OA16772A - Bispecific binding molecules binding to DII4 and Ang2. - Google Patents

Bispecific binding molecules binding to DII4 and Ang2. Download PDF

Info

Publication number
OA16772A
OA16772A OA1201300406 OA16772A OA 16772 A OA16772 A OA 16772A OA 1201300406 OA1201300406 OA 1201300406 OA 16772 A OA16772 A OA 16772A
Authority
OA
OAPI
Prior art keywords
amino acid
seq
binding
dii4
nos
Prior art date
Application number
OA1201300406
Inventor
Andreas Gschwind
Rene Georg Ott
Joachim Boucneau
Marie-Ange Buyse
Erik Depla
Original Assignee
Boehringer Ingelheim International Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Boehringer Ingelheim International Gmbh filed Critical Boehringer Ingelheim International Gmbh
Publication of OA16772A publication Critical patent/OA16772A/en

Links

Abstract

Bispecific binding molecules binding to both Dll4 and Ang2, preferably in the form of immunoglobulin single variable domains like VHHs and domain antibodies, pharmaceutical compositions containing the same and their use in the treatment of diseases that are associated with Dll4-and/or Ang2-mediated effects on angiogenesis are disclosed. Further, nucleic acids encoding bispecific binding molecules, host cells and methods for preparing same are also described.

Description

FIELD OF THE INVENTION
The invention relates to the field of human therapy, in particular cancer therapy and agents and compositions useful in such therapy.
BACKGROUND OF THE INVENTION
When tumors reach a critical size of approximately 1 mm3 they become dépendent on angiogenesis for maintaîning blood supply with oxygen and nutritients to allow for îo further growth. Anti-angiogenesis thérapies hâve become an important treatment option for several types of tumors. These thérapies hâve focused on blocking the VEGF pathway (Ferrara étal., Nat Rev Drug Discov, 2004 May;3(5):391-400.) by neutralizing VEGF (Avastin) or its receptors (Sutent and Sorafinib). Recent studies in mice hâve shown, that Angiopoietin2 (Ang2), a ligand of the Tie2 receptor, controls 15 vascular remodeling by enabling the functions of other angiogenic factors, such as
VEGF. Ang2 is primarily expressed by endothélial cells, strongly induced by hypoxia and other angiogenic factors and has been demonstrated to regulate tumor vessel plasticity, allowing vessels to respond to VEGF and FGF2 (Augustin et al., Nat Rev Mol Cell Biol. 2009 Mar;10(3):165-77.). Consistent with this rôle, the délétion or inhibition of Ang2 results in reduced angiogenesis (Falcôn et al., Am J Pathol. 2009 Nov;175(5):2159-70.). Elevated Ang2 sérum concentrations hâve been reported for patients with colorectal cancer, NSCLC and melanoma (Goede et al., Br J Cancer. 2010 Oct26; 103(9):1407-14),(Park étal., Chest. 2007 Jul;132(1 ): 200-6.),(Helfrich et al., Clin Cancer Res. 2009 Feb 15;15(4):1384-92.). In CRC cancer Ang2 sérum levels correlate with therapeutic response to anti-VEGF therapy.
The Ang-Tie System consists of 2 receptors (Tie1 and Tie2) and 3 ligands (Ang1, Ang2 and Ang4) (Augustin et al., Nat Rev Mol Cell Biol. 2009 Mar;10(3):165-77.). Tie2, Ang1 and Ang2 are the best studied members of this family, Tie1 is an orphan receptor and the rôle of Ang4 for vascular remodelling still needs to be defined. Ang2 and Ang1 médiate opposing functions upon Tie2 binding and activation. Ang2mediated Tie2 activation results in endothélial cell activation, pericyte dissociation, vessel leakage and induction of vessel sprouting. In contrastto Ang2, Ang1 signaling maintains vessel integrity by recruitment of pericytes, thereby maintaining endothélial cell quiescence.
Angiopoietin 2 (Ang2) is a secreted, 66 kDa ligand for the Tie2 receptor tyrosine kinase (Augustin et al., Nat Rev Mol Cell Biol. 2009 Mar;10(3):165-77.). Ang2 consists of an N-terminal coiled-coil domain and a C-terminal fibrinogen-like domain, the latter is required for Tie2 interaction. Ang2 is primarily expressed by endothélial cells and strongly induced by hypoxia and other angiogenic factors, including VEGF. Tie2 is found on endothélial cells, haematopoietic stem cells and tumor cells. Ang2Tie2 has been demonstrated to regulate tumor vessel plasticity, allowing vessels to respond to VEGF and FGF2.
In vitro Ang2 has been shown to act as a modest mitogen, chemoattractant and inducerof tube formation in human umbilical vein endothélial cells (HUVEC). Ang2 induces tyrosine phosphorylation of ectopically expressed Tie2 in fibroblasts and promûtes downstream signaling events, such as phosphorylation of ERK-MAPK, AKT and FAK in HUVEC. An antagonistic rôle of Ang2 in Ang1-induced endothélial cell responses has been described.
Ang2 -deficiency has been shown to resuit in a profound lymphatic patterning defect in mice. Although the loss of Ang2 is dispensable for embryonic vascular development, Ang2 -déficient mice hâve persistent vascular defects in the retina and kidney. Together with the dynamic pattern of Ang2 expression at sites of angiogenesis (for example ovary), these findings indicate that Ang2 controls vascular remodeling by enabling the functions of other angiogenic factors, such as VEGF.
The Ang2-Tie2 system exerts crucial rôles during the angiogenic switch and later stages of tumor angiogenesis. Ang2 expression is strongly up-regulated in the tumorassociated endothélium. Reduced growth of tumors has been observed when implanted into Ang2 -déficient mice, especially during early stages of tumor growth. Therapeutic blocking of Ang2 with Ang2 mAbs has shown broad efficacy in a variety of tumor xenograft models.
As summarized în US 2008/0014196, angiogenesis is implicated in the pathogenesis of a number of disorders, including solid tumors and metastasis. σ,
-216772
In the case of tumor growth, angiogenesis appears to be crucial for the transition from hyperplasia to neoplasia, and for providing nourishment for the growth and metastasis of the tumor. Folkman et al., Nature 339 -58 (1989), which allows the tumor cells to acquire a growth advantage compared to the normal cells. Therefore, anti-angiogenesis thérapies hâve become an important treatment option for several types of tumors. These thérapies hâve focused on blocking the VEGF pathway (Ferrara et al., Nat Rev Drug Discov. 2004 May;3(5):391-400.
The Notch signaling pathway is important for cell-cell communication, which involves gene régulation mechanisms that control multiple cell différentiation processes during embryonic development and in adult organisms. Notch signaling is dysregulated in many cancers, e.g. in T-cell acute lymphoblastic leukemia and in solid tumors (Sharma étal. 2007, Cell Cycle 6 (8): 927-30; Shih et al., Cancer Res. 2007 Mar 1 ;67(5): 1879-82).
