CDR1 comprises SEQ ID NO: 149, the LC-CDR2 comprises SEQ ID 10 NO: 144 and the LC-CDR3 comprises SEQ ID NO: 146.

Group IB mAbs

In another aspect, the invention provides an isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 comprising: (a) a heavy chain variable région 15 comprising a HC-CDR1 set forth as SEQ ID NO:213 (SYGXX, wherein X at position 4 is M or I and wherein X at position 5 is S or T); a HC-CDR2 set forth as SEQ ID NO:74; and a HC-CDR3 set forth as SEQ ID NO:214 (GGXAXDY, wherein X at position 3 is E or D and wherein X at position 5 is M or L); and (b) a light chain variable région comprising a LCCDR1 set forth as SEQ ID NO:215 (KSSQSLLDSXXKTYLX , wherein X at position 10 is 20 D, E or A; wherein X at position 11 is G or A; and wherein X at position 16 is N or S); a LCCDR2 set forth as SEQ ID NO: 155; and a LC-CDR3 set forth as SEQ ID NO:216 (WQGTHFPXT, wherein X at position 8 is W or Y).

In one embodiment, the HC-CDR1 of the heavy chain variable région according to (a) comprises SEQ ID NO:72 (SYGMS). In one embodiment, the HC-CDRI comprises SEQ ID 25 NO:79 (SYGIT). In one embodiment, the HC-CDR3 comprises SEQ ID NO:76 (GGEAMDY). In one embodiment, the HC-CDR3 comprises SEQ ID NO:82 (GGDALDY). In one embodiment, the LC-CDR1 comprises SEQ ID NO: 153 (KSSQSLLDSDGKTYLN); SEQ ID NO:26I (KSSQSLLDSEGKTYLN), SEQ ID NO:262 (KSSQSLLDSAGKTYLN) or SEQ ID NO:263 (KSSQSLLDSDAKTYLN). In one embodiment, the LC-CDRI comprises 30 SEQ ID NO:263 (KSSQSLLDSDAKTYLN). In one embodiment, the LC-CDRI comprises SEQ ID NO: 152. In one embodiment, the LC-CDR3 comprises SEQ ID NO: 159 (KSSQSLLDSDGKTYLS).

154

In one embodiment, the LC-CDR3 comprises SEQ ID NO: 160 (WQGTHFPYT). In one embodiment, the HC-CDR1 comprises SEQ ID NO:72, the HC-CDR2 comprises SEQ ID NO:74, the HC-CDR3 comprises SEQ ID NO:76, and the LC-CDR1 comprises SEQ ID NO: 153, SEQ ID NO:261, SEQ ID NO:262 or SEQ ID NO:263; the LC-CDR2 comprises SEQ ID NO: 155 and the LC-CDR3 comprises SEQ ID NO: 157.

In one embodiment, the HC-CDR comprises SEQ ID NO:72, the HC-CDR2 comprises SEQ ID NO:74, the HC-CDR3 comprises SEQ ID NO:76, and the LC-CDRI comprises SEQ ID NO: 153 or SEQ ID NO:263, the LC-CDR2 comprises SEQ ID NO: 155, and the LC-CDR3 comprises SEQ ID NO: 157.

In one embodiment, the HC-CDR1 comprises SEQ ID NO:79, the HC-CDR2 comprises SEQ ID NO:74, the HC-CDR3 comprises SEQ ID NO:82, and the LC-CDRI comprises SEQ ID NO: 159, the LC-CDR2 comprises SEQ ID NO: 155 and the LC-CDR3 comprises SEQ ID NO: 160.

Group IC mAbs

In one aspect, the présent invention provides an isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 comprising (a) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:84 (GKWIE); a HC-CDR2 set forth as SEQ ID NO:86 (EILPGTGSTNYNEKFKG) or SEQ ID NO:275 (EILPGTGSTNYAQKFQG); and a HC-CDR3 set forth as SEQ ID NO:88 (SEDV); and (b) a light chain variable région comprising a LC-CDRI set forth as SEQ ID NO: 142 (KSSQSLLNSRTRKNYLA), SEQ ID NO:257 (KSSQSLLQSRTRKNYLA); SEQ ID NO:258 (K.SSQSLLASRTRKNYLA); or SEQ ID NO:259 (KSSQSLLNTRTRKNYLA), a LC-CDR2 set forth as SEQ ID NO: 144 ( WASTRES); and a LC-CDR3 set forth as SEQ ID NO: 161 (KQSYNIPT). In one embodiment, the LC-CDRI comprises SEQ ID NO:258.

Group II mAbs

In one aspect, the présent invention provides an isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 comprising: (a) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:91 (GYWIE); a HC-CDR2 set forth as SEQ ID NO:93 (EMLPGSGSTHYNEKFKG), and a HC-CDR3 set forth as SEQ ID NO:95 (SIDY); and (b) a light chain variable région comprising a LC-CDRI set forth as SEQ ID

155

NO: 163 (RSSQSLVQSNGNTYLH), a LC-CDR2 set forth as SEQ ID NO: 165 (KVSNRFS) and a LC-CDR3 set forth as SEQ ID NO: 167 (SQSTHVPPT).

Group III mAbs

In another aspect, the présent invention provides an isolated antibody, or antigenbinding fragment thereof, that binds to MASP-3 comprising: (a) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO: 109 (RVHFAIRDTNYWMQ), a HC-CDR2 set forth as SEQ ID NO:110 (AIYPGNGDTSYNQKFKG), a HC-CDR3 set forth as SEQ ID NO:112 (GSHYFDY); and a light chain variable région comprising a LC-CDRI set forth as SEQ ID NO: 182 (RASQSIGTSIH), a LC-CDR2 set forth as SEQ ID NO: 184 (YASESIS) and a LC-CDR3 set forth as SEQ ID NO: 186 (QQSNSWPYT); or (b) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO: 125 (DYYMN), a HC-CDR2 set forth as SEQ ID NO: 127 (DVNPNNDGTTYNQKFKG), a HC-CDR3 set forth as SEQ ID NO: 129 (CPFYYLGKGTHFDY); and a light chain variable région comprising a LC-CDRI set forth as SEQ ID NO: 196 (RASQDISNFLN), a LC-CDR2 set forth as SEQ ID NO: 198 (YTSRLHS) and a LC-CDR3 set forth as SEQ ID NO:200 (QQGFTLPWT); or (c) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO: 137 a HC-CDR2 set forth as SEQ ID NO: 138, a HC-CDR3 set forth as SEQ ID NO: 140; and a light chain variable région comprising a LC-CDRI set forth as SEQ ID NO:206, a LCCDR2 set forth as SEQ ID NO:207 and a LC-CDR3 set forth as SEQ ID NO:208; or (d) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:98, a HC-CDR2 set forth as SEQ ID NO:99, a HC-CDR3 set forth as SEQ ID NO: 101; and a light chain variable région comprising a LC-CDRI set forth as SEQ ID NO: 169, a LC-CDR2 set forth as SEQ ID NO:17I and a LC-CDR3 set forth as SEQ ID NO:173; or (e) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO: 103, a HC-CDR2 set forth as SEQ ID NO: 105, a HC-CDR3 set forth as SEQ ID NO: 107; and a light chain variable région comprising a LC-CDRI set forth as SEQ ID NO:176, a LCCDR2 set forth as SEQ ID NO: 178 and a LC-CDR3 set forth as SEQ ID NO: 193; or (f) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:114, a HC-CDR2 set forth as SEQ ID NO:116, a HC-CDR3 set forth as SEQ ID NO:118; and a light chain variable région comprising a LC-CDRI set forth as SEQ ID NO: 188, a LCCDR2 set forth as SEQ ID NO: 178 and a LC-CDR3 set forth as SEQ ID NO: 190; or

156 (g) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:114, a HC-CDR2 set forth as SEQ ID NO:121, a HC-CDR3 set forth as SEQ ID NO:123; and a light chain variable région comprising a LC-CDR1 set forth as SEQ ID NO: 191, a LCCDR2 set forth as SEQ ID NO:178 and a LC-CDR3 set forth as SEQ ID NO:193; or (h) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO: 132, a HC-CDR2 set forth as SEQ ID NO: 133, a HC-CDR3 set forth as SEQ ID NO: 135; and a light chain variable région comprsing a LC-CDR1 set forth as SEQ ID NO:203, a LCCDR2 set forth as SEQ ID NO: 165 and a LC-CDR3 set forth as SEQ ID NO:204.

Heavy Chain and Light Chain Variable Régions

In one embodiment, the invention provides a high affinity MASP-3 inhibitory antibody comprising a heavy chain variable région comprising or consisting of a sequence which is at least 80%, 85%, 90%, 95%, 98%, 99% identical to any of SEQ ID NO:s 24-39, 248-249, 251-252, 254-255 or wherein the antibody comprises a heavy chain variable région comprising SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:254 or SEQ ID NO:255.

In one embodiment, the invention provides a high affinity MASP-3 inhibitory antibody comprising a light chain variable région comprising or consisting of a sequence which is at least 80%, 85%, 90%, 95%, 98%, 99% identical to any of SEQ ID NO:s 40-54, 250, 253, 256, 278, 279, or 280 or wherein the antibody comprises a light chain variable région comprising SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:250, SEQ ID NO:253, SEQ ID NO:256, SEQ ID NO:278, SEQ ID NO:279 or SEQ ID NO:280.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:24, SEQ ID NO:248 or SEQ ID NO:249 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:40, SEQ ID NO:250 or SEQ ID NO:278.

157

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:25 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:41.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:26 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:42.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:27 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:42.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:28, SEQ ID NO:251 or SEQ ID NO:252 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:43, SEQ ID NO:253 or SEQ ID NO:279.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:29 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:44.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:30, SEQ ID NO:254 or SEQ ID NO:255 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:45, SEQ ID NO:256 or SEQ ID NO:280.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:31 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:46.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:32 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:47.

158

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:33 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:48.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:34 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:49.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:35 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:50.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:36 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQIDNO:51.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:37 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ IDNO:52.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:38 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:53.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:39 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:54.

Cross-competition of high affinity MASP-3 antibodies

As described herein, the high affinity MASP-3 inhibitory antibodies disclosed herein recognize overlapping epitopes within the serine protease domain of MASP-3. As described

159 in Example 18, shown in FIGURES 61A-E and 62-67, and summarized in TABLES 4 and 28, cross-competition analysis and pepscan binding analysis shows that the high affinity MASP-3 inhibitory antibodies cross-compete and bind to common epitopes located within the MASP-3 serine protease domain. Thus, in one embodiment, the invention provides high affinitiy MASP-3 inhibitory antibodies that specifically recognize an epitope or part thereof within the serine protease domain of human MASP-3 recognised by one or more selected from the group consisting of:

a monoclonal antibody comprising a-heavy chain variable région set forth as SEQ ID NO:24 and a light chain variable région set forth as SEQ ID NO:40;

a monoclonal antibody comprising a heavy chain variable région set forth as SEQ ID NO:25 and a light chain variable région set forth as SEQ IDNO:41;

a monoclonal antibody comprising a heavy chain variable région set forth as SEQ ID NO:26 and a light chain variable région set forth as SEQ ID NO:42;

a monoclonal antibody comprising a heavy chain variable région set forth as SEQ ID NO:27 and a light chain variable région set forth as SEQ ID NO:42;

a monoclonal antibody comprising a heavy chain variable région set forth as SEQ ID NO:28 and a light chain variable région set forth as SEQ ID NO:43;

a monoclonal antibody comprising a heavy chain variable région set forth as SEQ ID NO:29 and a light chain variable région set forth as SEQ ID NO:44;

a monoclonal antibody comprising a heavy chain variable région set forth as SEQ ID NO:30 and a light chain variable région set forth as SEQ ID NO:45;

a monoclonal antibody comprising a heavy chain variable région set forth as SEQ ID NO:31 and a light chain variable région set forth as SEQ ID NO:46;

a monoclonal antibody comprising a heavy chain variable région set forth as SEQ ID NO:32 and a light chain variable région set forth as SEQ ID NO:47;

a monoclonal antibody comprising a heavy chain variable région set forth as SEQ ID NO:33 and a light chain variable région set forth as SEQ IDNO:48;

a monoclonal antibody comprising a heavy chain variable région set forth as SEQ ID NO:34 and a light chain variable région set forth as SEQ ID NO:49;

a monoclonal antibody comprising a heavy chain variable région set forth as SEQ ID NO:35 and a light chain variable région set forth as SEQ IDNO:50;

160 a monoclonal antibody comprising a heavy chain variable région set forth as SEQ ID NO:36 and a light chain variable région set forth as SEQ ID NO:51;

a monoclonal antibody comprising a heavy chain variable région set forth as SEQ ID NO:37 and a light chain variable région set forth as SEQ ID NO:52;

a monoclonal antibody comprising a heavy chain variable région set forth as SEQ ID NO:38 and a light chain variable région set forth as SEQ ID NO:53; and a monoclonal antibody comprising a heavy chain variable région set forth as SEQ ID NO:39 and a light chain variable région set forth as SEQ ID NO:54.

According to the présent invention, when a given antibody recognises at least part of an epitope recognised by another given antibody, these two antibodies are said to recognise the same or overlapping epitopes.

Different assays available to the person skilled in the art may be used to détermine whether an antibody (also designated test antibody) recognises the same or an overlapping epitope as a particular monoclonal antibody (also designated reference antibody). Preferably, the assay involves the steps of:

• Providing MASP-3 or a fragment thereof comprising the epitope recognised by the reference antibody • Add the test antibody and the reference antibody to the said MASP-3, wherein either the test antibody or the reference antibody is labelled with a détectable label. Altematively, both antibodies may be labeled with different détectable labels • Detecting the presence of the détectable label at MASP-3 • Thereby detecting whether the test antibody may displace the reference antibody

If the reference antibody is displaced, the test antibody recognises the same or an overlapping epitope as the reference antibody. Thus, if the reference antibody is labeled with a détectable label, then a low détectable signal at MASP-3 is indicative of displacement of the reference antibody. If the test antibody is labelled with a détectable label, then a high détectable signal at MASP-3 is indicative of displacement of the reference antibody. The MASP-3 fragment may preferably be immobilised on a solid support enabling facile

161 handling. The détectable label may be any directly or indirectly détectable label, such as an enzyme, a radioactive isotope, a heavy métal, a coloured compound or a fluorescent compound. In Example 18 in the section “Compétition Binding Analysis” herein below describes an exemplary method of determining whether a test antibody recognises the same or an overlapping epitope as a reference antibody is described. The person skilled in the art may easily adapt said method to the particular antibodies in question.

The MASP-3 antibodies useful in this aspect of the invention include monoclonal or recombinant antibodies derived from any antibody producing mammal and may be multispecific (i.e., bispecific or trispecific), chimeric, humanized, fully human, anti-idiotype, and antibody fragments. Antibody fragments include Fab, Fab', F(ab)2, F(ab')2, Fv fragments, scFv fragments and single-chain antibodies as further described herein.

MASP-3 antibodies can be screened for the ability to inhibit alternative pathway complément activation system using the assays described herein. The inhibition of alternative pathway complément activation is characterized by at least oneor more of the following changes in a component ofthe complément system that occurs as a resuit of administration of a high affinity MASP-3 inhibitory antibody in accordance with various embodiments of the invention: inhibition of hemolysis and/or opsonization; inhibition of lectin-independent conversion of factor B; inhibition of lectin-independent conversion of factor D, inhibition of MASP-3 serine protease substrate-specific cleavage; the réduction of hemolysis or the réduction of C3 cleavage and C3b surface déposition; the réduction of Factor B and Bb déposition on an activating surface; the réduction of resting levels (in circulation, and without the experimental addition of an activating surface) of active Factor D relative to pro-Factor D; the réduction of levels of active Factor D relative to pro-Factor D in response to an activating surface; and/or the production of resting and surface-induced levels of fluid-phase Ba, Bb, C3b, or C3a.

MASP-3 antibodies with reduced effector function

In some embodiments of this aspect of the invention, the high affinity MASP-3 inhibitory antibodies described herein hâve reduced effector function in order to reduce inflammation that may arise from the activation of the classical complément pathway. The ability of IgG molécules to trigger the classical complément pathway has been shown to

162 résidé within the Fc portion of the molécule (Duncan, A.R., et al., Nature 332:738-740 (1988)). IgG molécules in which the Fc portion of the molécule has been removed by enzymatic cleavage are devoid of this effector function (see Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Accordingly, antibodies with reduced effector function can be generated as the resuit of lacking the Fc portion of the molécule by having a genetically engineered Fc sequence that minimizes effector function, or being of either the human IgG2 or IgGq isotype.

Antibodies with reduced effector function can be produced by standard molecular biological manipulation of the Fc portion of the IgG heavy chains as described in Jolliffe étal., Int'l Rev. Immunol. 70:241-250, (1993), and Rodrigues et al., J.

Immunol. 757:6954-6961, (1998). Antibodies with reduced effector function also include human IgG2 and IgG4 isotypes that hâve a reduced ability to activate complément and/or interact with Fc receptors (Ravetch, J.V., et al., Annu. Rev. Immunol. 9:457-492, (1991); Isaacs, J.D., étal., J. Immunol. 745:3062-3071, 1992; van de Winkel, J.G., étal., Immunol. Today 74:215-221, (1993)). Humanized or fully human antibodies spécifie to human MASP1, MASP-2 or MASP-3 (including dual, pan, bispecific or trispecific antibodies) comprised of IgG2 or IgG4 isotypes can be produced by one of several methods known to one of ordinary skilled in the art, as described in Vaughan, T.J., et al., Nature Biotechnical 76:535-539, (1998).

Production of high affinity MASP-3 inhibitory antibodies

MASP-3 antibodies can be produced using MASP-3 polypeptides (e.g., full-Iength MASP-3) or using antigenic MASP- 3 epitope-bearing peptides (e.g., a portion of the MASP-3 polypeptide), for example as described in Example 14 herein below. Immunogenic peptides may be as small as five amino acid residues. The MASP-3 peptides and polypeptides used to raise antibodies may be isolated as natural polypeptides, or recombinant or synthetic peptides and catalytically inactive recombinant polypeptides. Antigens useful for producing MASP-3 antibodies also include fusion polypeptides, such as fusions of a MASP-3 polypeptide or a portion thereof with an immunoglobulin polypeptide or with maltose-binding protein. The polypeptide immunogen may be a full-Iength molécule or a portion thereof. If the polypeptide portion is hapten-Iike, such portion may be

163 advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine sérum albumin (BSA) or tetanus toxoid) for immunization.

Monoclonal antibodies

As used herein, the modifier monoclonal indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be obtained using any technique that provides for the production of antibody molécules by continuous cell lines in culture, such as the hybridoma method described by Kohler, G., étal., Nature 256:495, (1975), or they may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567 to Cabilly). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson, T., et al., Nature 352:624-628, (1991), and Marks, J.D., et al., J. Mol. Biol. 222:581-597, (1991). Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

For example, monoclonal antibodies can be obtained by injecting a suitable mammal (e.g., a BALB/c mouse) with a composition comprising a MASP-3 polypeptide, or portion thereof. After a predetermined period of time, splénocytes are removed from the mouse and suspended in a cell culture medium. The splénocytes are then fused with an immortal cell line to form a hybridoma. The formed hybridomas are grown in cell culture and screened for their ability to produce a monoclonal antibody against MASP-3. (See also Current Protocols in Immunology, Vol. 1., John Wiley & Sons, pages 2.5.1-2.6.7, 1991.)

Human monoclonal antibodies may be obtained through the use of transgenic mice that hâve been engineered to produce spécifie human antibodies in response to antigenic challenge. In this technique, éléments of the human immunoglobulin heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous immunoglobulin heavy chain and light chain loci. The transgenic mice can synthesize human antibodies spécifie for human antigens, such as the MASP-2 antigens described herein, and the mice can be used to produce human MASP-2 antibody-secreting hybridomas by fusing B-cells from such animais to suitable myeloma cell lines using conventional Kohler-Milstein technology. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green, L.L., et al., Nature

164

Genet. 7:13, 1994; Lonberg, N., et al., Nature 368:856, 1994; and Taylor, L.D., et al., Int. Immun. 6:579, 1994.

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affïnity 5 chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., Purification of immunoglobulin G (IgG), in Methods in Molecular Biology, The Humana Press, Inc., Vol. 10, pages 79-104, 1992).

Once produced, monoclonal antibodies are first tested for spécifie MASP-3 binding 10 or, where desired, dual MASP-1/3, MASP-2/3 or MASP-1/2 binding. Methods for determining whether an antibody binds to a protein antigen and/or the affïnity for an antibody to a protein antigen are known in the art. For example, the binding of an antibody to a protein antigen can be detected and/or quantifïed using a variety of techniques such as, but not limited to, Western blot, dot blot, plasmon surface résonance method (e.g., BIAcore 15 System; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ), or enzyme-Iinked immunosorbent assays (ELISA). See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.; Benny K. C. Lo (2004) Antibody Engineering: Methods and Protocols, Humana Press (ISBN: 1588290921); Borrebaek (1992) Antibody Engineering, A Practical Guide, W.H.

Freeman and Co., NY; Borrebaek (1995) Antibody Engineering, 2^nd Edition, Oxford University Press, NY, Oxford; Johne et al. (1993), Immunol. Meth. 160:191-198; Jonsson et al. (1993) Ann. Biol. Clin. 51: 19-26; and Jonsson et al. (1991) Biotechniques 11:620-627. See also, U.S. Patent No. 6,355,245.

The affïnity of MASP-3 monoclonal antibodies can be readily determined by one of 25 ordinary skill in the art (see, e.g., Scatchard, A., NY Acad. Sci. 51:660-672, 1949). In one embodiment, the MASP-3 monoclonal antibodies useful for the methods of the invention bind to MASP-3 with a binding affïnity of <100 nM, preferably <10 nM, preferably <2 nM, and most preferably with high affïnity of <500 pM.

Once antibodies are identified that specifïcally bind to MASP-3, the MASP-3 30 antibodies are tested for the ability to function as an alternative pathway inhibitor in one of several functional assays, such as, for example, the inhibition of alternative pathway complément activation is characterized by at least one or more of the following changes in a

165 component of the complément System that occurs as a resuit of administration of a high affinity MASP-3 inhibitory antibody in accordance with various embodiments of the invention: inhibition of hemolysis and/or opsonization; inhibition of lectin-independent conversion of factor B; inhibition of lectin-independent conversion of factor D, inhibition of MASP-3 serine protease substrate-specifîc cleavage; the réduction of hemolysis or the réduction of C3 cleavage and C3b surface déposition; the réduction of Factor B and Bb déposition on an activating surface; the réduction of resting levels (in circulation, and without the experimental addition of an activating surface) of active Factor D relative to pro-Factor D; the réduction of levels of active Factor D relative to pro-Factor D in response to an activating surface; the réduction in production of resting and surface-induced levels of fluidphase Ba, Bb, C3b, or C3a; and/or the the réduction in déposition of factor P.

Chimeric/humanized antibodies

Monoclonal antibodies useful in the method of the invention include chimeric antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies (U.S. Patent No. 4,816,567, to Cabilly; and Morrison, S.L., étal., Proc. Nat'l Acad. Sci. USA 57:6851-6855, (1984)).

One form of a chimeric antibody useful in the invention is a humanized monoclonal MASP-3 antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies, which contain minimal sequence derived from non-human immunoglobulin. Humanized monoclonal antibodies are produced by transferring the non-human (e.g., mouse) complementarity determining régions (CDR), from the heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typically, residues of human antibodies are then substituted in the framework régions of the non-human counterparts. Furthermore, humanized antibodies may comprise residues that are not found in the récipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially ail of at least one, and typically two, variable domains in which ali or substantially ail ofthe hypervariable

166 .

loops correspond to those of a non-human immunoglobulin and ail or substantially ail of the Fv framework régions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant région (Fc), typically that of a human immunoglobulin. For further details, see Jones, P.T., 5 étal., Nature 321:522-525, (1986); Reichmann, L., étal., Nature 332:323-329, (1988); and

Presta, Curr. Op. Struct. Biol. 2:593-596, (1992).

The humanized antibodies useful in the invention include human monoclonal antibodies including at least a MASP-3 binding CDR3 région. In addition, the Fc portions may be replaced so as to produce IgA or IgM as well as human IgG antibodies. Such 10 humanized antibodies will hâve particular clinical utility because they will specifically recognize human MASP-3 but will not evoke an immune response in humans against the antibody itself. Consequently, they are better suited for in vivo administration in humans, especially when repeated or long-term administration is necessary

Techniques for producing humanized monoclonal antibodies are also described, for 15 example, by Jones, P.T., étal., Nature 321:522, (1986); Carter, P., étal., Proc. Nat'l. Acad.

Sci. USA 89:4285, (1992); Sandhu, J.S., Crit. Rev. Biotech. 12:431, (1992); Singer, I.I., et al., J. Immun. 750:2844, (1993); Sudhir (ed.), Antibody Engineering Protocols, Humana Press, Inc., (1995); Kelley, Engineering Therapeutic Antibodies, in Protein Engineering: Principles and Practice, Cleland et al. (eds.), John Wiley & Sons, Inc., pages 399-434, 20 (1996); and by U.S. Patent No. 5,693,762, to Queen, 1997. In addition, there are commercial entities that will synthesize humanized antibodies from spécifie murine antibody régions, such as Protein Design Labs (Mountain View, CA).

Recombinant antibodies

MASP-3 antibodies can also be made using recombinant methods. For example, 25 human antibodies can be made using human immunoglobulin expression libraries (available for example, from Stratagene, Corp., La Jolla, CA) to produce fragments of human antibodies (Vh, V_l, Fv, Factor D, Fab or F(ab')2). These fragments are then used to construct whole human antibodies using techniques similar to those for producing chimeric antibodies.

Immunoglobulin fragments

The MASP-3 inhibitory agents useful in the method of the invention encompass not only intact immunoglobulin molécules but also the well-known fragments including Fab,

167

Fab', F(ab)2, F(ab')2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molécules and multispecific (e.g., bispecific and trispecific) antibodies formed from antibody fragments.

It is well known in the art that only a small portion of an antibody molécule, the paratope, is involved in the binding of the antibody to its epitope (see, e.g., Clark, W.R., The Experimental Foundations of Modem Immunology, Wiley & Sons, Inc., NY, 1986). The pFc' and Fc régions of the antibody are effectors ofthe classical complément pathway but are not involved in antigen binding. An antibody from which the pFc' région has been enzymatically cleaved, or which has been produced without the pFc' région, is designated an F(ab')2 fragment and retains both of the antigen binding sites of an intact antibody. An isolated F(ab')2 fragment is referred to as a bivalent monoclonal fragment because of its two antigen binding sites. Similarly, an antibody from which the Fc région has been enzymatically cleaved, or which has been produced without the Fc région, is designated a Fab fragment, and retains one of the antigen binding sites of an intact antibody molécule.

Antibody fragments can be obtained by proteolytic hydrolysis, such as by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2· This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab’ monovalent fragments. Optionally, the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that resuit from cleavage of disulfïde linkages. As an alternative, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, U.S. Patent No. 4,331,647 to Goldenberg; Nisonoff, A., étal., Arch. Biochem. Biophys. 89:230, (1960); Porter, R.R., Biochem. J. 73:119, (1959); Edelman, étal., in Methods in Enzymology 7:422, Academie Press, (1967); and by Coligan at pages 2.8.1-2.8.10 and 2.10-2.10.4.

In some embodiments, the use of antibody fragments lacking the Fc région are preferred to avoid activation of the classical complément pathway which is initiated upon binding Fc to the Fcy receptor. There are several methods by which one can produce a monoclonal antibody that avoids Fcy receptor interactions. For example, the Fc région of a monoclonal antibody can be removed Chemically using partial digestion by proteolytic

168 enzymes (such as ficin digestion), thereby generating, for example, antigen-binding antibody fragments such as Fab or F(ab)2 fragments (Mariani, M., étal., Mol. Immunol. 28:69-1\, (1991)). Alternatively, the human γ4 IgG isotype, which does not bind Fcy receptors, can be used during construction of a humanized antibody as described herein. Antibodies, single chain antibodies and antigen-binding domains that lack the Fc domain can also be engineered using recombinant techniques described herein.

Single-chain antibody fragments

Alternatively, one can create single peptide chain binding molécules spécifie for MASP-3 in which the heavy and light chain Fv régions are connected. The Fv fragments may be connected by a peptide linker to form a single-chain antigen binding protein (scFv). These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the Vpj and domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are described for example, by Whitlow, et al., Methods: A Companion to Methods in Enzymology 2:97, (1991); Bird, étal., Science 242:423, (1988); U.S. Patent No. 4,946,778, to Ladner; Pack, P., et al., Bio/Technology 77:1271, (1993).

As an illustrative example, a MASP-3-specific scFv can be obtained by exposing lymphocytes to MASP-3 polypeptide in vitro and selecting antibody display libraries in phage or similar vectors (for example, through the use of immobilized or labeled MASP-3 protein or peptide). Genes encoding polypeptides having potential MASP-3 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage or on bacteria such as E. coli. These random peptide display libraries can be used to screen for peptides which interact with MASP-3. Techniques for creating and screening such random peptide display libraries are well known in the art (U.S. Patent No. 5,223,409, to Lardner; U.S. Patent No. 4,946,778, to Ladner; U.S. Patent No. 5,403,484, to Lardner; U.S. Patent No. 5,571,698, to Lardner; and Kay étal., Phage Display of Peptides and Proteins Academie Press, Inc., 1996) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from CLONTECH Laboratories, Inc. (Palo

169

Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.).

Another form of a MASP-3 antibody fragment useful in this aspect of the invention is a peptide coding for a single complementarity-determining région (CDR) that binds to an epitope on a MASP-3 antigen and inhibits alternative complément pathway activation.

CDR peptides (minimal récognition units) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable région from RNA of antibody-producing cells (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, (1991); Courtenay-Luck, Genetic Manipulation of Monoclonal Antibodies, in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press, (1995); and Ward étal., Genetic Manipulation and Expression of Antibodies, in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), page 137, Wiley-Liss, Inc., 1995).

The high affinity MASP-3 inhibitory antibodies described herein are administered to a subject in need thereof to inhibit alternative pathway activation. In some embodiments, the high affinity MASP-3 inhibitory antibody is a humanized monoclonal MASP-3 antibody. optionally with reduced effector function.

Bispecific antibodies

The high affinity MASP-3 inhibitory antibodies useful in the method of the invention encompass multispecific (i.e., bispecific and trispecific) antibodies. Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that hâve binding specificities for at least two different antigens. In one embodiment, the compositions and methods comprise the use of a bispecific antibody comprising a binding specificity for the serine protease domain of MASP-3 and a binding specificity for MASP-2 (e.g., binding to at least one of CCP1-CCP2 or serine protease domain of MASP-2). In another embodiment, the method comprises the use of a bispecific antibody comprising a binding specificity for the serine protease domain of MASP-3 and a binding specificity for MASP-1 (e.g., binding to the serine protease domain of MASP-1). In another embodiment, the method comprises the use of a trispecific antibody comprising a binding specificity for MASP-3 (e.g., binding to the serine protease domain of MASP-3), a binding specificity for MASP-2 (e.g., binding to at least one of CCP1-CCP2 or

170 serine protease domain of MASP-2) and a binding specificity for MASP-1 (e.g., binding to the serine protease domain of MASP-1).

Methods for making bispecific antibodies are within the purview of those skilled in the art. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains hâve different specificities (Milstein and Cuello, Nature 305:537-539 (1983)). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, including at least part of the hinge, Ch2, and Ch3 régions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are cotransfected into a suitable host organism. For further details of illustrative currently known methods for generating bispecific antibodies see, e.g., Suresh et al., Methods in Enzymology 121:210 (1986); WO96/27011; Brennan et al., Science 229:81 (1985); Shalaby et al., J. Exp. Med. 175:217-225 (1992); Kostelny et al., J. Immunol. 148(5):1547-1553 (1992); Hollinger et al. Proc. Natl. Acad. Sci USA 90:6444-6448 (1993); Gruber et al., J. Immunol. 152:5368 (1994); and Tutt et al., J. Immunol. 147:60 (1991). Bispecific antibodies also include crosslinked or heteroconjugate antibodies. Heteroconjugate antibodies may be made using any convenient cross -linking methods. Suitable crosslinking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture hâve also been described. For example, bispecific antibodies hâve been produced using leucine zippers. (See, e.g., Kostelny et al. J. Immunol. 148(5):1547-1553 (1992)). The diabody technology described by Hollinger et al. Proc. Natl. Acad. Sci USA 90:6444-6448 (1993), has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Bispecific diabodies, as opposed to bispecific whole antibodies, may also be particularly useful because they can be

171 readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.

Another strategy for making bispecific antibody fragments by the use of single-chain Fv (scFv) dimers has also been reported. (See, e.g., Gruber et al. J. Immunol., 152:5368 (1994)). Altematively, the antibodies can be linear antibodies as described in, e.g., Zapata et al., Protein Eng. 8(10):1057-1062 (1995). Briefly described, these antibodies comprise a pair of tandem Factor D segments (Vh-ChI-Vh-ChI) which form a pair of antigen binding régions. Linear antibodies can be bispecific or monospecific. The methods of the invention also embrace the use of variant forms of bispecific antibodies such as the tetravalent dual variable domain immunoglobulin (DVD-Ig) molécules described in Wu et al., Nat Biotechnol 25:1290-1297 (2007). The DVD-Ig molécules are designed such that two different light chain variable domains (VL) from two different parent antibodies are linked in tandem directly or via a short linker by recombinant DNA techniques, followed by the light chain constant domain. Methods for generating DVD-Ig molécules from two parent antibodies are further described in, e.g., WO08/024188 and WO07/024715, the disclosures of each of which are incorporated herein by reference in their entirety.

XVIII. PHARMACEUTICAL COMPOSITIONS AND DELIVERY METHODS DOSING

In another aspect, the invention provides compositions comprising high affinity MASP-3 inhibitory antibodies for inhibiting the adverse effects of alternative pathway complément activation in a subject in need thereof, such as, for example, a subject suffering from an alternative pathway-related disease or condition, such as, for example a hemolytic disease, such as PNH, or a disease or disorder selected from the group consisting of agerelated macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS) or thrombotic thrombocytopénie purpura (TTP)), asthma, dense deposit disease, pauci-immune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis,

172 neuromyelitis optica, Behcet’s disease, multiple sclerosis (MS), Guillain Barre Syndrome, Alzheimer’s disease, Amylotrophic latéral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy, Uveitis, Chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD), hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis, Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy and Myasthenia Gravis.

The methods of this aspect of the invention comprises administering to the subject a composition comprising an amount of a high affinity MASP-3 inhibitory antibody effective to inhibit alternative pathway complément activation and a pharmaceutically acceptable carrier. In some embodiments, the method further comprises administering a composition comprising a MASP-2 inhibitory agent. The high affinity MASP-3 inhibitory antibodies and MASP-2 inhibitory agents can be administered to a subject in need thereof, at therapeutically effective doses to treat or ameliorate conditions associated with alternative pathway complément activation, and optionally also MASP-2-dependent complément activation. A therapeutically effective dose refers to the amount ofthe MASP-3 inhibitory antibody, or a combination of a MASP-3 inhibitory antibody and a MASP-2 inhibitory agent sufficient to resuit in amelioration of symptoms of the condition. The inhibition of alternative pathway complément activation is characterized by at least oneor more of the following changes in a component of the complément System that occurs as a resuit of administration of a high affinity MASP-3 inhibitory antibody in accordance with various embodiments of the invention: inhibition of hemolysis and/or opsonization; inhibition of lectin-independent conversion of factor B; inhibition of lectin-independent conversion of factor D, inhibition of MASP-3 serine protease substrate-specific cleavage; the réduction of hemolysis or the réduction of C3 cleavage and C3b surface déposition; the réduction of Factor B and Bb déposition on an activating surface; the réduction of resting levels (in circulation, and without the experimental addition of an activating surface) of active Factor D relative to pro-Factor D; the réduction of levels of active Factor D relative to pro-Factor D in response to an activating surface; and/or the the réduction in the production of resting and surface-induced levels of fluid-phase Ba, Bb, C3b, or C3a.

Toxicity and therapeutic efficacy of MASP-3 and MASP-2 inhibitory agents can be determined by standard pharmaceutical procedures employing experimental animal models.

173

Using such animal models, the NOAEL (no observed adverse effect level) and the MED (the minimally effective dose) can be determined using standard methods. The dose ratio between NOAEL and MED effects is the therapeutic ratio, which is expressed as the ratio NOAEL/MED. MASP-3 inhibitory agents and MASP-2 inhibitory agents that exhibit large 5 therapeutic ratios or indices are most preferred. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of the MASP-3 inhibitory agent and MASP-2 inhibitory agent preferably lies within a range of circulating concentrations that include the MED with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the 10 route of administration utilized.

For any compound formulation, the therapeutically effective dose can be estimated using animal models. For example, a dose may be formulated in an animal model to achieve a circulating plasma concentration range that includes the MED. Quantitative levels of the MASP-3 inhibitory agent or MASP-2 inhibitory agent in plasma may also be measured, for 15 example, by high performance liquid chromatography. '

In addition to toxicity studies, effective dosage may also be estimated based on the amount of target MASP protein présent in a living subject and the binding affinity of the MASP-3 or MASP-2 inhibitory agent.

It has been reported that MASP-1 levels in normal human subjects is présent in sérum 20 in levels in the range of from 1.48 to 12.83 pg/mL (Terai I. et al, Clin Exp Immunol 110:317323 (1997); Theil et al., Clin. Exp. Immunol. 169:38 (2012)). The mean sérum MASP-3 concentrations in normal human subjects has been reported to be in the range of about 2.0 to 12.9 pg/mL (Skjoedt M et al., Immunobiology 215(11):921-31 (2010); Degn et al., J. Immunol Methods, 361-37 (2010); Csuka et al., Mol. Immunol. 54:271 (2013). It has been 25 shown that MASP-2 levels in normal human subjects is présent in sérum in low levels in the range of 500 ng/mL, and MASP-2 levels in a particular subject can be determined using a quantitative assay for MASP-2 described in Moller-Kristensen M., et al., J. Immunol. Methods 252:159-167 (2003) and Csuka et al., Mol. Immunol. 54:271 (2013).

Generally, the dosage of administered compositions comprising MASP-3 inhibitory 30 agents or MASP-2 inhibitory agents varies depending on such factors as the subject's âge, weight, height, sex, general medical condition, and previous medical history. As an illustration, MASP-3 inhibitory agents or MASP-2 inhibitory agents (such as MASP-3

174 antibodies, MASP-1 antibodies or MASP-2 antibodies), can be administered in dosage ranges from about 0.010 to 100.0 mg/kg, preferably 0.010 to 10 mg/kg, preferably 0.010 to 1.0 mg/kg, more preferably 0.010 to 0.1 mg/kg of the subject body weight. In some embodiments, MASP-2 inhibitory agents (such as MASP-2 antibodies) are administered in dosage ranges from about preferably 0.010 to 10 mg/kg, preferably 0.010 to 1.0 mg/kg, more preferably 0.010 to 0.1 mg/kg of the subject body weight. In some embodiments, MASP-1 inhibitory agents (such as MASP-1 antibodies) or MASP-3 inhibitory agents (such as MASP3 antibodies) are administered in dosage ranges from about 0.010 to 100.0 mg/kg, preferably 0.010 to 10 mg/kg, such as form about 1 mg/kg to about 10 mg/kg, preferably 0.010 to 1.0 mg/kg, more preferably 0.010 to 0.1 mg/kg of the subject body weight.

Therapeutic efïicacy of MASP-3 inhibitory compositions, optionally in combination with MASP-2 inhibitory compositions, or of MASP-1 inhibitory compositions, optionally in combination with MASP-2 inhibitory compositions, and methods of the présent invention in a given subject, and appropriate dosages, can be determined in accordance with complément assays well known to those of skill in the art. Complément generates numerous spécifie products. During the last decade, sensitive and spécifie assays hâve been developed and are available commercially for most of these activation products, including the small activation fragments C3a, C4a, and C5a and the large activation fragments iC3b, C4d, Bb, and sC5b-9. Most of these assays utilize monoclonal antibodies that react with new antigens (neoantigens) exposed on the fragment, but not on the native proteins from which they are formed, making these assays very simple and spécifie. Most rely on ELISA technology, although radioimmunoassay is still sometimes used for C3a and C5a. These latter assays measure both the unprocessed fragments and their 'desArg' fragments, which are the major forms found in the circulation. Unprocessed fragments and C5adesArg are rapidly cleared by binding to cell surface receptors and are hence présent in very low concentrations, whereas C3adesArg does not bind to cells and accumulâtes in plasma. Measurement ofC3a provides a sensitive, pathway-independent indicator of complément activation. Alternative pathway activation can be assessed by measuring the Bb fragment and/or measurement of factor D activation. Détection of the fluid-phase product of membrane attack pathway activation, sC5b-9, provides evidence that complément is being activated to completion. Because both the lectin and classical pathways generate the same activation products, C4a and C4d, measurement of

175 these two fragments does not provide any information about which of these two pathways has generated the activation products.

The inhibition of the alternative pathway in a mammalian subject is characterized by at least one or more of the following in the mammalian subject after treatment with a high affïnity MASP-3 inhibitory antibody disclosed herein: inhibition of Factor D maturation; inhibition of the alternative pathway when administered to the subject at a molar ratio of from about 1:1 to about 2.5:1 (MASP-3 target to mAb); the classical pathway is not inhibited; inhibition of hemolysis and/or opsonization; a réduction of hemolysis or the réduction of C3 cleavage and C3b surface déposition; a réduction of Factor B and Bb déposition on an activating surface; a réduction of resting levels (in circulation, and without the experimental addition of an activating surface) of active Factor D relative to pro-Factor D; a réduction of levels of active Factor D relative to pro-Factor D in response to an activating surface; and/or a réduction of the production of resting and surface-induced levels of fluid-phase Ba, Bb, C3b, or C3a.

The inhibition of MASP-2-dependent complément activation is characterized by at least one of the following changes in a component of the complément System that occurs as a resuit of administration ofa MASP-2 inhibitory agent in accordance with the methods ofthe invention: the inhibition of the génération or production of MASP-2-dependent complément activation System products C4b, C3a, C5a and/or C5b-9 (MAC) (measured, for example, as described in measured, for example, as described in Example 2 of US Patent No. 7,919,094), the réduction of C4 cleavage and C4b déposition or the réduction of C3 cleavage and C3b déposition.

Pharmaceutical carriers and delivery vehicles

In general, the MASP-3 inhibitory antibody compositions, or compositions comprising a combination of MASP-2 and MASP-3 inhibitory agents, may be combined with any other selected therapeutic agents, are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the MASP-3 inhibitory antibody or the MASP-2 inhibitory agent (and any other therapeutic agents combined therewith). Exemplary pharmaceutically acceptable carriers for peptides are described in U.S. PatentNo. 5,211,657 to Yamada. The MASP-3 antibodies useful in the invention, as described herein, may be formulated into préparations in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules,

176 powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parentéral or surgical administration. The invention also contemplâtes local administration ofthe compositions by coating medical devices and the like.