DII4 (or Delta like 4 or delta-like ligand 4) is a member of the Delta family of Notch ligands. The extracellular domain of DII4 is composed of an N-terminal domain, a Delta/Serrate/Lag-2 (DSL) domain, and a tandem of eight epidermal growth factor (EGF)-like repeats. Generally, the EGF domains are recognized as comprising amino acid residues 218-251 (EGF-1; domain 1 ), 252-282 (EGF-2; domain 2), 284-322 (EGF-3; domain 3), 324-360 (EGF-4; domain 4), and 362-400 (EGF-5; domain 5), with the DSL domain at about amino acid residues 173-217 and the N-terminal domain at about amino acid residues 27-172 of hDII4 (WO 2008/076379).
It has been reported that DII4 exhibits highly sélective expression by vascular endothélium, in partîcular in arterial endothélium (Shutter étal. (2000) Genes Develop. 14:1313-1318). Recent studies in mice hâve shown that DII4 is induced by VEGF and is a négative feedback regulator that restreins vascular sprouting and branching. Consistent with this rôle, the délétion or inhibition of DII4 results in excessive angiogenesis (Scehnet étal., Blood. 2007 Jun 1 ;109(11 ):4753-60). This unrestrained angiogenesis paradoxically decreases tumor growth due to the formation of non-productive vasculature, even in tumors résistant to anti-VEGF thérapies (Thurston étal., Nat Rev Cancer. 2007 May;7(5):327-31;
WO 2007/070671 ; Noguera-Troise et al., Nature. 2006 Dec 21 ; 444(7122)). Furthermore, the combined inhibition of VEGF and DII4 is shown to provide superior anti-tumor activity compared to anti-VEGF alone in xenograft models of multiple
-316772 tumor types (Noguera-Troise étal., Nature. 2006 Dec 21; 444(7122):1032-7;
Ridgway étal., Nature. 2006 Dec 21 ;444(7122): 1083-7).
Due to these results, DII4 is being considered a promising target for cancer therapy, and several biological compounds that target DII4 are in (pre-)clinical development hâve been described: REGN-421 (= SAR153192; Regeneron, Sanofi-Aventis; W02008076379) and OPM-21M18 (OncoMed) (Hoey étal., Cell Stem Cell. 2009 Aug 7; 5(2):168-77), both fully human DII4 antibodies; YW152F (Genentech), a humanized DII4 antibody (Ridgway et al., Nature. 2006 Dec 21 ; 444(7122):1083-7); DII4-Fc (Regeneron, Sanofi-Aventis), a recombinant fusion protein composed of the extracellular région of D1I4 and the Fc région of human IgG 1 (Noguera-Troise et al., Nature. 2006 Dec 21 ;444(7122)).
However, the state-of-the art monoclonal antibodies (MAbs) and fusion proteins hâve several shortcomings in view of their therapeutic application: To prevent their dégradation, they must be stored at near freezing températures. Also, since they are quickly digested in the gut, they are not suited for oral administration. Another major restriction of MAbs for cancer therapy is poor transport, which results in low concentrations and a lack of targeting of ail cells in a tumor.
It has been an object of the présent invention to provide novel anti-angiogenic binding molécules for human therapy.
It has been a further object of the invention to provide methods for the prévention, treatment, alleviation and/or diagnosis of such diseases, disorders or conditions, involving the use and/or administration of such binding molécules and compositions comprising them. In particular, it is has been an object ofthe invention to provide such pharmacologically active binding molécules, compositions and/or methods that provide advantages compared to the agents, compositions and/or methods currently used and/or known in the art. These advantages include improved therapeutic and/or pharmacological properties and/or other advantageous properties, e.g, for manufacturing purposes, especially as compared to conventional antibodies as those described above, or fragments thereof. w—
-416772
BRIEF SUMMARY OF THE INVENTION
According to a first aspect, there are provided bispecific binding molécules, preferably bispecific immunoglobulins, preferably immunoglobulin single variable domains like VHHs and domain antibodies, which comprises at least one DLL4binding component and at least one Ang2-binding component in a single molécule. These bispecific binding molécules may preferably comprise a further binding component, preferably a binding omponent binding to sérum albumin.
More specifically, a bispecific binding molécule of the invention essentially comprises (i) at least one DII4-binding component specifically binding to at least one epitope of DII4 and (ii) at least one Ang2-binding component specifically binding to at least an epitope of Ang2, wherein the components are linked to each other in such a way that they simultaneously bind to DII4 and Ang2 or that they bind to either DII4 or Ang2 at a time.
According to preferred aspects of the invention, the two components comprise one or more immunoglobulin single variable domains that may be, independently of each other, VHHs or domain antibodies, and/or any other sort of immunoglobulin single variable domains, such as VL domains, as defined herein, provided that each of these immunoglobulin single variable domains will bind the antigen, i.e. DII4 or Ang2, respectively.
According to a preferred embodiment, the immunoglobulin single variable domains are ofthe same type, in particular, ail immunoglobulin single variable domains are VHHs or domain antibodies.
According to a particularly preferred embodiment, ail immunoglobulin single variable domains are VHHs, preferably humanized (or “sequence-optimized”, as defined herein) VHHs. Accordingly, the invention relates to bispecific binding molécules comprising an (optionally humanized or sequence-optimized) anti-DII4 VHH and an (optionally humanized or sequence-optimized) anti-Ang2 VHH.
However, it will be clear to the skilled person that the teaching herein may be applied analogously to bispecific binding molécules including other anti-DII4 or anti-Ang2 immunoglobulin single variable domains, such as domain antibodies.
-516772
In another aspect, the invention relates to nucleic acids encoding the bispecific binding molécules of the invention as well as host cells containing same.
The invention further relates to a product or composition containing or comprising at least one bispecific binding molécule of the invention and optionally one or more further components of such compositions.
The invention further relates to methods for preparing or generating the bispecific binding molécules, nucleic acids, host cells, products and compositions described herein.
The invention further relates to applications and uses of the bispecific binding molécules, nucleic acids, host cells, products and compositions described herein, as well as to methods for the prévention and/or treatment for diseases and disorders that can be modulated by inhibition of DII4.
It has been found that the Ang2-binding component of the bispecific binding molécules according to the présent invention binds to Ang2 with a potency at least 5,000 times higher, preferably 10,000 times higher than to Ang1 or Ang4. This will largely avoid blocking activation of Ang1-mediated signalling, which would counter the intended anti-angiogenetic effect.
It has further been found that the DLL4-binding component of the bi-specific binding molécules according to the présent invention binds to DLL4-A with an affinity of at least 1,000 times higher than to DII1, Jaggedl and preferably also against Jagged2. Due to this selectivity unwanted side reactions can be avoided.
In a preferred embodiment the bispecific binding molécules of the présent invention are provided as linked VHH domains. Such molécules are significantly smaller than conventional antibodies and hâve thus the potential for penetrating into a tumor deeper than such conventional antibodies. This benefit is further accentuated by the spécifie sequences disclosed herein after being free of glycosylation sites.
Further, due to the bispecific nature (DII4- and Ang2-binding components in one molécule) the tumor pénétration of both functionalities will be necessarily equal, which will ensure that the bénéficiai effects of the combined antagonism of DII4 and
Ang2 will be provided within the whole depth of pénétration of the tumor. This is an advantage over the combination of individual antagonists against these targets, since
-616772 the depth of pénétration of individual antagonists will always vary to some degree.
Another advantage of a preferred bispecific binding molécules of the présent invention is their increased sérum half-like due to a sérum albumin binding component such as a sérum albumin binding molécule as described herein.
These and other aspects, embodiments, advantages and applications of the invention will become clear from the further description hereinbelow.
DEFINITIONS
Unless indicated or defined otherwise, ail terms used hâve their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, Molecular Cloning: A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, Genes IV, Oxford University Press, New York, (1990), and Roitt étal., Immunology (2nd Ed.), Gower Medical Publishing, London, New York (1989), as well as to the general background art cited herein; Furthermore, unless indicated otherwise, ail methods, steps, techniques and manipulations that are not specifically described in detail can be performed and hâve been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks, to the general background art referred to above and to the further references cited therein.
The term “bispecific binding molécule refers to a molécule comprising at least one Ang2-binding molécule (or “Ang2-binding component”) and at least one DII4-binding molécule (or DII4-binding component”). A bispecific binding molécule may contain more than one Ang2-binding molécule and/or more than one DII4-binding molécule,
i.e. in the case that the bispecific binding molécule contains a biparatopic (as defined below) Ang2-binding molécule and/or a biparatopic DII4-binding molécule, in the part of the molécule that binds to Ang2 or to DII4, i.e. in its “Ang2-binding component (or anti-Ang2 component) or DII4-binding component (or anti-DII4 component), respectively. The word “bispecific in this context is however not to be construed as to exclude further binding components with binding specificity to molécules other than DII4 and Ang2 from the bispecific binding molécule. Non-limiting examples of such further binding components are binding components binding to sérum albumin.
-716772
Unless indicated otherwise, the terms immunoglobulin and immunoglobulin sequence - whether used herein to refer to a heavy chain antibody or to a conventional 4-chain antibody - are used as general terms to include both the fullsize antibody, the individual chains thereof, as well as ail parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as VHH domains or VH/VL domains, respectively). In addition, the term sequence as used herein (for example in terms like immunoglobulin sequence, antibody sequence, (single) variable domain sequence, VHH sequence or protein sequence), should generally be understood to include both the relevant amino acid sequence as well as nucleic acid sequences or nucléotide sequences encoding the same, unless the context requires a more limited interprétation.
The term domain (of a polypeptide or protein) as used herein refers to a folded protein structure which has the ability to retain its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrète functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.
The term immunoglobulin domain as used herein refers to a globular région of an antibody chain (such as e.g. a chain of a conventional 4-chain antibody or of a heavy chain antibody), or to a polypeptide that essentially consists of such a globular région. Immunoglobulin domains are characterized in that they retain the immunoglobulin fold characteristic of antibody molécules, which consists of a 2-layer sandwich of about 7 antiparallel beta-strands arranged in two beta-sheets, optionally stabilized by a conserved disulphide bond. An immunoglobulin domain comprises (a) variable domain(s), i.e., one or more immunoglobulin variable domains.
The term immunoglobulin variable domain as used herein means an immunoglobulin domain essentially consisting of four framework régions which are referred to in the art and hereînbelow as framework région 1 or FR1; as framework région 2 orFR2; as framework région 3 or FR3; and as framework région 4 or FR4, respectively; which framework régions are interrupted by three complementarity determining régions or CDRs, which are referred to in the art and hereînbelow as complementarity determining région 1or CDR1; as complementarity determining région 2 or CDR2; and as complementarity
-816772 determining région 3 or CDR3, respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1 CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the immunoglobulin variable domain(s) that confer specificity to an antibody for the antigen by carrying the antigen-binding site. In the context of the présent invention immunoglobulin single variable domains like VHHs and domain antîbodies are preferred.
The term immunoglobulin single variable domain as used herein means an immunoglobulin variable domain which is capable of specifically binding to an epitope of the antigen without pairing with an additional variable immunoglobulin domain. One example of immunoglobulin single variable domains in the meaning of the présent invention are domain antîbodies, such as the immunoglobulin single variable domains VH and VL (VH domains and VL domains). Another example of immunoglobulin single variable domains are VHH domains (or simply VHHs) from camelids, as defined hereinafter.
In view of the above définition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molécule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (ail known in the art) derived from such conventional 4chain antibody, would normally not be regarded as an immunoglobulin single variable domain, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e. by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen.
VHH domains, also known as VHHs, VhH domains, VHH antibody fragments, and VHH antîbodies, hâve originally been described as the antigen binding immunoglobulin (variable) domain of heavy chain antîbodies (i.e. of antîbodies devoid of light chains; Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: Naturally occurring antîbodies devoid of light chains; Nature 363, 446-448 (1993)). The term VHH domain has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are présent in conventional 4-chain antibodies (which are referred to herein as VH domains or VH domains) and from the light
-916772 chain variable domains that are présent in conventional 4-chain antibodies (which are referred to herein as VL domains or VL domains). VHH domains can specifically bind to an epitope without an additional antigen binding domain (as opposed to VH or VL domains in a conventional 4-chain antibody, in which case the epitope is recognized by a VL domain together with a VH domain). VHH domains are small, robust and efficient antigen récognition units formed by a single immunoglobulin domain.
In the context ofthe présent invention, the terms VHH domain, VHH, VhH domain, VHH antibody fragment, VHH antibody, as well as Nanobody® and Nanobody® domain (Nanobody being a trademark of the company Ablynx N.V.; Ghent; Belgium) are used interchangeably and are représentatives of immunoglobulin single variable domains (having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and specifically binding to an epitope without requiring the presence of a second immunoglobulin variable domain), and which are distinguished from VH domains by the so-called hallmark residues, as defined in e.g. W02009/109635, Fig. 1.