Suitable carriers for parentéral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, stérile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the préparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting example, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles. Suitable hydrogel and micelle delivery Systems include the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexes disclosed in WO 2004/009664 A2 and the PEO and PEO/cyclodextrin complexes disclosed in U.S. Patent Application Publication No. 2002/0019369 Al. Such hydrogels may be injected locally at the site of intended action, or subcutaneously or intramuscularly to form a sustained release depot.

Compositions of the présent invention may be formulated for delivery subcutaneously, intra-muscularly, intravenously, intra-arterially or as an inhalant.

For intra-articular delivery, the MASP-3 inhibitory antibody, optionally in combination with a MASP-2 inhibitory agent may be carried in above-described liquid or gel carriers that are injectable, above-described sustained-release delivery vehicles that are injectable, or a hyaluronic acid or hyaluronic acid dérivative.

For oral administration of non-peptidergic agents, the MASP-3 inhibitory antibody, optionally in combination with a MASP-2 inhibitory agent may be carried in an inert Aller or diluent such as sucrose, comstarch, or cellulose.

177

For topical administration, the MASP-3 inhibitory antibody, optionally in combination with a MASP-2 inhibitory agent may be carried in ointment, lotion, cream, gel, drop, suppository, spray, liquid or powder, or in gel or microcapsular delivery Systems via a transdermal patch.

Various nasal and pulmonary delivery Systems, including aérosols, metered-dose inhalers, dry powder inhalers, and nebulizers, are being developed and may suitably be adapted for delivery of the présent invention in an aérosol, inhalant, or nebulized delivery vehicle, respectively.

For intrathecal (IT) or intracerebroventricular (ICV) delivery, appropriately stérile delivery Systems (e.g., liquids; gels, suspensions, etc.) can be used to administer the présent invention.

The compositions of the présent invention may also include biocompatible excipients, such as dispersing or wetting agents, suspending agents, diluents, buffers, pénétration enhancers, emulsifiers, binders, thickeners, flavoring agents (for oral administration).

Pharmaceutical carriers for antibodies and peptides

More specifically with respect to high affinity MASP-3 inhibitory antibodies, as described herein, exemplary formulations can be parenterally administered as injectable dosages of a solution or suspension of the compound in a physiologically acceptable diluent with a pharmaceutical carrier that can be a stérile liquid such as water, oils, saline, glycerol or éthanol. Additionally, auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be présent in compositions comprising MASP-3 antibodies. Additional components of pharmaceutical compositions include Petroleum (such as of animal, vegetable or synthetic origin), for example, soybean oil and minerai oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers for injectable solutions.

The MASP-3 antibodies can also be administered in the form of a depot injection or implant préparation that can be formulated in such a manner as to permit a sustained or pulsatile release of the active agents.

XVIX. MODES OF ADMINISTRATION

The pharmaceutical compositions comprising the MASP-3 inhibitory antibodies, optionally in combination with MASP-2 inhibitory agents may be administered in a number of ways depending on whether a local or systemic mode of administration is most appropriate

178 for the condition being treated. Further, the compositions of the présent invention can be delivered by coating or incorporating the compositions on or into an implantable medical device.

Systemic delivery

As used herein, the terms systemic delivery and systemic administration are intended to include but are not limited to oral and parentéral routes including intramuscular (IM), subcutaneous, intravenous (IV), intraarterial, inhalational, sublingual, buccal, topical, transdermal, nasal, rectal, vaginal and other routes of administration that effectively resuit in dispersement of the delivered agent to a single or multiple sites of intended therapeutic action. Preferred routes of systemic delivery for the présent compositions include intravenous, intramuscular, subcutaneous, intraarterial and inhalational. It will be appreciated that the exact systemic administration route for selected agents utilized in particular compositions of the présent invention will be determined in part to account for the agent's susceptibility to metabolic transformation pathways associated with a given route of administration. For example, peptidergic agents may be most suitably administered by routes other than oral.

The MASP-3 inhibitory antibodies, as described herein, can be delivered into a subject in need thereof by any suitable means. Methods of delivery of MASP-3 antibodies and polypeptides include administration by oral, pulmonary, parentéral (e.g., intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation (such as via a fine powder formulation), transdermal, nasal, vaginal, rectal, or sublingual routes of administration, and can be formulated in dosage forms appropriate for each route of administration.

By way of représentative example, MASP-3 inhibitory antibodies and peptides can be introduced into a living body by application to a bodily membrane capable of absorbing the polypeptides, for example the nasal, gastrointestinal and rectal membranes. The polypeptides are typically applied to the absorptive membrane in conjunction with a perméation enhancer. (See, e.g., Lee, V.H.L., Crit. Rev. Ther. Drug Carrier Sys. 5:69, (1988); Lee, V.H.L., J. ControlledRelease 13:2\3, (1990); Lee, V.H.L., Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, A.G., et al., J. Controlled Release /3:241, (1990). For example, STDHF is a synthetic dérivative of fusidic acid, a stéroïdal surfactant

179 that is similar in structure to the bile salts, and has been used as a perméation enhancer for nasal delivery. (Lee, W.A., Biopharm. 22, Nov./Dec. 1990.)

The MASP-3 inhibitory antibodies as described herein may be introduced in association with another molécule, such as a lipid, to protect the polypeptides from enzymatic dégradation. For example, the covalent attachment of polymers, especially polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in the body and thus prolong half-life (Fuertges, F., et al., J. Controlled Release 77:139, (1990)). Many polymer Systems hâve been reported for protein delivery (Bae, Y.H., et al., J. Controlled Release 9:27\, (1989); Hori, R., et al., Pharm. Res.6:%\3, (1989); Yamakawa, I., et al., J. Pharm. Sci. 79:505, (1990); Yoshihiro, L, et al., J. ControlledRelease 70:195, (1989); Asano, M., étal., J. Controlled Release 9:111, (1989); Rosenblatt, J., étal., J. Controlled Release 9:\95, (1989); Makino, K., J. Controlled Release 72:235, (1990); Takakura, Y., et al., J. Pharm. Sci. 75:117, (1989); Takakura, Y., et al., J. Pharm. Sci. 75:219, (1989)).

Recently, liposomes hâve been developed with improved sérum stability and circulation half-times (see, e.g., U.S. Patent No. 5,741,516, to Webb). Furthermore, various methods of liposome and liposome-Iike préparations as potentia! drug carriers hâve been reviewed (see, e.g., U.S. Patent No. 5,567,434, to Szoka; U.S. Patent No. 5,552,157, to Yagi; U.S. Patent No. 5,565,213, to Nakamori; U.S. Patent No. 5,738,868, to Shinkarenko; and U.S. Patent No. 5,795,587, to Gao).

For transdermal applications, the MASP-3 inhibitory antibodies, as described herein, may be combined with other suitable ingrédients, such as carriers and/or adjuvants. There are no limitations on the nature of such other ingrédients, except that they must be pharmaceutically acceptable for their intended administration, and cannot dégradé the activity of the active ingrédients of the composition. Examples of suitable vehicles include ointments, creams, gels, or suspensions, with or without purified collagen. The MASP-3 inhibitory antibodies may also be impregnated into transdermal patches, plasters, and bandages, preferably in liquid or semi-liquid form.

The compositions of the présent invention may be systemically administered on a periodic basis at intervals determined to maintain a desired level of therapeutic effect. For example, compositions may be administered, such as by subcutaneous injection, every two to four weeks or at less frequent intervals. The dosage regimen will be determined by the physician considering various factors that may influence the action of the combination of

180 agents. These factors will include the extent of progress of the condition being treated, the patient's âge, sex and weight, and other clinical factors. The dosage for each individual agent will vary as a function of the MASP-3 inhibitory antibody or the MASP-2 inhibitory agent that is included in the composition, as well as the presence and nature of any drug delivery vehicle (e.g., a sustained release delivery vehicle). In addition, the dosage quantity may be adjusted to account for variation in the frequency of administration and the pharmacokinetic behavior of the delivered agent(s).

Local delivery

As used herein, the term local encompasses application of a drug in or around a site of intended localized action, and may include for example topical delivery to the skin or other affected tissues, ophthalmic delivery, intrathecal (IT), intracerebroventricular (ICV), intra-articular, intracavity, intracranial or intravesicular administration, placement or irrigation. Local administration may be preferred to enable administration of a lower dose, to avoid systemic side effects, and for more accurate control of the timing of delivery and concentration ofthe active agents at the site of local delivery. Local administration provides a known concentration at the target site, regardless of interpatient variability in metabolism, blood flow, etc. Improved dosage control is also provided by the direct mode of delivery.

Local delivery of a MASP-3 inhibitory antibody or a MASP-2 inhibitory agent may be achieved in the context of surgical methods for treating a disease or condition, such as for example during procedures such as arterial bypass surgery, atherectomy, laser procedures, ultrasonic procedures, balloon angioplasty and stent placement. For example, a MASP-3 inhibitory antibody or a MASP-2 inhibitory agent can be administered to a subject in conjunction with a balloon angioplasty procedure. A balloon angioplasty procedure involves inserting a cathéter having a deflated balloon into an artery. The deflated balloon is positioned in proximity to the atherosclerotic plaque and is inflated such that the plaque is compressed against the vascular wall. As a resuit, the balloon surface is in contact with the layer of vascular endothélial cells on the surface of the blood vessel. The MASP-3 inhibitory antibody or MASP-2 inhibitory agent may be attached to the balloon angioplasty cathéter in a manner that permits release of the agent at the site of the atherosclerotic plaque. The agent may be attached to the balloon cathéter in accordance with standard procedures known in the art. For example, the agent may be stored in a compartment of the balloon cathéter until the balloon is inflated, at which point it is released into the local environment. Altematively, the

181 agent may be impregnated on the balloon surface, such that it contacts the cells ofthe arterial wall as the balloon is inflated. The agent may also be delivered in a perforated balloon cathéter such as those disclosed in Flugelman, M.Y., étal., Circulation 55:1110-1117, (1992). See also published PCT Application WO 95/23161 for an exemplary procedure for attaching a therapeutic protein to a balloon angioplasty cathéter. Likewise, the MASP-3 inhibitory agent or MASP-2 inhibitory agent may be included in a gel or polymeric coating applied to a stent, or may be incorporated into the material of the stent, such that the stent elutes the MASP-3 inhibitory agent or MASP-2 inhibitory agent after vascular placement.

MASP-3 inhibitory antibodies used in the treatment of arthritides and other musculoskeletal disorders may be locally delivered by intra-articular injection. Such compositions may suitably include a sustained release delivery vehicle. As a further example of instances in which local delivery may be desired, MASP-3 inhibitory compositions used in the treatment of urogénital conditions may be suitably instilled intravesically or within another urogénital structure.

XX. TREATMENT REGIMENS

In prophylactic applications, the pharmaceutical compositions are administered to a subject susceptible to, or otherwise at risk of, an alternative pathway associated disease or disorder, for example, an alternative pathway disease or disorder selected from the group consisting of paroxysmal noctumal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS) and thrombotic thrombocytopénie purpura (TTP)), asthma, dense deposit disease, pauci-immune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behçet’s disease, multiple sclerosis, Guillain Barre Syndrome, Alzheimer’s disease, Amylotrophic latéral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy, Uveitis, Chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD), hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis, Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy and Myasthenia Gravis., in an amount sufficient to eliminate or reduce the risk of developing

182 symptoms of the condition. In therapeutic applications, the pharmaceutical compositions are administered to a subject suspected of, or already suffering from, an alternative pathwayrelated disease or disorder, such as an alternative pathway disease or disorder selected from the group consisting of paroxysmal noctumal hemoglobinuria (PNH), age-related macular 5 degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS) or thrombotic thrombocytopénie purpura (TTP)), asthma, dense deposit disease, pauci-immune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, 10 neuromyelitis optica, Behcet’s disease, multiple sclerosis, Guillain Barre Syndrome, Alzheimer’s disease, Amylotrophic latéral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy, Uveitis, Chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD), hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS), Acute Respiratory 15 Distress Syndrome (ARDS), ANCA vasculitis, Anti-phospholipîd syndrome, Atherosclerosis, IgA Nephropathy and Myasthenia Gravis, in a therapeutically effective amount sufficient to relieve, or at least partially reduce, the symptoms of the condition.

In one embodiment, the pharmaceutical composition comprising a high affïnity MASP-3 inhibitory antibody is administered to a subject suffering from, or at risk for 20 developing PNH. In accordance with this the subject's red blood cells are opsonized by fragments of C3 in the absence of the composition, and administration of the composition to the subject increases the survival of red blood cells in the subject. In one embodiment, the subject exhibits one or more symptoms in the absence of the composition selected from the group consisting of (i) below normal levels of hemoglobin, (ii) below normal levels of 25 platelets; (iii) above normal levels of réticulocytes, and (iv) above normal levels of bilirubin, and administration of the composition to the subject improves at least one or more of the symptoms, resulting in (i) increased, normal, or nearly normal levels of hemoglobin (ii) increased, normal or nearly normal levels of platelets, (iii) decreased, normal or nearly normal levels of réticulocytes, and/or (iv) decreased, normal or nearly normal levels of 30 bilirubin.

In both prophylactic and therapeutic regimens for the treatment, prévention or réduction in severity of a disease or condition selected from the group consisting of

183 paroxysmal noctumal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS) or thrombotic thrombocytopénie purpura (TTP)), asthma, dense deposit disease, pauci-immune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonîa, endophthalmitis, neuromyelitis optica and Behcet’s disease, compositions comprising high affinity MASP-3 inhibitory antibodies and optionally MASP-2 inhibitory agents may be administered in several dosages until a sufficient therapeutic outcome has been achieved in the subject. In one embodiment of the invention, the high affinity MASP-3 inhibitory antibody and/or MASP-2 inhibitory agent may be administered to an adult patient (e.g., an average adult weight of 70 kg) in a dosage of from 0.1 mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, more suitably 10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg and still more suitably from 50.0 mg to 500 mg, or 10 to 200 mg. For pédiatrie patients, dosage can be adjusted in proportion to the patient’s weight.

Application of the high affinity MASP-3 inhibitory antibodies and optional MASP-2 inhibitory compositions of the présent invention may be carried out by a single administration of the composition (e.g., a single composition comprising MASP-3 and optionally MASP-2 inhibitory agents, or bispecific or dual inhibitory agents, or co-administration of separate compositions), or a limited sequence of administrations, for treatment of an alternative pathway-related disease or disorder, such as a disease or disorder selected form the group consisting of paroxysmal noctumal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion înjury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS) or thrombotic thrombocytopénie purpura (TTP)), asthma, dense deposit disease, pauci-immune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonîa, endophthalmitis, neuromyelitis optica, Behcet’s disease, multiple sclerosis, Guillaîn Barre Syndrome, Alzheimer’s disease, Amylotrophic latéral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy, Uveitis, Chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD), hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS),

184

ANCA vasculitis, Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy and Myasthenia Gravis.

Alternatively, the composition may be administered at periodic intervals such as daily, biweekly, weekly, every other week, monthly or bimonthly over an extended period of time for as determined by a physician for optimal therapeutic effect.

In some embodiments, a first composition comprising at least one high affinity MASP-3 inhibitory antibody and a second composition comprising at least one MASP-2 inhibitory agent are administered to a subject suffering from, or at risk for developing a disease or condition selected from the group consisting of paroxysmal noctumal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemoiytic urémie syndrome (HUS), atypical hemoiytic urémie syndrome (aHUS) or thrombotic thrombocytopénie purpura (TTP)), asthma, dense deposit disease, pauciimmune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behcet’s disease, multiple sclerosis, Guillain Barre Syndrome, Alzheimer’s disease, Amylotrophic latéral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy, Uveitis, Chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, Graftversus-host disease (GVHD), hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis, Antiphospholipid syndrome, Atherosclerosis, IgA Nephropathy and Myasthenia Gravis.

In one embodiment, the first composition comprising at least one high affinity MASP3 inhibitory antibody and a second composition comprising at least one MASP-2 inhibitory agent are administered simultaneously (i.e., within a time séparation of no more than about 15 minutes or less, such as no more than any of 10, 5 or 1 minute). In one embodiment, the first composition comprising at least one high affinity MASP-3 inhibitory antibody and a second composition comprising at least one MASP-2 inhibitory agent are administered sequentially (i.e., the first composition is administered either prior to or after the administration of the second composition, wherein the time séparation of administration is more than 15 minutes). In some embodiments, the first composition comprising at least one high affinity MASP-3 inhibitory antibody and a second composition comprising at least one MASP-2 inhibitory agent are administered concurrently (i.e., the administration period ofthe

185 first composition overlaps with the administration of the second composition). For example, in some embodiments, the first composition and/or the second composition are administered for a period of at least one, two, three or four weeks or longer. In one embodiment, at least one high affinity MASP-3 inhibitory antibody and at least one MASP-2 inhibitory agent are combined in a unit dosage form. In one embodiment, a first composition comprising at least one high affinity MASP-3 inhibitory antibody and a second composition comprising at least one MASP-2 inhibitory agent are packaged together in a kit for use in treatment of an alternative pathway-related disease or condition, such as paroxysmal noctumal hemoglobinuria (PNH), age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS) or thrombotic thrombocytopénie purpura (TTP)), asthma, dense deposit disease, pauciimmune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behcet’s disease, multiple sclerosis, Guillain Barre Syndrome, Alzheimer’s disease, Amylotrophic latéral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy, Uveitis, Chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, Graftversus-host disease (GVHD), hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis, Antiphospholipid syndrome, Atherosclerosis, IgA Nephropathy or Myasthenia Gravis.

In some embodiments, the subject suffering from PNH, age-related macular degeneration (AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS) or thrombotic thrombocytopénie purpura (TTP)), asthma, dense deposit disease, pauci-immune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behcet’s disease, multiple sclerosis, Guillain Barre Syndrome, Alzheimer’s disease, Amylotrophic latéral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy, Uveitis, Chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD), hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis, Anti-phospholipid syndrome, Atherosclerosis,

186

IgA Nephropathy and Myasthenia Gravis has previously undergone, or is currently undergoing treatment with a terminal complément inhibitor that inhibits cleavage of complément protein C5. In some embodiments, the method comprises administering to the subject a composition of the invention comprising a high affinity MASP-3 inhibitory 5 antibody and optionally a MASP-2 inhibitor and further administering to the subject a terminal complément inhibitor that inhibits cleavage of complément protein C5. In some embodiments, the terminal complément inhibitor is a humanized anti-C5 antibody or antigenbinding fragment thereof. In some embodiments, the terminal complément inhibitor is eculizumab.

XXI. EXAMPLES

The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention. Ail literature citations herein are expressly incorporated by reference.

EXAMPLE 1

This Example demonstrates that MASP-2 déficient mice are protected from Neisseria meningitidis induced mortality after infection with either N. meningitidis serogroup A or N. meningitidis serogroup B.

Methods:

MASP-2 knockout mice (MASP-2 KO mice) were generated as described in Example 1 of US 7,919,094, hereby incorporated herein by reference. 10-week-old MASP-2 KO mice (n=10) and wild-type (WT) C57/BL6 mice (n=10) were inoculated by intraperitoneal (i.p.) injection with a dosage of 2.6 x 10? CFU of N. meningitidis serogroup A Z2491 in a volume 25 of 100 μΙ. The infective dose was administered to mice in conjunction with iron dextran at a final concentration of 400 mg/kg. Survival of the mice after infection was monitored over a 72-hour time period.

In a separate experiment, 10-week-old MASP-2 KO mice (n=10) and WT C57/BL6 mice (n=10) were inoculated by i.p. injection with a dosage of 6 x 10⁶ CFU of N. 30 meningitidis serogroup B strain MC58 in a volume of 100 pL. The infective dose was administered to mice in conjunction with iron dextran at a final dose of 400 mg/kg. Survival

187 of the mice after infection was monitored over a 72-hour time period. An illness score was also determined for the WT and MASP-2 KO mice during the 72-hour time period after infection, based on the illness scoring parameters described below in TABLE 5, which is based on the scheme of Fransen et al. (2010) with slight modifications.

TABLE 5: Illness Scoring associated with clinical signs in infected mice

Signs	Score
Normal	0
Slightly ruffled fur	1
Ruffled fur, slow and sticky eyes	2
Ruffled fur, léthargie and eyes shut	3
Very sick and no movement after stimulation	4
Dead	5

Blood samples were taken from the mice at hourly intervals after infection and analyzed to détermine the sérum level (log cfu/mL) of N. meningitidis in order to verify infection and détermine the rate of clearance of the bacteria from the sérum.

Results:

FIGURE 6 is a Kaplan-Meyer plot graphically illustrating the percent survival of MASP-2 KO and WT mice after administration of an infective dose of 2.6 x 10⁷ cfu of N. meningitidis serogroup A Z2491. As shown in FIGURE 6, 100% of the MASP-2 KO mice survived throughout the 72-hour period after infection. In contrast, only 80% of the WT mice (p=0.012) were still alive 24 hours after infection, and only 50% of the WT mice were still alive at 72 hours after infection. These results demonstrate that MASP-2-deficient mice are protected from N. meningitidis serogroup A Z2491-induced mortality. '

FIGURE 7 is a Kaplan-Meyer plot graphically illustrating the percent survival of MASP-2 KO and WT mice after administration of an infective dose of 6 x 10⁶ cfu of N. meningitidis serogroup B strain MC58. As shown in FIGURE 7, 90% of the MASP-2 KO mice survived throughout the 72-hour period after infection. In contrast, only 20% of the WT mice (p=0.0022) were still alive 24 hours after infection. These results demonstrate that MASP-2-deficient mice are protected from N. meningitidis serogroup B strain MC58-induced mortality.

FIGURE 8 graphically illustrâtes the log cfu/mL of N. meningitidis serogroup B strain MC58 recovered at different time points in blood samples taken from the MASP-2 KO and WT mice after i.p. infection with 6x10⁶ cfu of N. meningitidis serogroup B strain MC58

188 (n=3 at different time points for both groups of mice). The results are expressed as Means±SEM. As shown in FIGURE 8, in WT mice the level of N. meningitidis in the blood reached a peak of about 6.0 log cfu/mL at 24 hours after infection and dropped to about 4.0 log cfu/mL by 36 hours after infection. In contrast, in the MASP-2 KO mice, the level of N. 5 meningitidis reached a peak of about 4.0 log cfu/mL at 12 hours after infection and dropped to about 1.0 log cfu/mL by 36 hours after infection (the symbol * indicates p<0.05; the symbol ** indicates p=0.0043). These results demonstrate that although the MASP-2 KO mice were infected with the same dose of N. meningitidis serogroup B strain MC58 as the WT mice, the MASP-2 KO mice hâve enhanced clearance of bacteraemia as compared to 10 WT.

FIGURE 9 graphically illustrâtes the average illness score of MASP-2 KO and WT mice at 3, 6, 12 and 24 hours after infection with 6x10⁶ cfu of N. meningitidis serogroup B strain MC58. As shown in FIGURE 9, the MASP-2-defïcient mice showed high résistance to the infection, with much lower illness scores at 6 hours (symbol * indicates p=0.0411), 15 12 hours (symbol ** indicates p=0.0049) and 24 hours (symbol *** indicates p=0.0049) after infection, as compared to WT mice. The results in FIGURE 9 are expressed as means±SEM.

In summary, the results in this Example demonstrate that MASP-2-deficient mice are protected from N. meningitides-induced mortality after infection with either N. meningitidis 20 serogroup A or N. meningitidis serogroup B.

EXAMPLE 2

This Example demonstrates that the administration of MASP-2 antibody after infection with N. meningitidis increases the survival of mice infected with N. meningitidis.

Background/Rationale:

As described in Example 24 of US Patent 7,919,094, incorporated herein by reference, rat MASP-2 protein was utilized to pan a Fab phage display library, from which Fab2 #11 was identified as a functionally active antibody. Full-Iength antibodies of the rat IgG2c and mouse IgG2a isotypes were generated from Fab2 #11. The full-Iength MASP-2 30 antibody ofthe mouse IgG2a isotype was characterized for pharmacodynamie parameters (as described in Example 38 of US Patent 7,919,094).

189

In this Exampie, the mouse MASP-2 full-length antibody derived from Fab2 #11 was analyzed in the mouse model ofAL meningitidis infection.

Methods:

The mouse IgG2a full-length MASP-2 antibody isotype derived from Fab2 #11, generated as described above, was tested in the mouse model of N. meningitidis infection as follows.

1. Administration of ntouse-MASP-2 nionoclonal antibodies (MoAb) after infection 9-week-old C57/BL6 Charles River mice were treated with inhibitory mouse MASP-2 antibody (1.0 mg/kg) (n=12) or control isotype antibody (n=10) at 3 hours after i.p. injection with a high dose (4x10⁶ cfu) ofN. meningitidis serogroup B strain MC58.

Results:

FIGURE 10 is a Kaplan-Meyer plot graphically îllustrating the percent survival of mice after administration of an infective dose of 4x10⁶ cfu of N. meningitidis serogroup B strain MC58, followed by administration 3 hours post-infection of either inhibitory MASP-2 antibody (1.0 mg/kg) or control isotype antibody. As shown in FIGURE 10, 90% of the mice treated with MASP-2 antibody survived throughout the 72-hour period after infection. In contrast, only 50% of the mice treated with isotype control antibody survived throughout the 72-hour period after infection. The symbol * indicates p=0.0301, as determined by comparison of the two survival curves.

These results demonstrate that administration of a MASP-2 antibody is effective to treat and improve survival in subjects infected with N. meningitidis.

As demonstrated herein, the use of MASP-2 antibody in the treatment of a subject infected with N. meningitidis is effective when administered within 3 hours post-infection, and is expected to be effective within 24 hours to 48 hours after infection. Meningococcal disease (either meningococcemia or meningitis) is a medical emergency, and therapy will typically be initiated immediately if meningococcal disease is suspected (i.e., before N. meningitidis is positively identified as the etiological agent).

In view of the results in the MASP-2 KO mouse demonstrated in EXAMPLE 1, it is believed that administration of MASP-2 antibody prior to infection with N. meningitidis would also be effective to prevent or ameliorate the severity of infection.

190

EXAMPLE 3

This Example demonstrates the complement-dependent killing of N. meningitidis in human sera is MASP-3-dependent.

Rationale:

Patients with decreased sérum levels of functional MBL display increased susceptibility to récurrent bacterial and fungal infections (Kilpatrick et al., Biochim Biophys Acta 1572:401-413 (2002)). It is known that N. meningitidis is recognized by MBL, and it has been shown that MBL-deficient sera do not lyse N. meningitidis.

In view of the results described in Examples 1 and 2, a sériés of experiments were carried out to détermine the efficacy of administration of MASP-2 antibody to treat N. meningitidis infection in complement-deficient and control human sera. Experiments were carried out in a high concentration of sérum (20%) in .order to preserve the complément pathway.

Methods:

1. Sérum bactericidal activity in various complement-deficient human sera and in human sera treated with human MASP-2 antibody

The following complement-deficient human sera and control human sera were used in this experiment:

TABLE 6: Human sérum samples tested (as shown in FIGURE 11)

Sample	Sérum type
A	Normal human sera (NHS) + human MASP-2 Ab
B	NHS + isotype control Ab
C	MBL -/- human sérum
D	NHS
E	Heat-Inactivated (HI) NHS

A recombinant antibody against human MASP-2 was isolated from a combinatorial Antibody Library (Knappik, A., et al., J. Mol. Biol. 296:51-^6 (2000)), using recombinant human MASP-2A as an antigen (Chen, C.B. and Wallis, J. Biol. Chem. 276:25894-25902 (2001)). An anti-human scFv fragment that potently inhibited lectin pathway-mediated activation of C4 and C3 in human plasma (ICso~2O nM) was identified and converted to a full-length human IgG4 antibody.

191

N. meningitidis serogroup B-MC58 was incubated with the different sera show in TABLE 6, each at a sérum concentration of 20%, with or without the addition of inhibitory human MASP-2 antibody (3 pg in 100 μΐ total volume) at 37°C with shaking. Samples were taken at the following time points: 0-, 30-, 60- and 90-minute intervals, plated out and then viable counts were determined. Heat-inactivated human sérum was used as a négative control.

Results:

FIGURE 11 graphically illustrâtes the log cfu/mL of viable counts of N. meningitidis serogroup B-MC58 recovered at different time points in the human sera samples shown in 10 TABLE 6. TABLE 7 provides the Student’s t-test results for FIGURE 11.

TABLE 7: Student's t-test Results for FIGURE 11 (time point 60 minutes)

	Mean Diff. (Log)	Significant? P<0.05?	P value summary
A vs B	-0.3678	Yes	***(0.0002)
A vs C	-1.1053	Yes	***(p<0.0001)
A vs D	-0.2111	Yes	**(0.0012)
C vs D	1.9	Yes	***(p<0.0001)

As shown in FIGURE 11 and TABLE 7, complement-dependent killing of N. 15 meningitidis in human 20% sérum was significantly enhanced by the addition of the human MASP-2 inhibitory antibody.

2. Sérum bactericidal activity in various complement-deficient human sera

The following complement-deficient human sera and control human sera were used in 20 this experiment:

TABLE 8: Human sérum samples tested (as shown in FIGURE 12'

Sample	Sérum Type
A	Normal human sérum (NHS)
B	Heat-inactivated NHS
C	MBL -/-

192

D	MASP-3 -/- (MASP-1 +)

Note: The MASP-3 -/- (MASP-1 +) sérum in sample D was taken from a subject with 3MC syndrome, which is a unifying term for the overlapping Camevale, Mingarelli, Malpuech and Michels syndromes. As further described in Example 4, the mutations in exon 12 of the MASP-1/3 gene render the serine protease domain of MASP-3, but not MASP-1 dysfunctional. As described in Example 10, pro-factor D is preferentially présent in 3MC sérum, whereas activated factor D is preferentially présent in normal human sérum.

N. meningitidis serogroup B-MC58 was incubated with different complementdeficient human sera, each at a sérum concentration of 20%, at 37°C with shaking. Samples were taken at the following time points: 0-, 15-, 30-, 45-, 60-, 90- and 120-minute intervals, plated out and then viable counts were determined. Heat-inactivated human sérum was used as a négative control.

Results:

FIGURE 12 graphically illustrâtes the log cfu/mL of viable counts of N. meningitidis serogroup B-MC58 recovered at different time points in the human sera samples shown in TABLE 8. As shown in FIGURE 12, the WT (NHS) sérum has the highest level of bactericidal activity for N. meningitidis. In contrast, the MBL -/- and MASP-3 -/- (which is MASP-1-sufficient) human sera do not hâve àny bactericidal activity. These results indicate that complement-dependent killing ofN. meningitidis in human 20% (v/v) sérum is MASP-3and MBL-dependent. TABLE 9 provides the Student’s t-test results for FIGURE 12.

TABLE 9: Student’s t-test Results for FIGURE 12

Comparison	Time Point (min)	Mean Diff. (Log)	Significant? P<0.05?	P value Summary
A vs B	60	-0.8325	Yes	***(p<0.0001)
A vs B	90	-1.600	Yes	***(p<0.0001)
A vs C	60	-1.1489	Yes	***(p<0.0001)
A vs C	90	-1.822	Yes	***(p<0.0001)
A vs D	60	-1.323	Yes	***(0.0005)
A vs D	90	-2.185	Yes	***(p<0.0001)

193

In summary, the results shown in FIGURE 12 and TABLE 9 demonstrate that complement-dependent killing of N. meningilidis in 20% human sérum is MASP-3- and MBL-dependent.

3. Complement-dependent killing of N. meningilidis in 20% (v/v) mouse sera déficient of MASP-2, MASP-1/3 or MBL A/C.

The following complement-deficient mouse sera and control mouse sera were used in this experiment:

TABLE 10: Mouse sérum samples tested (as shown in FIGURE 13)

Sample	Sérum Type
A	WT
B	MASP-2 -/-
C	MASP-1/3 -/-
D	MBL A/C -/-
E	WT heat-inactivated (HIS)

N. meningilidis serogroup B-MC58 was incubated with different complementdeficient mouse sera, each at a sérum concentration of 20%, at 37°C with shaking. Samples were taken at the following time points: 0-, 15-, 30-, 60-, 90- and 120-minute intervals, plated out and then viable counts were determined. Heat-inactivated human sérum was used as a négative control.

Results:

FIGURE 13 graphically illustrâtes the log cfu/mL of viable counts of N. meningilidis serogroup B-MC58 recovered at different time points in the mouse sérum samples shown in TABLE 10. As shown in FIGURE 13, the MASP-2 -/- mouse sera hâve a higher level of bactericidal activity for N. meningilidis than WT mouse sera. In contrast, the MASP-1/3 -/mouse sera do not hâve any bactericidal activity. The symbol ** indicates p=0.0058, the symbol *** indicates p=0.001. TABLE 11 provides the Studenfs t-test results for FIGURE 13.

TABLE 11: Student's t-test Results for FIGURE 13

Comparison

Time point

Mean Diff.

Significant?

P value summary

194

		(LOG)	(p<0.05)?
A vs. B	60 min.	0.39	yes	** (0.0058)
A vs. B	90 min.	0.6741	yes	*** (0.001)

In summary, the results in this Example demonstrate that MASP-2 -/- sérum has a higher level of bactericidal activity for Ai meningitidis than WT sérum and that complementdependent killing ofA/ meningitidis in 20% sérum is MASP-3- and MBL-dependent.

EXAMPLE 4

This Example describes a sériés of experiments that were carried out to détermine the mechanism of the MASP-3-dependent résistance to N. meningitidis infection observed in MASP-2 KO mice, as described in Examples 1-3.

Rationale:

In order to détermine the mechanism of MASP-3-dependent résistance to N. meningitidis infection observed in MASP-2 KO mice (described in Examples 1-3 above), a sériés of experiments were carried out as follows.

1. MASP-l/3-deflcient mice are not déficient of lectin pathway functional activity (also referred to as “LEA-2”)

Methods:

In order to détermine whether MASP-l/3-deficient mice are déficient of lectin pathway functional activity (also referred to as LEA-2), an assay was carried out to measure the kinetics of C3 convertase activity in plasma from various complement-deficient mouse strains tested under lectin activation pathway-specific assay conditions (1% plasma), as described in Schwaeble W. et al., PNAS vol 108(18):7523-7528 (2011), hereby incorporated herein by reference.

Plasma was tested from WT, C4-/-, MASP-1/3-/-; Factor B-/-, and MASP-2-/- mice as follows.

To measure C3 activation, microtiter plates were coated with mannan (I pg/well), zymosan (1 pg/well) in coating buffer (15 mM NaiCoj, 35 mM NaHCCh), or immune complexes, generated in situ by coating with 1% human sérum albumin (HSA) in coating

195 buffer then adding sheep anti-HAS sérum (2 pg/mL) in TBS (lOmM Tris, 140 mM NaCl, pH 7.4) with 0.05% Tween 20 and 5 mM CaF Plates were blocked with 0.1% HSA in TBS and washed three times with TBS/Tween20/Ca⁺⁺. Plasma samples were diluted in 4 mM barbital, 145 mM NaCl, 2 mM CaCh, 1 mM MgCb, pH 7.4, added to the plates and incubated for 1.5 5 h at 37°C. After washing, bound C3b was detected using rabbit anti-human C3c (Dako), followed by alkaline phosphatase-conjugated goat anti-rabbit IgG and p-nitrophenyl phosphate.

. Results: .

The kinetics of C3 activation (as measured by C3b déposition on mannan-coated 10 plates with 1% sérum) under lectin pathway-specific conditions is shown in FIGURE 14. No C3 cleavage was seen in MASP-2-/- plasma. Factor B-/- (Factor B -/-) plasma cleaved C3 at half the rate of WT plasma, likely due to the loss of the amplification loop. A significant delay in the lectin pathway-dependent conversion ofC3 to C3b was seen in C4-/(Ti/2=33min) as well as in MASP-1/3-/- déficient plasma (Ti/2=49 min). This delay of C3 15 activation in MASP-1/3-/- plasma has been shown to be MASP-1- rather than MASP-3dependent. (See Takahashi M. et al., J Immunol 180:6132-6138 (2008)). These results demonstrate that MASP-l/3-deficient mice are not déficient of lectin pathway functional activity (also referred to as “LEA-2”).

2. Effect ofhereditary MASP-3 deficiency on alternativepathway activation.

Rationale:

The effect of hereditary MASP-3 deficiency on alternative pathway activation was determined by testing sérum of a MASP-3-defïcient patient with 3MC syndrome caused by a frame-shift mutation in the exon encoding the serine protease of MASP-3. The 3MC syndrome is a unifying term for the overlapping Cameavale, Mingarelli, Malpuech and 25 Michels syndromes. These rare autosomal récessive disorders exhibit a spectrum of developmental features, including characteristic facial dysmorphism, cleft lip and/or palate, craniosynostosis, leaming disability and génital, limb and vesicorenal abnormalities. Rooryck et al.. Nature Genetics 43:197-203 (2011) studied 11 families with 3MC syndrome and identified two mutated genes, COLEC11 and MASP-1. The mutations in the MASP-1 30 gene render the exon encoding the serine protease domain of MASP-3, but not the exons encoding the serine protease of MASP-1, dysfunctional. Therefore, 3MC patients with

196 mutations in the exon encoding the serine protease of MASP-3 are déficient of MASP-3 but sufficient in MASP-1.

Methods:

MASP-3-deficient sérum was obtained from a 3MC patient, the mother and father of the 3MC patient (both heterozygous for the allele bearing a mutation that renders the exon encoding the MASP-3 serine protease domain dysfunctional), as well as from a C4-deficient patient (déficient in both human C4 genes) and an MBL-deficient subject. An alternative pathway assay was carried out under traditional AP-specific conditions (BBS/ Mg^/EGTA, without Ca⁴/ wherein BBS = barbital buffered saline containing sucrose), as described in Bitter-Suermann et al., Eur. J. Immunol 11:291-295 (1981)), on zymosan-coated microtiter plates at sérum concentrations ranging from 0.5 to 25% and C3b déposition was measured over time.

Results:

FIGURE 15 graphically illustrâtes the level of alternative pathway-driven C3b déposition on zymosan-coated microtiter plates as a function of sérum concentration in sérum samples obtained from MASP-3-deficient, C4-deficient and MBL-deficient subjects. As shown in FIGURE 15, MASP-3-deficient patient sérum has residual alternative pathway (AP) activity at high sérum concentrations (25%, 12.5%, 6.25% sérum concentrations), but a significantly higher AP50 (i.e., 9.8% of sérum needed to achieve 50% of maximum C3 déposition).

FIGURE 16 graphically illustrâtes the level of alternative pathway-driven C3b déposition on zymosan-coated microtiter plates under “traditional” alternative pathwayspecific (AP-specific) conditions (i.e., BBS/EGTA/Mg⁺⁺ without Ca⁺⁺) as a function of time in 10% human sérum samples obtained from MASP-3-deficient, C4-deficient and MBLdeficient human subjects.

TABLE 12 below summarizes the AP50 results shown in FIGURE 15 and the halftimes for C3b déposition shown in FIGURE 16.

TABLE 12: Summary of Results shown in FIGURES 15 and 16

Sérum type	AP50 (%)	T1/2 (min)
MASP-3-deficient (3MC patient)	9.8	37.4

197

Mother of 3MC patient (heterozygous)	4.3	17.2
Father of 3MC patient (heterozygous)	4.3	20.9
C4-deficient	4.0	11.6
MBL-deficient	4.8	11.0

Note: In BBS/ Mg^/EGTA buffer, the lectin pathway-mediated effects are déficient due to absence of Ca^ in this buffer.

In summary, under the conditions of these assays, the alternative pathway is 5 significantly compromised in the 3MC patient.

3. Measurement of C3b déposition on mannan, zymosan and S. pneumonia D39 in mouse sera déficient of MASP-2 or MASP-1/3.

Methods:

C3b déposition was measured on mannan, zymosan and S. pneumonia D39-coated microtiter plates using mouse sérum concentrations ranging from 0% to 20% obtained from MASP-2-/-, MASP-1/3-/- and WT mice. The C3b déposition assays were carried out under either “traditional” alternative pathway-specific conditions (i.e. BBS/EGTA/Mg⁺⁺ without Ca©, or under physiological conditions allowing both the lectin pathway and the alternative 15 pathway to function (i.e., BBS/Mg⁺⁺/Ca⁺⁺).

Results:

FIGURE 17A graphically illustrâtes the level of C3b déposition on mannan-coated microtiter plates as a function of sérum concentration in sérum samples obtained from WT, MASP-2-deficient, and MASP-l/3-deficient mice under traditional alternative pathway20 spécifie conditions (i.e., BBS/EGTA/Mg^ without Ca©, or under physiological conditions allowing both the lectin pathway and the alternative pathway to function (BBS/Mg^/Ca©. FIGURE 17B graphically illustrâtes the level of C3b déposition on zymosan-coated microtiter plates as a function of sérum concentration in sérum samples from WT, MASP-2deficient, and MASP-l/3-deficient mice under traditional AP-specific conditions (i.e., 25 BBS/EGTA/Mg⁺⁺ without Ca©, or under physiological conditions allowing both the lectin pathway and the alternative pathway to function (BBS/Mg^/Ca©. FIGURE 17C graphically illustrâtes the level of C3b déposition on S. pneumoniae D39-coated microtiter

198 plates as a function of sérum concentration in sérum samples from WT, MASP-2-deficient, and MASP-1 /3-deficient mice under traditional AP-specific conditions (i.e., BBS/EGTA/Mg⁺⁺ without Ca⁺⁺), or under physiological conditions allowing both the lectin pathway and the alternative pathway to function (BBS/Mg⁺⁺/Ca⁺⁺).

FIGURE 18A graphically illustrâtes the results of a C3b déposition assay in highly diluted sera carried out on mannan-coated microtiter plates under traditional AP-specific conditions (i.e. BBS/EGTA/Mg⁺⁺ without Ca⁺⁺) or under physiological conditions allowing both the lectin pathway and the alternative pathway to function (BBS/Mg⁺⁺/Ca⁺⁺), using sérum concentrations ranging from 0 % up to 1.25%. FIGURE 18B graphically illustrâtes the results of a C3b déposition assay carried out on zymosan-coated microtiter plates under traditional AP-specific conditions (i.e. BBS/EGTA/Mg^4-1· without Ca^{4 1}·) or under physiological conditions allowing both the lectin pathway and the alternative pathway to function (BBS/EGTA/Mg⁺⁺/Ca⁺⁺), using sérum concentrations ranging from 0 % up to 1.25%. FIGURE 18C graphically illustrâtes the results of a C3b déposition assay carried out on S. pneumoniae D39-coated microtiter plates under traditional AP-specific conditions (i.e. BBS/EGTA/Mg⁺⁺ without Ca^{4 1-}) or under physiological conditions allowing both the lectin pathway and the alternative pathway to function (BBS/EGTA/Mg⁺⁺/Ca⁺⁺), using sérum concentrations ranging from 0 % up to 1.25%.