The amino acid residues of a immunoglobulin single variable domain, e.g. a VHH, are numbered according to the general numbering for Vh domains given by Kabat et al. (Sequence of proteins of immunological interest, US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to VHH domains from Camelids, as shown e.g. in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999). According to this numbering,
- FR1 comprises the amino acid residues at positions 1-30,
- CDR1 comprises the amino acid residues at positions 31-35,
- FR2 comprises the amino acids at positions 36-49,
- CDR2 comprises the amino acid residues at positions 50-65,
- FR3 comprises the amino acid residues at positions 66-94,
- CDR3 comprises the amino acid residues at positions 95-102, and
- FR4 comprises the amino acid residues at positions 103-113.
However, it should be noted that - as is well known in the art for Vh domains and for VHH domains - the total number of amino acid residues in each of the CDRs may
-1016772 vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.
Alternative methods for numbering the amino acid residues of VH domains, which methods can also be applied in an analogous manner to VHH domains, are known in the art. However, in the présent description, claims and figures, the numbering according to Kabat and applied to VHH domains as described above will be followed, unless indicated otherwise.
The total number of amino acid residues in a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.
Immunoglobuîin single variable domains, e.g. VHHs and domain antibodies, according to the preferred embodiments of the invention, hâve a number of unique structural characteristics and functional properties which makes them highly advantageous for use in therapy as functional antigen-binding molécules. In particular, and without being limited thereto, VHH domains (which hâve been designed by nature to functionally bind to an antigen without pairing with a light chain variable domain) can function as single, relatively small, functional antigenbinding structural units.
Due to their unique properties, immunoglobuîin single variable domains, as defined herein, like VHHs or VHs (or VLs) - either alone or as part of a larger polypeptide, e.g. a biparatopîc molécule - offer a number of significant advantages:
• only a single domain is required to bind an antigen with high affinity and with high selectivity, so that there is no need to hâve two separate domains présent, nor to assure that these two domains are présent in the right spacial conformation and configuration (i.e. through the use of especially designed linkers, as with scFv's);
-1116772 • immunoglobulin single variable domains can be expressed from a single nucleic acid molécule and do not require any post-translational modification (like glycosylation;
• immunoglobulin single variable domains can easily be engineered into multivalent and multispecific formats (as further discussed herein);
• immunoglobulin single variable domains hâve high specificity and affinity for their target, low inhérent toxicity and can be administered via alternative routes than infusion or injection;
• immunoglobulin single variable domains are highly stable to heat, pH, proteases and other denaturing agents or conditions and, thus, may be prepared, stored or transported without the use of réfrigération equipments;
• immunoglobulin single variable domains are easy and relatively inexpensive to préparé, both on small scale and on a manufacturing scale. For example, immunoglobulin single variable domains can be produced using microbial fermentation (e.g. as further described below) and do not require the use of mammalian expression Systems, as with for example conventional antibodies;
• immunoglobulin single variable domains are relatively small (approximately kDa, or 10 times smallerthan a conventional IgG) compared to conventional 4-chain antibodies and antigen-binding fragments thereof, and therefore show high(er) pénétration into tissues (including but not limited to solid tumors and other dense tissues) and can be administered in higher doses than such conventional 4-chain antibodies and antigen-binding fragments thereof;
• VHHs hâve spécifie so-called cavity-binding properties” (inter alia due to their extended CDR3 loop, compared to VH domains from 4-chain antibodies) and can therefore also access targets and epitopes not accessible to conventional
4-chain antibodies and antigen-binding fragments thereof;
• VHHs hâve the particular advantage that they are highly soluble and very stable and do not hâve a tendency to aggregate (as with the mouse-derived antigen-binding domains described by Ward et al., Nature 341: 544-546 (1989)).
-1216772
The immunoglobulin single variable domains of the invention are not limited with respect to a spécifie biological source from which they hâve been obtained or to a spécifie method of préparation. For example, obtaining VHHs may include the following steps:
(1 ) isolating the VHH domain of a naturally occurring heavy chain antibody; or screening a library comprising heavy chain antibodies or VHHs and isolating VHHs therefrom;
(2) expressing a nucleic acid molécule encoding a VHH with the naturally occurring sequence;
(3) humanizing (as described herein) a VHH, optionally after affinity maturation, with a naturally occurring sequence or expressing a nucleic acid encoding such humanized VHH;
(4) camelizing (as described below) a immunoglobulin single variable heavy domain from a naturally occurring antibody from an animal species, in particular a species of mammal, such as from a human being, or expressing a nucleic acid molécule encoding such camelized domain;
(5) camelizing a VH, or expressing a nucleic acid molécule encoding such a camelized VH;
(6) using techniques for preparing synthetically or semi-synthetically proteins, polypeptides or other amino acid sequences;
(7) preparing a nucleic acid molécule encoding a VHH domain using techniques for nucleic acid synthesis, followed by expression of the nucleic acid thus obtained;
(8) subjecting heavy chain antibodies or VHHs to affinity maturation, to mutagenesis (e.g. random mutagenesis or site-directed mutagenesis) and/or any other technique(s) in order to increase the affinity and/or specificity of the VHH; and/or (9) combinations or sélections of the foregoing steps.
Suitable methods and techniques for performing the above-described steps are known in the art and will be clear to the skilled person. By way of example, methods of obtaining VHH domains binding to a spécifie antigen or epitope hâve been described in W02006/040153 and W02006/122786.
-1316772
According to spécifie embodiments, the immunoglobulin single variable domains of the invention or présent in the polypeptides of the invention are VHH domains with an amino acid sequence that essentially corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been humanized or sequenceoptimized (optionally after affinity-maturation), i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence by one or more of the amino acid residues that occur at the corresponding position(s) in a variable heavy domain of a conventional 4-chain antibody from a human being. This can be performed using methods known in the art, which can by routinely used by the skilled person.
A humanized VHH domain may contain one or more fully human framework région sequences, and, in an even more spécifie embodiment, may contain human framework région sequences derived from the human germline Vh3 sequences DP29, DP-47, DP-51, or parts thereof, or be highly homologous thereto, optionally combined with JH sequences, such as JH5. Thus, a humanization protocol may comprise the replacement of any of the VHH residues with the corresponding framework 1,2 and 3 (FRI, FR2 and FR3) residues of germline VH genes such as DP 47. DP 29 and DP 51 ) either alone or in combination. Suitable framework régions (FR) of the immunoglobulin single variable domains of the invention can be selected from those as set out e.g. in WO 2006/004678 and specifically, include the so-called KERE and GLEW classes. Examples are immunoglobulin single variable domains having the amino acid sequence G-L-E-W at about positions 44 to 47, and their respective humanized counterparts. A humanized VHH domain may contain one or more fully human framework région sequences.
By way of example, a humanizing substitution for VHHs belonging to the 103 P,R,Sgroup and/or the GLEW-group (as defined below) is 108Q to 108L. Methods for humanizing immunoglobulin single variable domains are known in the art.
Binding immunoglobulin single variable domains with improved properties in view of therapeutic application, e.g. enhanced affinity or decreased immunogenicity, may be obtained from individual binding molécules by techniques known in the art, such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, humanizing, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping
-1416772 primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing, also termed sequence optimization”, as described herein. Reference is, for example, made to standard handbooks, as well as to the further description and Examples.
If appropriate, a binding molécule with increased affinity may be obtained by affinitymaturation of another binding molécule, the latter representing, with respect to the affinity-matured molécule, the parent binding molécule.
Methods of obtaining VHHs that bind to a spécifie antigen or epitope hâve been described earlier, e.g. in W02006/040153 and W02006/122786. As also described therein in detail, VHH domains derived from camelids can be humanized (also termed “sequence-optimized” herein, sequence-optimizing may, in addition to humanization, encompass an additional modification of the sequence by one or more mutations that furnish the VHH with improved properties, such as the removal of potential post translational modification sites) by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being. A humanized VHH domain can contain one or more fully human framework région sequences, and, in an even more spécifie embodiment, can contain human framework région sequences derived from DP-29, DP-47, DP-51, or parts thereof, optionally combined with JH sequences, such as JH5.
Domain antibodies, also known as Dabs and dAbs (the terms Domain Antibodies and dAbs being used as trademarks by the GlaxoSmithKIine group of companies) hâve been described in e.g. Ward, E.S., étal.: Binding activities of a répertoire of single immunoglobulin variable domains secreted from Escherichia coli; Nature 341: 544-546 (1989); Holt, L.J. étal.: Domain antibodies: proteins for therapy; TRENDS in Biotechnology 21(11): 484-490 (2003); and W02003/002609.
Domain antibodies essentially correspond to the VH or VL domains of antibodies from non-camelid mammals, in particular human 4-chain antibodies. In order to bind an epitope as a single antigen binding domain, i.e. without being paired with a VL or
VH domain, respectîvely, spécifie sélection for such antigen binding properties is required, e.g. by using libraries of human single VH or VL domain sequences.
-1516772
Domain antibodies have, like VHHs, a molecular weight of approximately 13 to approximately 16 kDa and, if derived from fully human sequences, do not require humanization for e.g. therapeutical use in humans. As in the case of VHH domains, they are well expressed also in prokaryotic expression Systems, providing a significant réduction in overall manufacturing cost.
Furthermore, it will also be clear to the skilled person that it is possible to graft one or more of the CDR's mentioned above onto other scaffolds, including but not limited to human scaffolds or non-immunoglobulin scaffolds. Suitable scaffolds and techniques for such CDR grafting are known in the art.
The terms epitope and antigenic déterminant, which can be used interchangeably, refer to the part of a macromolecule, such as a polypeptide, that is recognized by antigen-binding molécules, such as conventional antibodies or the polypeptides of the invention, and more particularly by the antigen-binding site of said molécules. Epitopes define the minimum binding site for an immunoglobulin, and thus represent the target of specificity of an immunoglobulin.
A polypeptide (such as an immunoglobulin, an antibody, an immunoglobulin single variable domain of the invention, or generally an antigen-binding molécule or a fragment thereof) that can bind to or specifically bind to, that has affinity for and/or that has specificity for*' a certain epitope, antigen or protein (or for at Ieast one part, fragment or epitope thereof) is said to be against1 or directed against' said epitope, antigen or protein or is a binding molécule with respect to such epitope, antigen or protein. In this context, a DII4-binding component may also be referred to as DM-neutralizing”.
Generally, the term specificity refers to the number of different types of antigens or epitopes to which a particular antigen-binding molécule or antigen-binding protein (such as an immunoglobulin single variable domain of the invention) molécule can bind. The specificity of an antigen-binding molécule can be determined based on its affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (KD), is a measure for the binding strength between an epitope and an antigen-binding site on the antigenbinding protein: the lesser the value of the KD, the stronger the binding strength between an epitope and the antigen-binding molécule (alternatively, the affinity can
-1616772 also be expressed as the affinity constant (KA), which is 1/KD). As will be clear to the skilled person (for example on the basis of the further disclosure herein), affinity can be determined in a manner known per se, depending on the spécifie antigen of interest. Avidity is the measure of the strength of binding between an antigen-binding moiecule (such as an immunoglobulin, an antibody, an immunoglobulin single variable domain or a polypeptides containing it and the pertinent antigen. Avidity is related to both the affinity between an epitope and its antigen binding site on the antigen-binding moiecule and the number of pertinent binding sites présent on the antigen-binding moiecule.
The part of an antigen-binding moiecule that recognizes the epitope is called a paratope.
Unless indicated otherwise, the term “DII4-binding moiecule or Ang2-binding molecuie” includes anti-DII4 or anti-Ang2 antibodies, anti-DII4 antibody or anti-Ang2 antibody fragments, “anti-DII4 antibody-like molécules or anti-Ang2 antibody-like molécules, as defined herein, and conjugates with any of these. Antibodies include, but are not limited to, monoclonal and chimerized monoclonal antibodies. The term „antibody encompasses complété immunoglobulins, like monoclonal antibodies produced by recombinant expression in host cells, as well as antibody fragments or “antibody-like molécules”, including single-chain antibodies and linear antibodies, socalled “SMIPs” (“Small Modular Immunopharmaceuticals”), as e.g described in WO 02/056910; Antibody-like molécules include immunoglobulin single variable domains, as defined herein. Other examples for antibody-like molécules are immunoglobulin super family antibodies (IgSF), or CDR-grafted molécules.
Ang2-binding moiecule” or DII4-binding molecuie respectively, refers to both monovalent target-binding molécules (i.e. molécules that bind to one epitope of the respective target) as well as to bi- or multivalent binding molécules (i.e. binding molécules that bind to more than one epitope, e.g. biparatopic molécules as defined hereinbelow). Ang2(or DII4)-binding molécules containing more than one Ang2(or DII4)-binding immunoglobulin single variable domain are also termed formatted binding molécules, they may, within the target-binding component, in addition to the immunoglobulin single variable domains, comprise linkers and/or moieties with effector functions, e.g. half-life-extending moieties like albumin-binding
-1716772 immunoglobulin single variable domains, and/or a fusion partner like sérum albumin and/or an attached polymer like PEG.
The term biparatopic Ang2(or DII4)-binding moléculeor biparatopic immunoglobulin single variable domain as used herein shall mean a binding molécule comprising a first immunoglobulin single variable domain and a second immunoglobulin single variable domain as herein defined, wherein the two molécules bind to two non-overlapping epitopes of the respective antigen. The biparatopic binding molécules are composed of immunoglobulin single variable domains which hâve different specificities with respect to the epitope. The part of an antigen-binding molécule (such as an antibody or an immunoglobulin single variable domain of the invention) that recognizes the epitope is called a paratope.