As shown in FIGURES 18A-C, C3b déposition assays were also carried out under traditional alternative pathway-specific conditions (i.e. BBS/EGTA/Mg^4-1· without Ca^{4 1-}) or under physiological conditions allowing both the lectin pathway and the alternative pathway to function (BBS/Mg⁺⁺/Ca⁺⁺), using higher dilutions ranging from 0 % up to 1.25% sérum on mannan-coated plates (FIGURE 18A); zymosan-coated plates (FIGURE 18B) and S. pneumoniae D39-coated plates (FIGURE 18C). The alternative pathway tails off under higher sérum dilutions, so the activity observed in the MASP-1/3-deficient sérum in the presence of Ca^4-1· is MASP-2-mediated LP activity, and the activity in MASP-2-deficient sérum in the presence of Ca^{4 1-} is MASP-I/3-mediated residual activation of the AP.

Discussion:

The results described in this Example demonstrate that a MASP-2 inhibitor (or MASP-2 KO) provides significant protection from N. meningitidis infection by promoting MASP-3-driven alternative pathway activation. The results of the mouse sérum bacteriolysis assays and the human sérum bacteriolysis assays further show, by monitoring the sérum

199 bactericîdal activity against N. meningitidis, that bactericidal activity against N. meningitidis is absent in MBL-deficient (mouse MBL A and MBL C double-deficient and human MBLdeficient sera).

FIGURE 1 illustrâtes the new understanding of the lectin pathway and alternative pathway based on the results provided herein. FIGURE 1 delineates the rôle of LEA-2 in both opsonization and lysis. While MASP-2 is the initiator of “downstream” C3b déposition (and résultant opsonization) in multiple lectin-dependent settings physiologically (FIGURE 18A, 18B, 18C), it also plays a rôle in lysis of serum-sensitive bacteria. As illustrated in FIGURE 1, the proposed molecular mechanism responsible for the increased bactericidal activity of MASP-2-deficient or MASP-2-depleted serum/plasma for serum-sensitive pathogens such as N. meningitidis is that, for the lysis of bacteria, lectin pathway récognition complexes associated with MASP-1 and MASP-3 hâve to bind in close proximity to each other on the bacterial surface, thereby allowing MASP-1 to cleave MASP-3. In contrast to MASP-1 and MASP-2, MASP-3 is not an auto-activating enzyme, but, in many instances, requires activation/cleavage by MASP-1 to be converted into its enzymatically active form.

As further shown in FIGURE 1, activated MASP-3 can then cleave C3b-bound factor B on the pathogen surface to initiate the alternative pathway activation cascade by formation of the enzymatically active alternative pathway C3 and C5 convertase C3bBb and C3bBb(C3b)n, respectively. MASP-2-bearing lectin-pathway activation complexes hâve no part in the activation of MASP-3 and, in the absence or after déplétion of MASP-2, ail-1 ectin pathway activation complexes will either be loaded with MASP-1 or MASP-3. Therefore, in the absence of MASP-2, the likelihood is markedly increased that on the microbial surface MASP-1 and MASP-3-bearing lectin-pathway activation complexes will corne to sit in close proximity to each other, leading to more MASP-3 being activated and thereby leading to a higher rate of MASP-3-mediated cleavage of C3b-bound factor B to form the alternative pathway C3 and C5 convertases C3bBb and C3bBb(C3b)n on the microbial surface. This leads to the activation of the terminal activation cascades C5b-C9 that forms the Membrane Attack Complex, composed of surface-bound C5b associated with C6, C5bC6 associated with C7, C5bC6C7 associated with C8, and C5bC6C7C8, leading to the polymerization of C9 that inserts into the bacterial surface structure and forms a pore in the bacterial wall, which will lead to osmolytic killing ofthe complement-targeted bacterium.

200

The core of this novel concept is that the data provided herein clearly show that the lectin-pathway activation complexes drive the two distinct activation routes, as illustrated in FIGURE 1.

201

EXAMPLE 5

This Example demonstrates the inhibitory effect of MASP-2 deficiency and/or MASP-3 deficiency on lysis of red blood cells from blood samples obtained from a mouse model of paroxysmal noctumal hemoglobinuria (PNH).

Background/Rationale:

Paroxysmal noctumal hemoglobinuria (PNH), also referred to as MarchiafavaMicheli syndrome, is an acquired, potentially life-threatening disease of the blood, characterized by complement-induced intravascular hemolytic anémia. The hallmark of PNH is the chronic complement-mediated intravascular hemolysis that is a conséquence of 10 unregulated activation of the alternative pathway of complément due to the absence of the complément regulators CD55 and CD59 on PNH érythrocytes, with subséquent hemoglobinuria and anémia. Lindorfer, M.A., et al., Blood 175(11) (2010), Risitano, A.M, Mini-Reviews in Médicinal Chemistry, 11:528-535 (2011). Anémia in PNH is due to destruction of red blood cells in the bloodstream. Symptoms of PNH include red urine, due 15 to appearance of hemoglobin in the urine, back pain, fatigue, shortness of breath and thrombosis. PNH may develop on its own, referred to as primary PNH or in the context of other bone marrow disorders such as aplastic anémia, referred to as secondary PNH. Treatment for PNH includes blood transfusion for anémia, anticoagulation for thrombosis and the use of the monoclonal antibody eculizumab (Soliris®), which protects blood cells against 20 immune destruction by inhibiting the complément System (Hillmen P. et al., N. Engl. J. Med. 350(6):552-9 (2004)). Eculizumab (Soliris®) is a humanized monoclonal antibody that targets the complément component C5, blocking its cleavage by C5 convertases, thereby preventing the production of C5a and the assembly of MAC. Treatment of PNH patients with eculizumab has resulted in a réduction of intravascular hemolysis, as measured by lactate 25 dehydrogenase (LDH), leading to hemoglobin stabilization and transfusion independence in about half of the patients (Hillmen P, et al., Mini-Reviews in Médicinal Chemistry, vol 11(6) (2011)). While nearly ail patients undergoing therapy with eculizumab achieve normal or almost normal LDH levels (due to control of intravascular hemolysis), only about one third of the patients reach a hemoglobin value about 11 gr/dL, and the remaining patients on 30 eculizumab continue to exhibit moderate to severe (i.e., transfusion-dependent) anémia, in about equal proportions (Risitano A.M. et al., Blood 113:4094-100 (2009)). As described in Risitano et al., Mini-Reviews in Médicinal Chemistry 11:528-535 (2011), it was

202 .

demonstrated that PNH patients on eculizumab contained C3 fragments bound to a substantial portion of their PNH érythrocytes (while untreated patients did not), leading to the conclusion that membrane-bound C3 fragments work as opsonins on PNH érythrocytes, resulting in their entrapment in the réticuloendothélial cells through spécifie C3 receptors and subséquent extravascular hemolysis. Therefore, therapeutic strategies in addition to the use of eculizumab are needed for those patients developing C3 fragment-mediated extravascular hemolysis because they continue to require red cell transfusions.

This Example describes methods to assess the effect of MASP-2- and MASP-3deficient sérum on lysis of red blood cells from blood samples obtained from a mouse model of PNH and demonstrates the efficacy of MASP-2 inhibition and/or MASP-3 inhibition to treat subjects suffering from PNH, and also supports the use of inhibitors of MASP-2 and/or inhibitors of MASP-3 (including dual or bispecific MASP-2/MASP-3 inhibitors) to ameliorate the effects of C3 fragment-mediated extravascular hemolysis in PNH subjects undergoing therapy with a C5 inhibitor such as eculizumab.

Methods: PNH animal model:

Blood samples were obtained from gene-targeted mice with deficiencies of Crry and C3 (Crry/C3-/-) and CD55/CD59-deficient mice. These mice are missing the respective surface complément regulators on their érythrocytes and these érythrocytes are, therefore, susceptible to spontaneous complément autolysis as are PNH human blood cells.

In order to sensitize these érythrocytes even more, these cells were used with and without coating by mannan and then tested for hemolysis in WT C56/BL6 plasma, MBL null plasma, MASP-2 -/- plasma, MASP-1/3 -I- plasma, human NHS, human MBL -I- plasma, and NHS treated with human MASP-2 antibody.

203

L Hemolysis assay of Crry/C3 and CD55/CD59 double-defîcient murine érythrocytes in MASP-2-defîcient/depleted sera and Controls

Day 1. Préparation of murine RBC (± mannan coating).

Materials included: fresh mouse blood, BBS/Mg^++/ Ca⁺⁺ (4.4 mM barbituric acid, 1.8 5 mM sodium barbitone, 145 mM NaCl, pH 7.4, 5mM Mg**, 5mM Ca⁺⁺), chromium chloride, CrCh-ôHiO (0.5mg/mL in BBS/Mg⁺⁺/Ca⁺⁺) and mannan, 100 pg/mL in BBS/Mg⁺⁺/Ca⁺⁺.

Whole blood (2 mL) was spun down for 1-2 min at 2000xg in a refrigerated centrifuge at 4°C. The plasma and bufify coat were aspirated off. The sample was then washed 3x by re-suspending RBC pellet in 2 mL ice-cold BBS/gelatin/Mg⁺⁺/Ca⁺⁺ and 10 repeating centrifugation step. After the third wash, the pellet was re-suspended in 4 mL BBS/Mg⁺⁺/Ca⁺⁺. A 2 mL aliquot of the RBC was set aside as an uncoated control. To the remaining 2 mL, 2 mL CrC13 and 2 mL mannan were added and the sample was incubated with gentle mixing at RT for 5 minutes. The reaction was terminated by adding 7.5 mL BBS/gelatin/MgWCaW The sample was spun down as above, re-suspended in 2 mL 15 BBS/gelatin/MgWCa^ and washed a further two times as above, then stored at 4°C.

Day 2. Hemolysis assay

Materials included BBS/gelatin/Mg⁺⁺/Ca⁺⁺ (as above), test sera, 96-well roundbottomed and flat-bottomed plates and a spectrophotometer that reads 96-well plates at 410414 nm.

The concentration of the RBC was first determined and the cells were adjusted to

10⁹/mL, and stored at this concentration. Before use, the cells were diluted in assay buffer to 10⁸/mL, and then 100 pL per well was used. Hemolysis was measured at 410-414 nm (allowing for greater sensitivity than 541 nm). Dilutions of test sera were prepared in ice-cold BBS/gelatin/Mg⁺⁺/Ca⁺⁺. lOOpL of each sérum dilution was pipetted into round-bottomed 25 plate. 100 pL of appropriately diluted RBC préparation was added (i.e., 10⁸/mL), incubated at 37°C for about 1 hour, and observed for lysis. (The plates may be photographed at this point.) The plate was then spun down at maximum speed for 5 minutes. 100 pL of the fluid phase was aspirated, transferred to flat-bottom plates, and the OD was recorded at 410-414 nm. The RBC pellets were retained (these can be subsequently lysed with water to obtain an 30 inverse resuit).

Experimcnt #1

204

Fresh blood was obtained from CD55/CD59 double-deficient mice and blood of Crry/C3 double-deficient mice and érythrocytes were prepared as described in detail in the above protocol. The cells were split and half of the cells were coated with mannan and the other half were left untreated, adjusting the final concentration to 10^/mL, of which 100 pL was used in the hemolysis assay, which was carried out as described above.

Results of Experiment #1: The lectin pathway is involved in érythrocyte lysis in the PNH animal model

In an initial experiment, it was determined that non-coated WT mouse érythrocytes were not lysed in any mouse sérum. It was further determined that mannan-coated Crry-/mouse érythrocytes were slowly lysed (more than 3 hours at 37 degrees) in WT mouse sérum, but they were not lysed in MBL null sérum. (Data not shown).

It was determined that mannan-coated Crry-/- mouse érythrocytes were rapidly lysed in human sérum but not in heat-inactivated NHS. Importantly, mannan-coated Crry-/- mouse érythrocytes were lysed in NHS diluted down to 1/640 (i.e., 1/40, 1/80, 1/160, 1/320 and 1/640 dilutions ail lysed). (Data not shown). In this dilution, the alternative pathway does not work (AP functional activity is significantly reduced below 8% sérum concentration).

Conclusions from Experiment #1

Mannan-coated Crry-/- mouse érythrocytes are very well lysed in highly diluted human sérum with MBL but not in that without MBL. The efficient lysis in every sérum concentration tested implies that the alternative pathway is not involved or needed for this lysis. The inability of MBL-deficient mouse sérum and human sérum to lyse the mannancoated Crry-/- mouse érythrocytes indicates that the classical pathway also has nothing to do with the lysis observed. As lectin pathway récognition molécules are required (i.e., MBL), this lysis is mediated by the lectin pathway.

Experiment #2

Fresh blood was obtained from the Crry/C3 and CD55/CD59 double-deficient mice and mannan-coated Crry-/- mouse érythrocytes were analyzed in the haemolysis assay as described above in the presence of the following human sérum: MASP-3 -/-; MBL null; WT; NHS pretreated with human MASP-2 antibody; and heat-inactivated NHS as a control.

205

Results of Experiment #2; MASP-2 inhibitors and MASP-3 deficiency prevents érythrocyte lysis in PNH animal model

With the mannan-coated Crry-/- mouse érythrocytes, NHS was incubated in the dilutions diluted down to 1/640 (i.e., 1/40, 1/80, 1/160, 1/320 and 1/640), human MBL-/5 sérum, human MASP-3-deficient sérum (from 3MC patient), and NHS pretreated with MASP-2 mAb, and heat-inactivated NHS as a control.

The ELISA microtiter plate was spun down and the non-lysed érythrocytes were collected on the bottom of the round-well plate. The supematant of each well was collected and the amount of hemoglobin released from the lysed érythrocytes was measured by reading 10 the OD415 nm in an ELI SA reader.

It was observed that MASP-3-/- sérum did not lyse mannan-coated mouse érythrocytes at ail. In the control heat-inactivated NHS (négative control), as expected, no lysis was observed. MBL-/- human sérum lysed mannan-coated mouse érythrocytes at 1/8 and 1/16 dilutions. MASP-2-antibody-pretreated NHS lysed mannan-coated mouse 15 érythrocytes at 1/8 and 1/16 dilutions while WT human sérum lysed mannan-coated mouse érythrocytes down to dilutions of 1/32.

FIGURE 19 graphically illustrâtes hemolysis (as measured by hemoglobin release of lysed mouse érythrocytes (Crry/C3-/-) into the supematant measured by photometry) of mannan-coated murine érythrocytes by human sérum over a range of sérum dilutions in 20 sérum from MASP-3-/-, heat-inactivated (HI) NHS, MBL-/-, NHS pretreated with MASP-2 antibody, and NHS control.

FIGURE 20 graphically illustrâtes hemolysis (as measured by hemoglobin release of lysed mouse érythrocytes (Crry/C3-/-) into the supematant measured by photometry) of mannan-coated murine érythrocytes by human sérum over a range of sérum concentration in 25 sérum from MASP-3-/-, heat-inactivated (HI) NHS, MBL-/-, NHS pretreated with MASP-2 antibody, and NHS control.

From the results shown in FIGURE 19 and 20, it is demonstrated that inhibiting MASP-3 will prevent any complement-mediated lysis of sensitized érythrocytes with déficient protection from autologous complément activation. MASP-2 inhibition with MASP30 2 antibody significantly shifted the CH50 and was protective to some extent, but MASP-3 inhibition was more effective.

Experiment #3

206

Non-coated Crry-/- mouse érythrocytes obtained from fresh blood from the Crry/C3 and CD55/CD59 double-deficient mice were analyzed in the hemolysis assay as described above in the presence of the following sera: MASP-3-/-; MBL-/-; WT; NHS pretreated with human MASP-2 antibody, and heat-inactivated NHS as a control.

Results:

FIGURE 21 graphically illustrâtes hemolysis (as measured by hemoglobin release of lysed WT mouse érythrocytes into the supematant measured by photometry) of non-coated murine érythrocytes over a range of sérum concentrations in human sera from a 3MC (MASP-3-/-) patient, heat inactivated (HI) NHS, MBL-/-, NHS pretreated with MASP-2 10 antibody, and NHS control. As shown in FIGURE 21 and summarized in TABLE 13, it is demonstrated that inhibiting MASP-3 inhibits complement-mediated lysis of non-sensitized WT mouse érythrocytes.

FIGURE 22 graphically illustrâtes hemolysis (as measured by hemoglobin release of lysed mouse érythrocytes (CD55/59 -/-) into the supematant measured by photometry) of 15 non-coated murine érythrocytes by human sérum over a range of sérum concentrations in human sera from heat-inactivated (HI) NHS, MBL-/-, NHS pretreated with MASP-2 antibody, and NHS control. As shown in FIGURE 22 and summarized in TABLE 13, it is demonstrated that inhibiting MASP-2 was protective to a limited extent.

20	TABLE 13: CH50 values expressed as sérum concentrations
	Sérum	WT	CD55/59 -/-
3MC patient	No lysis	No lysis
Heat-inactivated NHS	No lysis	No lysis
MBL AO/XX donor (MBL déficient)	7.2%	2.1%
NHS + MASP-2 antibody	5.4%	1.5%
NHS	3.1%	0.73%

Note: “CH50” is the point at which complement-mediated hemolysis reachs 50%.

In summary, the results in this Example demonstrate that inhibiting MASP-3 prevents any complément lysis of sensitized and non-sensitized érythrocytes with déficient protection 25 from autologous complément activation. MASP-2 inhibition also is protective to some

207 extent. Therefore, MASP-2 and MASP-3 inhibitors alone or in combination (i.e., coadministered, administered sequentially) or MASP-2/MASP-3 bispecific or dual inhibitors may be used to treat subjects suffering from PNH, and may also be used to ameliorate (i.e., inhibit, prevent or reduce the severity of) extravascular hemolysis in PNH patients undergoing treatment with a C5 inhibitor such as eculizumab (Soliris®).

EXAMPLE 6

This Example describes a hemolysis assay testing mannan-coated rabbit érythrocytes for lysis in the presence of WT or MASP-1/3-/- mouse sera.

Methods:

.Hemolysis assay of rabbit RBC (mannan coated) in mouse MASP-l/3-deflcient sera and JVT control sera

Day L Préparation of rabbit RBC.

Materials included: fresh rabbit blood, BBS/ Mg⁺⁺/Ca⁺⁺ (4.4 mM barbituric acid, 1.8 mM sodium barbitone, 145 mM NaCl, pH 7.4, 5 mM Mg⁺⁺, 5 mM Ca⁴”⁸), BBS/ Mg⁺⁺/Ca⁺⁺ with 0.1% gelatin, chromium chloride contained in buffer; i.e., CrCh.6 H2O (0.5 mg /mL in BBS/ Mg^/Ca*^4-) and mannan, 100 pg/mL in BBS/ Mg^/Ca*^4-.

1. Rabbit whole blood (2 mL) was split into two 1.5 mL eppendorf tubes and centrifuged for 3 minutes at 8000 rpm (approximately 5.9 ref) in a refrigerated eppendorf centrifuge at 4°C. The RBC pellet was washed three times after re-suspending in ice-cold BBS/Mg⁺⁺/Ca⁺⁺. After the third wash, the pellet was re-suspended in 4 mL BBS/Mg⁺⁺/Ca⁺⁺. Two mL of this aliquot were added to a 15-mL falcon tube to be used as the uncoated control. The remaining 2 mL of the RBCs aliquot were diluted in 2 mL of CrCh buffer, 2 mL of the mannan solution were added and the suspension was incubated at room température for 5 minutes with gentle mixing. The reaction was terminated by adding 7.5 mL of BBS/0.1% gelatin/Mg⁺⁺/Ca⁺⁺ to the mixture. The érythrocytes were pelleted and the RBCs were washed twice with BBS/0.1% gelatin/Mg⁺⁺/Ca⁺⁺ as described above. The RBCs suspension was stored in BBS/0.1% gelatin/ Mg^/Ca^at 4°C.

2. 100 pL of suspended RBCs were diluted with 1.4 mL water and spun down at

8000 rpm (approximately 5.9 ref) for 3 minutes and the OD of the supematant was adjusted to 0.7 at 541 nm (an OD of 0.7 at 541nm corresponds to approximately 10⁹ erythrocytes/mL).

208

3. The re-suspended RBCs were diluted with BBS/0.1% gelatin/Mg⁺⁺/Ca⁺⁺ to a concentration of 10⁸ /mL.

4. Dilutions of the test sera were prepared in ice-cold BBS/gelatin/ MgXCa⁴^ and 100 pL of each sérum dilution were pipetted into the corresponding well of round-bottom 5 plate. 100 pL of appropriately diluted RBC (108/mL) were added to each well. As a control for complété lysis, purified water (100 pL) was mixed with the diluted RBC (100 pL) to cause 100% lysis, while BBS/0.1% gelatin/ Mg^/Ca⁴^ without sérum (100 pL) was used as a négative control. The plate was then incubated for 1 hour at 37°C.

5. The round-bottom plate was centrifuged at 3250 rpm for 5 minutes. The 10 supernatant from each well (100 pL) was transferred into the corresponding wells of a flatbottom plate and OD was read in an ELISA reader at 415-490nm. Results are reported as the ratio of the OD at 415 nm to that at 49 Onm.

Results:

FIGURE 23 graphically illustrâtes hemolysis (as measured by hemoglobin release of 15 lysed rabbit érythrocytes into the supernatant measured by photometry) of mannan-coated rabbit érythrocytes by mouse sérum over a range of sérum concentrations in sérum from MASP-1/3-/- and WT control. As shown in FIGURE 23, it is demonstrated that inhibiting MASP-3 prevents complement-mediated lysis of mannan-coated WT rabbit érythrocytes. These results further support the use of MASP-3 inhibitors for the treatment of one or more 20 aspects of PNH as described in Example 5.

EXAMPLE 7

This Example describes the génération of MASP-1 and MASP-3 monoclonal antibodies using an in vitro system comprising a modified DT40 cell line, DTLacO.

Background/Rationale:

Antibodies against human MASP-1 and MASP-3 were generated using an in vitro system comprising a modified DT40 cell line, DTLacO, that permits réversible induction of diversification of a particular polypeptide, as further described in WO2009029315 and LJS2010093033. DT40 is a chicken B cell line that is known to constitutively mutate its 30 heavy and light chain immunoglobulin (Ig) genes in culture. Like other B cells, this constitutive mutagenesis targets mutations to the V région of Ig genes, and thus, the CDRs of the expressed antibody molécules. Constitutive mutagenesis in DT40 cells takes place by

209 gene conversion using as donor sequences an array of non-functional V gene segments (pseudo-V genes; ψΥ) situated upstream of each functional V région. Délétion of the ψν région was previously shown to cause a switch in the mechanism of diversification from gene conversion to somatic hypermutation, the mechanism commonly observed in human B cells.

The DT40 chicken B cell lymphoma line has been shown to be a promising starting point for antibody évolution ex vivo (Cumbers, S.J. et al. Nat Biotechnol 20, 1129-1134 (2002); Seo, H. et al. Nat Biotechnol 23, 731-735 (2005)). DT40 cells proliferate robustly in culture, with . an 8-10 hour doubling time (compared to 20-24 hr for human B cell fines), and they support very efficient homologous gene targeting (Buerstedde, J.M. et al. EmboJ9, 921-927 (1990)).

DT40 cells command enormous potential V région sequence diversity given that they can access two distinct physiological pathways for diversification, gene conversion and somatic hypermutation, which create templated and nontemplated mutations, respectively (Maizels, N. Annu Rev Genet 39, 23-46 (2005)). Diversified heavy and light chain immunoglobulins (Igs) are expressed in the form of a cell-surface displayed IgM. Surface IgM has a bivalent form, structurally similar to an IgG molécule. Cells that display IgM with specificity for a particular antigen can be isolated by binding either immobilized soluble or membrane displayed versions of the antigen. However, utility of DT40 cells for antibody évolution has been limited in practice because — as in other transformed B cell fines — diversification occurs at less than 1% the physiological rate.

In the system used in this example, as described in WO2009029315 and

US2010093033, the DT40 cells were engineered to accelerate the rate of Ig gene diversification without sacrificing the capacity for further genetic modification or the potential for both gene conversion and somatic hypermutation to contribute to mutagenesis. Two key modifications to DT40 were made to increase the rate of diversification and, consequently, the complexity of binding specificities in our fibrary of cells. First, Ig gene diversification was put under the control of the potent E. coli lactose operator/repressor regulatory network. Multimers consisting of approximately 100 polymerized repeats of the potent E. coli lactose operator (PolyLacO) were inserted upstream of the rearranged and expressed IgÀ and IgH genes by homologous gene targeting. Regulatory factors fused to lactose repressor protein (Lacl) can then be tethered to the LacO regulatory éléments to regulate diversification, taking advantage of the high affinity (kD=10'¹⁴ M) of lactose

210 repressor for operator DNA. DT40 PolyLacO-λκ cells, in which PolyLacO was integrated only at IgX, exhibited a 5-fold increase in Ig gene diversification rate relative to the parental DT40 cells prior to any engineering (Cummings, W.J. et al. PLoS Biol 5, e246 (2007)). Diversification was further elevated in cells engineered to carry PolyLacO targeted to both 5 the IgX and the IgH genes (DTLacO). DTLacO cells were demonstrated to hâve diversification rates 2.5- to 9.2-fold elevated relative to the 2.8% characteristic of the parental DT40 PolyLacO-ÀR LacI-HPl line. Thus, targeting PolyLacO éléments to both the heavy and light chain genes accelerated diversification 21.7-fold relative to the DT40 parental cell line. Tethering regulatory factors to the Ig loci not only alters the frequency of mutagenesis, 10 but also can change the pathway of mutagenesis creating a larger collection of unique sequence changes (Cummings et al. 2007; Cummings et al. 2008). Second, a diverse collection of sequence starting points for the tethered factor-accelerated Ig gene diversification was generated. These diverse sequence starting points were added to DTLacO by targeting rearranged Ig heavy-chain variable régions, isolated from a two month old chick, 15 to the heavy chain locus. The addition of these heavy chain variable régions created a répertoire of 10⁷ new starting points for antibody diversification. Building these new starting points into the DTLacO cell line permits the identification of clones that bind a particular target, and then rapid affinity maturation by the tethered factors. Following affinity maturation, a full-length, recombinant chimeric IgG is made by cloning the matured, 20 rearranged heavy- and light-chain variable sequences (VH and VX; consisting of chicken framework régions and the complementarity determining régions or CDRs) into expression vectors containing human IgGl and lambda constant régions. These recombinant mAbs are suitable for in vitro and in vivo applications, and they serve as the starting point for humanization. .

Methods:

Sélection for MASP-1 and MASPS antigen binding.

Initial sélections were performed by binding DTLacO populations diversified by gene targeting to beads complexed with human MASP-1 (SEQ ID NO:8) and MASP-3 antigen (SEQ ID NO:2); and subséquent sélections by FACS, using fluorescence-labeled soluble 30 antigen (Cumbers, S.J. et al. Nat Biotechnol 20, 1129-1134 (2002); Seo, H. et al. Nat Biotechnol 23, 731-735 (2005). Because of the conserved amino acid sequence in the alpha

211 chain that is shared between MASP-1 and MASP-3 (shown in FIGURE 2), and the distinct beta chain sequences (shown in FIGURE 2), separate, parallel screens for binders to MASP1 and MASP-3 were carried out to identify MASP-1 spécifie mAbs, MASP-3 spécifie mAbs and also mAbs capable of binding to both MASP-1 and MASP-3 (dual-specific). Two forms 5 of antigen were used to select and screen for binders. First, recombinant MASP-1 or MASP3, either full-length or a fragment, fused to an Fc domain were bound to Dynal magnetic Protein G beads or used in FACS-based sélections using a PECy5-labeled anti-human IgG(Fc) secondary antibody. Altematively, recombinant versions of MASP-1 or MASP-3 proteins were directly labeled with Dylight flours and used for sélections and screening.

Binding and affinity.

Recombinant antibodies were generated by cloning PCR-amplified V régions into a vector that supported expression of human IgGl in 293F cells (Yabuki et al., PLoS ONE, 7(4):e36032 (2012)). Saturation binding kinetics were determined by staining DTLacO cells expressing antibody binding MASP-1 or MASP-3 with various concentrations of fluorescent15 labeled soluble antigen. Functional assays for MASP-3 spécifie activity including MASP-3dependent C3b déposition and MASP-3-dependent factor D cleavage were carried out as described in Examples 8 and 9, respectively. A functional assay for MASP-1-spécifie activity, namely the inhibition of MASP-1-dépendent C3b déposition was carried out as described below.

Results:

Numerous MASP-1 and MASP-3 binding antibodies were generated using the methods described above. Binding, as demonstrated by FACS analysis, is described for the représentative clones M3J5 and M3M1, which were isolated in screens for MASP-3 binders.

FIGURE 24A is a FACS histogram of MASP-3 antigen/antibody binding for 25 DTLacO clone M3J5. FIGURE 24B is a FACS histogram of MASP-3 antigen/antibody binding for DTLacO clone M3M1. In FIGURES 24A and 24B the gray filled curves are IgGl-stained négative control, and thick black curves are MASP-3-staining.

FIGURE 25 graphically illustrâtes a saturation binding curve of clone M3J5 (Clone 5) for the MASP-3 antigen. As shown in FIGURE 25, the apparent binding affinity of the 30 M3J5 antibody for MASP-3 is about 31 nM.

Sequence analysis of identified clones was performed using standard methods. Ail clones were compared to the common (DT40) VH and VL sequences and to each other.

212

Sequences for the two afore-mentioned clones, M3J5 and M3M1 are provided in an alignment with two additional représentative clones, D14 and 1E10, which were identified in screens for CCP1-CCP2-SP fragments of MASP-1 and MASP-3, respectively. D14 and 1 El0 bind régions common to both MASP-1 and MASP-3.

FIGURE 26A is an amino acid sequence alignment of the VH régions of M3J5,

M3M1, D14 and 1E10 to the chicken DT40 VH sequence.

FIGURE 26B is an amino acid sequence alignment of the VL régions of M3J5, M3M1, D14 and 1E10 to the chicken DT40 VL sequence.

The VH and VL amino acid sequence of each clone is provided below.

Heavy Chain Variable Région (VH) sequences

FIGURE 26A shows an amino acid alignment of the heavy-Chain Variable Région (VH) sequences for the parent DTLacO (SEQ ID N0:300), the MASP-3-binding clones M3J5 (SEQ ID NO:30I), and M3M1 (SEQ ID NO:302), and the MASP-l/MASP-3 dual binding clones D14 (SEQ ID NO:306), and 1E10 (SEQ ID NO:308).

¹⁵ .

The Rabat CDRs in the VH sequences below are located at the following amino acid positions: Hl:aa 31-35; H2:aa 50-62; and H3:aa 95-102.

The Chothia CDRs in the VH sequences below are located at the following amino acid positions: Hl: aa 26-32; H2: aa 52-56; and H3: aa 95-101.

Parent DTLacO VH: (SEQ ID NQ:300)

AVTLDESGGGLQTPGGALSLVCKASGFTFSSNAMGWVRQAPGKGLEWVAGIDDDG SGTRYAPAVKGRATISRDNGQSTLRLQLNNLRAEDTGTYYCTKCAYSSGCDYEGGYI DAWGHGTEV1VSS

Clone M3J5 VH: (SEQ ID NO:3Q1)

AVTLDESGGGLQTPGGGLSLVCKASGFTFSSYAMGWMRQAPGKGLEYVAGIRSDGS FTLYATAVKGRATISRDNGQSTVRLQLNNLRAEDTATYFCTRSGNVGDIDAWGHGT EVIVSS .

Clone M3M1 VH: (SEQ ID NO:3Q2)

213

AVTLDESGGGLQTPGGGLSLVCKASGFDFSSYQMNWIRQAPGKGLEFVAAINRFGN STGHGAAVKGRVTISRDDGQSTVRLQLSNLRAEDTATYYCAKGVYGYCGSYSCCG VDTIDAWGHGTEVIVSS

Clone D14 VH: (SEQ ID NO:3Q6)

AVTLDESGGGLQTPGGALSLVCKASGFTFSSYAMHWVRQAPGKGLEWVAGIYKSG AGTNYAPAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAKTTGSGCSSGYRAE YIDAWGHGTEVIVSS

Clone 1 El0 VH: (SEQ ID NO:3Q8)

AVTLDESGGGLQTPGGALSLVCKASGFTFSSYDMVWVRQAPGKGLEFVAGISRNDG RYTEYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTATYYCARDAGGSAYWFDAG QIDAWGHGTEVIVSS

Light Chain Variable Région (VL) sequences

FIGURE 26B shows an amino acid alignment of the light-Chain Variable Région (VL) sequences for the parent DTLacO (SEQ ID NO:303) and the MASP-3-binding clones M3J5 (SEQ ID NO:304), and M3M1 (SEQ ID NO:305), and the MASP-l/MASP-3 dual binding clones D14 (SEQ ID NO:307) and I El0 (SEQ ID NO:309).

Parent DTLacO VL: (SEQ ID NO:3Q3)

ALTQPASVSANLGGTVKITCSGGGSYAGSYYYGWYQQKSPGSAPVTVIYDNDKRPS DIPSRFSGSLSGSTNTLTITGVRADDEAVYFCGSADNSGAAFGAGTTLTVL

Clone M3J5 VL: (SEQ IDNO:3Q4)

ALTQPASVSANPGETVKITCSGGYSGYAGSYYYGWYQQKAPGSAPVTLIYYNNKRP SDIPSRFSGSLSGSTNTLTITGVRADDEAVYFCGSADNSGAAFGAGTTLTVL

Clone M3M1 VL: (SEQ ID NO:3Q5)

ALTQPASVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYYNNKRPS DIPSRFSGSLSGSTNTLTITGVRADDEAVYFCGSADNSGAAFGAGTTLTVL

214

Clone D14 VL: (SEQ ID NO:3Q7)

ALTQPASVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYYNNKRPS DIPSRFSGSLSGSTNTLTITGVRADDEAVYFCGSADNSGAAFGAGTTLTVL

Clone 1E10 VL: (SEQ IDNO:3Q9)

ALTQPASVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYYNNKRPS dipsrfsgslsgstntltitgvraddeavyfcgsadnsgaafgagttltvl

LEA-2 (MASP-2-ilependent) Functional Assay

MASP-1 contributes to LEA-2 via its ability to activate MASP-2 (see FIGURE 1). The Wieslab® Complément System Screen MBL assay (Euro Diagnostica, Malmô, Sweden) measures C5b-C9 déposition under conditions that isolate LEA-2-dependent activation (i.e., traditional lectin pathway activity). The assay was carried out according to the manufacturer’³ instructions with représentative clone 1E10 tested as a final concentration of 400 nM.

FIGURE 27 is a bar graph showing the inhibitory activity of the mAb 1E10 in comparison to the positive sérum provided with the assay kit, as well as an isotype control antibody. As shown in FIGURE 27, mAb 1E10 demonstrates partial inhibition of LEA-2dependent activation (via inhibition of MASP-1-dépendent activation of MASP-2), whereas the isotype control antibody does not. Stronger inhibition should be achieved by continued affinity maturation of this antibody for MASP-1 binding using the tethered factors in the DTLacO system.

LEA-1 (MASP-3-dependent) Function Assays for représentative mAbs are described below in Examples 8 and 9.

Summary of Results:

The above results showed that the DTLacO platform permitted rapid ex vivo discovery of MASP-1 and MASP-3 monoclonal antibodies with inhibitory properties on LEA-I (as shown below in Examples 8 and 9) and on LEA-2 (as shown in this Example).

EXAMPLE 8

Analysis of the complément pathway in 3MC sérum with S. aureus

Background/Rationale:

215

It was determined that MASP-3 is not activated through exposure to non-immobilized fiuid-phase mannan, zymosan A or N-acetyl cysteine either in the presence or absence of normal human sérum. However, it was determined that recombinant and native MASP-3 are activated on the surface of heat-inactivated 5. aureus in the presence and absence of normal human sérum (NHS) or heat-inactivated human sérum (HIS) (data not shown). It was also determined that C3b déposition occurs on the surface of 5. aureus in the presence of normal human sérum, and that the déposition can be monitored using a flow cytometer. Therefore, the alternative pathway (AP) response to 5. aureus was measured as described in this Example as a means of assessing the contribution of MASP-3 to LEA-1.

Methods:

Recombinant MASP-3: polynucleotide sequences encoding full length recombinant human MASP-3, a truncated serine protease (SP) active version of MASP-3 (CCP1-CCP2SP), and a SP-inactivated form of MASP-3 (S679A) were cloned into the pTriEx7 mammalian expression vector (Invivogen). The resulting expression constructs encode the full length MASP-3 or the CCP1-CCP2-SP fragment with an amino-terminal Streptag and a carboxy-terminal Hise tag. The expression constructs were transfected into Freestyle 293-F or Expi293F cells (Invitrogen) according to the protocols provided by the manufacturer. After three to four days of culture in 5% CO2 at 37°C, recombinant proteins were purified utilizing Streptactin affinity chromatography.

Recombinant MASP-1: the full length or truncated CCP1-CCP2-SP forms of recombinant MASP-1 with or without the stabilizing R504Q (Dobo et al., J. Immunol 183:1207, 2009) or SP inactivating (S646A) mutations and bearing an amino-terminal Steptag and a carboxy-terminal His6 tag were generated as described for recombinant MASP3 above.

1. C3b déposition and factor B cleavage on S. aureus in 3MC (human) sérum

An initial experiment was carried out to demonstrate that the flow cytometry assay is able to detect the presence or absence of AP-driven C3b déposition (AP-C3b) as follows. Five percent of the following sera: normal human sérum, factor B (Factor B)- depleted human sérum, factor D-depleted human sérum and properdin-depleted human sérum (obtained from Complément Technology, Tyler, Texas, USA) were mixed with test antibody in either Mg⁺⁺/EGTA buffer or EDTA at 4°C ovemight. Heat-killed 5. aureus (10⁸/reaction) was added to each mixture to a total volume of 100 pL and rotated at 37°C for 40 minutes.

216

Bacteria were washed in washing buffer, the bacterial pellet was re-suspended in washing buffer and a 80 pL aliquot of each sample was analyzed for C3b déposition on the bacterial surface, which was detected with anti-human C3c (Dako, UK) using flow cytometry.

The results of the flow cytometry détection of C3b are shown in FIGURE 28A. As shown in FIGURE 28A, panel 1, normal human sérum in the presence of EDTA, which is known to inactivate the AP, no C3b déposition was observed (négative control). In normal human sérum treated with Mg⁺⁺/EGTA, only lectin-independent complément pathways can function. In panel 2, Mg⁺⁺/EGTA buffer is used, therefore the AP is active, and AP-driven C3b déposition is observed (positive control). As shown in panel 3, 4 and 5, in factor Bdepleted, factor D-depleted and properdin-depleted sérum, respectively, no alternative pathway driven C3b déposition is observed, as expected. These results demonstrate that the assay is capable of detecting AP-dependent C3b déposition.

A C3b déposition on S. aureus assay was carried out as described above to assess the ability of recombinant MASP-3 to reconstitute the AP (LEA-1) in human 3MC sérum, which is déficient in MASP-3 (Rooryck C, et al., Nat Genet. 43(3):197-203 (2011)). The following combinations of reagents were tested.

1.5% normal human sérum +EDTA

2. 5% normal human sérum +Mg/EGTA

3. 5% human 3MC (MASP-3⁷’) sérum + Mg^EGTA

4. 5% human 3MC (MASP-3⁷’) sérum + Mg⁺⁺/EGTA plus active full-length rMASP3

5. 5% human 3MC (MASP-3⁷’) sérum + Mg^/EGTA plus truncated active rMASP-3 (CCP1/CCP2/SP)

6. 5% human 3MC (MASP-3⁷’) sérum + Mg⁺⁺/EGTA plus inactive rMASP-3 (S679A)

7. 5% human 3MC (MASP-3⁷’) sérum + Mg^/EGTA plus active full length rMASP-1

The various mixtures of 5% sérum and recombinant proteins (5 pg of each) as shown above were incubated in the specified buffer conditions (either Mg⁺⁺/EGTA buffer or EDTA) at 4°C ovemight. After the incubation ovemight, 10⁸ heat-killed 5. auretis were added to each mixture in a total volume of 100 pL and rotated at 37°C for 40 minutes. Bacteria were

217 washed and re-suspended in washing buffer and an 80 μΐ aliquot of each sample was analyzed for C3b déposition by FACS. The remaining 20 pL aliquot of each sample was used to measure factor B cleavage by Western blot using anti-factor B antibody as described below.

The results ofthe flow cytometery détection of C3b are shown in FIGURE 28B. Panel numbers correspond to the numbers designated for each of the reagent combination outlined above. The négative control (panel 1) and positive control (panel 2) show the absence and presence of C3b déposition, as expected. Panel 3 shows that AP-driven C3b déposition is absent in 3MC sérum. Panels 4 and 5 show that active full length rMASP-3 (panel 4) and active rMASP-3 (CCP1-CCP2-SP) (panel 5) both restore AP-driven C3b déposition in 3MC sérum. Panel 6 shows that inactive rMASP-3 (S679A) does not restore AP-driven C3b déposition in 3MC sérum. Panel 7 shows that rMASP-1 does not restore AP-driven C3b déposition in 3MC sérum.

Taken together, these results demonstrate that MASP-3 is required for AP-driven C3b 15 déposition on S. aureus in human sérum.

MASP-3-dependent Activation of Factor B

In order to analyze MASP-3-dependent activation of Factor B, the various mixtures of 5% sérum (either normal human sérum or 3MC patient sérum) and recombinant proteins as shown above were assayed as described above. From each reaction mixture, 20 pL were 20 removed and added to protein sample loading buffer. The samples were heated at 70°C for minutes and loaded onto an SDS-PAGE gel. Western blot analysis was performed using a Factor B polyclonal antibody (R&D Systems). Activation of Factor B was apparent by the formation of two lower molecular weight cleavage products (Bb and Ba) derived from the higher molecular weight pro-Factor B protein.

FIGURE 29 shows the results of a Western blot analysis to détermine factor B cleavage in response to S. aureus in 3MC sérum in the presence or absence of rMASP-3. As shown in lane 1, the normal human sérum in the presence of EDTA (négative control) demonstrates very little Factor B cleavage relative to normal human sérum in the presence of Mg^/EGTA, shown in lane 2 (positive control). As shown in lane 3, 3MC sérum demonstrates very little

Factor B cleavage in the presence of Mg⁺⁺/EGTA. However, as shown in lane 4, Factor B cleavage is restored by the addition and pre-incubation of full-length, recombinant MASP-3 protein (5 pg) to the 3MC sérum.

218

Assay to déterminé the effect of rMASPS on pro-factor D in factor B/C3(H2O) Cleavage

The following assay was carried out to détermine the minimal requirement for MASP3-dependent activation/cleavage of factor B.

C3(H2O) (200ng), purified plasm factor B (20 pg), recombinant pro-factor D (200 ng) and recombinant human MASP-3 (200 ng) were mixed together in various combinations (as shown in FIGURE 30), in a total volume of 100 pL in BBS/Ca**/ Mg** and incubated at 30°C for 30 minutes. The reaction was stopped by adding 25 uL of SDS loading dye containing 5% 2-mercaptoethanol. After boiling at 95°C for 10 minutes under shaking (300 rpm), the mixture was spun down at 1400 rpm for 5 minutes and 20 uL of the supematant was loaded and separated on a 10% SDS gel. The gel was stained with Coomassie brilliant blue.

Results:

FIGURE 30 shows a Comassie-stained SDS-PAGE gel in which factor B cleavage is analyzed. As shown in lane 1, factor B cleavage is most optimal in the presence of C3, MASP-3 and pro-factor D. As shown in lane 2, C3 is absolutely required; however, as shown in lanes 4 and 5, either MASP-3 or pro-factor D are able to médiate factor B cleavage, as long as C3 is présent.

Analysis ofthe ability of MASPS mAbs to inhibit MASPS-dependent AP-driven C3b déposition

As described in this Example it was demonstrated that MASP-3 is required for AP-driven C3b déposition on 5. aureus in human sérum. Therefore, the following assay was carried out to détermine if a représentative MASP-3 mAb identified as described in Example 7, could inhibit activity of MASP-3. Active, recombinant MASP-3 (CCP1-CCP2-SP) fragment protein (250 ng) was pre-incubated with an isotype control mAb, mAblA5 (control obtained from the DTLacO platform that does not bind MASP-3 or MASP-1), or mAbD14 (binds MASP-3) at three different concentrations (0.5, 2 and 4 μΜ) for 1 hour on ice. The enzymemAb mixture was exposed to 5% 3MC sérum (MASP-3 déficient) and 5xl0⁷ heat-killed 5. aureus in a final reaction volume of 50 μΕ. The reactions were incubated at 37°C for 30 minutes, and then stained for the détection of C3b déposition. The stained bacterial cells were analyzed by a flow cytometer.

219

FIGURE 31 graphically illustrâtes the mean fluorescent intensities (MFI) of C3b staining obtained from the three antibodies plotted as a function of mAb concentration in 3MC sérum with the presence of rMASP-3. As shown in FIGURE 31, mAbD14 demonstrates inhibition of C3b déposition in a concentration-dependent manner. In contrast, neither of the control mAbs inhibited C3b déposition. These results demonstrate that mAbD14 is able to inhibit MASP-3-dependent C3b déposition. Improved inhibitory activity for mAbD14 is expected following continued affinity maturation of this antibody for MASP3 binding using the tethered factors in the DTLacO system.

Summary of Results:

In summary, the results in this Example demonstrate a clear defect of the AP in sérum déficient for MASP-3. Thus, MASP-3 has been demonstrated to make a critical contribution to the AP, using factor B activation and C3b déposition as functional end-points. Furthermore, addition of functional, recombinant MASP-3, including the catalytically-active C-terminal portion of MASP-3 corrects the defect in factor B activation and C3b déposition in the sérum from the 3MC patient. Conversely, as further demonstrated in this Example, addition of a MASP-3 antibody (e.g., mAbD14) in 3MC sérum with rMASP-3 inhibits APdriven C3b déposition. A direct rôle of MASP-3 in Factor B activation, and therefore the AP, is demonstrated by the observation that recombinant MASP-3, along with C3, is sufficient to activate recombinant factor B.

EXAMPLE 9

This Example demonstrates that MASP-1 and MASP-3 activate factor D.

Methods:

Recombinant MASP-1 and MASP-3 were tested for their ability to cleave two different recombinant versions of pro-factor D. The first version (pro-factor D-His) lacks an N-terminal tag, but has a C-terminal His tag. Thus, this version of pro-factor D contains the 5 amino acid pro-peptide that is removed by cleavage during activation. The second version (ST-pro-factor D-His) has a Strep-Tagll sequence on the N-terminus, thus increasing the cleaved N-terminal fragment to 15 amino acids. ST-pro-factor D also contains a Hîsô tag at the C-terminus. The increased length of the propeptide of ST-pro-factor D-His improves the

220 resolution between the cleaved and uncleaved forms by SDS-PAGE compared to the resolution possible with the pro-factor D-HIS form.

Recombinant MASP-1 or MASP-3 proteins (2 pg) was added to either pro-factor DHis or ST-pro-factor D-His substrates (100 ng) and incubated for 1 hour at 37°C. The 5 reactions were electrophoresed on a 12% Bis-Tris gel to résolve pro-factor D and the active factor D cleavage product. The resolved proteins were transferred to a PVDF membrane and analyzed by Western blot by détection with a biotinylated factor D antibody (R&D Systems).

Results:

FIGURE 32 shows the Western blot analysis of pro-factor D substrate cleavage.

TABLE 14: Lane Description for Western Blot shown in FIGURE 32

Experimental conditions	Lane 1	Lane 2	Lane 3	Lane 4	Lane 5
Pro-Factor D	+	+	+	+	+
rMASP-3 (full-length)	-	+	—	—	—
rMASP-3a (S679A)	-	-	+	-	-
rMASP-lA (S646A)	-	-	-	. +	-
rMASP-1 (CCP-1CCP2-SP)	-	-	-		+

As shown in FIGURE 32, only full length MASP-3 (lane 2) and the MASP-1 CCP115 CCP2-SP) fragment (lane 5) cleaved ST-pro-factor D-His6. The catalytically-inactive full length MASP-3 (S679A; lane 3) and MASP-1 (S646A; lane 3) failed to cleave either substrate. Identical results were obtained with the pro-factor D-His6 polypeptide (not shown). The comparison of a molar excess of MASP-1 (CCP1-CCP2-SP) relative to MASP-3 suggests that MASP-3 is a more effective catalyst of pro-factor D cleavage than is MASP-1, 20 as least under the condtions described herein.

Conclusions: Both MASP-1 and MASP-3 are capable of cleaving and activating factor D. This activity directly connects LEA-1 with the activation of the AP. More specifically,

221 activation of factor D by MASP-1 or MASP-3 will lead to factor B activation, C3b déposition, and likely opsonization and/or lysis.

Assay for Inhibition of MASP-3-dependent Cleavage of pro-factor D with MASP-3 antibodies

An assay was carried out to détermine the inhibitory effect of représentative MASP-3 and MASP-1 mAbs, identified as described in Example 7, on MASP-3-dependent factor D cleavage as follows. Active, recombinant MASP-3 protein (80 ng) was pre-incubated with 1 pg of représentative mAbs D14, M3M1 and a control antibody (which binds specifically to MASP-1, but not to MASP-3) at room température for 15 minutes. Pro-factor D with an Nterminal Strep-tag (ST-pro-factor D-His, 70 ng) was added and the mixture was incubated at 37°C for 75 minutes. The reactions were then electrophoresed, blotted and stained with antifactor D as described above.

FIGURE 33 is a Western blot showing the partial inhibitory activity of the mAbs D14 and M3M1 in comparison to a control reaction containing only MASP-3 and ST-profactor D-His (no mAb; lane 1), as well as a control reaction containing a mAb obtained from the DTLacO library that binds MASP-1, but not MASP-3 (lane 4). As shown in FIGURE 33, in the absence of an inhibitory antibody, MASP-3 cleaves approximately 50% of profactor D into factor D (lane 1). The control MASP-1 spécifie antibody (lane 4) does not change the ratio of pro-factor D to factor D. In contrast, as shown in lanes 2 and 3, both mAb D14 and mAb M3M1 inhibit MASP-3-dependent cleavage of pro-factor D to factor D, resulting in a réduction in factor D generated.

Conclusions: These results demonstrate that MASP-3 mAbs D14 and M3M1 are able to inhibit MASP-3-dependent factor D cleavage. Improved inhibitory activity for mAbD14 and mAb M3M1 is expected following continued affinity maturation of these antibodies for MASP-3 binding using the tethered factors in the DTLacO system.

EXAMPLE 10

This Example demonstrates that MASP-3 deficiency prevents complement-mediated lysis of mannan-coated WT rabbit érythrocytes.

Background/Rationale:

As described in Examples 5 and 6 herein, the effect of MASP-2- and MASP-3deficient sérum on lysis of red blood cells from blood samples obtained from a mouse model

222 of PNH demonstrated the efficacy of MASP-2 inhibition and/or MASP-3 inhibition to treat subjects suffering from PNH, and also supported the use of inhibitors of MASP-2 and/or inhibitors of MASP-3 (including dual or bi-specific MASP-2/MASP-3 inhibitors) to ameliorate the effects of C3 fragment-mediated extravascular hemolysis in PNH subjects undergoing therapy with a C5 inhibitor such as eculizumab.

As described in this Example, C3b déposition experiments and hemolysis experiments were carried out in MASP-3 déficient sérum from additional 3MC patients, confirming the results obtained in Examples 5 and 6. In addition, experiments were carried out which demonstrated that addition of rMASP-3 to 3MC sérum was able to reconstitute C3b déposition and hemolytic activity.

Methods;

MASP-3-deficient sérum was obtained from three different 3MC patients as follows: 3MC Patient 1: contains an allele bearing a mutation that renders the exon encoding the MASP-3 serine protease domain dysfunctional, supplied along with the mother and father of the 3MC patient (both heterozygous for the allele bearing a mutation that renders the exon encoding the MASP-3 serine protease domain dysfunctional), 3MC Patient 2: Has C1489T (H497Y) mutation in exon 12 of MASP-1, the exon that encodes the serine protease domain of MASP-3, resulting in nonfunctional MASP-3, but functional MASP-1 proteins.

3MC Patient 3: Has a confirmed defect in the MASP-1 gene, resulting in nonfunctional MASP-3 and nonfunctional MASP-1 proteins.

Experiment #1; C3b Déposition Assay

An AP assay was carried out under traditional AP-specific conditions (BBS/ Mg^/EGTA, without Ca⁺⁺, wherein BBS= barbital buffered saline containing sucrose), as described in Bitter-Suermann et al., Eur. J. Immunol 11:291-295 (1981)), on zymosan-coated microtiter plates at sérum concentrations ranging from 0.5 to 25% and C3b déposition was measured over time.

Results;

FIGURE 34 graphically illustrâtes the level of AP-driven C3b déposition on zymosan-coated microtiter plates as a function of sérum concentration in sérum samples obtained from MASP-3-deficient (3MC), C4-deficient and MBL-deficient subjects. As shown in FIGURE 34, and summarized below in TABLE 15, MASP-3-deficient patient sera . 223 from Patient 2 and Patient 3 hâve residual AP activity at high concentrations (25%, 12.5%, 6.25% sérum concentrations), but a significantly higher AP50 (i.e., 8.2% and 12.3% of sérum needed to achieve 50% of maximum C3 déposition).

FIGURE 35A graphically illustrâtes the level of AP-driven C3b déposition on zymosan-coated microtiter plates under “traditional” AP-specific conditions (i.e., BBS/EGTA/Mg⁺⁺ without Ca⁺⁺) as a function oftime in 10% human sérum samples obtained from MASP-3 déficient, C4-deficient and MBL-deficient human subjects.

TABLE 15 below summarizes the AP50 results shown in FIGURE 34 and the halftimes for C3b déposition shown in FIGURE 35A.

TABLE 15: Summary of Results shown in FIGURES 34 and 35A
Sérum type	APso (%)	T1/2 (min)
Normal	4.5	26.3
MBL-deficient (MBL-/-)	5.7	27.5
C4-deficient (C4-/-)	5.1	28.6
3 MC (Patient 3)	8.2	58.2
3 MC (Patient 2)	12.3	72.4

Note: In BBS/Mg⁺⁺/EGTA buffer, the lectin pathway-mediated effects are déficient due to absence of Ca⁺⁺ in this buffer.

Experiment #2: Analysis of pro-factor D cleavage in 3MC patient sera by Western

Blot

Methods: Sérum was obtained from 3MC patient #2 (MASP-3 (-/-), MASP-1 (+/+)) and from 3MC patient #3 (MASP-3 (-/-), MASP-1 (-/-)). The patient sera, along with sera from normal donors (W), were separated by SDS-polyacrylamide gel and the resolved 20 proteins were blotted to a polyvinylidine fluoride membrane. Human pro-factor D (25,040 Da) and/or mature factor D (24,405 Da) were detected with a human factor D-specific antibody.

Results: The results of the Western blot are shown in FIGURE 35B. As shown in FIGURE 35B, in the sera from normal donors (W), the factor D antibody detected a protein 25 of a size consistent with mature factor D (24,405 Da). As further shown in FIGURE 35B, the factor D antibody detected a slightly larger protein in the sera from 3MC patient #2 (P2) and

224

3MC patient #3 (P3), consistent with the presence of pro-factor D (25,040 Da) in these 3MC patients.

Experiment #3: Wieslab Complément Assays with 3MC patient sera

Methods: Sera obtained from 3MC patient #2 (MASP-3 (-/-), MASP-1 (+/+)) and from 3MC patient #3 (MASP-3 (-/-), MASP-1 (-/-)) were also tested for classical, lectin and alternative pathway activity using the Wieslab Complément System Screen (EuroDiagnostica, Malmô, Sweden) according to the manufacturées instructions. Normal human sérum was tested in paralle! as a control.

Results: FIGURE 35C graphically illustrâtes the results of the Weislab classical, lectin and alternative pathway assays with plasma obtained from 3MC patient #2, 3MC patient #3, and normal human sérum. As shown in FIGURE 35C, under conditions of the Wieslab assay, the classical, alternative, and MBL (lectin) pathways are ail functional in the normal human sérum. In sérum from 3MC patient #2 (MASP-3 (-/-), MASP-1 (+/+)), the classical pathway and lectin pathway are functional, however there is no détectable alternative pathway activity. In sérum from 3MC patient #3 (MASP-3 (-/-), MASP-1 (-/-)), the classical pathway is functional, however there is no détectable lectin pathway activity and no détectable alternative pathway activity.

The resuit in FIGURES 35B and 35C further support our understanding of the rôle of MASP-1 and MASP-3 in the LEA-I and LEA-2 pathways. Specifïcally, the absence ofthe alternative pathway with a nearly fully functional lectin pathway in sérum from Patient 2, who lacks only MASP-3, confirms that MASP-3 is essential for activation of the alternative pathway. Sérum from Patient 3, who lacks both MASP-1 and MASP-3, has lost the ability to activate the lectin pathway as well as the alternative pathway. This resuit confions the requirement of MASP-1 for a functional LEA-2 pathway, and is consistent with Example 7, .

and the literature demonstrating that MASP-1 activâtes MASP-2. The apparent inability of both sera to activate pro-factor D is also consistent with the data described in Example 9 demonstrating that MASP-3 cleaves pro-factor D. These observations are consistent with the LEA-1 and LEA-2 pathways as diagrammed in Figure 1.

Experiment #4; Hemolysis assay testing mannan-coated rabbit érythrocytes for lysis in the presence of human normal or 3MC sérum (in the absence of Ca⁴^)

Methods:

225

Préparation of rabbit RBC in the absence of Ca** (i.e., by using EGTA)

Rabbit whole blood (2 mL) was split into two 1.5 mL eppendorf tubes and centrifuged for 3 minutes at 8000 rpm (approximately 5.9 rcf) in a refrigerated eppendorf centrifuge at 4°C. The RBC pellet was washed three times after re-suspending in ice-cold BBS/ Mg**/Ca** (4.4 mM barbituric acid, 1.8 mM sodium barbitone, 145 mM NaCl, pH 7.4, 5 mM Mg**, 5 mM Ca**). After the third wash, the pellet was re-suspended in 4 mL BBS/ Mg**/Ca**. The érythrocytes were pelleted and the RBCs were washed with BBS/0.1% gelatin/Mg**/Ca** as described above. The RBCs suspension was stored in BBS/0.1% gelatin/ Mg**/Ca**at 4°C. Then, 100 pL of suspended RBCs were diluted with 1.4 mL water and spun down at 8000 rpm (approximately 5.9 rcf) for 3 minutes and the OD of the supematant was adjusted to 0.7 at 541nm (an OD of 0.7 at 541nm corresponds to approximately 10⁹ erythrocytes/ml). After that, 1 mL of the resuspended RBCs at OD 0.7 were added to 9 ml of BBS/Mg**/EGTA in order to achieve a concentration of 10⁸ erythrocytes/ml. Dilutions of the test sera or plasma were prepared in ice-cold BBS, Mg**, EGTA and 100 pL of each sérum or plasma dilution was pipetted into the corresponding well of round-bottom plate. 100 pL of appropriately diluted RBC (10⁸ erythrocytes/ml) were added to each well. Nano-water was used to produce the positive control (100% lysis), while a dilution with BBS/Mg**/EGTA without sérum or plasma was used as a négative control. The plate was then incubated for 1 hour at 37°C. The round bottom plate was spun down at 3750 rpm for 5 minutes. Then, 100 pL of the supematant from each well was transferred into the corresponding wells of a flat-bottom plate and OD was read at 415-490 nm.

Results:

FIGURE 36 graphically illustrâtes the percent hemolysis (as measured by hemoglobin release of lysed rabbit érythrocytes into the supematant measured by photometry) of mannan-coated rabbit érythrocytes over a range of sérum concentrations in sérum from normal subjects and from two 3MC patients (Patient 2 and Patient 3), measured in the absence of Ca**. As shown in FIGURE 36, it is demonstrated that MASP-3 deficiency reduces the percentage of complement-mediated lysis of mannan-coated érythrocytes as compared to normal human sérum. The différences between the two curves from the normal human sérum and the two curves from the 3MC patients is significant (p=0.013, Friedman test).

TABLE 16 below summarizes the AP50 results shown in FIGURE 36.

226

TABLE 16: Summary of Results shown in FIGURE 36

Sérum type	AP50 (%)
Normal human sérum #1	7.1
Normal human sérum #2	8.6
3MC Patient #2	11.9
3MC Patient #3	14.3

It is noted that when the sérum samples shown in TABLE 16 were pooled, the AP50 value for normal human sérum = 7.9 and the AP50 value for 3MC sérum = 12.8 (p=0.031, Wilcox matched-pairs signed rank test).

Experiment #5: Reconstitution of human 3MC sérum by recombinant MASP-3 restores AP-driven C3b déposition on zymosan coated plates .

Methods:

An AP assay was carried out under traditional AP-specific conditions (BBS/Mg⁺⁺/EGTA, without Ca⁺⁺, wherein BBS=barbital buffered saline containing sucrose), as described in Bitter-Suermann et al., Eur. J. Immunoî 11:291-295 (1981)), on zymosancoated microtiter plates in the following sérum samples (1) 5% human sérum from 3MC Patient #2 with full length active rMASP-3 added in at a range of 0 to 20 pg/mL; (2) 10% human sérum from 3MC Patient #2 with full length active rMASP-3 added in at a range of 0 to 20 pg/mL; and (3) 5% human sérum from 3MC Patient #2 with inactive rMASP-3A (S679A) added in at a range of 0 to 20 pg/mL.

Results:

FIGURE 37 graphically illustrâtes the level of AP-driven C3b déposition on zymosan-coated microtiter plates as a function of the concentration of rMASP-3 protein added to sérum samples obtained from human 3MC Patient #2 (MASP-3-deficient). As shown in FIGURE 37, active recombinant MASP-3 protein reconstitutes AP-driven C3b déposition on zymosan-coated plates in a concentration-dependent manner. As further shown in FIGURE 37, no C3b déposition was observed in the 3MC sérum containing inactive rMASP-3 (S679A).

227

Experiment #6: Reconstitution of human 3MC sérum by recombinant MASP-3 restores hemolytic activity in 3MC patient sérum

Methods:

A hemolytic assay was carried out using rabbit RBC using the methods described above in Experiment #2 with the following test sera at a range of 0 to 12% sérum: (1) normal human sérum; (2) 3MC patient sérum; (3) 3MC patient sérum plus active full length rMASP3 (20 pg/ml); and (4) heat-inactivated human sérum.

Results:

FIGURE 38 graphically illustrâtes the percent hemolysis (as measured by hemoglobin release of lysed rabbit érythrocytes into the supernatant measured by photometry) of mannan-coated rabbit érythrocytes over a range of sérum concentrations in (1) normal human sérum; (2) 3MC patient sérum; (3) 3MC patient sérum plus active fui! length rMASP-3 (20 pg/ml); and (4) heat-inactivated human sérum, measured in the absence of Ca⁺⁺. As shown in FIGURE 38, the percent lysis of rabbit RBC is significantly increased in 3MC sérum including rMASP-3 as compared to the percent lysis in 3MC sérum without rMASP-3 (p=0.0006).

FIGURE 39 graphically illustrâtes the percentage of rabbit érythrocyte lysis in 7% human sérum from 3MC Patient 2 and from 3MC Patient 3 containing active rMASP-3 at a concentration range of 0 to 110 pg/ml in BBS/Mg^/EGTA. As shown in FIGURE 39, the percentage of rabbit RBC lysis is restored with the amount of rMASP-3 in a concentrationdependent manner up to 100% activity.

Experiment #7: Sérum of MASP-3 déficient (3MC) patient has functional MASP-2 if MBL is présent '

Methods:

A C3b déposition assay was carried out using Mannan-coated ELISA plates under to examine whether 3MC sérum is déficient in LEA-2. Citrate plasma was diluted in BBS buffer in serial dilutions (starting at 1:80, 1:160, 1: 320, 1:640, 1:1280, 1:2560) and plated on Mannan-coated plates. Deposited C3b was detected using a chicken anti-human C3b assay. LEA-2 driven C3b déposition (the plasma dilutions are to high for the AP and LEA-1 to work) on Mannan-coated ELISA plates was evaluated as a function of human sérum concentration in sérum from a normal human subject (NHS), from two 3MC patients (Patient 2 and Patient 3), from the parents of Patient 3 and from a MBL-deficient subject.

228

Results:

FIGURE 40 graphically illustrâtes the level of LEA-2-driven (i.e., MASP-2-driven) C3b déposition on Mannan-coated ELISA plates as a function of the concentration of human sérum diluted in BBS buffer, for sérum from a normal human subject (NHS), from two 3MC patients (Patient 2 and Patient 3), from the parents of Patient 3 and from a MBL-deficient subject. These data indicate that Patient 2 is MBL sufficient. However, Patient 3 and the mother of Patient 3 are MBL déficient, and therefore their sérum does not deposit C3b on Mannan via LEA-2. Replacement of MBL in these sera restores LEA-2 mediated C3b déposition in the sérum of Patient 3 (who is homozygous for the SNP leading to MASP-3 deficiency) and his mother (who is heterozygous for the mutant MASP-3 allele) (data not shown). This finding demonstrates that 3MC sérum is not déficient in LEA-2, but rather appears to hâve functional MASP-2.

Overall summary and Conclusions:

These results demonstrate that MASP-3 deficiency in human sérum results in loss of AP activity, as manifested in reduced C3b déposition on zymosan-coated wells and reduced rabbit érythrocyte lysis. The AP can be restored in both assays by supplementing the sera with functional, recombinant human MASP-3.

EXAMPLE 11

This Example demonstrates that a chimeric mouse V region/human IgG4 constant région anti-human MASP-3 monoclonal antibody (mAb M3-1, also referred to as mAb 13B1 ) is a potent inhibitor of MASP-3-mediated Alternative Pathway Complément (APC) Activation.

Methods:

Génération of a chimeric mouse V region/human IgG constant région antihuman MASP-3 monoclonal antibody (mAb M3-1)

A murine anti-human MASP-3 inhibitory antibody (mAb M3-1) was generated by immunizing MASP-1/3 knockout mice with the human MASP-3 CCP1-CCP2-SP domain (aa 301-728 of SEQ ID NO:2) (see also Example 14). Briefly described, splénocytes from the

229 immunized mice were fused with P3/NSl/l-Ag4-l and supematants from resulting hybridoma clones were screened for the production of antibodies that bind to human MASP-3 and for the ability to block MASP-3-mediated cleavage of complément pro-factor D (proCFD) to factor D (CFD). Monoclonal antibody (mAb) variable régions were isolated by RTPCR, sequenced and cloned into human IgG4 expression vectors. Chimeric monoclonal antibodies were expressed in transiently transfected HEK293T cells, purified and tested for binding affinity to mouse and human MASP-3 and for the ability to inhibit MASP-3mediated cleavage of pro-CFD to CFD.

The MASP-3 inhibitory monoclonal antibody M3-1 (13B1) comprises a heavy chain variable région (VH) set forth as SEQ ID NO:30 and a light chain variable région (VL) set forth as SEQ ID NO:45. The sequences of the variable régions of the M3-1 monoclonal antibody are provided below:

Heavy Chain Variable Région

Presented below is the heavy chain variable région (VH) sequence for mAb M3-1. The Kabat CDRs (31-35 (Hl), 50-65 (H2) and 95-102 (H3) are underlined, which correspond to amino acid residues 31-35 (Hl), 50-66 (H2) and 99-102 (H3) of SEQ ID NO:30.

mAb M3-1 heavy chain variable région (VH) (SEQ ID NO:3Q)

QVQLKQSGAELMKPGASVKLSCKATGYTFTGKW1EWVKQRPGHGLEWIGEILPGTGSTNYN EKFKGKATFTADSSSNTAYMOLSSLTTEDSAMYYCLRSEDVWGTGTTVTVSS

Light Chain Variable Région

Presented below is the light chain variable région (VL) sequence for mAb M3-1. The Kabat CDRs (24-34 (Hl), 50-56 (H2) and 89-97 (H3) are underlined, which correspond to amino acid residues 24-40 (Ll); 56-62 (L2) and 95-102 (L3) of SEQ ID NO:45. These régions are the same whether numbered by the Kabat or Chothia system.

mAb M3-1 light chain variable région (VL) (SEQ ID NO:45)

230

DIVMTOSPSSLAVSAGEKVTMSCKSSOSLLNSRTRKNYLAWYOOKPGOSPKLLIYWASTRES GVPDRFTGSGSGTDFTLTISSVOAEDLAVYYCKOSYNIPTFGGGTKLEIKR mAb M3-1 VH CDRs

VHCDR1: GKWIE (SEQ ID NO:84)

VHCDR2: EILPGTGSTNYNEKFKG (SEQ IDNO:86)

VHCDR3: SEDV (SEQ ID NO:88) mAb M3-1 VL CDRs

VLCDR1: KSSQSLLNSRTRKNYLA (SEQ ID NO:142)

VLCDR2: WASTRES (SEQ ID NO: 144)

VLCDR3: KQSYNIPT (SEQ IDNO:161)

As shown above, MASP-3 monoclonal antibody M3-I comprises (a) a heavy chain variable région comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable région comprising (i) VLCDR1 comprising SEQ ID NO: 142, (ii) VLCDR2 comprising SEQ ID NO: 144 and (iii) VLCDR3 comprising SEQ ID NO: 161.

Binding of mAb M3-1 to recombinant forms of human and mouse MASP-3

A monovalent Fab version of M3-1 was tested for binding to recombinant, full-length human and mouse MASP-3 protein in an ELISA experiment. Binding affinity déterminations were made by coating 96-weIl plates with an anti-MASP-3 capture antibody that binds the protein from multiple species. The capture antibody has been shown to bind the CCP1-CCP2 région of MASP-1 and MASP-3. Full-length versions of human and mouse protein were immobilized on ELISA plates coated with the capture antibody, and varying concentrations of M3-I Fab were allowed to bind to the target protein in separate wells. Bound M3-I was detected using an anti-kappa light chain antibody that is conjugated to HRP (Novus Biologicals NBP1-75064), and was visualized with the TMB substrate reagent set (BD

231

Biosciences 555214).

FIGURE 41 graphically illustrâtes a représentative example of a binding experiment that was performed with human MASP-3 in which the M3-1 Fab (also referred to as 13B1) shows an apparent binding affinity (EC50) of about 0.117 nM to the human protein.

FIGURE 42 graphically illustrâtes a représentative example of a binding experiment that was performed with mouse MASP-3 in which the M3-1 Fab shows an apparent binding affinity (EC50) of about 0.214 nM to the mouse protein.

These results demonstrate that mAb M3-1 ( 13B1 ) has a high binding affinity for both human and mouse MASP-3.

Démonstration that mAb M3-1 is capable of inhibiting Alternative Pathway Complément (APC) Activation and Measurement of the in vitro potency of mAb M3-1

As described in the présent disclosure, it has been determined that MASP-3 is a key regulator of the APC, at least in part due to its requirement for the activation of CFD, a central APC enzyme. As also described in the présent disclosure, MASP-3 circulâtes in the body at a relatively low concentration and has a slow catabolic rate, allowing for long-lasting inhibition of the pro-inflammatory pathway through intravenous, subcutaneous and oral routes of MASP-3 antibody administration. The following experiment was carried out to détermine the efficacy of mAb M3-1 for inhibiting MASP-3-mediated CFD maturation and inhibition of APC in human sérum. Normal human sérum contains predominantly active or processed (i.e., mature) CFD, so we performed experiments in which CFD-depleted human sérum (Complément Technology A336) was reconstituted with a recombinant, unprocessed form of CFD (pro-CFD). Thus, in this experimental System, APC activation requires the processing of pro-CFD into active CFD.

The APC was induced by the addition of zymosan particles, which function as an activating surface for complément déposition. Varying concentrations of mAb M3-1 were added to the sérum prior to the addition of recombinant pro-CFD and zymosan. The mixtures were incubated at 37°C for 75 minutes, and the APC activity was measured by the flow

232 cytométrie détection of complément factor Bb (Quidel A252) on the surface of the zymosan particles.

FIGURE 43 graphically illustrâtes the level of complément factor Bb déposition on zymosan particles (determined by flow cytométrie détection measured in MFI units) in the 5 presence of varying concentrations of mAb M3-1 in CFD-depleted human sérum. As shown in FIGURE 43, mAb M3-1 shows potent inhibition of the APC in 10% human sérum, with an IC50 of 0.311 nM in this experimental example.

These results demonstrate that MASP-3 plays a key rôle in APC activation in an in vitro model in human sérum, and further demonstrate that mAb M3-1 is a potent inhibitor of 10 the APC.

Inhibition of the APC by mAb M3-1 in vivo:

In order to détermine the efficacy of mAb M3-1 for inhibiting the APC in vivo, a group of mice (n = 4) received a single intravenous tail vein injection of 10 mg/kg mAb M31. Blood collected from the animais was used to préparé sérum, providing a matrix for the 15 flow cytométrie assessment of APC activity in an ex vivo assay measuring the level of C3 (also C3b and iC3b) déposition on zymosan particles. Sérum prepared from blood harvested at a pre-dose timepoint and multiple post-dose time points (96 hrs, 1 week, and 2 weeks) was diluted to 7.5% and zymosan particles were added to induce the APC. Antibody-treated mice were compared to a group of control mice (n = 4) that were given a single intravenous dose of 20 vehicle.

FIGURE 44 graphically illustrâtes the level of C3 déposition on zymosan particles at various time points after a single dose of mAb M3-1 (10 mg/kg i.v.) in wild-type mice. As shown in FIGURE 44, in the pre-dose time point the two conditions show comparable levels of APC activity. At 96 hours and the two later time points, the mAb M3-1 treated group 25 shows essentially complété APC inhibition, while the APC activity of the vehicle-treated group remains unabated. As shown in FIGURE 44, a single dose of mAb M3-1 administered intravenously to mice led to near-complete ablation of systemic APC activity for at least 14 days.

233 '

These results demonstrate that mAb M3-1 is a potent inhibitor of the APC in vivo in a mouse model.

EXAMPLE 12

This Example demonstrates that chimeric mouse V region/human lgG4 constant région anti-human MASP-3 monoclonal antibody (mAb M3-1, also referred to as mAb 13B1 ) provides a clear benefit to survival of red blood cells lacking Crry in a mouse model associated with paroxysmal noctumal hemoglobinuria (PNH).

Methods:

The chimeric mouse V region/human IgG4 constant région anti-human MASP-3 monoclonal antibody (mAb M3-1) was generated as described in Example 11 and Example 14. As further described in Example 11, it was determined that mAb M3-1 is a potent inhibitor of the APC in a mouse model in vivo. This Example describes the analysis of mAb M3-1 for efficacy in a murine model associated with PNH.

Analysis of mAb M3-1 for efficacy in a murine model associated with PNH

In a mouse model associated with PNH, red blood cells (RBCs) from Crry-defïcient mice lacking the major cell surface repressor of the APC in mouse were obtained for use as donor cells. RBCs obtained from a wild-type (WT) donor mouse were run in parallel. These donor RBCs were differentially labeled with fluorescent lipophilie dyes (Sigma): WT (red), and Crry- (green). In two different experiments, the labelled WT and Crry- donor cells were mixed 1:1 and injected intravenously into wild-type récipient mice and percent WT and Crrydefïcient RBC survival (relative to the early time point) in the récipient mice were determined by flow cytométrie assessment of 20,000 live cell events. In the first experiment, multiple pre-dose treatments of mAb M3-1 antibody were given, and the effect ofthe mAb M3-1 was compared to that of another inhibitory complément antibody mAb BB5.1 (available from Hycult Biotech), which is a C5 inhibitory antibody that has shown efficacy in multiple mouse studies (Wang et al., PNAS vol 92:8955-8959, 1995; Hugen et al., Kidney Int 71(7):646-54,

2007). Administration of a C5 inhibitor is the current standard of treatment for human

234 patients with PNH. In the second experiment, a single pre-treatment dose of mAb M3-1 was evaluated.

In the first experiment, three different groups of mice (n = 4 per condition) were assessed: vehicle-treated condition, mAb M3-1-treated condition, and mAb BB5.1 (mAb 5 blocking mouse C5)-treated condition. Labeled cells were injected into mice on “day 0”, and multiple doses of both M3-1 and BB5.1 were administered as follows: mAb M3-1 was administered intravenously (10 mg/kg) on days -11, -4, -1, and +6. The mAb BB5.1 was administered by intraperitoneal injection (40 mg/kg) on days -1, +3, +6, and +10. The vehicle treatment followed the same dosing schedule as mAb M3-1.

FIGURE 45 graphically illustrâtes the percent survival of donor RBCs (WT or Crry-) over a period of 14 days in WT récipient mice treated with mAb M3-1 (10 mg/kg on days 11, 04, -1 and +6), mAb BB5.1 treated, or vehicle treated mice. As shown in FIGURE 45, compared to WT RBCs that showed survival typical of RBCs in mice in the vehicle-treated animais, Crry-deficient RBCs had rapid clearance (more than 75% cleared within 24 hours).

Treatment of mice with mAb BB5.1 provided no improvement over vehicle treatment in Crry-deficient RBC survival. In contrast, mAb M3-1 treatment caused a dramatic improvement of Crry-deficient RBC survival over both mAb BB5.1 and vehicle-treated animais. The protective effect of mAb M3-1 was observed throughout the duration of the experiment.

In the second study, differentially labeled WT (red)- and Crry- (green) RBCs were evaluated in two different groups of WT mice (n = 4 per condition): vehicle-treated and mAb M3-1-treated. A single dose of either vehicle or antibody (20 mg/kg) was given to the récipient mice by intravenous administration six days (day -6) before the labeled donor cells were injected into the récipient mice. The labeled donor RBCs were then analyzed for 25 percent survival in the récipient mice at incrémental time points after injection over a 16-day period.

FIGURE 46 graphically illustrâtes the percent survival of donor RBCs (WT or Crry-) over a period of 16 days in WT récipient mice treated with a single dose of mAb M3-1 (20 mg/kg on day -6) or vehicle-treated mice. As shown in FIGURE 46, a single pre-treatment

235 dose of mAb M3-1 demonstrated improved survival of Crry- RBCs as compared to the survival of Crry- RBCs in vehicle-treated mice. At 96 hours post injection, approximately 90% of the vehicle-treated WT RBCs survived under the control conditions, whereas only 5% of the Crry- RBCs survived in the vehicle-treated WT mice. In contrast to the vehicle-treated mice, 40% of the Crry- RBCs survived in the mice treated with mAb M3-1.

Taken together, these results demonstrate that the MASP-3 inhibitory antibody mAb M3-1 provides a clear benefit to survival of RBCs lacking Crry, a key surface complément inhibitor in a mouse model associated with PNH.

EXAMPLE 13

This Example describes a study demonstrating that a chimeric MASP-3 inhibitory monoclonal antibody (mAb M3-1, also referred to as mAb 13B1) reduces clinical scores in collagen antibody-induced arthritis (CAIA), a murine model of rheumatoid arthritis (RA).

Background/Rational e:

CAIA is a well-established animal model of arthritis. In additional to providing insight into RA, the pathology of the CAIA model has an established connection with the APC. Banda and coworkers hâve demonstrated improved outcomes in the CAIA model in mice carrying defîciencies in components of the APC, such as factor B and factor D (Banda et al., J. Immunol vol 177:1904-1912, 2006 and Banda et al., Clinical & Exp Imunol vol 159:100-108, 2009). APC mouse knock-outs demonstrate lower arthritis (disease) scores, lower incidence, and less C3 and factor H déposition in synovium and surrounding tissues relative to WT Controls. Additionally, disease activity scores, complément C3 tissue déposition in the joint, and histopathologie injury scores were markedly decreased in MASP1/3 knock-out mice (Banda et al., J Immunol vol 185:5598-5606, 2010). Therefore, the MASP-3 inhibitory antibody mAb M3-1 was analyzed for efficacy in the CAIA.

Methods:

The chimeric MASP-3 monoclonal antibody (mAb M3-1) was generated as described in

236

Example 11 and Example 14. As further described in Example 11, it was determined that mAb M3-1 is a potent inhibitor of the APC in a mouse model in vivo.

mAb M3-1 was tested in the CAIA model as follows. Wild-type mice (n=7) were injected intravenously with 3 mg of a cocktail of anti-collagen antibodies on day 0. The mice were 5 dosed intraperitoneally with E. coli lipopolysaccharide (LPS) (25 pg/mouse) on day +3. As described in Nandakumar et al. (Am J Pathol 163(5):1827-1837, 2003), arthritis typically · occurs in this model on days +3 through +10. Terminal sérum samples were collected on day +14. mAb M3-1 (5 mg/kg and 20 mg/kg) was dosed on days -12, -5, +1 and day +7. Vehicle (PBS) was injected as a négative control.

Clinical scores were evaluated for each mouse on ail 4 paws on study days 0 through 14 using the following scoring standards:

0= normal

1= 1 hind and/or fore paw joint affected or minimal diffuse erythema and swelling 2= 2 hind and/or fore paw joints affected or mild diffuse erythema and swelling

3= 3 hind and/or fore paw joints affected or moderate diffuse erythema and swelling

4= marked diffuse erythema and swelling, or 4 digit joints affected

5= severe diffuse erythema and severe swelling of entire paw, unable to flex digits.

The incidence = % mice within a treatment group showing arthritic symptoms was 20 also determined.

The results are shown in FIGURE 47 (clinical scores) and FIGURE 48 (incidence of arthritis). FIGURE 47 graphically illustrâtes the clinical scores ofthe mice treated with mAb M3-1 (5 mg/kg or 20 mg/kg) or vehicle over a 14-day time course. FIGURE 48 graphically illustrâtes the percent incidence of arthritis of the mice treated with mAb M3-1 (5 mg/kg or 25 20 mg/kg) or vehicle over a 14-day time course. As shown in FIGURE 47, mAb M3-1 demonstrates a clear therapeutic benefit for both endpoints starting at day 5 and lasting throughout the duration of the study. As shown in FIGURE 48, while the incidence of disease reached 100% in the vehicle-treated animais, two-thirds ofthe animais in the 5 mg/kg mAb M3-1 condition remained disease-free. Additionally, only one ofthe animais (i.e., only one in a total n = 7) demonstrated any arthritic symptoms in the 20 mg/kg mAb M3-1 condition.

237

The results of this study demonstrate that the MASP-3 inhibitory antibody mAb M3-1 provides a clear therapeutic benefit in the CAIA model, a well-established murine model of rheumatoid arthritis (RA) and a model strongly linked to APC activation. As shown in Example 11, a single dose of mAb M3-1 administered intravenously to mice led to nearcomplete ablation of systemic APC activity for at least 14 days. As shown in this Example, in the animal model induced by administration of auto-antibodies against mouse connective tissue, mAb M3-1 reduced the incidence and severity of clinical arthritis scores in a dosedependent fashion. Compared to control-treated animais, mAb M3-1 reduced the incidence and severity of the disease by approximately 80% at the highest dose tested. Therefore, it is expected that administration of a MASP-3 inhibitory antibody, such as mAb M3-1 will be an effective therapy in patients suffering from arthritis, such as rheumatoid arthritis, osteoarthritis, juvénile rheumatoid arthritis, infection-related arthritis, psoriatic arthritis, as well as ankylosing spondylitis and Bechcet’s disease.

EXAMPLE 14

This Example describes the génération of high affinity anti-human MASP-3 inhibitory antibodies.

Background/Rationale:

A limited number of antibodies spécifie for MASP-3 hâve been described (Thiel et al., Mol. Immunol. 43:122, 2006; Moller-Kristensen et al., Int. Immunol. 19:141, 2006; Skjoedt et al., Immunobiol 215:921, 2010). These antibodies were useful for détection assays such as Western blotting, immunoprécipitation, and as capture or détection reagents in ELISA assays. However, the antibodies described in Thiel et al., 2006, Moller-Kristensen et al., 2006 and Skjoedt et aL, 2010 hâve not been found to inhibit MASP-3 catalytic activity.

MASP-3 antibodies were also generated previously, as described in Example 7 herein (also published as Example 15 in WO2013/192240) by screening a chicken antibody library in a modified DT40 cell line, DTLacO, for MASP-3 binding molécules. These antibodies bound to human MASP-3 in the nanomolar range with an EC50 between 10 nM and 100 nM and partially inhibited cleavage of pro-CFD by MASP-3.

238

This Example describes the génération of anti-human MASP-3 inhibitory antibodies with unusually strong binding affinity (i.e., subnanomolar binding affinity, ranging from <500 pM to 20 pM). The antibodies described in this Example specifically bind to human MASP-3 with high affinity (e.g., < 500 pM), inhibit Factor D maturation, and do not bind to human MASP-1 (SEQ ID NO:8).

Methods:

1. Génération of chimeric mouse V region/human IgG constant région anti-human MASP-3 monoclonal antibodies

Seven to fourteen-week old C57BL/6, MASP-1/3 knockout mice were immunized with either the human MASP-3 CCP1/CCP2/SP polypeptide (amino acid residues 299-728 of SEQ ID NO:2) including a StrepTag II epitope tag on the N-terminus; or were immunized with the human MASP-3 SP domain (amino acid residues 450-728 of SEQ ID NO:2), including StrepTaglI on the N-terminus, using the Sigma Adjuvant System (Sigma-Aldrich, St Louis, MO). The mice were injected intraperitoneally with 50 pg of immunogen per mouse. The immunized mice were boosted 14 days later with additional immunogen in adjuvant. Thereafter, for several weeks, the mice were boosted every 14 to 21 days with immunogen in PBS. Sérum samples from the mice were periodically prepared from tail bleeds and tested by ELISA for the presence of antigen-specific antibodies. Mice with a significant antibody titer received a pre-fusion immunogen boost in PBS four days prior to splenic fusion. Three days prior to the fusion, the mice were treated subcutaneously at the base of the tail with 50 pg of a anti-CD40 agonist mAb in PBS (R&D Systems, Minneapolis, MN) to increase B cells numbers (see Rycyzyn et ai., Hybridoma 27:25-30, 2008). The mice were sacrificed and the spleen cells were harvested and fused to a selected murine myeloma cell line P3/NSI/1-AG4-1 (NS-1) (ATCC No. TIB18) using 50% polyethylene glycol or 50% polyethylene glycol plus 10% DMSO. The fusions generated hybridoma cells which were plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin and thymidine) medium to inhibit prolifération of non-fused cells, myeloma hybrids and spleen hybrids. After hybridoma sélection, the culture supernatants were assayed for MASP-3 binding (ELISA) and inhibition of pro-Factor D activation. The positive hybridomas were

239 identified and subcloned by serial dilution methods.