A formatted binding molécule may, albeit less preferred, also comprise two identical immunoglobulin single variable domains or two different immunoglobulin single variable domains that recognize the same or overlapping epitopes or their respective antigen. In this case, with respect to VEGF, the two immunoglobulin single variable domains may bind to the same or an overlapping epitope in each of the two monomers that form the VEGF dimer.
Typically, the binding molécules of the invention will bind with a dissociation constant (Ko) of 10E-5 to 10E-14 moles/liter (M) or less, and preferably 10E-7 to 10E-14 moles/liter (M) or less, more preferably 10E-8 to 10E-14 moles/liter, and even more preferably 10E-11 to 10E-13, as measured e.g. in a Biacore or in a Kinexa assay), and/or with an association constant (Ka) of at least 10E7 ME-1, preferably at least 10E8 ME-1, more preferably at least 10E9 ME-1, such as at least 10E11 ME-1. Any Ko value greater than 10E-4 M is generally considered to indicate non-specific binding. Preferably, a polypeptide of the invention will bind to the desired antigen, i.e. VEGF or DII4, respectively, with a Kd less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Spécifie binding of an antigen-binding protein to an antigen or epitope can be determined in any suitable manner known per se, including, for example, the assays described herein, Scatchard analysis and/or compétitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich compétition assays, and the different variants thereof known per se in the art. y,—
-1816772
Amino acid residues will be indicated according to the standard three-letter or oneletter amino acid code, as generally known and agreed upon in the art. When comparing two amino acid sequences, the term amino acid différence refers to insertions, délétions or substitutions of the indicated number of amino acid residues at a position of the reference sequence, compared to a second sequence. In case of substitution(s), such substitution(s) will preferably be conservative amino acid substitution(s), which means that an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example from WO 98/49185, wherein conservative amino acid substitutions preferably are substitutions in which one amino acid within the following groups (i) - (v) is substituted by another amino acid residue within the same group: (i) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (iii) polar, positively charged residues: His, Arg and Lys; (iv) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues:
Phe, Tyr and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu;Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into SerjTrp into Tyr; Tyr into Trp or into Phe; Val into Ile or into Leu.
A polypeptide or nucleic acid molécule is considered to be (in) essentially isolated (form) - for example, when compared to its native biological source and/or the reaction medium or cultivation medium from which it has been obtained - when it has been separated from at least one other component with which it is usually associated in said source or medium, such as another protein/polypeptide, another nucleic acid, another biological component or macromolecule or at least one contaminant, impurity or minor component. In particular, a polypeptide or nucleic acid molécule is considered essentially isolated when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 10O-fold, and up to 1000-fold or more. A polypeptide or nucleic acid molécule that is in essentially isolated form is preferably
-1916772 essentially homogeneous, as determined using a suitable technique, such as a suitable chromatographical technique, such as polyacrylamide gel electrophoresis.
Sequence identitÿ' between two DII4-binding molécule sequences or between two Ang2-binding molécule sequences indicates the percentage of amino acids that are identical between the sequences. It may be calculated or determined as described in paragraph f) on pages 49 and 50 of WO 2008/020079. Sequence similarity indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions.
Alternative methods for numbering the amino acid residues of Vh domains, which methods can also be applied in an analogous manner to VHH domains, are known in the art. However, in the présent description, claims and figures, the numbering according to Kabat and applied to VHH domains as described above will be followed, unless indicated otherwise.
An affinity-matured DII4-binding molécule or Ang2-binding molécule, in particular a VHH or a domain antibody, has one or more alterations in one or more CDRs which resuit in an improved affinity for DII4 or Ang2, as compared to the respective parent DII4-binding molécule or Ang2-binding molécule. Afffinity-matured DII4-binding molécules or Ang2-binding molécules ofthe invention may be prepared by methods known in the art, for example, as described by Marks et al., 1992, Biotechnology 10:779-783, or Barbas, étal., 1994, Proc. Nat. Acad. Sci, USA 91: 3809-3813.; Shier étal., 1995, Gene 169:147-155; Yelton étal., 1995, Immunol. 155:1994-2004; Jackson étal., 1995, J. Immunol. 154(7):3310-9; and Hawkins étal., 1992, J. Mol. Biol. 226(3): 889 896; KS Johnson and RE Hawkins, Affinity maturation of antibodies using phage display, Oxford University Press 1996.
For the présent invention, an amino acid sequences of SEQ ID NO: x: includes, if not otherwise stated, an amino acid sequence that is 100% identical with the sequence shown in the respective SEQ ID NO: x;
a) amino acid sequences that have at least 80% amino acid identitÿ with the sequence shown in the respective SEQ ID NO: x;
b) amino acid sequences that have 3, 2, or 1 amino acid différences with the sequence shown in the respective SEQ ID NO: x. ν'—-2016772
The terms cancer and cancerous refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proiiferation. Examples of cancer to be treated with a bispecific binding molécule of the invention, include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers, as suggested for treatment with DII4 antagonists in US 2008/0014196, include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma ofthe lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endométrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, gastric cancer, melanoma, and various types of head and neck cancer. Dysrégulation of angiogenesis can lead to many disorders that can be treated by compositions and methods of the invention. These disorders include both nonneoplastic and neoplastic conditions. Neoplasties include but are not limited those described above.
Non-neoplastic disorders include, but are not limited to, as suggested for treatment with DII4 antagonists in US 2008/0014196, undesired or aberrant hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriatic plaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, diabetic and other proliférative rétinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, diabetic macular edema, corneal neovascularization, corneal graft neovascularization, corneal graft rejection, retinal/choroidal neovascularization, neovascularization ofthe angle (rubeosis), ocular neovascular disease, vascular restenosis, arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, acute lung injury/ ARDS, sepsis, primary pulmonary hypertension, malignant pulmonary effusions, cérébral edema (e.g., associated with acute stroke/ closed head injury/ trauma), synovial inflammation, pannus formation in RA, myositis ossificans, hypertropic bone formation, osteoarthritis (OA), refractory ascites, polycystic ovarian disease, endometriosis, 3rd spacing of fluid diseases (pancreatitis, compartment syndrome, burns, bowel disease), uterine fibroids, prématuré labor,
-2116772 chronic inflammation such as IBD (Crohn's disease and ulcerative colitis), rénal allograft rejection, inflammatory bowel disease, nephrotic syndrome, undesired or aberrant tissue mass growth (non-cancer), hémophilie joints, hypertrophie scars, inhibition of hair growth, Osier-Weber syndrome, pyogénie granuloma retrolental fibroplasias, scleroderma, trachoma, vascular adhesions, synovitis, dermatitis, preeclampsia, ascites, pericardial effusion (such as that associated with pericarditis), and pleural effusion.