TABLE 17: Summary of Fusion Experiments
Fusion	Immunogen: Human MASP-3	Total hybridomas	MASP-3 Binding hybridomas	MASP-3 Functional hybridomas
1	SP	434	38	10
2	SP	279	13	0
3	CCP1/CCP2/SP	348	40	2
4	CCP1/CCP2/SP	319	60	2
5	CCP1/CCP2/SP	651	152	1
6	CCP1/CCP2/SP	1297	ND	1

Note: “ND” means this fusion was only screened for functional inhibition of pro-CFD activation.

Results:

As shown in TABLE 17, a total of 3328 hybridomas from immunized MASP1/3 KO mice were screened, of which >303 were found to bind to MASP-3 and of which 16 were found to bind to MASP-3 and to inhibit pro-CFD activation. mAb M3-1 (13B1 ) described in Example 10 11 is one of the 16 functional MASP-3 inhibitory antibodies described in TABLE 17. As described in Example 15, it was determined that ail 16 functional MASP-3 inhibitory antibodies bind to human MASP-3 with unusually strong binding affinity (< 500 pM).

Discussion:

This Example describes the génération of antibodies that inhibit human MASP-3 with unusually strong binding affinity (i.e., subnanomolar binding affinity, ranging from <500 pM to 20 pM) by immunizing MASP1/3 knockout mice. The antibodies described in this Example specifically bind to human MASP-3 with high afïinity (e.g., < 500 pM), inhibit Factor D maturation, and do not bind to human MASP-1. As described herein, the amino acid sequences of human, mouse and chicken MASP-3 revealed that the SP domain of MASP-3 is highly conserved, especially in the active site (see FIGURES 4 and 5). It is likely that the ability to generate MASP-3 inhibitory antibodies with unusually strong binding

240 affinity in MASP1/3 KO mice, as described in this example, is due in part to avoidance of immunological tolérance that may hamper the génération of highly potent MASP-3 catalytic site-specific antibodies in wild-type animais.

EXAMPLE 15

This Example describes the cloning and sequence analysis of high affinity anti-human MASP-3 inhibitory mAbs.

Methods:

Cloning and purification of recombinant antibodies:

The heavy chain and light chain variable régions were cloned from the hybridomas described in Examples 11 and 14 using RT-PCR and were sequenced. Mouse-human chimeric mAbs consisting of the mouse mAb variable régions fused to the human IgG4 heavy chain (SEQ ID NO:311) and kappa light chain (SEQ ID NO:313) constant régions were produced as recombinant proteins in Expi293F cells. The IgG4 constant hinge région (SEQ ID NO:311) contains the stabilizing S228P amino acid substitution. In one embodiment, the chimeric mAbs were fused to the human IgG4 constant hinge région (SEQ ID NO:3I2) which contains the S228P amino acid substitution and also a mutation that promûtes FcRn interations at low pH.

The sequences ofthe heavy chain variable régions and light chain variable régions are shown in FIGURES 49A and 49B, respectively (“SIN” = “SEQ ID NO:” in FIGURE 49A and FIGURE 49B), and are included below. The complementarity régions (CDRs) and framework régions (FRs) of each are provided in TABLES 18-22 below.

FIGURE 50A is a dendrogram of the VH régions of high affinity anti-human MASP-3 inhibitory mAbs generated in MASP1/3 KO mice. FIGURE 50B is a dendrogram of the VL régions of high affinity anti-human MASP-3 inhibitory mAbs generated in MASP 1/3 KO

241 mice. As shown in FIGURES 50A and 50B, several groups of related antibodies were identified.

Presented below is the heavy chain variable région (VH) sequence for each high affinity

MASP-3 inhibitory antibody. The Kabat CDRs are underlined.

Heavy Chain Variable Régions:

4D5 VH: SEQ ID NO:24

QVQLKQSGPELVKPGASVKLSCKASGYTFTTDDTN WVKQRPGQGLEWIGWIYPRDD RTKYNDKFKDKATLTVDTSSNTAYMDLHSLTSEDSAVYFCSSLEDTYWGOGTLVAV

SS

1F3 VH: SEQ ID NO:25

QVQLKQSGPELVKPGASVKLSCKASG YTFTSNDIN WVKORPGOGLEWIGWIYPRDG SIKYNEKFTDKATLTVDVSSSTAYMELHSLTSEDSAVYFCSGVEDSYWGOGTLVTVS

S

4B6 VH: SEQ ID NO:26

QVQLKQSCiPELVKPGASVKLSCKASGYTFTSNDINWVKORPGOGLEWIGWIYPRDG TTKYNEEFTDKATLTVDVSSSTAFMELHSLTSEDSAVYFCSSVEDSYWGOGTLVTVS

S

IA 10 VH: SEQ ID NO:27

QVQLKQSGPELVKPGASVKLSCKASGYTFTSNDINWVKORPGOGLEWIGWIYPRDG TTKYNEKFTDKATLTVDVSSSTAFMELHRLTSEDSAVYFCSSVEDSYWGOGTLVTVS

S

10D12 VH: SEP ID NO:28

Q1QLVQSGPELKKPGETVKISCKASGY1FTSYGMSWVROAPGKGLKWMGWINTYSG VPTYADDFKGRFAFSLETSARTPYLOINNLKNEDTATYFCARGGEAMDYWGOGTSV

TVSS

242

35C1 VH:SEPIDNP:29

QIQLVQSGPELKTPGETVK1SCKASGYIFTSYGITWVKQAPGKGLKWMGWINTYSGV PTYADDFKGRFAFSLETSASTAYLQINNLKNEDTTTYFCTRGGDALDYWGQGTSVT

VSS

13B1 VH: SEP ID NP:30

QVQLKQSGAELMKPGASVKLSCKATGYTFTGKWIEWVKQRPGHGLEWIGEILPGTG STNYNEKFKGKATFTADSSSNTAYMQLSSLTTEDSAMYYCLRSEDVWGTGTTVTVS

S

1G4 VH: SEP ID NO:31

QVQLKQSGAELMKPGASVKLACKATGYTFTGYW1EWIKQRPGQGLEWIGEMLPGS GSTHYNEKFKGKATFTADTSSNTAYMPLSGLTTEDSAIYYCVRS1DYWGPGTTLTVS

S

1E7 VH: SEP 1DNO:32

PVQLKQSGPELARPWASVKISCQAFYTFSRRVHFA1RDTNYWMQWVKQRPGQGLE WIGAIYPGNGDTSYNPKFKGKATLTADKSSSTAYMPLSSLTSEDSAVYYCASGSHYF DYWGPGTTLTVSS

2D7 VH: SEP ID NP:33

EVQLQQSGPELVKPGASVKVSCKASGYTLTDYYMNWVKQSHGKSLEW1GDVNPNN DGTTYNPKFKGRATLTVDKSSNTASMELRSLTSEDSAVYYCAICPFYYLGKGTHFD YWGQGTSLTVSS

49C11 VH: SEP ID NP:34

EVQLQQSGPVLVKPGASGKMSCKASGYKFTDYYMIWVKQSHGKSLEWIGVIKIYNG GTSYNPKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARGPSLYDYDPYWYFD VWGTGTTVTVSS

15D9 VH: SEP ID NP:35

243 pVpLKpSGTELMKPGASVNLSCKASGYTFTAYWlEWVKpRPGHGLEWIGEILPGSG TTNYNENFKDRATFTADTSSNTAYMOLSSLTSEDSAIYYCARSYYYASRWFAFWGQ

GTLVTVSS

2F5 VH: SEP ID NO:36

EVQLQQPGAELVKPGASVKMSCKASGYTFTSYW1TWVKQRPGQGLEWIGDIYPGSG STNYNEKFKSKATLTVDTSSSTAYMpLSSLTSEDSAVYYCARRRYYATAWFAYWG QGTLVTVSS

1B11 VH: SEP ID NO:37 pVpLKpSGAELVRPGASVKLSCKASGYTFTDYYINWVKpRPGpGLEWIARIYPGSG NTYYNEKFKGKATLTAEKSSSTAYMPLSSLTSEDSAVYFCARNYYISSPWFAYWGP

GTLVTVSS

2F2 VH: SEP ID NP:38 pVpLKpSGAELVTPGASVKMSCKASGYTFTTYPIEWMKpNHGKSLEWIGNFHPYND DTKYNEKFKGKATLTVEKSSNTVYLELSRLTSDDSAVYFCARRVYYSYFWFGYWG HGTLVTVSS

11B6 VH: SEP ID NP:39 pVpLKpSGAELVKPGASVKMSCKASGYTFTTYPIEWMKpNHGKSLEWIGNFHPYN GDSKYNEKFKGKATLTVEKSSSTVYLELSRLPSADSAIYYCARRHYAASPWFAHWG pGTLVTVSS

TABLE 18: MASP-3 Antibody VH Sequences (CDRs and	7R régions, Kabat)
Antibody	HC FRI	HC CDR1
4D5	QVQLKQSGPELVKPGASVKLSCKASGYTFT (SEQ IDNO:55)	TDDIN (SEQ IDNO:56)
1F3	QVQLKQSGPELVKPGASVKLSCKASGYTFT (SEQ IDNO:55)	SNDIN (SEQIDNO:62)
4B6	QVQLKQSGPELVKPGASVKLSCKASGYTFT (SEQ IDNO:55)	SNDIN (SEQ IDNO:62)
1A10	QVQLKQSGPELVKPGASVKLSCKASGYTFT (SEQ ID NO:55)	SNDIN (SEQ IDNO:62)

244

10D12	QIQLVQSGPELKKPGETVKISCKASGY1FT (SEQIDNO:71)	SYGMS (SEQIDNO:72)
35C1	QIQLVQSGPELKTPGETVKISCKASGYIFT (SEQ IDNO:78)	SYGIT (SEQIDNO:79)
13B1	QVQLKQSGAELMKPGASVKLSCKATGYTFT (SEQ ID NO:83)	GKWIE (SEQIDNO:84)
1G4	QVQLKQSGAELMKPGASVKLACKATGYTFT (SEQ ID NO:90)	GYWIE (SEQIDNO:91)
2F5	EVQLQQPGAELVKPGASVKMSCKASGYTFT (SEQIDNO:97)	SYWIT (SEQ IDNO:98)
1B11	QVQLKQSGAELVRPGASVKLSCKASGYTFT (SEQ IDNO:102)	DYYIN (SEQ ID NO: 103)
1E7	QVQLKQSGPELARPWASVKISCQAFYTFSR (SEQ ID NO:108)	RVHFAIRDTNYWMQ (SEQ IDNO:109)
2F2	QVQLKQSGAELVTPGASVKMSCKASGYTFT (SEQ ID NO:113)	TYPIE (SEQ ID NO: 114)
11B6	QVQLKQSGAELVKPGASVKMSCKASGYTFT (SEQ 1DNO:120)	TYPIE (SEQIDNO:II4)
2D7	EVQLQQSGPELVKPGASVKVSCKASGYTLT (SEQ IDNO:I24)	DYYMN (SEQ ID NO: 125)
49C11	EVQLQQSGPVLVKPGASGKMSCKASGYKFT (SEQ IDNO:131)	DYYMI (SEQID NO:132)
15D9	QVQLKQSGTELMKPGASVNLSCKASGYTFT (SEQ ID NO:136)	AYWIE (SEQ IDNO:137)
Antibody	HC FR2	HCCDR2
4D5	WVKQRPGQGLEWIG (SEQ ID NO:57)	WIYPRDDRTKYNDKFKD (SEQIDNO:58)
1F3	WVKQRPGQGLEWIG (SEQ ID NO:57)	WIYPRDGSIKYNEKFTD (SEQIDNO:63)
4B6	WVKQRPGQGLEWIG (SEQ ID NO:57) ·	WIYPRDGTTKYNEEFTD (SEQ ID NO:67)
1A10	WVKQRPGQGLEWIG (SEQIDNO:57)	WIYPRDGTTKYNEKFTD (SEQIDNO:69)
10D12	WVRQAPGKGLKWMG (SEQ IDNO:73)	WINTYSGVPTYADDFKG (SEQIDNO:74)
35C1	WVKQAPGKGLKWMG (SEQ ID NO:80)	WINTYSGVPTYADDFKG (SEQIDNO:74)

245

13B1	WVKQRPGHGLEWIG (SEQIDNO:85)	EILPGTGSTNYNEKFKG (SEQIDNO:86)
1G4	WIKQRPGQGLEWIG (SEQ IDNO:92)	EMLPGSGSTHYNEKFKG (SEQIDNO:93)
2F5	WVKQRPGQGLEWIG (SEQIDNO:57)	DIYPGSGSTNYNEKFKS (SEQ IDNO:99)
1B11	WVKQRPGQGLEWIA (SEQ [D NO: 104)	RIYPGSGNTYYNEKFKG (SEQ ID NO: 105)
1E7	WVKQRPGQGLEWIG (SEQ ID NO:57)	AIYPGNGDTSYNQKFKG (SEQ IDNO:IIO)
2F2	WMKQNHGKSLEWIG (SEQ IDNO:1I5)	NFHPYNDDTKYNEKFKG (SEQ ID NO:116)
11B6	WMKQNHGKSLEWIG (SEQ IDNO:II5)	NFHPYNGDSKYNEKFKG (SEQ IDNO:I2I)
2D7	WVKQSHGKSLEWIG (SEQ ID NO: 126)	DVNPNNDGTTYNQKFKG (SEQ IDNO:I27)
49C11	WVKQSHGKSLEWIG (SEQ ID NO:I26)	VIKIYNGGTSYNQKFKG (SEQ ID NO:I33)
15D9	WVKQRPGHGLEWIG (SEQ IDNO:85)	EILPGSGTTNYNENFKD (SEQ ID NO:138)
Antibody	HCFR3	HC CDR3
4D5	KATLTVDTSSNTAYMDLHSLTSEDSAVYFCSS (SEQIDNO:59)	LEDTY (SEQ IDNO:60)
1F3	KATLTVDVSSSTAYMELHSLTSEDSAVYFCSG (SEQ IDNO:64)	VEDSY (SEQ ID NO:65)
4B6	KATLTVDVSSSTAFMELHSLTSEDSAVYFCSS (SEQ IDNO:68)	VEDSY (SEQ IDNO:65)
1A10	KATLTVDVSSSTAFMELHRLTSEDSAVYFCSS (SEQ ID NO:70)	VEDSY (SEQIDNO:65)
10D12	RFAFSLETSARTPYLQINNLKNEDTATYFCAR (SEQ IDNO:75)	GGEAMDY (SEQ IDNO:76)
35C1	RFAFSLETSASTAYLQINNLKNEDTTTYFCTR (SEQ IDNO:8I)	GGDALDY (SEQ ID NO:82)
13B1	KATFTADSSSNTAYMQLSSLTTEDSAMYYCLR (SEQ ID NO:87)	SEDV (SEQ IDNO:88)
1G4	KATFTADTSSNTAYMQLSGLTTEDSAIYYCVR (SEQIDNO:94)	SID Y (SEQ ID NO:95)

246

2F5	KATLTVDTSSSTAYMQLSSLTSEDSAVYYCAR (SEQ ID NO: 100)	RRYYATAWFAY (SEQ ID NO: 101)
1B11	KATLTAEKSSSTAYMQLSSLTSEDSAVYFCAR (SEQ ID NO: 106)	NYY1SSPWFAY (SEQ ID NO: 107)
1E7	KATLTADKSSSTAYMQLSSLTSEDSAVYYCAS (SEQ ID NO:111)	GSHYFDY (SEQ ID NO: 112)
2F2	KATLTVEKSSNTVYLELSRLTSDDSAVYFCAR (SEQ ID NO: 117)	RVYYSYFWFGY (SEQ ID NO: 118)
11B6	KATLTVEKSSSTVYLELSRLPSADSAIYYCAR (SEQ ID NO: 122)	RHYAASPWFAH (SEQ ID NO: 123)
2D7	RATLTVDKSSNTASMELRSLTSEDSAVYYCAI (SEQ IDNO:I28	CPFYYLGKGTHFDY (SEQ ID NO:129)
49C11	KATLTVDKSSSTAYMELNSLTSEDSAVYYCAR (SEQ ID NO: 134)	GPSLYDYDPYWYFDV ( SEQ ID NO:135)
15D9	RATFTADTSSNTAYMQLSSLTSEDSA1YYCAR (SEQ ID NO: 139)	SYYYASRWFAF (SEQ ID NO: 140)
Antibody	HC FR4
4D5	WGQGTLVAVSS (SEQ IDNO:61)
1F3	WGQGTLVTVSS (SEQ ID NO:66)
4B6	WGQGTLVTVSS (SEQIDNO:66)
1A10	WGQGTLVTVSS (SEQIDNO:66)
10D12	WGQGTSVTVSS (SEQ 1DNO:77)
35C1	WGQGTSVTVSS (SEQ ID NO:77)
13B1	WGTGTTVTVSS (SEQIDNO:89)
1G4	WGQGTTLTVSS (SEQIDNO:96)
2F5	WGQGTLVTVSS (SEQ ID NO:66)
1B11	WGQGTLVTVSS

247

	(SEQ ID NO:66)
1E7	WGQGTTLTVSS (SEQ ID NO:96)
2F2	WGHGTLVTVSS (SEQ IDNO:119)
11B6	WGQGTLVTVSS (SEQ ID NO:66)
2D7	WGQGTSLTVSS (SEQ IDNO:I30)
49C11	WGTGTTVTVSS (SEQ IDNO:89)
15D9	WGQGTLVTVSS (SEQIDNO:66)

Presented below are the light chain variable région (VL) sequences for the high affinity MASP-3 inhibitory antibodies. The Kabat CDRs are underlined. These régions are the same whether numbered by the Kabat or Chothia system.

Light Chain Variable Régions:

4D5 VL: SEQ IDNQ:40

D1VMTQSPSSLAVSAGEKVTMTCKSS0SLLNSRTRKNYLAWY00KPG0SPKLLIYW ASTRESGVPDRFTGSGSGTDFSLTISSVQAEDLAVYYCKOSYNLYTFGGGTKLEIKR 10

1F3 VL: SEQ 1DNO:41

DIVMTQSPSSLAVSAGERVTMSCKSSOSLLISRTRKNYLSWYOQKPGOSPKLLIYWA STRESGVPDRFTGSGSGTDFTLT1SSVOAEDLAVYYCKOSYNLYTFGGGTKLE1KR

4B6 VL: SEQ IDNO:42 (SAME for 1A10 VL)

D1VMTQSPSSLAVSAGEKVTMSCKSSOSLL1SRTRKNYLSWYOOKPGOSPKLLIYWA STRESGVPDRFTGSGSGTDFTLTISSVOAEDLAVYYCKQSYNLYTFGGGTKLEIKR

10D12 VL: SEQ 1DNO:43

248 nvr MTQTPT ΤΓ SVTIGQPASISCKSSPSLLDSDGKTYLN WLLPRPGPSPKRLIYLVSK ldsgvpdrftgsgsgtdftlkisrveaedlgvyycwqgthfpwtfgggtkleikr

35C1 VL: SEP IDNP:44

DIVMTQAPI ΤΓ svtigqpasisckssqslldsdgktylswllorpgospkrliylvskl dsgvpdrftgsgsgtdftlkisrveaedlgvyycwqgthfpytfgggtkleikr

13B1 VL: SEP ID NP:45

D1VMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYW astresgvpdrftgsgsgtdftltissvpaedlavyyckpsyniptfgggtkleikr

1G4 VL: SEP ID NP:46 .

DVLMTpTPLSLPVSLGEpASISCRSSQSLVQSNGNTYLHWYLpKPGpSPKLLIYKVS nrfsgvpdrfsgsgsgtdftlkisrveaedlgvyfcsqsthvpptfgggtkleikr

IΕ7 VL: SEP ID NP:47

DIQLTQSPAILSVSPGERVSFSCRASPSIGTSIHWYPPRTNGSPRLLIKYASESISGIPSR FSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPYTFGGGTKLEIKR

2D7 VL: SEP ID NP:48

DIQMTQTPASLSASLGDRVTISCRASPDISNFLNWYPPKPNGTVKLLVFYTSRLHSG VPSRFSGSGSGAEHSLTISNLEpEDVATYFCpQGFTLPWTFGGGTKVEIKR

49C11 VL:SEP ID NP:49

DVLMTQTPLSLPVSLGDPASFSCRSSPSLIHSNGNTYLHWYLPKPGPSPKLLIYKVSN RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSPSTHVPWTFGGGTKLE1KR

15D9 VL: SEP IDNP:50

DIVMTQSQKFMSTSIGDRVSVTCRASPNVGPNLAWYPPKPGPSPKALIYSASYRFSG

VPDRFTGSGSGTDFTLTISNVPSEDLAEYFCPPYNRYPFTFGSGTKLEIKR

249

2F5 VL: SEQ IDNO:51

DIVMTOSOKFMSTSVGDRVSITCKASQNVGTAVAWYOOKPGOSPKLLISSASNRYT GVPDRFTGSGSGTDFTLT1SNMQSEDVADYFCOOYNSYPLTFGAGTKLELKR

1B11 VL:SEQIDNO:52

DIVMTOSOKFMSTSVGDRVSVTCKASQNVGPNVAWYOOKPGOSPKALIYSASYRYS GVPDRFTGSGSGTDFTLTISNVOSEDLADYFCOQYNRYPLTFGAGTKLELKR

2F2 VL: SEQ IDNO:53

DIVMTOSOKFMSTSVGDRVNVTCKASQNVGTHVAWYOOKPGOSPKALIYSASYRY SGVPDRFTGSGSGTDFTLTISNVOSEDLAEYFCOQYNSYPRALTFGAGTKLELKR

11B6 VL: SEQ ID NO:54

DIVMTOSOKFMSTSVGDRVNVTCKASQNVGPTVAWYOOKPGOSPKALIYSASYRYS

GVPDRFTGSGSGTDFTLTISNVHSEDLAEYFCOOYNSYPFTFGSGTKLEIKR

TABLE 19: MASP-3 Antibody VL Sequences (CDRs and FR régions, Kabat and Chothia)

Antibody	LC FRI	LC CDR1
4D5	DIVMTQSPSSLAVSAGEKVTMTC (SEQ ID NO:141)	KSSQSLLNSRTRKNYLA (SEQID NO: 142)
1F3	DIVMTQSPSSLAVSAGERVTMSC (SEQ ID NO: 148)	KSSQSLLISRTRKNYLS (SEQID NO: 149)
4B6	DIVMTQSPSSLAVSAGEKVTMSC (SEQID NO: 151)	KSSQSLLISRTRKNYLS (SEQID NO: 149)
1A10*	[used 4B6 LC: SEQ IDNO:151]	[used 4B6 LC: SEQ ID NO: 149]
10D12	DVLMTQTPLTLSVTIGQPASISC (SEQ ID NO: 152)	KSSQSLLDSDGKTYLN (SEQID NO: 153)
35C1	DIVMTQAPLTLSVTIGQPASISC (SEQID NO: 158)	KSSQSLLDSDGKTYLS (SEQID NO: 159)
13B1	DIVMTQSPSSLAVSAGEKVTMSC (SEQ IDNO:151)	KSSQSLLNSRTRKNYLA (SEQ IDNO:142)
1G4	DVLMTQTPLSLPVSLGEQASISC (SEQID NO: 162)	RSSQSLVQSNGNTYLH (SEQ IDNO:163)

250

2F5	DIVMTQSQKFMSTSVGDRVSITC (SEQ IDNO:168)	KASQNVGTAVA (SEQ ID NO: 169)
1B11	DIVMTQSQKFMSTSVGDRVSVTC (SEQ IDNO:175)	KASQNVGPNVA (SEQ ID NO: 176)
1E7	DIQLTQSPA1LSVSPGERVSFSC (SEQ IDNO:18I)	RASQSIGTSIH (SEQ ID NO: 182)
2F2	DIVMTQSQKFMSTSVGDRVNVTC (SEQ IDNO:187)	KASQNVGTHVA (SEQ IDNO:188)
11B6	DIVMTQSQKFMSTSVGDRVNVTC (SEQ IDNO:I87)	KASQNVGPTVA (SEQ IDNO:191)
2D7	DIQMTQTPASLSASLGDRVTISC (SEQ ID NO: 195)	RASQD1SNFLN (SEQ ID NO: 196)
49C11	DVLMTQTPLSLPVSLGDQASFSC (SEQ ID NO:202)	RSSQSLIHSNGNTYLH (SEQ ID NO:203)
15D9	DIVMTQSQKFMSTSIGDRVSVTC (SEQ IDNQ:205)	RASQNVGPNLA (SEQ ID NQ:206)
Antibody	LC FR2	LC CDR2
4D5	WYQQKPGQSPKLLIY (SEQ IDNO:143)	WASTRES (SEQ ID NO: 144)
1F3	WYQQKPGQSPKLLIY (SEQ IDNO:143)	WASTRES (SEQ IDNO:144)
4B6	WYQQKPGQSPKLLIY (SEQ ID NO: 143)	WASTRES (SEQ ID NO: 144)
1A10	[used 4B6 LC: SEQ IDNO:143]	[used 4B6 LC: SEQ 1DNO:144]
10D12	WLLQRPGQSPKRLIY (SEQ IDNO:154)	LVSKLDS (SEQ ID NO: 155)
35C1	WLLQRPGQSPKRLIY (SEQ IDNO:I54)	LVSKLDS (SEQ IDNO:155)
13B1	WYQQKPGQSPKLLIY (SEQ IDNO:143)	WASTRES (SEQ ID NO: 144)
1G4	WYLQKPGQSPKLLIY (SEQ IDNO:I64)	KVSNRFS (SEQ ID NO: 165)
2F5	WYQQKPGQSPKLLIS	SASNRYT

251

	(SEQ IDNO:170)	(SEQ 1DNO:171)
1B11	WYQQKPGQSPKALIY (SEQ ID NO: 177)	SASYRYS (SEQ 1DNO:I78)
1E7	WYQQRTNGSPRLLIK (SEQ ID NO: 183)	YASESIS (SEQ ID NO: 184)
2F2	WYQQKPGQSPKALIY (SEQ IDNO:177)	SASYRYS (SEQ IDNO:I78)
11B6	WYQQKPGQSPKALIY (SEQ IDNO:177)	SASYRYS (SEQ IDNO:178)
2D7	WYQQKPNGTVKLLVF (SEQ IDNO:197)	YTSRLHS (SEQ IDNO:198)
49C11	WYLQKPGQSPKLLIY (SEQ IDNO:164)	KVSNRFS (SEQ IDNO:165)
15D9	WYQQKPGQSPKALIY (SEQ IDNO:I77)	SASYRFS (SEQ ID NO:207)
Antibody	LC FR3	LC CDR3
4D5	GVPDRFTGSGSGTDFSLTISSVQAEDLAVYYC (SEQ IDNO:145)	KQSYNLYT (SEQ IDNO:146)
1F3	GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC (SEQ IDNO:150)	KQSYNLYT (SEQ ID NO: 146)
4B6	GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC (SEQ IDNO:150)	KQSYNLYT (SEQ IDNO:146)
1A10	[used 4B6 LC: SEQ ID NO: 1501	[used 4B6 LC: SEQ ID NO: 146]
10D12	GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC (SEQ IDNO:156)	WQGTHFPWT (SEQ IDNO:157)
35C1	GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC (SEQ IDNO:156)	WQGTHFPYT (SEQ IDNO:160)
13B1	GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC (SEQ IDNO:150)	KQSYNIPT (SEQ ID NO: 161)
1G4	GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ IDNO:166)	SQSTHVPPT (SEQ IDNO:167)
2F5	GVPDRFTGSGSGTDFTLTISNMQSEDVADYFC (SEQ IDNO:172)	QQYNSYPLT (SEQ IDNO:173)

252

1B11	GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC (SEQ ID NO: 179)	QQYNRYPLT (SEQ IDNO:180)
1E7	GIPSRFSGSGSGTDFTLSINSVESEDIADYYC (SEQ IDNO:185)	QQSNSWPYT (SEQ ID NO: 186)
2F2	GVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC (SEQ IDNO:189)	QQYNSYPRALT (SEQ IDNO:I90)
11B6	GVPDRFTGSGSGTDFTLTISNVHSEDLAEYFC (SEQ IDNO:I92)	QQYNSYPFT (SEQ ID NO: 193)
2D7	GVPSRFSGSGSGAEHSLTISNLEQEDVATYFC (SEQ IDNO:199)	QQGFTLPWT (SEQ ID N0:200)
49C11	GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ IDNO:I66)	SQSTHVPWT (SEQ ID NO:204)
15D9	GVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC (SEQ IDNO:I89)	QQYNRYPFT (SEQ ID NO:208)
Antibody	LC FR4
4D5	FGGGTKLEIKR (SEQ IDNO:147)
1F3	FGGGTKLEIKR (SEQ ID NO: 147)
4B6	FGGGTKLEIKR (SEQ IDNO:I47)
1A10	[used 4B6 LC: SEQ IDNO:147]
10D12	FGGGTKLEIKR (SEQ IDNO:147)
35C1	FGGGTKLEIKR (SEQ IDNO:I47)
13B1	FGGGTKLEIKR (SEQ IDNO:147)
1G4	FGGGTKLEIKR (SEQ IDNO:147)
2F5	FGAGTKLELKR (SEQ IDNO:I74)
1B11	FGAGTKLELKR

253

	(SEQ ID NO: 174)
1E7	FGGGTKLEIKR (SEQ ID NO: 147)
2F2	FGAGTKLELKR (SEQ ID NO: 174)
11B6	FGSGTKLEIKR (SEQ ID NO: 194)
2D7	FGGGTKVEIKR (SEQ IDNO:201)
49C11	FGGGTKLEIKR (SEQ ID NO: 147)
15D9	FGSGTKLEIKR (SEQ ID NO: 194)
*Note: the light chain for mAb 1A10 was not identified, so t	le light chain from 4B6 was used

with the 1A10 HC.

TABLE 20: Consensus Sequences for Group IA HC CDRs:

Antibody	Région	Sequence
4D5	HC-CDRI	TDDIN (SEQ ID NO:56)
IF3	HC-CDR1	SNDIN (SEQ IDNO:62)
4B6	HC-CDRI	SNDIN (SEQ IDNO:62)
1A10	HC-CDRI	SNDIN (SEQ ID NO:62)
Consensus	HC-CDRI	XXDIN (SEQ ID NO:209) wherein X at position 1 is S or T; and X at position 2 is N or D
4D5	HC-CDR2	WIYPRDDRTKYNDKFKD (SEQ ID NO:58)
1F3	HC-CDR2	WIYPRDGSIKYNEKFTD (SEQ ID NO:63)
4B6	HC-CDR2	WIYPRDGTTKYNEEFTD (SEQ ID NO:67)
1A10	HC-CDR2	W1YPRDGTTKYNEKFTD (SEQIDNO:69)
Consensus	HC-CDR2	WIYPRDXXXKYNXXFXD (SEQ ID NO:210) wherein X at position 7 is G or D; X at position 8 is S, T or R; X at position 9 is I or T; X at position 13 is E or D; X at position 14 is K or E; X at position 16 is T or K
4D5	HC-CDR3	LEDTY (SEQ ID NO:60)
1F3	HC-CDR3	VEDSY (SEQIDNO:65)
4B6	HC-CDR3	VEDSY (SEQIDNO:65)
1A10	HC-CDR3	VEDSY (SEQ ID NO:65)

254

Consensus

HC-CDR3

XEDXY (SEQ ID NO:211) wherein X at position 1 is L or V, and wherein X at position 4 is T or S

TABLE 21: Consensus Sequences for Group IA LC CDRs:

Antibody	Région	Sequence
4D5	LC-CDRI	KSSQSLLNSRTRKNYLA (SEQ ID NO: 142)
4D5-NQ	LC-CDRI	KSSQSLLQSRTRKNYLA (SEQ ID NO:257)
4D5-NA	LC-CDRI	KSSQSLLASRTRKNYLA (SEQ ID NO:258)
4D5-ST	LC-CDRI	KSSQSLLNTRTRKNYLA (SEQ ID NO:259)
1F3	LC-CDRI	KSSQSLLISRTRKNYLS (SEQ ID NO: 149)
4B6	LC-CDRI	KSSQSLLISRTRKNYLS (SEQ ID NO: 149)
Consensus*	LC-CDRI	KSSQSLLXXRTRKNYLX (SEQ ID NO:212) wherein X at position 8 is N, I, Q or A; wherein X at position 9 is S or T; and wherein X at position 17 is A or S
4D5	LC-CDR2	WASTRES (SEQ ID NO: 144)
1F3	LC-CDR2	WASTRES (SEQ ID NO: 144)
4B6	LC-CDR2	WASTRES (SEQ ID NO: 144)
Consensus	LC-CDR2	WASTRES (SEQ ID NO: 144)
4D5	LC-CDR3	KQSYNLYT (SEQ IDNO:146)
1F3	LC-CDR3	KQSYNLYT (SEQ ID NO: 146)
4B6	LC-CDR3	KQSYNLYT (SEQ IDNO:146)
Consensus	LC-CDR3	KQSYNLYT (SEQ ID NO: 146)

*Note: CDR-L1 consensus includes variants generated as described in Example 19.

TABLE 22 Consensus Sequences for Group IB HC CDRs;

Antibody	Région	Sequence
10D12	HC-CDR1	SYGMS (SEQ1DNO:72)
35C1	HC-CDR1	SYGIT (SEQIDNO:79)
Consensus	HC-CDR1	SYGXX (SEQ IDNO:213) wherein X at position 4 is M or I; and wherein X at position 5 is S or T
10D12	HC-CDR2	WINTYSGVPTYADDFKG (SEQ ID NO:74)
35C1	HC-CDR2	WINTYSGVPTYADDFKG (SEQIDNO:74)
Consensus	HC-CDR2	WINTYSGVPTYADDFKG (SEQ ID NO:74)
10D12	HC-CDR3	GGEAMDY (SEQ ID NO:76)
35CI	HC-CDR3	GGDALDY (SEQ ID NO:82)
Consensus	HC-CDR3	GGXAXDY (SEQ ID NO:214) wherein X at position 3 is E or D; and wherein X at position 5 is M or L

255

TABLE 23 Consensus Sequences for Group IB LC CDRs: ..........
Antibody	Région	Sequence
10D12	LC-CDR1	KSSQSLLDSDGKTYLN (SEQ ID NO:153)
10D12-DE	LC-CDR1	KSSQSLLDSEGKTYLN (SEQ ID NO:261 )
10D12-DA	LC-CDR1	KSSQSLLDSAGKTYLN (SEQ ID NO:262)
10D12-GA	LC-CDR1	KSSQSLLDSDAKTYLN (SEQ ID NO:263)
35C1	LC-CDRI	KSSQSLLDSDGKTYLS (SEQ ID NO: 159)
Consensus*	LC-CDR1	KSSQSLLDSXXKTYLX (SEQ ID NO:215) Wherein X at position 10 is D, E or A; Wherein X at position 11 is G or A; and wherein X at position 16 is N or S
10DI2	LC-CDR2	LVSKLDS (SEQID NO: 155)
35C1	LC-CDR2	LVSKLDS (SEQ ID NO:155)
Consensus	LC-CDR2	LVSKLDS (SEQID NO: 155)
I0D12	LC-CDR3	WQGTHFPWT (SEQ ID NO:157)
35C1	LC-CDR3	WQGTHFPYT (SEQ IDNO:160)
Consensus	LC-CDR3	WQGTHFPXT (SEQ ID NO: 216) Wherein X at position 8 is W or Y

*Note: CDR-L1 consensus includes variants generated as described in Example 19.

DNA encoding mouse mAb heavy and light chains:

SEQ ID NO:217: DNA encoding 4D5 heavy chain variable région (parental)

CAGGTGCAGCTGAAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTG AAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACAACCGACGATATAAACTGGG TGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTAGAG

ATGATAGAACTAAGTACAATGACAAGTTCAAGGACAAGGCCACATTGACTGTAG ACACATCTTCCAACACAGCGTACATGGACCTCCACAGCCTGACATCTGAGGACTC TGCGGTCTATTTCTGTTCAAGCCTCGAGGATACTTACTGGGGCCAAGGGACTCTG GTCGCTGTCTCTTCA

SEQ IDNO:218: DNA encoding 1F3 heavy chain variable région (parental)

CAGGTGCAGCTGAAGCAGTCTGGACCTGAGCTGGTG AAGCCTGGGGCTTCAGTG AAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACAAGTAACGATATAAACTGGG TGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTAGAG ATGGGAGTATTAAATATAATGAGAAATTCACGGACAAGGCCACATTGACAGTTG ACGTATCCTCCAGCACAGCGTACATGGAGCTCCACAGCCTGACATCTGAGGACTC

TGCGGTCTATTTCTGTTCAGGTGTCGAGGATTCTTACTGGGGCCAAGGGACTCTG GTCACTGTCTCTTCA

SEQ ID NO:219: DNA encoding 4B6 heavy chain variable région (parental)

256

CAGGTGCAGCTGAAGCAGTCTGGACCTGAACTGGTGAAGCCTGGGGCTTCAGTG AAATTGTCCTGCAAGGCTTCTGGCTACACCTTCACAAGTAACGATATAAACTGGG TGAAACAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTAGAG ATGGTACTACTAAGTACAATGAGGAGTTCACGGACAAGGCCACATTGACTGTTG

ACGTATCCTCCAGCACAGCGTTCATGGAGCTCCACAGCCTGACATCTGAGGACTC TGCTGTCTATTTCTGTTCAAGTGTCGAGGATTCTTACTGGGGCCAAGGGACTCTG GTCACTGTCTCTTCA

SEQ ID NO:22Q: DNA encoding 1A10 heavy chain variable région (parental) CAGGTGCAGCTGAAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTG

AAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACAAGTAACGATATAAACTGGG TGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTAGAG ATGGTACTACTAAGTACAATGAGAAGTTCACGGACAAGGCCACATTGACTGTTG ACGTATCCTCCAGCACAGCGTTCATGGAGCTCCACAGGCTGACATCTGAGGACTC TGCGGTCTATTTCTGTTCAAGTGTCGAGGATTCTTACTGGGGCCAAGGGACTCTG

GTCACTGTCTCTTCA

SEQIDNO:221: DNA encoding 10D12 heavy chain variable région (parental) CAGATCCAGTTGGTACAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTC AAGATCTCCTGCAAGGCTTCTGGGTATATTTTCACAAGCTATGGAATGAGCTGGG TGAGACAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACT 20 CTGGAGTGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGA

AACCTCTGCCAGAACTCCCTATTTGCAGATCAACAACCTCAAAAATGAGGACAC GGCTACATATTTCTGCGCAAGAGGGGGCGAAGCTATGGACTACTGGGGTCAAGG AACCTCAGTCACCGTCTCCTCA

SEQ ID NO:222: DNA encoding 35C1 heavy chain variable région (parental)

CAGATCCAGTTGGTACAGTCTGGACCTGAGCTGAAGACGCCAGGAGAGACAGTC AAGATCTCCTGCAAGGCTTCTGGGTATATCTTCACATCCTATGGAATTACCTGGG TGAAACAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACT CTGGAGTGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGA AACGTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACAC

GACTACATATTTCTGTACAAGAGGGGGTGATGCTTTGGACTACTGGGGTCAAGGA ACCTCAGTCACCGTCTCCTCA

SEQ ID NO:223: DNA encoding 13B1 heavy chain variable région (parental) CAGGTGCAGCTGAAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTG AAGCTTTCCTGCAAGGCTACTGGCTACACATTCACTGGCAAGTGGATAGAGTGGG 35 TAAAACAGAGGCCTGGACATGGCCTAGAGTGGATTGGAGAGATTTTACCTGGAA

CTGGTAGTACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAG ACTCATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACAACTGAAGACTC TGCTATGTATTATTGTTTAAGATCCGAGGATGTCTGGGGCACAGGGACCACGGTC ACCGTCTCCTCA

SEQ ID NO:224: DNA encoding 1G4 heavy chain variable région (parental) CAGGTGCAGCTGAAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTG AAGCTTGCCTGCAAGGCTACTGGCTACACATTCACTGGCTACTGGATAGAGTGGA TAAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATGTTACCTGGAA

257

GTGGTAGTACTCACTACAATGAGAAGTTCAAGGGTAAGGCCACATTCACTGCAG ATACATCCTCCAACACAGCCTACATGCAACTCAGCGGCCTGACAACTGAGGACT CTGCCATCTATTACTGTGTAAGAAGCATAGACTACTGGGGCCAAGGCACCACTCT CACAGTCTCCTCA

SEQ IDNO:225: DNA encoding 1E7 heavy chain variable région (parental)

CAGGTGCAGCTGAAGCAGTCTGGGCCTGAGCTGGCAAGGCCTTGGGCTTCAGTG AAGATATCCTGCCAGGCTTTCTACACCTTTTCCAGAAGGGTGCACTTTGCCATTA GG G ATACC A ACTACTGG ATGC AGTG G GTAA AACAG AG GCCTG G ACAG GGTCTG G AATGGATCGGGGCTATTTATCCTGGAAATGGTGATACTAGTTACAATCAGAAGTT CAAGGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCA ACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCATCCGGTAGC CACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

SEQ ID NO:226: DNA encoding 2D7 heavy chain variable région (parental)

GAGGTCCAGCTGCAACAATCTGGGCCTGAGCTGGTGAAGCCTGGGGCTTCAGTG AAG GTATCCTGTAAG GCTTCTG G ATAC ACG CTC ACTG ACTACTACATG A ACTGG G TGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATGTTAATCCTAACA ATGATGGTACTACCTACAACCAGAAATTCAAGGGCAGGGCCACATTGACTGTAG ACAAGTCTTCCAACACAGCCTCCATGGAGCTCCGCAGCCTGACATCTGAGGACTC TGCAGTCTACTACTGTGCAATATGCCCCTTTTATTACCTCGGTAAAGGGACCCAC TTTGACTACTGGGGCCAAGGCACCTCTCTCACAGTCTCCTCA

SEQ ID NO:227: DNA encoding 49C1 i heavy chain variable région (parental)

G AGGTCCAG CTGC A ACA ATCTG G ACCTGTGCTGGTG AAGCCTG GGGCTTC AG G G AAGATGTCCTGTAAGGCTTCTGGATACAAATTCACTGACTACTATATGATCTGGG TGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGTTATTAAAATTTATA ACGGTGGTACGAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTTG ACAAGTCCTCCAGCACAGCCTACATGGAGCTCAACAGCCTGACATCTGAGGACT CTGCAGTCTATTACTGTGCAAGAGGGCCATCTCTCTATGATTACGACCCTTACTG GTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO:228: DNA encoding 15D9 heavy chain variable région (parental)

CAGGTGCAGCTGAAGCAGTCTGGAACTGAGCTGATGAAGCCTGGGGCCTCAGTG AACCTTTCCTGCAAGGCTTCTGGCTACACATTCACTGCCTACTGGATAGAGTGGG TAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAA GTGGTACTACTAACTACAATGAGAACTTCAAGGACAGGGCCACATTCACTGCAG ATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACAAGTGAGGACT CTGCCATCTATTACTGTGCAAGATCCTATTACTACGCTAGTAGATGGTTTGCTTTC TGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA

SEQ ID NO:229: DNA encoding 2F5 heavy chain variable région (parental)

GAGGTCCAGCTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTG AAGATGTCCTGTAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGG TGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGATATTTATCCTGGTA GTGGTAGTACTAACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAG ACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTC

258

TGCGGTCTATTACTGTGCAAGAAGGAGATACTACGCTACGGCCTGGTTTGCTTAC TGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA

SEQ ID NO:23Q: DNA encoding 1B11 heavy chain variable région (parental)

CAGGTGCAGCTGAAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTG

AAGCTGTCCTGCAAGGCTTCTGGCTACACTTTCACTGACTACTATATAAACTGGG TGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGCAAGGATTTATCCTGGAA GTGGTAATACTTACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAG AAAAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTC TGCTGTCTATTTCTGTGCAAGAAATTACTACATTAGTAGTCCCTGGTTTGCTTACT

GGGGCCAAGGGACTCTGGTCACTGTCTCTTCA

SEQ ID NO:231: DNA encoding 2F2 heavy chain variable région (parental) CAGGTGCAGCTGAAGCAGTCTGGGGCTGAGCTAGTGACGCCTGGAGCCTCAGTG AAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACTACCTATCCTATAGAGTGGA TGAAACAGAATCATGGAAAGAGCCTAGAGTGGATTGGAAATTTTCATCCTTACA

ATGATGATACTAAGTACAATGAAAAGTTCAAGGGCAAGGCC ACATTGACTGTAG AAAAATCCTCTAACACAGTCTACTTGGAGCTCAGCCGATTAACATCTGATGACTC TGCTGTTTATTTCTGTGCAAGGAGGGTCTACTATAGTTACTTCTGGTTTGGTTACT GGGGCCACGGGACTCTGGTCACTGTCTCTTCA

SEQ ID NO:232: DNA encoding 11B6 heavy chain variable région (Parental)

CAGGTGCAGCTGAAGCAGTCTGGGGCTGAGCTAGTGAAACCTGGAGCCTCAGTG AAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACTACCTATCCTATAGAGTGGA TGAAGCAGAATCATGGGAAGAGCCTAGAGTGGATTGGAAATTTTCATCCTTACA ATGGTGATTCTAAGTACAATGAAAAGTTCAAGGGCAAGGCCACCTTGACTGTAG AAAAATCCTCTAGCACAGTCTACTTAGAACTCAGCCGATTACCATCTGCTGACTC

TGCTATTTATTACTGTGCAAGGAGGCACTACGCTGCTAGTCCCTGGTTTGCTCACT GGGGCCAAGGGACTCTGGTCACTGTCTCTTCA

DNA encoding light chain variable région (mouse mAbs):

SEQ ID NO:233: DNA encoding 4D5 light chain variable région (parental)

GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGG TCACTATGACCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGA ACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTA CTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCT

GGGACAGATTTCTCTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTT 35 ATTACTGCAAGCAATCTTATAATCTGTACACGTTCGGAGGGGGGACCAAGCTGG

AAATAAAACGG

SEQ ID NO:234: DNA encoding 1F3 light chain variable région (parental)

GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAGGG . TCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCATCAGTAGAACCCGAAAGA

ACTATTTGTCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTA CTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCT GGGACAGATTTCACTCTCACCATCAGCAGTGTACAGGCTGAAGACCTGGCAGTTT

259

ATTACTGCAAGCAATCTTATAATCTGTACACGTTCGGCGGGGGGACCAAGCTGGA AATAAAACGG

SEQ IDNO:235: DNA encoding 4B6/1A10 light chain variable région (parental)

GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGG

TCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCATCAGTAGAACCCGAAAGA ACTATTTGTCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTA TTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCT GGGACAGATTTCACTCTCACCATCAGCAGTGTACAGGCTGAAGACCTGGCAGTTT ATTACTGCAAACAATCTTATAATCTGTACACGTTCGGCGGGGGGACCAAGCTGGA

AATCAAACGG

SEQ IDNO:236: DNA encoding 10D12 light chain variable région (parental) GATGTTTTGATGACCCAAACTCCACTCACTTTGTCGGTTACCATTGGACAACCAG CCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATA TTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTG

GTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGG A CAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATT ATTGCTGGCAAGGTACACATTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGA AATCAAACGG

SEQ ID NO:237: DNA encoding 35C1 light chain variable région (parental)

GATATTGTGATGACGCAGGCTCCACTCACTTTGTCGGTTACCATTGGACAACCAG CCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATA TTTGAGTTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTG GTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGA CAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATT

ATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGG AAATAAAACGG

SEQ ID NO:238: DNA encoding 13B1 light chain variable région (parental)

GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGG TCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGA

ACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTA CTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCT GGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTT ATTACTGCAAGCAATCTTATAATATTCCGACGTTCGGTGGAGGCACCAAGCTGGA AATCAAACGG

SEQ ID NO:239: DNA encoding 1G4 light chain variable région (parental) GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGAACAAG CCTCCATCTCTTGCAGATCAAGTCAGAGCCTTGTACAAAGTAATGGAAACACCTA TTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAA GTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGA

CAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTT CTGCTCTCAAAGTACACATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAA ATCAAACGG

SEQ ID NO:24Q: DNA encoding IE7 light chain variable région (parental)

260

GACATCCAGCTGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAG TCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAAGCATACACTGGTATCA GCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATC TCTGG G ATCCCTTCC AG GTTTAGTGGC AGTG G ATC AG G G AC AGATTTTACTCTTA

GCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAGTAA TAGCTGGCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGG

SEQ IDNO:241: DNA encoding 2D7 light chain variable région (parental) GATATCCAGATGACACAGACTCCAGCCTCCCTGTCTGCCTCTCTGGGAGACAGAG

TCACCATCAGTTGTAGGGCAAGTCAGGACATTAGCAATTTTTTAAACTGGTATCA

ACAGAAACCGAATGGAACTGTTAAACTCCTAGTCTTCTACACATCAAGATTACAC TCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAGCAGAGCATTCTCTCA CCATTAGCAACCTGGAGCAGGAAGATGTTGCCACTTACTTTTGCCAACAGGGTTT TACGCTTCCGTGGACGTTCGGTGGGGGCACCAAGGTGGAAATCAAACGG

SEQ 1DNO:242: DNA encoding 49C11 light chain variable région (parental)

GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAG CCTCCTTCTCTTGCAGATCTAGTCAGAGCCTTATACACAGTAATGGAAACACCTA TTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAA GTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGA CAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTT

CTGCTCTCAAAGTACACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAA ATCAAACGG

SEQ 1DNO:243: DNA encoding 15D9 light chain variable région (parental) GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAATAGGAGACAGG GTCAGCGTCACCTGCAGGGCCAGTCAGAATGTGGGTCCCAATTTAGCCTGGTATC

AACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGATT CAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTC ACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATA ACAGGTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGG

SEQ 1DNO:244: DNA encoding 2F5 light chain variable région (parental)

GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGG GTCAGCATCACCTGCAAGGCCAGTCAGAATGTGGGTACTGCTGTAGCCTGGTATC AACAGAAACCAGGACAATCTCCTAAACTACTGATTTCCTCGGCATCCAATCGGTA CACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTC ACCATCAGTAATATGCAGTCTGAAGACGTGGCAGATTATTTCTGCCAGCAATATA

ACAGCTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG

SEQ ID NO:245: DNA encoding 1B11 light chain variable région (parental) GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACTTCAGTAGGAGACAGGG

TCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTCCTAATGTAGCCTGGTATCA ACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTAC

AGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCA CCATCAGCAATGTGCAGTCTGAAGACTTGGCAGACTATTTCTGTCAGCAATATAA CCGCTATCCTCTCACGTTCGGTGCTGGGACCAAACTGGAGCTGAAACGG

SEQ ID NO:246: DNA encoding 2F2 light chain variable région (parental)

261

GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGG GTCAACGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTCATGTAGCCTGGTATC AACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTA CAGTGGCGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTC

ACCATCAGCAATGTGCAGTCTGAAGACCTGGCAGAGTATTTCTGTCAGCAATATA ACAGCTATCCTCGAGCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAC

GG

SEQ ID NO:247: DNA encoding 11B6 light chain variable région (parental)

GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGG

GTCAACGTCACCTGCAAGGCCAGTCAGAATGTGGGTCCTACTGTAGCCTGGTATC AACAGAAACCAGGGCAATCTCCTAAAGCACTAATTTACTCGGCATCCTACCGGTA CAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTC ACCATCAGCAATGTGCACTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATA ACAGCTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGG

SEQ IDNQ:310: human IgG4 constant région

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR

EEQFNSTYRVVSVLTVLHQDWLNGK.EYK.CKVSNKGLPSS1EKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO:311 : human IgG4 constant région with S228P mutation

ASTKGPSVFPLAPCSRSTSESTAALGCLVK.DYFPEPVTVSWNSGALTSGVHTFPAVLQ 25 SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG

GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK.TTPPVLDSDGSFFL

YSRLTVDK.SRWQEGNVFSCSVMHEALHNHYTQK.SLSLSLGK

SEQ ID NO:312: human IgG4 constant région with S228P mutation and and also a mutation (Xtend) that promûtes FcRn interations at low pH

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTK.NQVSLTCLVK.GFYPSDIAVEWESNGQPENNYK.TTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK

SEQ ID NO:313: human IgK constant région TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

262

EXAMPLE 16

This Example describes functional characterization of recombinant purified high affinity MASP-3 inhibitory antibodies in several in vitro assays.

Methods:

The recombinant MASP-3 mAbs generated as described in Examples 11 and 14 were characterized for (i) binding to human MASP-3 and other species’ MASP-3; (ii) the ability to inhibit cleavage of an artificial substrate; (iii) the capacity to inhibit pro-factor D to factor D cleavage; (iv) inhibition of complément déposition in human sérum and (v) inhibition of rabbit érythrocyte lysis in human sérum as follows:

L Assays to Détermine Binding to human and mouse MASP-3

ELISA Assays:

MASP-3 Binding Assay with purified recombinant MASP-3 mAbs:

Human MASP-3:

A sandwich ELISA assay was carried out to measure binding of 16 purified recombinant MASP-3 antibodies to human MASP-3 (CCPI-CCP2-SP fragment) as follows. An ELISA plate was coated in carbonate/bi-carbonate buffer ovemight at 4°C with capture antibody aM3-259 at 4 pg/mL. aM3-259 is a high avidity recombinant, chimeric chicken-human MASP-3 mAb from chickens immunized with the CCP1-CCP2-SP région of human MASP20 3. Domain mapping studies revealed that aM3-259 binds the CCP1-CCP2 région of MASP-3 from multiple species, including human, cynomolgus monkey, mouse, rat and dog. As shown in FIGURE 51 C, aM3-259 also binds to MASP-1.

The plate was subsequently blocked with 1% BSA/PBS, washed in PBS and then incubated 25 for one hour at room température with MASP-3 CCP1-CCP2-SP (2 pg/mL). The plate was then washed (PBS-T, 0.05%) and the candidate MASP-3 antibodies were added followed by incubation for one hour at room température. The plate was washed (PBS-T, 0.05%) and a détection antibody was added (mouse anti-human kappa-HRP, SouthemBiotech #9230-05) for one hour at room température. After another wash (PBS-T, 0.05%) the plate was

263 developed (5 minutes) with OPT EIA TMB (BD Biosciences #555214). Absorbance reading at A450 was measured using the Spectramax M5e plate reader.

Results:

FIGURE 51A and FIGURE 51B graphically illustrate the avidities of MASP-3 mAbs (purified recombinant) for human MASP-3 (CCP1-CCP2-SP). As shown in FIGURE 51 A, FIGURE 51 B, and Table 24, the MASP-3 mAbs hâve high avidity for human MASP-3, ranging from 0.241 nM to 0.023 nM. These values are 10 to 100-fold lower than those reported for the previously described MASP-3 mAbs (see Example 7 herein, also published 10 as Example 15 in WO2013/192240).

MASP-3 mAb Binding Specificity:

To détermine the specificity of the high affinity MASP-3 mabs for MASP-3, binding experiments were carried out to measure binding of 16 purified recombinant MASP-3 15 antibodies to human MASP-1 and to human MASP-2. Binding was determined as described for the MASP-3 binding ELISA, except that recombinant MASP-1A (S646A, CCP1-CCP2SP fragment) and MASP-2 (CCP1-CCP2-SP fragment) were immobilized directly on the plate.

Results:

FIGURE 51C graphically illustrâtes the results of a binding experiment in which représentative purified recombinant high affinity human MASP-3 inhibitory antibodies are shown to be sélective for binding to MASP-3 and do not bind to human MASP-1.

FIGURE 51D graphically illustrâtes the results of a binding experiment in which 25 représentative purified recombinant high afïinity human MASP-3 inhibitory antibodies are shown to be sélective for binding to MASP-3 and do not bind to human MASP-2.

Mouse MASP-3:

Binding of the MASP-3 mAbs to mouse MASP-3 was measured as described above 30 for human MASP-3 except that recombinant, full-length mouse MASP-3 (SEQ ID NO:3) was captured on the plate with aM3-259. The négative control mAb used in both experiments

264 was mAb77, a recombinant, chimeric chicken-human mAb obtained from the same immunized chickens as aM3-259, however, mAb 77 does not bind mouse MASP-3.

Results:

FIGURE 52 graphically illustrâtes the avidities of représentative MASP-3 mAbs 5 (purified recombinant) for mouse full length MASP-3. As shown in FIGURE 52, most of the MASP-3 mAbs tested also hâve high avidity for mouse MASP-3.

The avidity values (EC50) of the 16 recombinant chimeric MASP-3 mAbs for human and mouse MASP-3 are summarized in TABLE 24.

TABLE 24: Binding Avidity of MASP-3 mAbs for human and mouse MASP-3 (FIGURES

5IA, 51Band 52)

Antibody clone	Antigen used to generate mAb	Human MASP-3 (CCP1-CCP2-SP) Binding Avidity (EC5o nM)	Mouse MASP-3 (full length) Binding Avidity (ECso nM)
ΙΑΙ0*	SP	0.241	0.15
1B11	SP	0.059	1.10
1E7 '	SP	0.112	117.00
1F3	SP	0.236	0.111
1G4	SP	0.177	3.70
2D7	SP	0.122	NA
2F2	SP	0.057	0.105
2F5	SP	0.073	0.102
4B6	SP	0.211	0.188
4D5	SP	0.058	0.098
I0D12	CCP1-CCP2-SP	0.089	0.081
11B6	CCP1-CCP2-SP	0.060	0.066
I3B1	CCP1-CCP2-SP	0.059	0.035
15D9	CCP1-CCP2-SP	0.074 -	0.092
35CI	CCP1-CCP2-SP	0.091	0.209
49C11	CCP1-CCP2-SP	0.069	0.064

265

Three of the MASP-3 mAbs- 13B1, 10D12 and 4D5 - were also tested for binding to recombinant cynomolgus monkey, dog, and rat MASP-3. These results are summarized below in Table 25.

TABLE 25: Summary of MASP-3 mAb Cross-Species Binding Experiments

Species of MASP-3	Ranking of Fab Binding
Human	13B1 (pM) = 10D12(pM) = 4D5 (pM)
Cynomolgus monkey	13B1 (pM)~4D5 (pM)> 10D12(pM)
Dog	13B1 (pM) > 10D12 (pM) » 4D5 (nM)
Rat	13B1 (pM) ~ 10D12 (pM) » 4D5 (nM)
Mouse	10D12(pM)> 13B1 (pM)»4D5 (nM)

As shown in TABLE 25, MASP-3 mAbs 13B1, 10D12 and 4D5 bind to ail five species of MASP-3 tested (human, mouse, rat, dog and cynomolgus monkey). While these mAbs bind 10 to human with high avidity (<500 pM), they bind to other species of MASP-3 with varying avidities.

2. Fluorogenic Tripeptide Cleavage Assay

Background/Rationale:

In addition to its known natural substrates (Iwaki et al., J. Immunol. 187:3751, 2011;

Cortesio and Jiang, Arch. Biochem. Biophys. 449:164-170, 2006), MASP-3 has been shown to hydrolyze various tripeptide substrates (Cortesio and Jiang, Ibid.). As very small substrates, these molécules can be used to map the catalytic site of the protease. Inhibition of tri-peptide cleavage is an indication that an inhibitory agent, such as an antibody, either directly blocks access of the small substrate to the catalytic site or causes a conformational shift in the SP domain that similarly déniés access. As such, the antibody can also be expected to block catalysis of the large natural substrates by interfering with the active site of the enzyme. Functionally, this would most closely approximate the MASP-3 null mouse or 3MC patient (déficient in MASP-3).

266

Methods:

Titrations of the recombinant mAbs (3-fold dilution from 666 nM to 0.91 nM) were incubated with MASP-3 CCP1-CCP2-SP (197 nM) for 15 minutes at room température. Tripeptide substrate BOC-V-P-R-AMC (t-Butyloxycarbonyl-Val-Pro-Arg-7-Amino-45 methylcoumarin) (R&D Systems, Cat. No. ES011) was added at a final concentration of 0.2 mM. Hydrolysis of the Arg-AMC amide bond releases AMC, a highly fluorescent group. Excitation 380nm/emission 460nm kinetic values were recorded every 5 minutes at 37°C for 70 minutes using the Spectramax M5e fluorescence plate reader.

Results:

FIGURE 53 graphically illustrâtes the results of the assay measuring inhibition of

MASP-3-dependent fluorogenic tripeptide cleavage with the MASP-3 monoclonal antibodies. As shown in FIGURE 53, the MASP-3 mAbs tested fall into three distinct groups:

1. MASP-3 mAbs that are strong inhibitors of peptide cleavage by MASP-3:

1A10 (29.77 nM), 1G4 (29.64 nM), 1F3 (32.99 nM), 4B6 (26.03 nM), 4D5 (27.54 15 nM), 10D12 (30.94 nM) and 13B1 (30.13 nM).

2. MASP-3 mAbs that are weak or very weak inhibitors of peptide cleavage by MASP3: 15D9, 11B6, 2F5, 1E7 and 2D7 .

3. MASP-3 mAbs that are neutral or appear to stimulate peptide cleavage by MASP-3: 1B11; 2F2; 77 (control mAb)

3. Inhibition of Pro-Factor D to Factor D Cleavage

Methods:

Active, recombinant human MASP-3 protein (240 ng per reaction) was pre-incubated with représentative MASP-3 mAbs and a control mAb (which binds to MASP-1 but not to MASP25 3) in GVB++ buffer with a total volume of 9 pL at room température for 15 minutes. 70 ng of pro-factor D with an N-terminal Strep-tag 11 epitope tag (ST-pro-factor D-His) was then added to each tube to make the final volume per reaction to 10 pL. The reactions were incubated in a thermocycler at 37°C for 6 hours. One tenth from each réaction was then electrophoresed on a 12% Bis-Tris gel to résolve pro-factor D and active factor D cleavage 30 product. The resolved proteins were transferred to a PVDF membrane and analyzed using

Western blot by détection with a biotinylated factor D antibody (R&D Systems).

267

Results:

FIGURE 54 shows a Western blot analysis demonstrating the ability of représentative MASP-3 mAbs to block recombinant MASP-3-mediated cleavage of pro-CFD to CFD in an in vitro assay. As shown in FIGURE 54, représentative high affinity MASP-3 inhibitory mAbs 13B1, 4B6, 1G4, 2D7, 10D12, 1A10, 4D5, 1E7, and 1F3 mouse-human chimeric mAbs showed partial to full inhibition of the pro-CFD cleavage in this assay. ’

4. Factor Bb Déposition on Zymosan Assay

Methods:

Varying concentrations of MASP-3 mAbs were added to 10% CFD-depleted human sérum (Complément Technology A336) and GVB + Mg/EGTA (20 nM) and incubated for 30 minutes on ice prior to the addition of recombinant ST-pro-factor D-His (2 pg/mL final) and zymosan (0.1 mg/mL final). The zymosan particles fùnction as an activating surface for complément déposition. The mixtures were incubated at 37°C and the APC activity was measured by the flow cytométrie détection of complément factor Bb (Quidel antibody A252) on the surface of the zymosan particles.

Results:'

FIGURE 55A graphically illustrâtes the level of factor Bb déposition on zymosan particles (determined by flow cytométrie détection measured in MFI units) in the presence of varying concentrations of MASP-3 mAbs 1F3, 1G4, 2D7 and 4B6 in factor D-depleted human sérum at 37°C for 70 minutes.

FIGURE 55B graphically illustrâtes the level of factor Bb déposition on zymosan particles (determined by flow cytométrie détection measured in MFI units) in the presence of varying concentrations of MASP-3 mAbs 4D5, 10D12 and 13B1 in CFD-depleted human sérum at 37°C for 70 minutes.

The results shown in FIGURES 55A and 55B are summarized below in TABLE 26.

TABLE 26: Inhibition of Factor Bb déposition on zymosan by MASP-3 mAbs (FIGURE 55A and FIGURE 55B)_________________________________

Antibody

Inhibition of Factor Bb Déposition on Zymosan

268

	(IC50 nM)
1F3	0.1
1G4	1.1
2D7	3.5
4B6	0.2
4D5	0.4
10D12	0.5
13B1	0.3

As shown in FIGURE 55A, FIGURE 55B and TABLE 26, the MASP-3 mAbs show potent inhibition of the APC in human sérum, with IC50 values ranging from 0.1 nM to 3.5 nM. These results demonstrate that MASP-3 plays a key rôle in APC activation in an in vitro model in human sérum, and further demonstrate that MASP-3 inhibitory antibodies are potent inhibitors of the APC. '

5. Assay to measure the ability of représentative MASP-3 mAbs to inhibit rabbit érythrocyte lysis

Methods:

To monitor the inhibition of the APC in another experimental context, we evaluated the ability of représentative MASP-3 mAbs to block the lysis of rabbit érythrocytes in human sérum. Varying concentrations of MASP-3 mAbs were added to 10% factor D-depleted human sérum and GVB + Mg/EGTA (20 nM) and incubated for 30 minutes on ice prior to the addition of recombinant ST-pro-factor B-His (2 pg/mL final) and érythrocytes (2.5x10⁸ cells/mL final). The mixtures were incubated at 37°C for 70 minutes and APC-mediated hemolysis was measured by diluting the reactions and measuring the absorbance (A405), which indicates levels of free hemoglobin.

Results: .

FIGURE 56A graphically illustrâtes the level of inhibition of rabbit érythrocyte lysis in the presence of varying concentrations of MASP-3 mAbs 1A10, 1F3, 4B6, 4D5, 1G4 and 2F2 in CFD-depleted human sérum. FIGURE 56B graphically illustrâtes the level of inhibition of rabbit érythrocyte lysis in the presence of varying concentrations of MASP-3

269 mAbs 1B11, 1E7, 1G4, 2D7 and 2F5 in CFD-depleted human sérum. The results are summarized in TABLE 27.

TABLE 27: Inhibition of Rabbit Erythrocyte Lysis by MASP-3 mAbs

As shown in FIGURE 56A, FIGURE 56B and TABLE 27, the MASP-3 mAbs show inhibition of the APC-driven hemolysis of rabbit érythrocytes, with IC50 values ranging from 0.1 nM to 5.4 nM. These results corroborate the observations of the MASP-3 antibodies in 10 the zymosan assay, and further demonstrate that MASP-3 inhibitory antibodies are potent inhibitors of the APC.

6. Inhibition of pro-Factor D cleavage in 3MC patient sérum

Methods:

A représentative recombinant MASP-3 mAb (4D5) was tested for the ability to block 15 recombinant MASP-3 cleavage (and activation) of pro-factor D originating from normal human sérum and sérum from 3MC Patient B (“Pat B”), an individual who has no détectable MASP-3 in the sérum and manifests a deficiency in the APC.

270

Normal human sérum and Patient B sérum (10% final) and GVB + Mg/EGTA (30 nM) were incubated with no enzyme or with active recombinant MASP-3 (rMASP-3; 0.5 pg/mL), inactive rMASP-3, or active rMASP-3 plus MASP-3 mAb 4D5 (500 nM final) on ice for 1 hour. Zymosan (0.1 mg/mL final) was added, and the mixtures were incubasted at 37°C. After 2 hours, the samples were centrifuged and the supematants were collected. The samples were immunoprecipitated with goat antibody raised against human Factor D (R&D Systems AF1824), heat denatured and treated with Peptide-N-Glycosidase (New England Biolabs P0704L). The captured and deglycosylated proteins were resolved with SDS-PAGE and the gels were electroblotted for Western blot analysis with a biotinylated anti-CFD (R&D Systems BAF1824) and High Sensitivity Streptavidin-HRP (Thermo Fischer Scientific 21130).

Results:

FIGURE 57 shows a Western blot analyzing the level of pro-factor D and factor Din 3MC Patient B sérum in the presence active rMASP-3, inactive rMASP-3, and active rMASP-3 plus mAb 4D5. As shown in FIGURE 57, normal human sérum contains predominately the mature form, while Patient B sérum principally contains the zymogen form of factor D. As further shown in FIGURE 57, active rMASP-3 in the presence of zymosan causes cleavage of pro-factor D in Patient 3 sérum, while the inactive (zymogen) form of MASP-3 does not. Finally, as shown in FIGURE 57, the MASP-3 mAb 4D5 blocks cleavage of pro-factor D in Patient 3 sérum in the presence of active rMASP-3. These results further demonstrate the rôle of MASP-3 in the cleavage of pro-factor D in the activation of the APC, and demonstrate that a MASP-3 inhibitory mAb is capable of blocking MASP-3 mediated pro-factor D cleavage and thereby blocking the APC.

EXAMPLE 17

Analysis of représentative MASP-3 inhibitory mAbs 10DI2 and 13B1 for the ability to inhibit the APC in vivo.

1. Inhibition of the APC by mAb M3-1 (13B1) and 10D12 in vivo:

Methods:

271

In order to détermine the efficacy of MASP-3 mAb 13B1 (M3-1) and 10D12 for inhibiting the APC in vivo, a group of mice (n = 4) received a single intravenous tail vein injection of 10 mg/kg mAb 13B1 and a second group of mice (n=4) received a single intravenous tail vein injection of 10 mg/kg mAb 10D12. Blood collected from the animais was used to préparé 5 sérum, providing a matrix for the flow cytométrie assessment of APC activity in an ex vivo assay measuring the level of C3 (also C3b and iC3b, Dako F020102-2) déposition on zymosan particles. Sérum prepared from blood harvested at a pre-dose timepoint and multiple post-dose time points (96 hrs, 1 week, and 2 weeks) was diluted to 7.5% and zymosan particles (0.1 mg/mL final) were added to induce the APC. Antibody-treated mice 10 were compared to a group of control mice (n = 4) that were given a single intravenous dose of vehicle.

Results:

FIGURE 58 graphically illustrâtes the level of C3 déposition on zymosan particles at various 15 time points after a single dose of mAb M3-1 (13B1), mAb 10D12, or vehicle in wild-type mice. As shown in FIGURE 58, in the pre-dose time point the three conditions show comparable levels of APC activity. At 96 hours and the two later time points, both mAbtreated groups show near-complete ablation of systemic APC activity, while the APC activity of the vehicle-treated group remains unabated.

These results demonstrate that MASP-3 mAb M3-1 (13B1) and mAb 10D12 are potent inhibitors of the APC in vivo in mouse.

2. Status of Factor B in mice treated with MASP-3 mAb 10D12

Methods:

During the conversion of Factor B zymogen to an active proteolytic enzyme, Factor B is 25 cleaved into the Ba (~30 kDa) and Bb (~60 kDa) fragments by Factor D. The status of the Ba fragment in mouse sérum obtained from mice treated with the MASP-3 mAb 10D12 was determined as follows.

272

Mice (n=4) were given two intravenous tail vein injections of 10 mg/kg mAb 10D12.The treatments occurred seven days apart and blood was collected from the animais three days after the second injection. A second set of four mice received a single intravenous dose of vehicle (PBS). The blood collected from both groups was used to préparé sérum, providing a matrix for complément activation. Zymosan particles (0.1 mg/mL final) were added to diluted sérum (7.5% final) and incubated for 35 minutes at 37°C.

Results:

As a measure of APC activation, FIGURE 59 shows a Western blot analyzing the status of the Ba fragment in mouse sérum obtained from mice treated with mAb 10D12 or PBS and stimulated with zymosan. Each lane in FIGURE 59 represents a different mouse, and the lanes altemate to show sérum from a représentative vehicle mouse adjacent to a MASP-3 mAb-treated mouse for the purposes of comparison. Two control conditions, from mice treated with vehicle or mAb 10D12 are shown in lanes 1 and 2, respectively (starting from the left side of the blot) as représentatives of the basal level of Ba présent in the sérum samples in the absence of zymosan. Lanes 3 to 10 ail show the level of Ba fragment présent after incubation with zymosan. In ail cases, the MASP-3 mAb-treated mice demonstrate a reduced level ofthe Ba fragment in comparison to the vehicle-treated animais.

3. Sérum from Mice Treated with mAb 10D12 inhibits hemolysis

Methods:

As another measure of APC inhibition by MASP-3 inhibitory antibodies, we evaluated the ability of the MASP-3 antibodies to block the lysis of rabbit érythrocytes in sérum from mice treated with représentative MASP-3 mAb 10D12 as compared to sérum from vehicle control treated mice.

Mice (n=4/group) were given three intravenous tail vein injections of vehicle control (PBS), 10 mg/kg MASP-3 mAb 10DI2, or 25 mg/kg MASP-3 mAb 10D12. The treatments occurred seven days apart from one another and blood was collected from the animais three days after the third injection. The blood was used to préparé sérum, providing a matrix for

273 hemolysis reactions. Erythrocytes (2.5 x 10⁸ cells/mL final) were added to 20% pooled sérum from four mice in GVB + Mg/EGTA (20 nM). The mixtures were incubated at 37°C and APC-mediated hemolysis was measured by diluting the reactions and measuring the absorbance (A405).

Results:

FIGURE 60 graphically illustrâtes the level of inhibition of hemolysis by 20% sérum from mice treated with MASP-3 mAb 10D12 (10 mg/kg or 25 mg/kg) or vehicle control treated mice. As shown in FIGURE 60, sérum from mice treated with MASP-3 mAb 10D12 at both 1 Omg/kg and 25 mg/kg demonstrated less overall hemolysis during the 1 hour test period as compared to vehicle-treated mice.

Overall Summary of Results:

As described in this Example, représentative high affinity MASP-3 inhibitory mAbs 13B1 and 10D12 inhibit the APC in vivo. As described in Example 12, it was determined that MASP-3 monoclonal antibody 13B1 (also referred to as mAb M3-1) provides a clear benefit to survival of red blood cells lacking Crry in a mouse model associated with paroxysmal nocturnal hemogloinuria (PNH). As described in Example 13, it was determined that MASP3 mAb M3-1 reduced the incidence and severity of clinical arthritis scores in a dosedependent fashion.

EXAMPLE 18

This Example describes the results of epitope binding analysis of high potency MASP-3 inhibitory mAbs.

L Compétition Binding Analysis

Methods:

well ELISA assay plates were coated with the capture antibody, aM3-259, an IgG4 isotype mAb which has been shown to bind the CCP1-CCP2 région of MASP-1 and MASP19469

274

3. The full-length human MASP-3 protein was immobilized on the plate via capture antibody aM3-259. In separate, non-coated wells, a 2-fold dilution sériés of one test MASP-3 mAb of an IgG4 isotype was mixed with a constant concentration of another test MASP-3 antibody of an IgGl isotype. The mixture was added to the coated wells and allowed to bind to the captured MASP-3. Potential compétition between the two antibodies was determined by the détection of the IgGl isoform using an HRP-conjugated antibody against the human IgGl hinge région (Southern Biotech 9052-05), and a TMB substrate reagent set (BD Biosciences 555214).

Results:

FIGURES 61A-61E graphically illustrate the results ofthe compétition binding analysis.

FIGURE 61A graphically illustrâtes the results of the compétition binding analysis to identify MASP-3 mAbs (IgG4) that block the interaction between mAb 4D5 (IgGl) and human MASP-3.

FIGURE 61B graphically illustrâtes the results of the compétition binding analysis to identify MASP-3 mAbs (IgG4) that block the interaction between mAb 10D12 (IgGl) and human MASP-3.

FIGURE 61C graphically illustrâtes the results of the compétition binding analysis to identify MASP-3 mAbs (IgG4) that block the interaction between mAb 13B1 (IgGl) and human MASP-3.

FIGURE 61D graphically illustrâtes the results of the compétition binding analysis to identify MASP-3 mAbs (IgG4) that block the interaction between mAb 1F3 (IgGl) and human MASP-3.

FIGURE 61E graphically illustrâtes the results of the compétition binding analysis to identify MASP-3 mAbs (IgG4) that block the interaction between mAb 1G4 (IgGl) and human MASP-3.

The data from FIGURES 61A to 61E is summarized below in TABLE 28.

275

These data indicate that MASP-3 mAbs 4D5, 10D12, 13B1, 1A10, 1F3 and 1G4 share a common epitope or overlapping epitopes on human MASP-3. Surprisingly, 1G4 has a very limited capacity to block the binding of the other five mAbs to MASP-3, but those mAbs almost completely block the binding of 1G4 itself to MASP-3.

2. Analysis of mAb binding to peptides representing linear and discontinuons MASP-3 epitopes

Methods:

Fourteen of the 16 MASP-3 mAbs were evaluated by Pepscan to identify the régions of MASP-3 to which they bind. To reconstruct both linear and potential discontinuous epitopes of the target molécule, a library of peptides was synthesized corresponding to amino acid residues 299 to 728 of SEQ ID NO:2 (human MASP-3). Amino acid residues 1-298 of MASP-3 were not présent in the immunogen and were not included in this analysis.

Pepscan epitope analsysis included use of the CLIPS technology, which structurally fixes peptides into defined three-dimensional structures (see Timmerman et al., J Mol Recog. 20:283-299, 2007 and Langedijk et al., Analytical Biochemistry 417:149-155, 2011). The binding of each antibody to each of the synthesized peptides was tested in a Pepscan-based ELISA.

Results:

The peptide binding results from Pepscan for each antibody analyzed is described below and summarized in TABLE 4, TABLE 28 and FIGURES 62-67.

Antibodies 1F3, 4B6, 4D5 and 1 Al0 (Group IA)

When tested under moderate stringency conditions, antibodies 1F3, 4B6, 4D5 and 1A10 bound discontinuous epitope mimics and also bound simple constrained and linear mimics. Data analysis demonstrates that antibodies IF3, 4B6, 4D5 and 1A10 ail dominately recognize peptide stretch 498VLRSQRRDTTVI509 (SEQ ID NO:9) of MASP-3. This peptide lies immediately adjacent to the active site histidine, H497. Data obtained for these antibodies with discontinuous mimics suggest that peptide stretches 544DFNIQNYNHDIALVQ558 (SEQ

276

ID NO:I I), 639GNYSVTENMFC649 (SEQ ID NO:13) and 7&4VSNYVDWVWE713 (SEQ ID NO: 14) of MASP-3 also contribute to the binding. Peptide 544DFNIQNYNHDIALVQ558 (SEQ ID NO:11) contains the active site aspartate (D553).

Antibody 10D12 (Group IB)

When tested under moderate stringency conditions, antibody 10D12 bound peptides with core sequence 498VLRSQRRDTTVI509 (SEQ ID NO:9) of MASP-3, the sequence adjacent to the active site histidine, H497.

Antibody 13B1 (Group IC)

When tested under moderate stringency conditions antibody 13B1 recognizes a discontinuons epitope comprising peptide stretches 494TAAHVLRSQRRDTTV508 (SEQ ID NO: 10) and 626PHAECKTSYESRS638 (SEQ ID NO: 12) of MASP-3, where peptide stretch 626PHAECKTSYESRS638 (SEQ ID NO: 12) appears to be the dominant part of the epitope as 15 it can also be bound in simple constrained form. The peptide 494TAAHVLRSQRRDTTV508 (SEQ ID NO: 10) includes the active site histidine, H497.

Antibody 1G4 (Group II)

When tested under low stringency conditions antibody IG4 recognizes a discontinuons 20 epitope comprising peptide stretches 454RNAEPGLFPWQ464 (SEQ ID NO: 17), 514EHVTVYLGLH523 (SEQ ID NO: 19) and ₆67AFV1FDDLSQRW₆78 (SEQ ID NO:23) of MASP-3, where peptide stretch 667AFVIFDDLSQRW678 (SEQ ID NO:23) is the dominant part of the epitope. The dominant peptide lies within three amino acids of the active site serine, S664.

Antibodies 1E7 and 2D7 (Group ΙΠΑ)

When tested under high and low stringency conditions, respectively, antibodies 1E7 and 2D7 recognize a discontinuons epitope comprising peptide stretches 454RNAEPGLFPWQ464 (SEQ ID NO: 17), 514EHVTVYLGLH523 (SEQ ID NO: 19) and 667AFVIFDDLSQRW678 (SEQ ID 30 NO:23) of MASP-3, where peptide stretch 667AFVIFDDLSQRW₆78 (SEQ ID NO:23) is the dominant part of the epitope and which lies within three amino acids of the active site serine, S664.

277

Antibodies 2F5 and 15D9 (Group IIIB)

When tested under low stringency conditions, antibodies 2F5 and 15D9 dominantly recognize a discontinuons epitope comprising peptide stretches 454RNAEPGLFPWQ464 (SEQ 5 ID NO: 17), 479KWFGSGALLSASWIL493 (SEQ ID NO: 18), 562PVPLGPHVMP571 (SEQ ID

NO:20) and 667AFVIFDDLSQRW₆78 (SEQ ID NO:23) of MASP-3. Peptides 479KWFGSGALLSASWIL493 (SEQ ID NO: 18) and ₆67AFVIFDDLSQRW₆78 (SEQ ID NO:23) localize within four or three amino acids of the active site residues H497 and S664, respectively.

Antibody 1B11 (Group IIIC)

When tested under moderate stringency conditions, antibody 1B11 recognizes a discontinuous epitope comprising peptide stretches 435ECGQPSRSLPSLV447 (SEQ ID NO: 16), 454RNAEPGLFPWQ4M (SEQ ID NO: 17), 583APHMLGL589 (SEQ ID NO:21) and 15 614SDVLQYVKLP623 (SEQ ID NO:22) of MASP-3.

TABLE 28: Summary of Epitope Binding Analysis

MASP-3 mAb Ref. No./Group	Peptide Binding Fragments (Epitopes) on human MASP-3 (w/leader)	Competes With	Peptide Cleavage Assay
4D5 Group IA	498VLRSQRRDTTVI509 (SIN:9) 544DFNIQNYNHDIALVQ558 (SIN: 11 ) 639GNYSVTENMFC649 (SIN: 13) 704VSNYVDWVWE713 (SIN: 14)	1F3, 1G4, 4D5, 10D12, 13B1	inhibits
IF3 Group IA	498VLRSQRRDTTVI509 (SIN:9) 544DFNIQNYNHDIALVQ558 (SIN:11 ) 639GNYSVTENMFC649 (SIN: 13) 704VSNYVDWVWE713 (SIN: 14)	1F3, 1G4, 4D5, 10D12, 13B1	inhibits
4B6 Group IA	49sVLRSQRRDTTVI509 (SIN:9) 544DFNIQNYNHDIALVQ558 (SIN: 11 ) 639GNYSVTENMFC649 (SIN: 13) 704VSNYVDWVWE713 (SIN: 14)	1F3, 1G4, 4D5, 10D12, 13B1	inhibits
ΙΑΙ0 Group IA	49sVLRSQRRDTTVI509 (SIN:9) 544DFNIQNYNHDIALVQ558 (SIN: 11)	1F3, 1G4, 4D5,	inhibits

278

	639GNYSVTENMFC649 (SIN: 13) 704VSNYVDWVWE713 (SIN: 14)	10D12, 13B1
10D12 Group IB	498VLRSQRRDTTVI509 (SIN:9)	1F3, 1G4, 4D5, 10D12, 13B1	inhibits
13B1 Group IC	494TAAHVLRSQRRDTTV5o8 (SIN: 10) 626PHAECKTSYESRS638 (SIN: 12)	1F3, 1G4, 4D5, 10D12, 13B1	inhibits
Group I core sequence	498VLRSQRRDTTV508 (SIN: 15)
1G4 Group IIcross competes with Group I and III	454RNAEPGLFP WQ464 (SIN: 17) 5i4EHVTVYLGLH523 (SIN: 19) 667AFVIFDDLSQRW678 (SIN:23)	1F3, IG4, 4D5, 10D12, 13B1	inhibits
1E7 Group IIIA	454RNAEPGLFP WQ4m (SIN: 17) 5i4EHVTVYLGLH523 (SIN: 19) 667AFVIFDDLSQRW678 (SIN:23)	1G4	Weakly inhibits
2D7 Group IIIA	454RNAEPGLFP WQ464 (SIN: 17) 5i4EHVTVYLGLH523 (SIN: 19) 667AFVIFDDLSQRW678 (SIN:23)		Weakly inhibits
2F5 Group IIIB	454RNAEPGLFP WQ464 (SIN: 17) 479KWFGSGALLSASWIL493(SIN 18) 562PVPLGPHVMPs7i (SIN:20) 667AFVIFDDLSQR W67s (SIN:23)		No effect
15D9 Group IIIB	454RNAEPGLFPWQ464(SIN:I7) 479KWFGSGALLSASWIL493(SIN 18) 562PVPLGPHVMP571 (SIN:20) 667AFVIFDDLSQRW678 (SIN:23)		No effect
1B11 Group IIIC	435ECGQPSRSLPSLV447 (SIN: 16) 454RNAEPGLFPWQ464 (SIN: 17) 583APHMLGL589 (SIN:21) 614SDVLQYVKLP623 (SIN:22)		stimulâtes

279

Core sequence for Group II and Group III	454RNAEPGLFPWQ464 (SIN:17)
2F2 Group IV	Binding epitope not determined	1F3, 4D5, 11B6, 2F2	stimulâtes
11B6 Group IV	Binding epitope not determined	1F3, 4D5, 11B6, 2F2	No effect

FIGURE 62 provides a schematic diagram showing the régions of contact on human MASP-3 by the MASP-3 mAbs, as determined by Pepscan Analysis. As shown in FIGURE 5 62, ail of the MASP-3 mAbs hâve régions of contact in the beta chain containing the SP domain of MASP-3. One mAb, 1B11, also has a région of contact between the CCP2 and SP domains in the alpha chain of MASP-3.