DETAILED DESCRIPTION OFTHE INVENTION
In a first aspect, the présent invention relates to a bispecific binding molécule comprising at least one DII4-binding component and at least one Ang2-binding component.
In a preferred embodiment, the présent invention relates to a bispecific binding molécule comprising at least one DII4-binding component and at least one Ang2binding component which further comprises at least a further binding component, preferably a sérum albumin binding component (sérum albumin binding molécule).
In a preferred embodiment, the sérum albumin binding component of the binding molécule of the présent invention is an isolated immunoglobulin single variable domain or a polypeptide containing one or more of said immunoglobulin single variable domains, wherein said immunoglobulin single variable domain consists of four framework régions and three complementarity determining régions CDR1, CDR2 and CDR3, respectively, and wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in SEQ ID NOs: 522, 525, 528, 531, 534, 537, or 540.
More preferably, said one or more immunoglobulin single variable domain of the sérum albumin binding component contain
a. a CDR3 with an amino acid sequence selected from a first group of amino acid sequences shown in SEQ ID NOs: SEQ IDs NOs: 522, 525, 528, 531, 534, 537, or 540;
b. a CDR1 with an amino acid sequences selected from a second group of amino acid sequences shown SEQ ID NOs: 520, 523, 526; 529, 532, 535, or 538; i/__
-2216772
c. a CDR2 with an amino acid sequences selected from a second group of amino acid sequences shown SEQ ID NOs: 521, 524, 527, 530, 533, 536, or 539.
In a more preferred embodiment, said one or more immunoglobuîin single variable domains of the sérum albumin binding component are VHHs, preferably having an amino acid sequence shown in SEQ ID NOs: 98 or 519.
According to preferred embodiments, said DII4-binding component and said Ang2-binding component comprise at least one DII4-binding immunoglobuîin single variable domain and at least one Ang2-binding immunoglobuîin single variable domain, respectively.
In a preferred aspect, said DII4-binding component and said Ang2-binding component each comprise at least one Ang2-binding immunoglobuîin single variable domain and at least one DII4-binding immunoglobuîin single variable domain, respectively, wherein each of said immunoglobuîin single variable domains has four framework régions and three complementarity determining régions CDR1, CDR2 and CDR3, respectively.
Thus, the anti-DII4 and/or the anti-Ang2 component contained in the bispecific binding molécules of the invention may include two (or more) anti-DII4 (or anti-Ang2, respectively) immunoglobuîin single variable domains, wherein the immunoglobuîin single variable domains are directed against different epitopes within the DII4 (or Ang2) target. Thus, the two immunoglobuîin single variable domains in a bispecific binding molécule will hâve different antigen specificity and therefore different CDR sequences.
Such bivalent binding molécules are also named biparatopic single domain antibody constructs (if the immunoglobuîin single variable domains consist or essentially consist of single domain antibodies), or biparatopic VHH constructs (if the immunoglobuîin single variable domains consist or essentially consist of VHHs), respectively, as the two immunoglobuîin single variable domains will include two different paratopes.
In the bispecific binding molécule of the invention, one or both of the binding molécules may be bivalent; e.g. the Ang2-binding component may be biparatopic and the DII4-binding component may be one immunoglobuîin single variable domain, or __
-2316772 the Ang2-binding component may be one immunoglobulin single variable domain and the DII4-binding component may be biparatopic.
In bispecific binding molécules of the invention, it is preferably the Ang2-binding component that contains a bivalent Ang2-binding immunoglobulin single variable domain, e.g. a biparatopic VHH.
The DII4-binding component comprises at least a variable domain with four framework régions and three complementarity determining régions CDR1, CDR2 and CDR3, respectively, wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in
a) SEQ ID NOs: 1 to 166 and 458,
b) SEQ ID NOs: 333 to 353, or
c) SEQID NOs: 375 to 395.
An amino acid sequence a), selected from a first group of SEQ ID NOs: 1 to166 and 458, is contained as partial sequence in a corresponding amino acid sequence selected from a second group of sequences shown in Table 5 and in SEQ ID NO: 167 to 332 and 459.
An amino acid sequence b), selected from a first group of SEQ ID NOs: 333 to 353, is contained as partial sequence in a corresponding sequence selected from a second group of sequences shown in Table 16-A and in SEQ ID NOs: 354 to 374.
An amino acid sequence c) selected from a first group of SEQ ID NOs: 375 to 395 is contained as partial sequence in a corresponding sequence selected from a second group of sequences shown in Table 16-B and in SEQ ID NOs: 396 to 416.
In a second aspect, said DII4-binding component is an isolated immunoglobulin single variable domain or a polypeptide containing one or more of said immunoglobulin single variable domains, wherein said immunoglobulin single variable domain consists of four framework régions and three complementarity determining régions CDR1, CDR2 and CDR3, respectively, and wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in
a) SEQ ID NOs: 1 to 166 and 458,
-2416772
b) SEQ ID NOs: 333 to 353, or
c) SEQ ID NOs: 375 to 395.
In a further aspect, said immunoglobulin single variable domain ofthe DII4-binding component contains
a) a CDR3 with an amino acid sequence selected from a first group of amino acid sequences shown in SEQ ID NOs: 1 to 166 and 458;
b) a CDR1 and a CDR2 with an amino acid sequence that is contained, as indicated in Table 5, as partial sequence in a sequence selected from a second group of amino acid sequences shown in SEQ ID NOs: 167 to 332 and 459;
wherein a SEQ ID NO: x of said first group, for SEQ ID Nos 1-166: corresponds to SEQ ID NO: y of said second group in that y = x +166.
In a further aspect said immunoglobulin single variable domain contains
a) a CDR3 with an amino acid sequence selected a said first group of amino acid sequences shown in SEQ ID NOs: 333 to 353;
b) a CDR1 and a CDR2 with an amino acid sequence that is contained, as indicated in Table 16-A, as a partial sequence in a sequence selected from a second group of sequences shown in SEQ ID NOs: 354 to 374;
wherein a SEQ ID NO: x of said first group corresponds to SEQ ID NO: y of said second group in that y = x +21.
In a further aspect said immunoglobulin single variable domain has
a) a CDR3 with an amino acid sequence selected a said first group of amino acid sequences shown in SEQ ID NOs: 375 to 395;
b) a CDR1 and a CDR2 with an amino acid sequence that is contained, as indicated in Table 16-B, as a partial sequence in a sequence selected from a second group of sequences shown in SEQ ID NOs: 396 to 416;
wherein a SEQ ID NO: x of said first group corresponds with SEQ ID NO; y of said second group in that y = x +21.
-2516772
In a preferred embodiment, the immunoglobulin single variable domain is a VHH.