FIGURES 63A to 67 show 3-D models illustrating the régions of contact of the high affinity MASP-3 mAbs on the CCPI/2/SP domains of human MASP-3, wherein the SP 10 domain active site of MASP-3 is facing towards the front and the catalytic triad is shown as side chains.

FIGURE 63A shows the régions of contact between human MASP-3 and high affinity MASP-3 mAbs 1F3, 4D5 and 1A10, including aa residues 498-509 (SEQ ID NO:9), aa residues 544-558 (SEQ ID NO:11), aa residues 639 to 649 (SEQ ID NO: 13) and aa residues 15 704 to 713 (SEQ ID NO:14).

FIGURE 63B shows the régions of contact between human MASP-3 and high affinity MASP-3 mAb 10D12, including aa residues 498 to 509 (SEQ ID NO:9).

FIGURE 64 shows the régions of contact between human MASP-3 and high affinity MASP-3 mAb 13B1, including aa residues 494 to 508 (SEQ ID NO: 10) and aa residues 626 20 to 638 (SEQ IDNO: 12).

FIGURE 65 shows the régions of contact between human MASP-3 and high affinity MASP-3 mAb 1B11, including aa residues 435 to 447 (SEQ ID NO: 16), aa residues 454 to

280

464 (SEQ ID NO: 17), aa residues 583 to 589 (SEQ ID NO:21) and aa residues 614 to 623 (SEQ ID NO:22).

FIGURE 66 shows the régions of contact between human MASP-3 and high affinity MASP-3 mAbs 1E7, 1G4 and 2D7, including aa residues 454 to 464 (SEQ ID NO: 17), aa residues 514 to 523 (SEQ ID NO: 19) and aa residues 667 to 678 (SEQ ID NO:23).

FIGURE 67 shows the régions of contact between human MASP-3 and high affinity MASP-3 mAbs 15D9 and 2F5, including aa residues 454 to 464 (SEQ ID NO: 17), aa residues 479 to 493 (SEQ ID NO: 18), aa residues 562 to 571 (SEQ ID NO:20), and aa residues 667 to 678 (SEQ ID NO:23).

In summary, conclusive binding profiles were obtained for 12 of the 14 antibodies. Ail 12 mapped antibodies recognized solvent exposed epitopes within the peptidase SI domain. The close proximity of a number of the epitope déterminants to residues for the active site catalytic triad (H497, D553, S664) is consistent with a model in which the high affinity inhibitory MASP-3 mAbs block enzymatic activity by interfering with the enzyme-substrate interaction.

EXAMPLE 19

This Example describes the humanization of représentative MASP-3 mAbs and engineering of potential post-translational modification sites.

Methods:

1. Humanization of Représentative High Affiinity MASP-3 mAbs

Methods:

To reduce immunogenicity risk, représentative high affinity MASP-3 inhibitory antibodies 4D5, 10D12 and 13B1 were humanized by a CDR-grafting method. CDRS of each MASP-3 antibody were grafted into the closest consensus human framework sequences. Some of the Vernier zone residues were modified by Quickchange site-directed mutagenesis (Agitent

281

Technologies). The resulting humanized VH and VL régions were transferred into pcDNA3.1-based human IgGl or IgG4 and IgK expression contructs, and the recombinant antibodies were expressed and purified as described above. Affïnity of the humanized antibodies was determined by ELIS A using monovalent Fab fragments, and potency was 5 assessed by C3 déposition assay using intact IgG4 formats.

Results:

Amino acid sequences of représentative humanized versions of the heavy chain variable régions and light chain variable régions for mAbs 4D5, 10D12 and 13B1 are provided below. The CDRs (Kabat) are underlined.

4D5:

h4D5 VH-14 (SEQ ID NO:248)

OVQLVQSGAEVKKPGASVKVSCKASGYTFTTDDINWVROAPGOGLEWIGWIYPRD DRTKYNDKFKDKATLTVDTSSNTAYMELSSLRSEDTAVYYCSSLEDTYWGOGTLVT VSS h4D5 VH-19 (SEQ ID NO:249)

OVQLVQSGAEVKKPGASVKVSCKASGYTFTTDDINWVROAPGOGLEWIGWIYPRD DRTKYNDKFKDRATLTVDTSSNTAYMELSSLRSEDTAVYYCSSLEDTYWGOGTLVT

VSS h4D5 VL-1 (SEP ID NO.-250)

DIVMTQSPDSLAVSLGERATINCKSSOSLLNSRTRKNYLAWYOQKPGOPPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCKOSYNLYTFGOGTKVE1KR

10D12:

h!0D12 VH-45 (SEQ ID NO:251)

282

QIOLVOSGSELKKPGASVKVSCKASGYIFTSYGMSWVROAPGKGLKWMGWINTYS GVPTYADDFKGRFVFSLDTSVRTPYLOISSLKAEDTAVYFCARGGEAMDYWGOGTL

VTVSS h!0D12 VH-49 (SEQ ID NO:252)

QIOLVOSGSELKKPGASVKVSCKASGYIFTSYGMSWVROAPGKGLKWMGWINTYS GVPTYADDFKGRFVFSLDTSVRTPYLOISSLKAEDTATYFCARGGEAMDYWGOGTL VTVSS hlOD12 VL-21 (SEQ ID NO:253)

DVLMTOTPLSLSVTPGOPASISCKSSOSLLDSDGKTYLNWLLORPGQSPKRLIYLVSK LDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPWTFGOGTKVEIKR

13B1 h!3Bl VH-9 (SEQ IDNO:254)

OVQLVQSGAEVKKPGASVKVSCKASGYTFTGKWIEWVROAPGOGLEWIGEILPGTG STNYAOKFQGRATFTADSSTSTAYMELSSLRSEDTAVYYCLRSEDVWGOGTLVTVS S h!3Bl VH-10 (SEQ ID NO:255)

QVQLVQSGAEVKK.PGASVKVSCKASGYTFTGKWIEWVROAPGOGLEWIGEILPGTG STNYNEKFKGRATFTADSSTSTAYMELSSLRSEDTAVYYCLRSEDVWGQGTLVTVSS h!3Bl VL-1 (SEQ IDNO:256)

283

DIVMTOSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYOOKPGOPPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCKOSYNIPTFGOGTKVEIKR

The affinity of représentative humanized 4D5, 10D12 and 13B1 antibodies for human 5 MASP-3 is shown below in TABLE 29.

TABLE 29: Binding of Représentative humanized MASP-3 mAbs to MASP-3

MASP-3 antibody clone (Fab format)	Binding to human MASP-3 ECso (nM)
4D5 Parental Fab	0.107
h4D5_14-l Fab (VH-14 and VL-1)	0.085 .
h4D5_19-l Fab (VH-19 and VL-1)	0.079
10D12 Parental Fab	0.108
hlOD12_45-21 Fab (VH-45 and VL-21)	0.108
hlOD12_49-21 Fab (VH-49 and VL-21)	0.115
13B1 Parental Fab	0.123
hl3Bl_9-l Fab (VH-9 and VL-1)	0.101
hl3Bl_10-l Fab (VH-10 and VL-1)	0.097

The percent identity of humanized framework sequences to those of human germline framework sequences:

h4D5_VH-14=90%; h4D5_VH-19=91 %; h4D5_VL-1=100%;

hlOD12_VH-45=92%; h!0D12_VH-49=91%; h!0D12_VL-21=93%;

284 h!3Bl_VH-9=95%; hl3Bl_VH-10=94%; h!3Bl_VL-l=100%

2. Mutagenesis of Représentative MASP-3 mAbs to remove Asn/Asp Modification Sites in CDR-1 of the Light Chain Variable Région of 4D5,10D12 and 13B1

Représentative high affinity MASP-3 inhibitory mAbs 4D5, 10D12 and 13B1 were analyzed for post-translational modification. Asparagine residues with a succeeding Glycine, Serine, Histidine, Alanine or Asparagine (“NG”, “NS”, “NH”, “NA”, or “NN” motif) are often susceptible to the hydrolysis of the amide group of Asparagine side-chain, or “deamidation.” Aspartic acid residues with a succeeding Glycine or Proline (“DG” or “DP” motif) are often susceptible to the interconversion, or “isomerization.” Such modifications resuit in charge heterogeneity and may affect antibody function if they occur in a binding interface. They also may increase risks of fragmentation, immunogeneticity and aggregation.

Potential post-translational modification motifs were identified in CDR-1 of the light chain variable régions of 4D5, 10D12and 13B1.

4D5 and 13B1 contained one possible Asn deamidation site in CDR1 of the light chain (shown as “NS” at positions 8 and 9 of SEQ ID NO: 142 underlined in TABLE 30 below. As further shown below in Table 30, 10D12 contained one possible Asp isomerization site in CDR1 ofthe light chain.

Variants of the humanized version of these MASP-3 mAbs were generated by site-directed 20 mutagenesis as shown in TABLE 30. The variants were expressed and purified as described . above. Affinity was determined by ELISA using monovalent Fab fragments, and potency was assessed by C3 déposition assay using intact IgG4 formats as described above.

TABLE 30: Variants of CDR-L1 for 4D5,10D12 and 13B1

Antibody	Région	Sequence
4D5 parent	LC-CDR1	KSSQSLLNSRTRKNYLA (SEQ ID NO: 142)
4D5-NQ mutant	LC-CDR1	KSSQSLLQSRTRKNYLA (SEQ ID NO:257)

285

4D5-NA mutant	LC-CDR1	KSSQSLLASRTRKNYLA (SEQ ID NO:258)
4D5-ST mutant	LC-CDR1	KSSQSLLNTRTRKNYLA (SEQ ID NO:259)
13B1 parent	LC-CDR1	KSSQSLLNSRTRKNYLA (SEQ ID NO: 142)
13B1-NQ	LC-CDR1	KSSQSLLQSRTRKNYLA (SEQ IDNO:257)
13B1-NA	LC-CDR1	KSSQSLLASRTRKNYLA (SEQ IDNO:258)
13B1-ST	LC-CDR1	KSSQSLLNTRTRKNYLA (SEQ IDNO:259)
Consensus for 4D5, 13B1 and variants	LC-CDR1	KSSQSLLXXRTRKNYLA (SEQ IDNO:260) Wherein X at position 8 is N, Q or A; and wherein X at position 9 is S or T
10D12 parent	LC-CDR1	KSSOSLLDSDGKTYLN (SEQ ID NO: 153)
10D12-DE mutant	LC-CDR1	KSSQSLLDSEGKTYLN (SEQ IDNO:261)
10D12-DA mutant	LC-CDR1	KSSQSLLDSAGKTYLN (SEQ ID NO:262)
10D12-GA mutant	LC-CDR1	KSSQSLLDSDAKTYLN (SEQ ID NO:263)
35CI	LC-CDR1	KSSQSLLDSDGKTYLS (SEQ ID NO: 159)
Consensus of 10D12, 35C1 and variants	LC-CDR1	KSSQSLLDSXXKTYLX (SEQ ID NO:215) Wherein X at position 10 is D, E or A; Wherein X at position 11 is G or A; and wherein X at position 16 is N or S

TABLE 31: Binding of mutagenized candidates of humanized 4D5, 10D12 and 13B1 mAbs to human MASP-3

MASP-3 antibody clone (Fab format)	Binding to human MASP-3 ECso (pM)
h4D5_19-l parental Fab (VH-19 and VL-1)	102
h4D5-19-l-NQ Fab (VH-19 and VL-1 -NQ)	732
h4D5-19-l-NA Fab (VH-19 and VL-1-NA)	122

286

h4D5-19-l-ST Fab (VH-19 and VL-l-ST)	151
hlOD12_45-21 parental Fab (VH-45 and VL-21)	108
h 10D12-45-21-DE Fab (VH-45 and VL-21-DE)	326
h 1OD12-45-21-DA Fab (VH-45 and VL-21-DA)	294
h 1OD12-45-21-GA Fab (VH-45 and VL-21-GA)	181
hl3Bl_10-l parental Fab (VH-10 and VL-1)	100
hl3Bl_10-l-NQ Fab (VH-10 and VL-l-NQ)	138
h!3Bl_10-l-NA Fab (VH-10 and VL-1-NA)	105
hl3Bl_10-l-ST Fab (VH-10 and VL-l-ST)	120

287

TABLE 32: MASP-3 Antibody humanized VH Sequences (CDRs and FR régions, Kabat)

Antibody	HC FRI	HCCDR1
4D5 parent (SIN:24)	QVQLKQSGPELVKPGASVKLSCKASGYTFT (SEQIDNO:55)	TDD1N (SEQ IDNO:56)
h4D5_VH-14 (SIN:248)	QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO:264)	TDD1N (SEQ1DNO:56)
h4D5_VH-19 (SIN:249)	QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO:264)	TDD1N (SEQ1DNO:56)
10D12 parent (SIN:28)	QIQLVQSGPELKKPGETVK1SCKASGYIFT (SEQIDNO:71)	SYGMS (SEQ ID NO:72)
h!0DI2_VH45 (SIN:251)	QIQLVQSGSELKKPGASVKVSCKASGYIFT (SEQ IDNO:269)	SYGMS (SEQIDNO:72) ·
hI0DI2-VH- 49 (SIN:252)	QIQLVQSGSELKKPGASVKVSCKASGYIFT (SEQ 1DNO:269)	SYGMS (SEQ1DNO:72)
13BI parent (S1N:3O)	OVOLKOSGAELMKPGASVKLSCKATGYTFT (SEQIDNO:83) .	GKWIE (SEQ IDNO:84)
h!3Bl_VH-9 (SIN:254)	QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO:273)	GKWIE (SEQ ID NO:84)
h!3Bl_VH10 (SIN:255)	QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ 1DNO:273)	GKWIE (SEQ 1DNO:84)
Antibody	HC FR2	HCCDR2
4D5 parent	WVKQRPGQGLEWIG (SEQIDNO:57)	WIYPRDDRTKYNDKFKD (SEQIDNO:58)
h4D5_VH-I4	WVRQAPGQGLEWIG (SEQ ID NO:265)	WIYPRDDRTKYNDKFKD (SEQIDNO:58)
h4D5_VH-19	WVRQAPGQGLEWIG (SEQ ID NO:265)	WIYPRDDRTKYNDKFKD (SEQIDNO:58)
10D12 parent	WVRQAPGKGLKWMG (SEQ 1DNO:73)	WINTYSGVPTYADDFKG (SEQ1DNO:74)
h!0D12_VH45	WVRQAPGKGLKWMG (SEQ IDNO:73)	W1NT YSGVPT Y ADDF KG (SEQ 1DNO:74)
h!0D12-VH- 49	WVRQAPGKGLKWMG (SEQ 1DNO:73)	WINTYSGVPTYADDFKG (SEQ ID NO:74)
13B1 parent	WVKQRPGHGLEWIG (SEQ1DNO:85)	E1LPGTGSTNYNEKFKG (SEQ1DNO:86)

288

h!3Bl_VH-9	WVRQAPGQGLEWIG (SEQ ID NO:274)	EILPGTGSTNYAQKFQG (SEQ ID NO:275)
hl3Bl_VH10	WVRQAPGQGLEWIG (SEQ ID NO:274)	EILPGTGSTNYNEKFKG (SEQIDNO:86)
Antibody	HC FR3	HC CDR3
4D5 parent	KATLTVDTSSNTAYMDLHSLTSEDSAVYFCSS (SEQIDNO:59)	LEDTY (SEQ1DNO:60)
h4D5_VH-14	KATLTVDTSSNTAYMELSSLRSEDTAVYYCSS (SEQ ID NO:266)	LEDTY (SEQ ID NO:60)
h4D5_VH-19	RATLTVDTSSNTAYMELSSLRSEDTAVYYCSS (SEQ ID NO:267)	LEDTY (SEQIDNO:60)
10D12 parent	RFAFSLETSARTPYLQINNLKNEDTAIYFCAR (SEQIDNO:75)	GGEAMDY (SEQ ID NO:76)
h!0D12_VH45	RFVFSLDTSVRTPYLQ1SSLKAEDTAVYFCAR (SEQ ID NO:270)	GGEAMDY (SEQ1DNO:76)
hlODI2-VH- 49	RFVFSLDTSVRTPYLQ1SSLKAEDTATYFCAR (SEQ ID NO:271)	GGEAMDY (SEQ ID NO:76)
13B1 parent	KATFTADSSSNTAYMQLSSLTTEDSAMYYCLR (SEQIDNO:87)	SEDV (SEQ ID NO:88)
hl3BI_VH-9	RATFTADSSTSTAYMELSSLRSEDTAVYYCLR (SEQ ID NO:276)	SEDV (SEQIDNO:88)
hl3Bl_VH10	RATFTADSSTSTAYMELSSLRSEDTAVYYCLR (SEQ ID NO:276)	SEDV (SEQ ID NO:88)
Antibody	HC FR4
4D5 parent	WGQGTLVAVSS (SEQIDNO:61)
h4D5_VH-14	WGQGTLVTVSS (SEQ ID NO:268)
h4D5_VH-19	WGQGTLVTVSS (SEQ ID NO:268)
10DI2 parent	WGQGTSVTVSS (SEQ ID NO:77)
hlOD12_VH45	WGQGTLVTVSS (SEQ 1DNO:272)

289

h!0DI2-VH- 49	WGQGTLVTVSS (SEQ IDNO:272)
13B1 parent	WGTGTTVTVSS (SEQ ID NO:89)
hI3BI_VH-9	WGQGTLVTVSS (SEQ ID NO:277)
hI3Bl_VH10	WGQGTLVTVSS (SEQ ID NO:277)

Représentative Humanized Light Chain Variable Régions with Variants:

h4D5 VL-1 -NA (SEQ ID NO:278)

DIVMTOSPDSLAVSLGERATINCKSSOSLLASRTRKNYLAWYOQKPGOPPKLLIYWA

STRESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCKQSYNLYTFGOGTKVEIKR h!0D12 VL-21-GA (SEQ ID NO:279)

DVLMTOTPLSLSVTPGOPASISCKSSOSLLDSDAKTYLNWLLQRPGOSPKRLIYLVSK

LDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWOGTHFPWTFGOGTKVE1KR h!3Bl VL-l-NA (SEQ ID NO:28Q)

DIVMTOSPDSLAVSLGERATINCKSSOSLLASRTRKNYLAWYOQKPGOPPKLLIYWA

STRESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCKQSYNIPTFGOGTKVE1KR

TABLE 33: MASP-3 Antibody humanized VL Sequences (CDRs and FR régions, Kabat) [plus variants in LC-CDRI]__________________________________________________________

Antibody	LC FRI	LC CDR1
4D5 parent (S1N:4O)	DIVMTOSPSSLAVSAGEKVTMTC (SEQ 1DNO:I4I)	KSSQSLLNSRTRKNYLA (SEQ IDNO:142)
h4D5_VL-l (SIN:250)	DIVMTQSPDSLAVSLGERATINC (SEQIDNO:281)	KSSQSLLNSRTRKNYLA (SEQ IDNO:142)
h4D5_VL-lNA (SIN:278)	DIVMTQSPDSLAVSLGERATINC (SEQ ID NO:28I)	KSSQSLLASRTRKNYLA (SEQ IDNO:258)

290

10DI2 parent (SIN:43)	DVLMTQTPLTLSVT1GQPAS1SC (SEQ ID NO:152)	KSSQSLLDSDGKTYLN (SEQ IDNO:153)
hlODI2_VL21 (SIN:253)	DVLMTQTPLSLSVTPGQPASISC (SEQ IDNO:285)	KSSQSLLDSDGKTYLN (SEQ IDNO:153)
h!0DI2_VL21-GA (SIN:279)	DVLMTQTPLSLSVTPGQPASISC (SEQ ID NO:285)	KSSQSLLDSDAKTYLN (SEQ IDNO:263)
13B1 parent	DIVMTQSPSSLAVSAGEKVTMSC (SEQID NO: 151)	KSSQSLLNSRTRKNYLA (SEQID NO: 142)
h!3BI_VL-l	DIVMTQSPDSLAVSLGERATINC (SEQ 1DNO:281)	KSSQSLLNSRTRKNYLA (SEQID NO: 142)
h!3BI_VL1-NA	DIVMTQSPDSLAVSLGERATINC (SEQ IDNO:281)	KSSQSLLASRTRKNYLA (SEQ IDNO:258)
Antibody	LC FR2	LC CDR2
4D5 parent	WYQQKPGQSPKLLIY (SEQ IDNO:I43)	WASTRES (SEQ ID NO:144)
h4D5_VL-l	WYQQKPGQPPKLLIY (SEQ IDNO:282)	WASTRES (SEQ ID NO:144)
h4D5_VL-l- NA	WYQQKPGQPPKLLIY (SEQ ID NO:282)	WASTRES (SEQID NO: 144)
10DI2 parent	WLLQRPGQSPKRLIY ' (SEQID NO: 154)	LVSKLDS (SEQ IDNO:155)
h!0DI2_VL21	WLLQRPGQSPKRLIY (SEQ IDNO:154)	LVSKLDS (SEQ IDNO:155)
h!0D12_VL21-GA	WLLQRPGQSPKRLIY (SEQID NO: 154)	LVSKLDS (SEQ ID NO:155)
13B1 parent	WYQQKPGQSPKLLIY (SEQ IDNO:143)	WASTRES (SEQ IDNO:I44)
h!3BI_VL-I	WYQQKPGQPPKLLIY (SEQ IDNO:282)	WASTRES (SEQ IDNO:I44)
h!3BI_VLI-NA	WYQQKPGQPPKLLIY (SEQ ID NO:282)	WASTRES (SEQ IDNO:144)
Antibody	LC FR3	LC CDR3
4D5 parent	GVPDRFTGSGSGTDFSLTISSVQAEDLAVYYC (SEQID NO: 145)	KQSYNLYT (SEQ IDNO:146)

291

h4D5_VL-l	GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ IDNO:283)	KQSYNLYT (SEQ ID NO: 146)
h4D5_VL-l- NA	GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO:283)	KQSYNLYT (SEQ IDNO:146)
10D12 parent	GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC (SEQ ID NO: 156)	WQGTHFPWT (SEQ IDNO:157)
h!0DI2_VL21	GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO:286)	WQGTHFPWT (SEQ ID NO: 157)
h!0D12_VL21-GA	GVPDRFSGSGSGTDFTLK1SRVEAEDVGVYYC (SEQ 1DNO:286)	WQGTHFPWT (SEQ ID NO: 157)
I3B1 parent	GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC (SEQ IDNO:150)	KQSYNIPT (SEQ IDNO:161)
h!3Bl_VL-l	GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ 1DNO:283)	KQSYNIPT (SEQ IDNO:16I)
hl3Bl_VLI-NA	GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO:283)	KQSYNIPT (SEQ IDNO:161)
Antibody	LCFR4
4D5 parent	FGGGTKLEIKR (SEQ ID NO: 147)
h4D5_VL-l	FGQGTKVEIKR (SEQ IDNO:284)
h4D5_VL-l- NA	FGQGTKVEIKR (SEQIDNO:284)
I0D12 parent	FGGGTKLEIKR (SEQ ID NO: 147)
hI0D12_VL21	FGQGTKVEIKR (SEQ ID NO:287)
hlOD12_VL21-GA	FGQGTKVEIKR (SEQIDNO:287)
13B1 parent	FGGGTKLEIKR (SEQ IDNO:147)
hl3Bl_VL-I	FGQGTKVEIKR (SEQ ID NO:284)

292 hl3Bl_VL- FGQGTKVEIKR 1-NA (SEQ ID NO:284)

EXAMPLE 20

Analysis of a représentative MASP-3 inhibitory mAb 13B1 in a mouse model of multiple sclerosis.

Background/Rationale: Experimental autoimmune encephalomyelitis (EAE), an acquired inflammatory and demyelinating autoimmune disorder, is an established animal model of multiple sclerosis (MS). Evidence suggesting that the APC plays a significant rôle in the development/progression of EAE was provided by reports that the disease is attenuated in mice treated with a Factor B-neutralizing antibody (Hu et al., Mol. Immunol. 54:302, 2013). This Example describes the analysis of a représentative high affinity MASP-3 inhibitory antibody, 13B1, in the EAE model.

Methods:

EAE Induction:

A kit for inducing EAE, purchased from Hooke Laboratories (Lawrence, MA) was used to induce EAE in this study. This kit contained the neuroantigen MOG35-55 in Complété Freund’s Adjuvant (CFA) as well as pertussis toxin.

wild-type C57 B1/6J female mice were used for this study and were acclimated to the facility for at least one week prior to EAE induction. The mice were approximately 10 weeks of âge at the time of induction. As shown in TABLE 34 below, at the time of induction, each mouse received two 100 pL subcutaneous (sc) injections of MOG35-55 and one intraperitoneal (ip) injection of 100 pL (400 ng) pertussis toxin. A second injection of pertussis toxin was administered 24 hours after the first.

Treatment: The 30 mice were divided into three groups of 10 and treated with an irrelevant isotype control mAb 10 mg/kg i.v.); mAb 13B1 (anti-MASP-3, 10 mg/kg i.v.) or mAb 1379 (anti-Factor B (Hu et al., Mol. Immunol. 54:302, 2013) 40 mg/kg i.p.). As shown in TABLE

293

34, dosing with the isotype control mAb and MASP-3 mAb 13B1 occurred weekly beginning at Day -16 and ending on Day +12. Dosing with mAb 1379 occurred every other day from Day +3 to Day +11, according to the dosing schedule described in Hu et al., Mole Immunol 54:302-308, (2013).

TABLE 34: Experimental Methods for EAE Experiment with MASP-3 mAb 13B1

Day of Administration	Pertussis Toxin 400 ng i.p.	MOG peptide 3555 250 pg	mAb 1379 (anti-Factor B) 40 mg i.p.	Isotype Control mAb 10 mg/kg i.v.	mAb 13B1 (anti-MASP-3) 10 mg/kg i.v.
-16				+	+
-9				+	+
-2				+	+
0	+	+
+1	+
+3			+
+5			+	+	+
+7			+
+9			+
+11			+
+12				+	+

Scoring: The mice were checked every other day until the emergence of symptoms, after which they were checked daily. The first signs of disease appeared 7-12 days after immunization, as expected. The mice were scored according to the scale shown below in 10 TABLE 35.

TABLE 35: EAE Model Scoring Criteria

Score	Clinical Observations
0.0	No obvious changes in motor functions of the mouse in comparison to non-immunized mice. When picked up by the base of the tail, the tail has tension and is erect. Hind legs are usually spread apart. When the mouse is walking, there is no gait or head tilting.
0.5	Tip oftail is limp. When the mouse is picked up by the base ofthe tail, the tail has tension except for the tip. Muscle straining is felt in the tail, while the tail continues to move.
1.0	Limp tail. When mouse is picked up by the base of the tail, instead of being erect, the whole tail drapes over finger. Hind legs are usually spread apart. No signs of tail

294

	movement are observed.
1.5	Limp tail and hind leg inhibition. When picked up by the base of the tail, the whole tail drapes over finger. When the mouse is dropped on a wire rack, at least one hind leg falls through consistently. Walking is very slightly wobbly.
2.0	Limp tail and weakness of hind legs. When picked up by the base of the tail, the legs are not spread apart, but held close together. When the mouse is observed walking, it has a clearly apparent wobbly walk. One foot may hâve toes dragging, but the other leg has no apparent inhibitions of movement; OR, Mouse appears to be at score 0.0, but there are obvious signs of head tilting when the walk is observed. The balance is poor.
2.5	Limp tail and dragging of hind legs. Both hind legs hâve some movement, but both are dragging at the feet (mouse trips on hind feet). - OR- No movement in one leg/completely dragging one leg, but movement in the other leg.- OR - EAE severity appears mild when picked up (as score 0.0-1.5), but there is a strong head tilt that causes the mouse to occasionally fall over.
3.0	Limp tail and complété paralysis of hind legs (most common). - OR - Limp tail and almost complété paralysis of hind legs. One or both hind legs are able to paddle, but neither hind leg is able to move forward of the hind hip. - OR - Limp tail with paralysis of one front and one hind leg. - OR - ALL of: Severe head tilting, walking only along the edges of the cage, pushing against the cage wall, spinning when picked up by base of tail.
3.5	Limp tail and complété paralysis of hind legs. In addition to: Mouse is moving around the cage, but when placed on its side, is unable to right itself. Hind legs are together on one side of body. - OR - Mouse is moving around the cage, but the hind quarters are fiat like a pancake, giving the ap'pearance of a hump in the front quarters ofthe mouse
4.0	Limp tail, complété hind leg and partial front leg paralysis. Mouse is minimally moving around the cage but appears alert and feeding. Often euthanasia is recommended after the mouse scores 4.0 for 2 days. However, with daily s.c. fluids some mice can recover to 3.5 or 3.0. When the mouse is euthanized because of severe paralysis, a score of 5.0 is entered for that mouse for the rest of the experiment.
4.5	Complété hind and partial front leg paralysis, no movement around the cage. Mouse is not alert. Mouse has minimal movement in the front legs. The mouse barely responds to contact. Euthanasia is recommended. When the mouse is euthanized because of severe paralysis. a score of 5.0 is entered for that mouse for the rest of the experiment.
5.0	Mouse is spontaneously rolling in the cage.

Results:

FIGURE 68 graphically illustrâtes the results of the EAE model in mice treated with either MASP-3 inhibitory mAb 13B1 (lOmg/kg), Factor B mAb 1379 (40 mg/kg) or isotype 5 control mAb (10 mg/kg), wherein downward pointing arrows indicate dosing of anti-Factor B antibody and upward pointing arrows indicates the last dose of mAb 13B1. As shown in FIGURE 68, mice treated with MASP-3 inhibitory mAb 13B1 and Factor B mAb 1379 exhibited an improvement in clinical symptoms scored according to the parameters shown in TABLE 35, as compared to isotype control.

295

In accordance with the foregoing, MASP-3 inhibitory antibodies, such as the high affinity MASP-3 inhibitory antibodies disclosed herein, are expected to be bénéficiai (neuroprotective or neuroregenerative) in the treatment and/or réhabilitation of a subject suffering from multiple sclerosis, Balo concentric sclerosis, neuromyelitis optica, Marburg multiple sclerosis, Schilder’s disease, Tumefactive multiple sclerosis and acute disseminated encephalomyelitis (ADM).

EXAMPLE 21

Pharmacodynamie Study with Représentative high affinity MASP-3 mAbs in Cynomolgus Monkeys.

Background/Rationale: As was demonstrated in rodent studies (FIGURE 44), a high affinity MASP-3 inhibitory antibody was capable of inhibiting steady-state (resting) pro-factor D maturation in vivo. This Example describes a study that was carried out in cynomolgus monkeys to détermine if représentative high affinity MASP-3 inhibitory mAbs are capable of inhibiting APC activity in a non-human primate.

Methods: To confirm that MASP-3 functions in the APC in a non-human primate, and that the high affinity MASP-3 antibodies are capable of inhibiting the APC in a non-human primate, 9 cynomolgus monkeys (3 animais per mAb condition) were given a single 5 mg/kg intravenous dose with one of three représentative high affinity MASP-3 inhibitory antibodies: h4D5X, hlOD12X, or hl3BlX. (“h” refers to humanized, “X” refers to the IgG4 constant hinge région (SEQ ID NO:312) containing the stabilizing S228P amino acid substitution and a mutation human IgG4 constant région with S228P mutation and also a mutation that promûtes FcRn interations at low pH). Plasma (EDTA) and sérum samples were collected at regular intervals over a period of three weeks or longer.

Two assays were employed to measure APC activity in the sera from treated monkeys. The first assay assessed levels of complément factor Bb deposited on zymosan beads added to diluted sérum. The second assay measured the fluid phase products of the zymosan-activated APC, complément factors Ba and Bb, as well as C3a.

296

Flow cytometry using the factor Bb antibody A252 (Quidel) was used to detect factor Bb deposited on zymosan. As a means for determining the background signal in the assay following complété inhibition of the APC, an aliquot of sérum (5% final, diluted in GVB + Mg/EGTA) prepared from MASP-3 mAb-treated cynomolgus monkeys was spiked with 300 5 nM of an inhibitory Factor D antibody. To détermine the degree of APC inhibition by the

MASP-3 mAb delivered intravenously to the monkey, another aliquot of diluted sérum was spiked with 300 nM of a neutral isotype control antibody (that has no APC inhibitory activity) before testing factor Bb déposition on zymosan. The spiked antibody-serum mixtures were incubated for 30 minutes on ice prior to the addition of zymosan (0.1 mg/mL 10 final). The mixtures were incubated at 37°C for 65 minutes, and the APC activity was measured by the flow cytométrie détection of complément factor Bb (Quidel antibody A252) on the surface of the zymosan particles.

For determining génération of the fluid phase markers Ba, Bb, and C3a, the APC was induced in ex vivo assays by incubating zymosan (1 mg/mL final) in sérum (5% final, diluted in GVB 15 + Mg/EGTA) prepared from anti-MASP-3 mAb-treated cynomolgus monkeys. The mixtures were incubated at 37°C for 40 minutes, and the APC activity was measured by ELISA-based détection of the complément end-points. Ba, Bb, and C3a were detected in the reaction supernatants using commercially available ELISA kits (Quidel). Absorbance values of ail tests were normalized by setting pre-treatment values as 100% activity, and a pre-treatment 20 sample incubated, but not exposed to zymosan, to 0%.

In order to relate the degree of APC inhibition to the antibody to target ratio in MASP-3 mAb-treated monkeys, sérum MASP-3 and inhibitory MASP-3 mAb levels were quantitated. Sérum MASP-3 was measured by a sandwich ELISA assay. The MASP-3 protein was captured on a plate with aM3-259 (described in Example 16). Sérum samples (diluted 1:40) 25 were first incubated with unlabeled (non-biotinylated) MASP-3 mAb, corresponding to the treatment mAb, at 37°C for 1 hour, then further diluted 1:250 (final 1:10,000) and added to the plate and incubated at 37°C for another hour. The plate was washed and a biotinylated version of mAb 10D12 was used as a détection antibody. The large dilution of sérum prior to the détection steps was used to uncouple target and treatment mAb, and to prevent 30 compétition between the treatment antibody and the détection antibody. After the plate was

297 washed multiple times, streptavidin-HRP was used for the final détection step. Absorbance values were collected at A450 with a plate reader. MASP-3 sérum concentrations were extrapolated from a standard curve created by assaying recombinant, full-Iength cyno MASP3 protein. The amount of anti-MASP-3 antibody présent in the sérum was detected using the Human Therapeutic IgG4 ELISA Kit (Cayman Chemicals), following the manufacturées instructions.

Western blot analysis was used to analyze the level of pro-Factor D and Factor D in sérum from a cynomolgus monkey over time (hours) after treatment with a single 5 mg/kg intravenous dose of mAb hl3BlX. Briefly described, the Western blot analysis was carried out by mixing 20 pL of cynomolgus plasma obtained at the different timepoints prior to treatment (-120hr, -24hr) and after treatment (72hr, 168hr, 336 hr, 504hr, 672hr and 840 hr) with PBS and 11.2 pL of anti-CFD antibody (0.5 pg/pL) in a total volume of 400pL at 4°C for 1 hour. 12 pL of Protein A/G Plus Agarose (Santa Cruz Biotech) was added and the mixture was incubated ovemight at 4°C. Immunoprecipitates were collected by centriguation at 1000 x g for 5 minutes at 4°C. The pellets were washed five times with PBS. After the final wash, the pellets were resuspended in 30 pL of Ix Glycoprotein Denaturing Buffer and the glycoprotein was denatured by heating the reaction at 100°C for 10 minutes. 10X G2 reaction buffer, 10% NP-40 and 2.5 pL Peptide-N-Glycosidase (New England Biolabs, P0704L) was added into each tube and the reaction was incubated at 37°C for 2 hours. The agarose beads were pelleted by centrifugation at 1000 x g for 5 minutes and 20 pL supematant was collected into new tubes. The captured and deglycosylated proteins were resolved with SDS-PAGE (NuPAGE 12% Bis-Tris Mini Gel) and the gels were electroblotted for Western blot analysis with a biotinylated anti-CFD (R&D Systems BAF1824) and Pierce ™ High Sensitivity Streptavidin-HRP (Thermo Fischer Scientific 21130).

Results:

FIGURE 69 graphically illustrâtes APC activityin sérum samples obtained from a group of three cynomolgus monkeys over time after a single treatment at time=0 with high affinity MASP-3 mAb hl3B!X. The figure shows the average MFI in a flow cytométrie . 298 assay detecting complément factor Bb on the surface of zymosan particles in 5% sérum spiked with either the APC-inhibiting fact D mAb or the neutral isotype control mAb. As shown in FIGURE 69, the animais demonstrate diminished APC activity as early as 4 hrs. If MASP-3 antibody treatment blocks the APC as effectively as Factor D inhibition, the two 5 spiked antibody conditions will demonstrate identical levels of inhibition of Bb déposition in post-dose samples, but not in the pre-dose (or time = 0; FIGURE 69) condition. As shown in FIGURE 69, by 72 hrs post-treatment, the APC activity is decreased to approximately that achieved by adding the Factor D mAb to the sérum samples. Nearly complété inhibition due to hl3BlX treatment, as experimentally determined by comparison with the spiked Factor D 10 antibody, persists until 336 hrs (14 days) post-dose. Thus, these results demonstrate that treatment with a high affinity MASP-3 inhibitory mAb provides a complété, sustained inhibition of the APC in a non-human primate.

FIGURE 70 graphically illustrâtes APC activity, as determined by Bb déposition on zymosan, in sérum samples obtained from groups of cynomolgus monkeys (3 animais per 15 group) treated with a single 5 mg/kg intravenous dose of high affinity MASP-3 inhibitory mAbs h4D5X, hlOD12X or hl3BlX. Bb déposition data was collected as described above. APC activity for the treatment timepoints was normalized by setting pre-treatment MFIs of samples spiked with the non-inhibitory, isotype control antibody as 100% activity, and a pretreatment sample incubated with 50 mM EDTA (to inhibit ail complément activity) to 0%.

The hl3BX treatment data used for FIGURE 70 are also reflected in FIGURE 69. As shown in FIGURE 70, treatment with ail three high affinity MASP-3 inhibitory antibodies resulted in greater than 95% inhibition of the APC. The h4D5X-, hlOD12X-, and hl3BlX-treated animais maintained at least 90% inhibition of the APC for 6.7, 11.7, and 16 days, respectively. Thus, these results demonstrate that treatment with these represensative high 25 affinity MASP-3 inhibitory mAbs provides sustained inhibition of the APC in a nonhuman primate with a single 5 mg/kg dose.

FIGURE 71A-C graphically illustrâtes additional measures of APC activity. Fluidphase Ba (FIGURE 71 A), Bb (FIGURE 71 B) and C3a (FIGURE 71C) were measured in zymosan-treated, diluted sérum samples obtained from groups of cynomolgus monkeys (3 30 animais per group) over time after treatment with a single 5 mg/kg intravenous dose of

299 h4D5X, hlOD12X, and hl3BlX as described above.

As shown in FIGURE 71A-C, single administrations of ail three high affinity MASP3 inhibitory antibodies resulted in inhibition of the APC, as defined by three different fluidphase endpoints. These data are consistent with level of APC inhibition demonstrated in the Bb déposition study of FIGURE 70, and further illustrate the efficacy of these mAbs to inhibit the pathway for multiple weeks.

FIGURE 72A-C graphically illustrâtes the relationship of APC activity, as determined by fluid-phase Ba production, relative to the molar ratio of monomeric MASP-3 and MASP-3 mAb antibody detected in sérum from monkeys treated with either h4D5X (FIGURE 72A), h!0D12X (FIGURE 72B) or h!3B!X (FIGURE 72C). Each panel in FIGURE 72A-C represents the data from one monkey. The monkey subjects used and sérum (or plasma) obtained in this study are the same as those described above (FIGURES 69, 70, and 71).

FIGURES 72A-C graphically illustrâtes the molar ratio of target (MASP-3) to the high affinity MASP-3 inhibitory antibodies h4D5X (FIGURE 72A), h!0D12X (FIGURE 72B) and M3B1X (FIGURE 72C) at the timepoints of complété APC inhibition, as measured by fluid-phase Ba. For reference purposes, the molar ratio of 1:1 target to antibody is shown as a dotted line in each graph. As shown in FIGURES 72A-C, target (MASP-3) to the high affinity MASP-3 inhibitory mAbs h4D5X, h!0D12X and h!3B!X at a molar ratio in the range of about 2:1 to about 2.5:1 (target to antibody) are sufficient to completely inhibit the APC. These data demonstrate that these three représentative MASP-3 inhibitory mAbs are potent, high-affinity MASP-3 inhibitory antibodies that are capable at inhibiting the APC when présent at molar concentrations less than the concentration of target. These levels of potency strongly indicate that the mAbs hâve the potential to be used clinically to treat diseases or indications caused by the APC.

FIGURE 73 shows a Western blot analyzing the level of pro-Factor D and Factor D in sérum from a cynomolgus monkey over time (hours) prior to and after treatment with a single 5 mg/kg intravenous dose of mAb hl3B!X. As shown in FIGURE 73, Factor D is présent in

300 plasma as pro-Factor D for at least 336 hours (14 days) following a single dose of mAb hl3BlX.

Summary of Results

As described in Example 11, a single dose administration of a high affinity MASP-3 inhibitory antibody, mAb 13B1, to mice led to near-complete ablation of systemic alternative pathway complément activity for at least 14 days. As further described in Example 12, in a study conducted in a well-established animal model associated with PNH it was demonstrated that mAb 13B1 significantly improved the survival of PNH-like red blood cells and protected 10 PNH-like red blood cells significantly better than did C5 inhibition. As described in Example

13, it was further demonstrated that mAb 13B1 reduced the incidence and severity of disease in a mouse model of arthritis. The results in this example demonstrate that représentative high affinity MASP-3 inhibitory mAbs 13B1, 10D12 and 4D5 are highly effective at blocking the alternative pathway in primates. Single dose administration of mAb 13B1, 15 10D12 or 4D5 to cynomolgus monkeys resulted in sustained ablation of systemic alternative pathway activity lasting for approximately 16 days. The extent of alternative pathway ablation in cynomolgus monkeys treated with high affinity MASP-3 inhibitory antibodies was comparable to that achieved by factor D blockade in vitro, indicating complété blockade of factor D conversion by the MASP-3 inhibitory antibodies. Therefore, high affinity 20 MASP-3 inhibitory mAbs hâve therapeutic utility in the treatment of patients suffering from diseases related to alternative pathway hyperactivity, such as, for example, paroxysmal noctumal hemoglobinuria (PNH), age-related macular degeneration (AMD, including wet and dry AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic urémie syndrome (HUS), atypical 25 hemolytic urémie syndrome (aHUS),thrombotic thrombocytopénie purpura (TTP) or transplant-associated TMA), asthma, dense deposit disease, pauci-immune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behcet’s disease, multiple sclerosis, Guillain Barre Syndrome, Alzheimer’s disease, Amylotrophic latéral sclerosis (ALS), lupus nephritis, systemic lupus 30 erythematosus (SLE), Diabetic retinopathy, Uveitis, Chronic obstructive pulmonary disease

301 (COPD), C3 glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD), hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis, Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy and Myasthenia Gravis.

VIL OTHER EMBODIMENTS

Ail publications, patent applications, and patents mentioned in this spécification are herein incorporated by reference.

Various modifications and variations of the described methods, compositions, and compounds, of the invention will be apparent to those skilled in the art without departing 10 from the scope and spirit of the invention. Although the invention has been described in connection with spécifie desired embodiments, it should be understood that the invention as claimed should not be unduly limited to such spécifie embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the fîelds of medicine, immunoïogy, pharmacology, oncology, or related fîelds are 15 intended to be within the scope of the invention.

Ih accordance with the foregoing', the invention features the following embodiments.

302

High Affinity MASP-3 inhibitory antibodies that bind one or more epitopes within the SP domain

IA. An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to the serine protease domain of human MASP-3 (amino acid residues 450 to 728 of SEQ ID NO:2) with high affinity (having a Kd of less than 500 pM), wherein the antibody or antigen-binding fragment thereof inhibits alternative pathway complément activation.

2A. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment is characterized by at least one or more of the following properties:

(a) inhibits pro-Factor D maturation;

(b) does not bind to human MASP-1 (SEQ IDNO:8);

(c) inhibits the alternative pathway at a molar ratio of from about 1:1 to about 2.5:1 (MASP-3 target to mAb) in a mammalian subject;

(d) does not inhibit the classical pathway.

(e) inhibition of hemolysis and/or opsonization;

^)- inhibition of MASP-3 serine^protease substrate-specific cléaVage; ' (g) a réduction of hefnolysis or the réduction of 03 cleavage and C3b surface déposition;

(h) a réduction of Factor B and Bb déposition on an activating surface;

(i) a réduction of resting levels (in circulation, and without the experimental addition of an activating surface) of active Factor D relative to pro-Factor D;

(j) a réduction of levels of active Factor D relative to pro-Factor D in response to an activating surface;

(k) a réduction of the production of resting and/or surface-induced levels of fluidphase Ba, Bb, C3b, or C3a and/or (I) a réduction in factor P déposition.

3A. The isolated antibody or antigen-binding fragment thereof of paragraph 1 or 2, wherein said antibody or antigen-binding fragment thereof specifically binds to an epitope located within the serine protease domain of human MASP-3, wherein said epitope is located within

303 at least one or more of: VLRSQRRDTTVI (SEQ ID NO:9), TAAHVLRSQRRDTTV (SEQ ID NO: 10), DFNIQNYNHDIALVQ (SEQ ID NO: 11), PHAECKTSYESRS (SEQ ID NO: 12), GNYSVTENMFC (SEQ ID NO: 13), VSNYVDWVWE (SEQ ID NO: 14) and/or VLRSQRRDTTV (SEQ ID NO: 15). [Group I]

4A. The antibody or antigen-binding fragment thereof of paragraph 3, wherein said antibody or antigen-binding fragment binds to an epitope within SEQ ID NO: 15. [includes ail group I abs]

5A. The antibody or antigen-binding fragment of paragraph 3, wherein said antibody or antigen-binding fragment binds to an epitope within SEQ ID NO:9. [10D12]

6A. The antibody or antigen-binding fragment of paragraph 3, wherein said antibody or antigen-binding fragment binds to an epitope within SEQ ID NO: 10. [ 13B1 ]

7A. The antibody or antigen-binding fragment of paragraph 6, wherein said antibody or antigen binding fragment also binds to an epitope within SEQ ID NO: 12. [ 13B1 ]

8A. The antibody or antigen-binidng fragment of paragraph 3, wherein said antibody or 20 antigen-binding fragment also binds to an epitope within SEQ ID NO: 10 and/or SEQ ID

NO:12. [13B1]

9A. The antibody or antigen-binding fragment of paragraph 3, wherein said antibody or antigen binding fragment binds to an epitope within SEQ IDNO:9. [1F3,4B6, 4D5, 1 Al0]

10A. The antibody or antigen-binding fragment of paragraph 7, wherein said antibody or antigen binding fragment also binds to an epitope within at least one of SEQ ID NO:11, SEQ IDNO: 13 and/or SEQ ID NO: 14. [1F3, 4B6,4D5, 1A10]

HA. The antibody or antigen-binding fragment of paragraph 7, wherein the antibody or antigen-binding fragment also binds to an epitope within at least one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13 and/or SEQ ID NO: 14. [1F3, 4B6, 4D5, 1A10]

304

12A. The antibody or antigen-binding fragment of paragraph 1 or 2, wherein said antibody binds to an epitope within at least one of: ECGQPSRSLPSLV (SEQ ID NO: 16), RNAEPGLFPWQ (SEQ ID NO: 17); KWFGSGALLSASWIL(SEQ ID NO: 18); EHVTVYLGLH (SEQ IDNO:19); PVPLGPHVMP (SEQ ID NO:20); APHMLGL (SEQ ID NO:21); SDVLQYVKLP (SEQ ID NO:22); and/or AFVIFDDLSQRW (SEQ ID NO:23). [group II and III]

13A. The antibody or antigen-binding fragment of paragraph 12, wherein said antibody or antigen-binding fragment binds to an epitope within SEQ ID NO: 17. [ail group II and III abs]

14A. The antibody or antigen-binding fragment of paragraph 13, wherein said antibody or antigen binding fragment also binds to an epitope within EHVTVYLGLH (SEQ ID NO: 19) and/or AFVIFDDLSQRW (SEQ ID NO:23). [1G4, 1E7, 2D7 15D9]

15A. The antibody or antigen-binding fragment of paragraph 14, wherein said antibody or antigen binding fragment also binds to an epitope within SEQ ID NO:23. [1G4, IE7, 2D7, 15D9, 2F5]

16A. The antibody or antigen-binding fragment of paragraph 14, wherein said antibody or antigen binding fragment also binds to an epitope within SEQ ID NO: 19 and/or SEQ ID NO:23.[Ig4, 1E7, 2D7]

17A. The antibody or antigen-binding fragment of paragraph 14, wherein said antibody or antigen-binding fragment also binds to an epitope within SEQ ID NO: 18, SEQ ID NO:20 and/or SEQ IDNO:23. [15D9, 2F5]

18A. The antibody or antigen-binding fragment of paragraph 14, wherein said antibody or antigen-binding fragment also binds to an epitope within at least one of SEQ ID NO: 18, SEQ ID NO:20 and/or SEQ ID NO:23 [15D9, 2F5].

305

19A. The antibody or antigen-binding fragment of paragraph 14, wherein said antibody or antigen-binding fragment also binds to an epitope within at least one of SEQ ID NO: 16, SEQ ID NO: 21 and/or SEQ ID NO:22. [ 1B11 ]

20A. The antibody or antigen-binding fragment of paragraph 14, wherein said antibody or antigen-binding fragment also binds to an epitope within at least one of SEQ ID NO:16, SEQ IDN0:21 and/or SEQ ID NO:22 [1B11].

2IA. The antibody or antigen binding fragment thereof of any one of paragraphs 1-20, 10 wherein the antibody or antigen-binding fragment is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, and an antigen-binding fragment of any of the foregoing.

22A. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-21, 15 wherein said antibody or antigen binding fragment thereof is selected from the group consisting of a single chain antibody, an ScFv, a Fab fragment, an Fab’ fragment, an F(ab’)2 fragment, a univalent antibody lacking a hinge région and a whole antibody.

23A. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-22, 20 further comprising an immunoglobulin constant région.

24A. The antibody or antigen binding fragment thereof of any one of paragraphs 1-23, wherein the antibody or antigen-binding fragment is humanized.

25A. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-24 wherein said antibody binds to the serine protease domain of human MASP-3 with an afïinity of less than 500 pM.

26A. The antibody or antigen-binding fragment thereof of any of paragraphs 1-25, wherein 30 said antibody inhibits alternative pathway activation in mammalian blood.

306

27A. A composition comprising the antibody or antigen-binding fragment of any of paragraphs 1A-26A and a pharmaceutically acceptable excipient.

A. Group IA High Affinity MASP-3 inhibitory antibodies That Bind one or more 5 epitopes within the SP domain (4D5, 4B6,1A10 plus 4D5 variants)

IB. An isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 comprising:

(a) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:209 (XXDIN, wherein X at position 1 is S or T and wherein X at position 2 is

N or D); a HC-CDR2 set forth as SEQ ID NO:210 (W1YPRDXXXKYNXXFXD, wherein X at position 7 is G or D; X at position 8 is S, T or R; X at position 9 is I or T; X at position 13 is E or D; X at position 14 is K or E; and X at position 16 is T or K); and a HC-CDR3 set forth as SEQ ID NO:211 (XEDXY, wherein X at position 1 is L or V, and wherein X at position 4 is T or S); and (b) a light chain variable région comprising a LC-CDRI set forth as SEQ ID NO:212 (KSSQSLLXXRTRKNYLX, wherein X at position 8 is N, I, Q or A; wherein X at position 9 is S or T; and wherein X at position 17 is A or S); a LC-CDR2 set forth as SEQ ID NO: 144 (WASTRES) and a LC-CDR3 set forth as SEQ ID NO: 146 (KQSYNLYT).

2B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the

HC-CDR1 of the heavy chain variable région according to (a) comprises SEQ ID NO:56 (TDDIN). [4D5 and variants]

3B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the 25 HC-CDR1 of the heavy chain variable région according to (a) comprises SEQ ID NO:62 (SNDIN). [1F3, 4B6 and 1A10]

307

4B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR2 of the heavy chain variable région according to (a) comprises SEQ ID NO:58 (WIYPRDDRTKYNDKFKD) [4D5 and variants].

5B. The isolated antibody or antigen-binding fragment thereof of paragraph I, wherein the HC-CDR2 of the heavy chain variable région according to (a) comprises SEQ ID NO:63 (WIYPRDGSIKYNEKFTD). [IF3]

6B. The isolated antibody or antigen-binding fragment thereof of paragraph I, wherein the HC-CDR2 of the heavy chain variable région according to (a) comprises SEQ ID NO:67 (WIYPRDGTTKYNEEFTD). [4B6]

7B. The isolated antibody or antigen-binding fragment thereof of paragraph I, wherein the HC-CDR2 of the heavy chain variable région according to (a) comprises SEQ ID NO:69 (WIYPRDGTTKYNEKFTD). [IAI0]

8B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR3 of the heavy chain variable région according to (a) comprises SEQ ID NO:60 (LEDTY)[4D5 and variants]

9B. The isolated antibody or antigen-binding fragment thereof of paragraph I, wherein the HC-CDR3 of the heavy chain variable région according to (a) comprises SEQ ID NO:65 (VEDSY). [IF3,4B6and 1A10]

10B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the LC-CDRI of the light chain variable région according to (b) comprises SEQ ID NO:142 (KSSQSLLNSRTRKNYLA); SEQ ID NO:257 (KSSQSLLQSRTRKNYLA), SEQ ID NO:258 (KSSQSLLASRTRKNYLA); or SEQ ID NO:259 (KSSQSLLNTRTRKNYLA). [4D5 and variants]

308

B. The isolated antibody or antigen-binding fragment thereof of paragraph 10, wherein the LC-CDR1 of the light chain variable région according to (b) comprises SEQ ID NO:258 (KSSQSLLASRTRKNYLA). [4D5 NA mutant]

12B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the

LC-CDR1 of the light chain variable région according to (b) comprises SEQ ID ΝΟΠ49 (KSSQSLLISRTRKNYLS). [1F3 and 4B6]

13B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the 10 HC-CDR1 comprises SEQ ID NO:56, the HC-CDR2 comprises SEQ ID NO:58, the HCCDR3 comprises SEQ ID NO:60 and wherein the LC-CDR1 comprises SEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259; wherein the LC-CDR2 comprises SEQ ID NO: 144 and wherein the LC-CDR3 comprises SEQ ID NO: 146. [ail 6 CDRs of4D5 with variants at LC-CDR1].

14B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR1 comprises SEQ ID NO:62, the HC-CDR2 comprises SEQ ID NO:63, SEQ ID NO:67 or SEQ ID NO:69, the HC-CDR3 comprises SEQ ID NO:65 and wherein the LCCDR1 comprises SEQ ID NO: 149, the LC-CDR2 comprises SEQ ID NO: 144 and the LC20 CDR3 comprises SEQ ID NO: 146. [ail 6 CDRS of 1F3, 4B6 and 1A10]

15B. The antibody or antigen binding fragment thereof of any one of paragraphe 1-14, wherein the antibody or antigen-binding fragment is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, and an 25 antigen-binding fragment of any of the foregoing.

16B. The antibody or antigen-binding fragment thereof of any one of paragraphe 1-15, wherein eaid antibody or antigen binding fragment thereof ie eelected from the group coneieting of a eingle chain antibody, an ScFv, a Fab fragment, an Fab’ fragment, an F(ab’)2 30 fragment, a univalent antibody lacking a hinge région and a whole antibody.

309

17B. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-16, further comprising an immunoglobulin constant région.

18B. The antibody or antigen binding fragment thereof of any one of paragraphs 1-17, 5 wherein the antibody or antigen-binding fragment is humanized.

19B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:24, SEQ ID NO:248 or 10 SEQ ID NO:249 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or

100% identical to SEQ ID NO:40, SEQ ID NO:250 or SEQ ID NO:278 [4D5 parental, humanized and modified versions].

20B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the 15 antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:25 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:41 [1F3].

21 B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:26 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:42 [4B6].

22B. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:27 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:42 30 [1A10].

310

23B. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-22 wherein said antibody binds to human MASP-3 with an affinity of less than 500 pM.

24B. The antibody or antigen-binding fragment thereof of any of paragraphs 1-23, wherein said antibody inhibits alternative pathway activation in mammalian blood.

25B. A composition comprising the antibody or antigen-binding fragment of any of paragraphs 1B-24B and a pharmaceutically acceptable excipient.

B. Group IB High Affinity MASP-3 inhibitory antibodies That Bind one or more epitopes within the SP domain (10D12,35C1 and 10D12 variants)

IC. An isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 comprising:

(a) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:213 (SYGXX, wherein X at position 4 is M or I and wherein X at position 5 is S or T); a HC-CDR2 set forth as SEQ ID NO:74; and a HC-CDR3 set forth as SEQ ID NO:214 (GGXAXDY, wherein X at position 3 is E or D and wherein X at position 5 is M or L); and (b) a light chain variable région comprising a LC-CDRI set forth as SEQ ID NO:215 (KSSQSLLDSXXKTYLX , wherein X at position 10 is D, E or A; wherein X at position 11 is G or A; and wherein X at position 16 is N or S); a LC-CDR2 set forth as SEQ ID NO: 155; and a LC-CDR3 set forth as SEQ ID NO:216 (WQGTHFPXT, wherein X at position 8 is W or Y).

2C. The isolated antibody or antigen-binding fragment thereof of paragraph I, wherein the HC-CDR1 of the heavy chain variable région according to (a) comprises SEQ ID NO:72 (SYGMS). [10D12 and variants]

311

3C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDRI of the heavy chain variable région according to (a) comprises SEQ ID NO:79 (SYGIT). [35C1]

4C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the

HC-CDR3 of the heavy chain variable région according to (a) comprises SEQ ID NO:76 (GGEAMDY). [10D12 and variants].

5C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the 10 HC-CDR3 of the heavy chain variable région according to (a) comprises SEQ ID NO:82 (GGDALDY). [35C1]

6C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the LC-CDR1 of the light chain variable région according to (b) comprises SEQ ID NO: 153 15 (KSSQSLLDSDGKTYLN); SEQ ID NO:261 (KSSQSLLDSEGKTYLN), SEQ ID NO:262 (KSSQSLLDSAGKTYLN) or SEQ ID NO:263 (KSSQSLLDSDAKTYLN). [10D12 and variants]

7C. The isolated antibody or antigen-binding fragment thereof of paragraph 6, wherein the 20 LC-CDR1 of the light chain variable région comprises SEQ ID NO:263 (KSSQSLLDSDAKTYLN). [10D12 GA variant]

8C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the LC-CDR1 ofthe light chain variable région comprises SEQ ID NO: 152. [35CI]

9C. The isolated antibody or antigen-binding fragment thereof of paragraph I, wherein the LC-CDR3 of the light chain variable région according to (b) comprises SEQ ID NO: 159 (KSSQSLLDSDGKTYLS). [ 10D12] '

312

10C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the LC-CDR3 of the light chain variable région according to (b) comprises SEQ ID NO: 160 (WQGTHFPYT). [35C1]

11C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the

HC-CDR1 comprises SEQ ID NO:72, the HC-CDR2 comprises SEQ ID NO:74, the HCCDR3 comprises SEQ ID NO:76, the LC-CDRI comprises SEQ ID NO: 153, SEQ ID NO:261, SEQ ID NO:262 or SEQ ID NO:263; the LC-CDR2 comprises SEQ ID NO:155 and the LC-CDR3 comprises SEQ ID NO:157. [ail 6 CDRs of 10D12 with variants at LC10 CDR1]

12C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the HC-CDR1 comprises SEQ ID NO:79, the HC-CDR2 comprises SEQ ID NO:74, the HCCDR3 comprises SEQ ID NO:82, the LC-CDRI comprises SEQ ID NO: 159, the LC-CDR2 comprises SEQ ID NO: 155 and the LC-CDR3 comprises SEQ ID NO: 160. [ail 6 CDRs of 35C1]

13C. The antibody or antigen binding fragment thereof of any one of paragraphe 1-12, wherein the antibody or antigen-binding fragment is selected from the group consisting of a 20 human antibody, a humanized antibody, a chimeric antibody, a murine antibody, and an antigen-binding fragment of any of the foregoing.

14C. The antibody or antigen-binding fragment thereof of any one of paragraphe 1-13, wherein eaid antibody or antigen binding fragment thereof ie eelected from the group 25 coneieting of a eingle chain antibody, an ScFv, a Fab fragment, an Fab’ fragment, an F(ab’)2 fragment, a univalent antibody lacking a hinge région and a whole antibody.

15C. The antibody or antigen-binding fragment thereof of any one of paragraphe 1-14, further comprieing an immunoglobulin conetant région.

16C. The antibody or antigen binding fragment thereof of any one of paragraphe 1-15, wherein the antibody or antigen-binding fragment ie humanized.

313

17C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:28, SEQ ID NO:251 or SEQ ID NO:252 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:43, SEQ ID NO:253 or SEQ ID NO:279 [10D12 parental, humanized and variants].

18C. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:29 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:44 [35C1].

19C. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-18 wherein said antibody binds to human MASP-3 with an affinity of less than 500 pM.

20C. The antibody or antigen-binding fragment thereof of any of paragraphs 1-19, wherein said antibody inhibits alternative pathway activation in mammalian blood.

21C. A composition comprising the antibody or antigen-binding fragment of any of paragraphs 1C-20C and a pharmaceutically acceptable excipient.

C. Group IC High Affinity MASP-3 inhibitory antibodies That Bind one or more epitopes within the SP domain (13B1 and variants)

ID. An isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 comprising:

(a) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:84 (GKWIE); a HC-CDR2 set forth as SEQ ID NO:86 (EILPGTGSTNYNEKFKG)

314 or SEQ ID NO:275 (EILPGTGSTNYAQKFQG); and a HC-CDR3 set forth as SEQ ID NO:88 (SEDV); and (b) a light chain variable région comprising a LC-CDR 1 set forth as SEQ ID NO: 142 (KSSQSLLNSRTRKNYLA), SEQ ID NO:257 (KSSQSLLQSRTRKNYLA); SEQ ID NO:258 (KSSQSLLASRTRKNYLA); or SEQ ID NO:259 (KSSQSLLNTRTRKNYLA), a LC-CDR2 set forth as SEQ ID NO: 144 ( WASTRES); and a LC-CDR3 set forth as SEQ ID NO: 161 (KQSYNIPT). [ail 6 CDRs of 13B1 and variants in LC-CDR1]

2D. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the LC-CDR1 comprises SEQ IDNO:258. [13B1 LC-CDR1 NA variant]

3D. The antibody or antigen binding fragment thereof of any one of paragraphs 1-2, wherein the antibody or antigen-binding fragment is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, and an antigenbinding fragment of any ofthe foregoing.

4D. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-3, wherein said antibody or antigen binding fragment thereof is selected from the group consisting of a single chain antibody, an ScFv, a Fab fragment, an Fab’ fragment, an F(ab’)2 fragment, a univalent antibody lacking a hinge région and a whole antibody.

5D. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-4, further comprising an immunoglobulin constant région.

6D. The antibody or antigen binding fragment thereof of any one of paragraphs 1-5, wherein the antibody or antigen-binding fragment is humanized.

7D. The isolated antibody or antigen-binding fragment of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:30, SEQ ID NO:254 or SEQ ID NO:255 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100%

315 identical to SEQ ID NO:45, SEQ ID NO:256 or SEQ ID NO:280 [13B1 parental, humanized and variants].

8D. The antibody or antigen-binding fragment thereof of any one of paragraphs 1 -7 wherein said antibody binds to human MASP-3 with an affînity of less than 500 pM.

9D. The antibody or antigen-binding fragment thereof of any of paragraphs 1-8, wherein said antibody inhibits alternative pathway activation in mammalian blood.

10D. A composition comprising the antibody or antigen-binding fragment of any of paragraphs 1D-9D and a pharmaceutically acceptable excipient.

D. Group II High Affinity MASP-3 inhibitory antibodies That Bind one or more epitopes within the SP domain (1G4)

IE. An isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 comprising:

(a) a heavy chain variable région comprising a HC-CDRI set forth as SEQ ID NO:9I (GYWIE); a HC-CDR2 set forth as SEQ ID NO:93 (EMLPGSGSTHYNEKFKG), and a HC-CDR3 set forth as SEQ ID NO:95 (SIDY); and (b) a light chain variable région comprising a LC-CDR1 set forth as SEQ ID NO: 163 (RSSQSLVQSNGNTYLH), a LC-CDR2 set forth as SEQ ID NO: 165 (KVSNRFS) and a LC-CDR3 set forth as SEQ ID NO: 167 (SQSTHVPPT).

2E. The antibody or antigen binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, and an antigen-binding fragment of any of the foregoing.

3E. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-2, wherein said antibody or antigen binding fragment thereof is selected from the group consisting of a

316 single chain antibody, an ScFv, a Fab fragment, an Fab’ fragment, an F(ab’)2 fragment, a univalent antibody lacking a hinge région and a whole antibody.

4E. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-3, further comprising an immunoglobulin constant région.

5E. The antibody or antigen binding fragment thereof of any one of paragraphs 1-4, wherein the antibody or antigen-binding fragment is humanized.

6E. The isolated antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen-binding fragment thereof comprise a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:3I and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:46 [1G4]. ·

7E. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-6 wherein said antibody binds to human MASP-3 with an affinity of less than 500 pM.

8E. The antibody or antigen-binding fragment thereof of any of paragraphs 1-7, wherein said antibody inhibits alternative pathway activation in mammalian blood.

9E. A composition comprising the antibody or antigen-binding fragment of any of paragraphs 1E-8E and a pharmaceutically acceptable excipient.

F. Group III High Affinity MASP-3 inhibitory antibodies that bind one or more epitopes within the SP domain (1E7,2D7,15D9,2F5,1B11,2F2,11B6)

1F. An isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 comprising:

(a) a heavy chain variable région comprising a HC-CDRI set forth as SEQ ID NO: 109 (RVHFAIRDTNYWMQ), a HC-CDR2 set forth as SEQ ID NO:110 (AIYPGNGDTSYNQKFKG), a HC-CDR3 set forth as SEQ ID NO: 112

317 (GSHYFDY); and a light chain variable région comprising a LC-CDR1 set forth as SEQ ID NO: 182 (RASQSIGTS1H), a LC-CDR2 set forth as SEQ ID NO: 184 (YASESIS) and a LC-CDR3 set forth as SEQ ID NO:186 (QQSNSWPYT) [1E7]; or (b) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO: 125 (DYYMN), a HC-CDR2 set forth as SEQ ID NO: 127 (DVNPNNDGTTYNQKFKG), a HC-CDR3 set forth as SEQ ID NO: 129 (CPFYYLGKGTHFDY); and a light chain variable région comprising a LC-CDR1 set forth as SEQ ID NO: 196 (RASQDISNFLN), a LC-CDR2 set forth as SEQ ID NO: 198 (YTSRLHS) and a LCCDR3 set forth as SEQ ID NO:200 (QQGFTLPWT) [2D7]; or (c) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO: 132, a HC-CDR2 set forth as SEQ ID NO: 133, a HC-CDR3 set forth as SEQ ID NO: 135; and a light chain variable région comprsing a LC-CDR1 set forth as SEQ ID NO:203, a LC-CDR2 set forth as SEQ ID NO: 165 and a LC-CDR3 set forth as SEQ ID NO:204 [49Cll];or (d) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO: 137 a HC-CDR2 set forth as SEQ ID NO: 138, a HC-CDR3 set forth as SEQ ID NO: 140; and a light chain variable région comprising a LC-CDR1 set forth as SEQ ID NO:206, a LC-CDR2 set forth as SEQ ID NO:207 and a LC-CDR3 set forth as SEQ ID NO:208 [15D9];or (e) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:98, a HC-CDR2 set forth as SEQ ID NO:99, a HC-CDR3 set forth as SEQ ID NO: 101; and a light chain variable région comprising a LC-CDR1 set forth as SEQ ID NO: 169, a LC-CDR2 set forth as SEQ ID NO:17I and a LC-CDR3 set forth as SEQ ID NO: 173. [2F5]; or (f) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO: 103, a HC-CDR2 set forth as SEQ ID NO: 105, a HC-CDR3 set forth as SEQ ID NO: 107; and a light chain variable région comprising a LC-CDR1 set forth as SEQ ID NO: 176, a LC-CDR2 set forth as SEQ ID NO: 178 and a LC-CDR3 set forth as SEQ ID NO:193 [1B11]; or (g) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:114, a HC-CDR2 set forth as SEQ ID NO:116, a HC-CDR3 set forth as SEQ ID NO:118; and a light chain variable région comprising a LC-CDR1 set forth as SEQ IDNO:188,

318 a LC-CDR2 set forth as SEQ ID NO: 178 and a LC-CDR3 set forth as SEQ ID NO: 190 [2F2]; or (h) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:114, a HC-CDR2 set forth as SEQ ID NO:121, a HC-CDR3 set forth as SEQ ID NO:123;

and a light chain variable région comprising a LC-CDRI set forth as SEQ ID NO:191, a LC-CDR2 set forth as SEQ ID NO: 178 and a LC-CDR3 set forth as SEQ ID NO:193. [11B6]

2F. The antibody or antigen binding fragment thereof of paragraph l(a)-(g), wherein the 10 antibody or antigen-binding fragment is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, and an antigenbinding fragment of any of the foregoing.

3F. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-2, wherein 15 said antibody or antigen binding fragment thereof is selected from the group consisting of a single chain antibody, an ScFv, a Fab fragment, an Fab’ fragment, an F(ab’)2 fragment, a univalent antibody lacking a hinge région and a whole antibody.

4F. The antibody or antigen-binding fragment thereof of any one of paragraphs 1 -3, further 20 comprising an immunoglobulin constant région.

5F. The antibody or antigen binding fragment thereof of any one of paragraphs 1-4, wherein the antibody or antigen-binding fragment is humanized.

6F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:32 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:47 [1E7].

7F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or antigen binding fragment thereof comprises a heavy chain comprising at least 80%, 85%,

319

90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:33 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:48 [2D7].

8F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or 5 antigen binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:34 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:49 [49C11].

9F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody or 10 antigen binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:35 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:50 [15D9]

10F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody 15 or antigen binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:36 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:51 [2F5].

11F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody 20 or antigen binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:37 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:52 [1B11].

12F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody 25 or antigen binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:38 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:53 [2F2].

13F. The antibody or antigen-binding fragment thereof of paragraph 1, wherein the antibody 30 or antigen binding fragment thereof comprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:39 and a light chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:54 [11B6].

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14F. The antibody or antigen-binding fragment thereof of any one of paragraphs 1-13 wherein said antibody binds to human MASP-3 with an affinity of less than 500 pM.

15F. The antibody or antigen-binding fragment thereof of any of paragraphs 1-14, wherein said antibody inhibits alternative pathway activation in mammalian blood.

16F. A composition comprising the antibody or antigen-binding fragment of any of paragraphs 1 F-15F and a pharmaceutically acceptable excipient.

E. Use of MASP-3 Inhibitory Antibodies for treatment of AP diseases

1. A method of inhibiting alternative pathway complément activation in a mammal, the method comprising administering to a mammal subject in need thereof an amount of a composition comprising a high affinity MASP-3 inhibitory antibody or antigenbinding fragment thereof suffïcient to inhibit alternative pathway complément pathway activation in the mammal.

2. The method of claim 1, wherein the antibody, or antigen binding fragment thereof binds to MASP-3 with an affinity of less than 500 pM.

3. The method of paragraph 1, wherein as a resuit of administering the composition comprising the antibody or antigen-binding fragment one or more of the following is présent in the mammalian subject:

(a) inhibition of Factor D maturation;

(b) inhibition of the alternative pathway when administered to the subject at a molar ratio of from about 1:1 to about 2.5:1 (MASP-3 target to mAb) (c) the classical pathway is not inhibited.

(d) inhibition of hemolysis and/or opsonization;

(e) a réduction of hemolysis or the réduction of C3 cleavage and C3b surface déposition;

321 (f) a réduction of Factor B and Bb déposition on an activating surface;

(g) a réduction of resting levels (in circulation, and without the experimental addition of an activating surface) of active Factor D relative to pro-Factor D;

(h) a réduction of levels of active Factor D relative to pro-Factor D in response to an activating surface; and/or (i) a réduction of the production of resting and surface-induced levels of fluid-phase Ba, Bb, C3b, or C3a.

4. The method of paragraph 1, wherein the antibody inhibits the alternative pathway at a molar ratio of from about 1:1 to about 2.5:1 (MASP-3 target to mAb)

5. The method of any of paragraphs 1-3 wherein the high affinity MASP-3 antibody characterized according to any of daims 27A, 25B, 21C, 10D, 9E or I6F.

6. The method of any of paragraphs 1-4, wherein the antibody or antigen binding fragment thereof selectively inhibits the alternative pathway without affecting the classical pathway activation.

7. The method of any of paragraphs 1-6 wherein the mammal subject is suffering from, or at risk of developing an altemative-pathway disease or disorder selected from the group consisting of paroxysmal noctumal hemoglobinuria (PNH), age-related macular degeneration (AMD, including wet and dry AMD), ischemia-reperfusion injury, arthritis, disseminated intravascular coagulation, thrombotic microangiopathy (including hemolytic urémie syndrome (HUS), atypical hemolytic urémie syndrome (aHUS),thrombotic thrombocytopénie purpura (TTP) or transplant-associated TMA), asthma, dense deposit disease, pauci-immune necrotizing crescentic glomerulonephritis, traumatic brain injury, aspiration pneumonia, endophthalmitis, neuromyelitis optica , Behçet’s disease, multiple sclerosis, Guillain Barre Syndrome, Alzheimer’s disease, Amylotrophic latéral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy, Uveitis, Chronic obstructive pulmonary disease (COPD), C3 glomerulopathy, transplant rejection, Graft-versushost disease (GVHD), hemodialysis, sepsis, Systemic inflammatory response

322 syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis, Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy and Myasthenia Gravis.

While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims (15)

1. An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to the serine protease domain of human MASP-3 (amino acid residues 450 to 728 of SEQ ID NO:2) with high affinity, wherein said antibody binds within at least one of:

(a) an epitope in human MASP-3 set forth as VLRSQRRDTTV (SEQ ID NO:15), TAAHVLRSQRRDTTV (SEQ ID N0:10), VLRSQRRDTTVI (SEQ ID NO:9), DFNIQNYNHDIALVQ (SEQ ID NO:11), PHAECKTSYESRS (SEQ ID NO:12), GNYSVTENMFC (SEQ ID NO:13), and/or VSNYVDWVWE (SEQ ID N0:14); or (b) an epitope in human MASP-3 set forth as ECGQPSRSLPSLV (SEQ ID NO:16), RNAEPGLFPWQ (SEQ ID NO:17); KWFGSGALLSASWIL (SEQ ID NO:18);

EHVTVYLGLH (SEQ ID NO:19); PVPLGPHVMP (SEQ ID N0:20); APHMLGL (SEQ ID NO:21); SDVLQYVKLP (SEQ ID NO:22); and/or AFVIFDDLSQRW (SEQ ID NO:23), wherein the antibody or antigen-binding fragment thereof inhibits alternative pathway complément activation.

2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment is characterized by at least one or more of the following properties:

(a) inhibits pro-Factor D maturation;

(b) does not bind to human MASP-1 (SEQ ID NO:8);

(c) inhibits the alternative pathway at a molar ratio of from about 1:1 to about 2.5:1 (MASP-3 target to mAb) in a mammalian subject;

(d) does not inhibit the classical pathway.

(e) inhibition of hemolysis and/or opsonization;

(f) inhibition of MASP-3 serine protease substrate-specific cleavage;

(g) a réduction of hemolysis or the réduction ofC3 cleavage and C3b surface déposition;

(h) a réduction of Factor B and Bb déposition on an activating surface;

(i) a réduction of resting levels (in circulation, and without the experimental addition of an activating surface) of active Factor D relative to pro-Factor D;

324 (j) a réduction of levels of active Factor D relative to pro-Factor D in response to an activating surface;

(k) a réduction of the production of resting and surface-induced levels of fluid-phase Ba, Bb, C3b, orC3a; and/or (l) a réduction in factor P déposition.

3. The antibody or antigen-binding fragment thereof of claim 1 (a), wherein said antibody or antigen-binding fragment binds to an epitope within SEQ ID NO: 15.

4. The antibody or antigen-binding fragment of claim l(a), wherein said antibody or antigen-binding fragment binds to an epitope within SEQ ID NO: 10.

5 CDR3 set forth as SEQ ID NO: 129 (CPFYYLGKGTHFDY); and a light chain variable région comprising a LC-CDRI set forth as SEQ IDNO:196 (RASQDISNFLN), a LC-CDR2 set forth as SEQ ID NO: 198 (YTSRLHS) and a LC-CDR3 set forth as SEQ ID NO:200 (QQGFTLPWT); or (d) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO: 132, a

5. The antibody or antigen-binding fragment of claim l(a), wherein said antibody or antigen-binding fragment binds to an epitope within SEQ ID NO:9. .

6. The antibody or antigen-binding fragment of claim 4, wherein said antibody or antigen binding fragment also binds to an epitope within SEQ ID NO: 12.

7. The antibody or antigen-binding fragment of claim 5, wherein said antibody or antigen binding fragment also binds to an epitope within at least one of SEQ ID NO:11, SEQ ID NO: 13 and/or SEQ IDN0:14.

8. The isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 according to claim 1 (a) comprising:

(a) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:84 (GKWIE); a HC-CDR2 set forth as SEQ ID NO:86 (EILPGTGSTNYNEKFKG) or SEQ ID NO:275 (EILPGTGSTNYAQKFQG); and a HC-CDR3 set forth as SEQ ID NO:88 (SEDV); and a light chain variable région comprising a LC-CDRI set forth as SEQ ID NO: 142 (KSSQSLLNSRTRKNYLA), SEQ ID NO:257 (KSSQSLLQSRTRKNYLA); SEQ ID NO:258 (KSSQSLLASRTRKNYLA); or SEQ ID NO:259 (KSSQSLLNTRTRKNYLA), a LC-CDR2 set forth as SEQ ID NO: 144 ( WASTRES); and a LC-CDR3 set forth as SEQ ID NO:I61 (KQSYNIPT); or (b) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:213 (SYGXX, wherein X at position 4 is M or I and wherein X at position 5 is S or T); a

325

HC-CDR2 set forth as SEQ ID NO:74; and a HC-CDR3 set forth as SEQ ID NO:214 (GGXAXDY, wherein X at position 3 is E or D and wherein X at position 5 is M or L); and a light chain variable région comprising a LC-CDR1 set forth as SEQ ID NO:215 (KSSQSLLDSXXKTYLX, wherein X at position 10 is D, E or A; wherein X at position 11 is G or A; and wherein X at position 16 is N or S); a LC-CDR2 set forth as SEQ ID NO: 155; and a LC-CDR3 set forth as SEQ ID NO:216 (WQGTHFPXT, wherein X at position 8 is W or Y; or (c) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:209 (XXDIN, wherein X at position 1 is S or T and wherein X at position 2 is N or D); a HC-CDR2 set forth as SEQ ID NO:210 (WIYPRDXXXKYNXXFXD, wherein X at position 7 is G or D; X at position 8 is S, T or R; X at position 9 is I or T; X at position 13 is E or D; X at position 14 is K or E; and X at position 16 is T or K); and a HCCDR3 set forth as SEQ ID NO:211 (XEDXY, wherein X at position 1 is L or V, and wherein X at position 4 is T or S); and a light chain variable région comprising a LCCDR1 set forth as SEQ IDNO:212 (KSSQSLLXXRTRKNYLX, wherein X at position 8 is N, I, Q or A; wherein X at position 9 is S or T; and wherein X at position 17 is A or S); a LC-CDR2 set forth as SEQ IDNO: 144 (WASTRES)and a LC-CDR3 set forth as SEQ IDNO: 146 (KQSYNLYT).

9. The isolated antibody, or antigen-binding fragment thereof, that binds to MASP-3 according to claim 1 (b) comprising:

(a) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:91 (GYWIE); a HC-CDR2 set forth as SEQ ID NO:93 (EMLPGSGSTHYNEKFKG), and a HCCDR3 set forth as SEQ ID NO:95 (SIDY); and a light chain variable région comprising a LCCDR1 set forth as SEQ IDNO: 163 (RSSQSLVQSNGNTYLH), a LC-CDR2 set forth as SEQ ID NO: 165 (KVSNRFS) and a LC-CDR3 set forth as SEQ ID NO: 167 (SQSTHVPPT); or (b) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO: 109 (RVHFAIRDTNYWMQ), a HC-CDR2 set forth as SEQ ID NO: 110 (AIYPGNGDTSYNQKFKG), a HC-CDR3 set forth as SEQ IDNO:112 (GSHYFDY); and a light chain variable région comprising a LC-CDR1 set forth as SEQ ID NO: 182

326 (RASQSIGTSIH), a LC-CDR2 set forth as SEQ ID NO: 184 (YASESIS) and a LC-CDR3 set forth as SEQ ID NO: 186 (QQSNSWPYT); or (c) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO: 125 (DYYMN), a HC-CDR2 set forth as SEQ ID NO: 127 (DVNPNNDGTTYNQKFKG), a HC-

10 21. The antibody or antigen-binding fragment of any of claims 1, 8 or 9 or a composition according to claim 20 comprising a high affinity MASP-3 inhibitory antibody or antigen-binding fragment thereof for use in a method of inhibiting alternative pathway complément activation in a mammal.

10. The antibody or antigen binding fragment thereof of any of Claims 1, 8 or 9, wherein the antibody or antigen-binding fragment is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, and an antigen-binding fragment of any of the foregoing.

10 HC-CDR2 set forth as SEQ ID NO:133, a HC-CDR3 set forth as SEQ ID NO:135; and a light chain variable région comprising a LC-CDRI set forth as SEQ ID NO:203, a LC-CDR2 set forth as SEQ ID NO: 165 and a LC-CDR3 set forth as SEQ ID NO:204; or (e) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO: 137 a HC-CDR2 set forth as SEQ ID NO:138, a HC-CDR3 set forth as SEQ ID NO: 140; and a light

15 chain variable région comprising a LC-CDRI set forth as SEQ ID NO:206, a LC-CDR2 set forth as SEQ IDNO:207 and a LC-CDR3 set forth as SEQ ID NO:208; or .

(f) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:98, a HC-CDR2 set forth as SEQ ID NO:99, a HC-CDR3 set forth as SEQ ID NO: 101; and a light chain variable région comprising a LC-CDRI set forth as SEQ ID NO: 169, a LC-CDR2 set

20 forth as SEQ ID NO: 171 and a LC-CDR3 set forth as SEQ ID NO: 173; or (g) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO: 103, a HC-CDR2 set forth as SEQ ID NO: 105, a HC-CDR3 set forth as SEQ ID NO: 107; and a light chain variable région comprising a LC-CDRI set forth as SEQ ID NO: 176, a LC-CDR2 set forth as SEQ ID NO: 178 and a LC-CDR3 set forth as SEQ ID NO: 193; or

25 (h) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO: 114, a

HC-CDR2 set forth as SEQ ID NO:116, a HC-CDR3 set forth as SEQ ID NO:118; and a light chain variable région comprising a LC-CDRI set forth as SEQ ID NO: 188, a LC-CDR2 set forth as SEQ ID NO: 178 and a LC-CDR3 set forth as SEQ ID NO: 190; or (i) a heavy chain variable région comprising a HC-CDR1 set forth as SEQ ID NO:114, a 30 HC-CDR2 set forth as SEQ ID NO: 121, a HC-CDR3 set forth as SEQ ID NO: 123; and a light

327 chain variable région comprising a LC-CDRI set forth as SEQ ID NO: 191, a LC-CDR2 set forth as SEQ ID NO: 178 and a LC-CDR3 set forth as SEQ ID NO: 193.

11. The antibody or antigen-binding fragment thereof of any of Claims 1, 8 or 9, wherein said antibody or antigen binding fragment thereof is selected from the group consisting of a single chain antibody, an ScFv, a Fab fragment, an Fab’ fragment, an F(ab’)2 fragment, a univalent antibody lacking a hinge région and a whole antibody.

12. The antibody or antigen-binding fragment thereof of any of Claims 1, 8 or 9, further comprising an immunoglobulin constant région.

13. The antibody or antigen binding fragment thereof of any one of claims 1, 8 or 9, wherein the antibody or antigen-binding fragment is humanized.

14. The antibody or antigen-binding fragment thereof of any of Claims 1, 8 or 9, wherein said antibody binds to the serine protease domain of human MASP-3 with an affinity of less than 500 pM.

15. The antibody or antigen-binding fragment thereof of any of Claims 1, 8 or 9, wherein said antibody inhibits alternative pathway activation in mammalian blood.

16. An isolated DNA sequence encoding the heavy and/or light chain variable régions of an antibody or antigen-binding fragment thereof from any one of claims 1, 8 or 9.

17. A cloning or expression vector comprising one or more DNA sequences of Claim 16.

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18. A host cell comprising one or more cloning or expression vectors of claim 17.

19. A process for producing the antibody or antigen-binding fragment of any of claims 1, 8 or 9 comprising culturing the host cell of Claim 59 and isolating the antibody or antigen-binding 5 fragment thereof. .

20. A composition comprising the antibody or antigen-binding fragment of any of claims 1, 8 or 9 and a pharmaceutically acceptable excipient.

15 22. Use of the antibody or antigen-binding fragment of any of claims 1, 8 or 9 in the manufacture of a médicament for inhibiting alternative pathway complément activation in a mammal.