In a further aspect, the VHH has an amino acid sequence selected from amino acid sequences shown in Table 5 and in SEQ ID NOs: 167 to 332 and 459.
The Ang2-binding component comprises at least a variable domain with four framework régions and three complementarity determining régions CDR1, CDR2 and CDR3, respectively, wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in SEQ ID NOs: 491, 494, 497, 500, 503, 506, 509, 512, 515, or 518.
In a second aspect, said Ang2-binding component is an isolated immunoglobulin single variable domain or a polypeptide containing one or more of said immunoglobulin single variable domains, wherein said immunoglobulin single variable domain consists of four framework régions and three complementarity determining régions CDR1, CDR2 and CDR3, respectively, and wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in SEQ ID NOs: 491, 494, 497, 500, 503, 506, 509, 512, 515, or 518
In a further aspect, said immunoglobulin single variable domain ofthe Ang2-binding component contains
a. a CDR3 with an amino acid sequence selected from a first group of amino acid sequences shown in SEQ ID NOs: SEQ IDs NOs: 491, 494, 497, 500, 503,
506, 509, 512, 515, or 518 (see also Table 36);
b. a CDR1 with an amino acid sequences that is contained, as indicated in Table 22-A or 28, as partial sequence in a sequence selected from a second group of amino acid sequences shown SEQ ID NOs: 489, 492, 495, 498, 501,504,
507, 510, 513, or 516 (see also Table 36);
c. a CDR2 with an amino acid sequences that is contained, as indicated in Table 22-A or 28, as partial sequence in a sequence selected from a second group of amino acid sequences shown SEQ ID NOs: 490, 493, 496, 499, 502, 505,
508, 511,514, or 517 (see also Table 36).
Preferably, the immunoglobulin single variable domain of the Ang2-binding component is a VHH, preferably having amino acid sequence selected from amino u
-2616772 acid sequences shown in SEQ ID NOs: 479, 480, 481,482, 483, 484, 485, 486, 487, or 488.
In another preferred embodiment, the immunoglobulin single variable domain of the Ang2-binding component has been obtained by affinity maturation or humanization of an immunoglobulin single variable domain as described herein.
Similarly, the présent invention also relates to a VHH which has been obtained by affinity maturation or humanization of a VHH of the Ang2-binding component as described herein.
The présent invention thus also relates to an Ang2-binding VHH with an amino acid sequence selected from acid sequences shown in SEQ ID NOs: 479, 480, 481, 482, 483, 484, 485, 486, 487, or 488.
DII4-and/or Ang2-binding components with improved properties in view of therapeutic application, e.g. enhanced affinity or decreased immunogenicity, may be obtained from individual DII4- or Ang2-binding components of the invention by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, humanizing, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing. Reference is, for example, made to standard handbooks, as well as to the further description and Examples.
Preferably, a DII4-binding component of the invention with increased affinity is obtained by affinity-maturation of another DII4-binding component, the latter representing, with respect to the affinity-matured molécule, the parent DII4-bînding component. The same holds true for the Ang2-binding component.
Thus, in yet another preferred embodiment, a DII4-or Ang2-binding molécule of the invention is an immunoglobulin single variable domain that has been obtained by affinity maturation of a parent immunoglobulin single variable domain defined above.
In yet another preferred embodiment, the invention relates to an immunoglobulin single variable domain obtained by affinity-maturation of a VHH.
-2716772
Suitable parent DII4-binding components for affinity maturation are, by way of example, the above-described VHHs with amino acid sequences shown in SEQ ID NOs: 167 to 332 and 459.
Suitable parent Ang2-binding components for affinity maturation are, by way of example, the above-described VHHs with amino acid sequences shown in SEQ ID NOs:479, 480, 481, 482, 483, or 484.
Accordingly, the invention also relates to Ang2-binding molécules that hâve been obtained by affinity maturation and/or sequence optimization of an above-defined VHH, e.g. to a VHH that has been obtained by sequence optimization of a VHH having an amino acid sequence shown as SEQ ID NOs: 482, 483, 484, 485, 486, 487, 488. The source amino acid sequences that were used to generate the latter VHHs are shown in SEQ ID NOs: 479, 480, or 481. Also these amino acid sequences are suitable Ang2-binding components that can be applied in the binding molécules of the présent invention.
As described herein, the binding molécule of the présent invention preferably comprises at least one sérum albumin binding component. Particularly preferred binding molécules thus hâve at least one DII4-binding component, at least one Ang2binding component and at least one sérum albumin binding component. The order of these three binding components could be any possible order such as the order set out in Figure 16 or 23, e.g., the DII4-, Ang2-or sérum albumin binding component can be N-terminal or C-terminal. Notably, “00042, 00045 or 00050 as referred to in the legend of Figure 16 stand for Ang2-binding components, while 00018 stands for a DII4-binding component and ALB11 stands for a sérum albumin binding component, None of them is to be construed to a spécifie sequence, but stands for a Ang2-, DII4- and sérum albumin binding component in general when used in the context of possible set-ups of binding molécules of the présent invention.
However, it is preferred that the sérum albumin binding component is in between the
DII4- and Ang2-binding component (or vice versa), while it is particularly preferred that at least one Ang2-binding component is N-terminal, followed by at least one sérum albumin binding component, followed by at least one DII4-binding component at the C-Terminus. This set-up is shown to be specifically useful. λ___-2816772
The présent invention relates thus in a preferred aspect to binding molécules comprising at least one DII4-binding component, at least one Ang2-binding component and at least one sérum albumin binding component having an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 460-478.
At least one binding component (Ang2, DII4 or sérum albumin) when used herein includes that a binding molécule ofthe présent invention may contain one, two, three, four or five Ang2-, DII4, and/or sérum albumin binding components (i.e., entities/units) which are preferably represented by an immunoglobulin singly variable domain as described herein.
ίο In yet another preferred embodiment, the invention relates to a DII4 immunoglobulin single variable domain that has been obtained by affinity maturation of a VHH with an amino acid sequence shown in SEQ ID NO: 197.
In yet another embodiment, said immunoglobulin single variable domain that is derived from a VHH with the amino acid sequence shown in SEQ ID NO: 197 is selected from immunoglobulin single variable domains with amino acid sequences shown in SEQ ID NOs: 354 to 374.
In a preferred embodiment, the immunoglobulin single variable domain is a VHH with an amino acid sequence shown in SEQ ID NO: 358.
In an even more preferred embodiment, the immunoglobulin single variable domain has been obtained by humanization of a VHH with an amino acid sequence shown in SEQ ID NO: 358.
In another preferred embodiment, the immunoglobulin single variable domain is a VHH with an amino acid sequence shown in SEQ ID NO: 356.
In an even more preferred embodiment, the invention relates to an immunoglobulin single variable domain that has been obtained by humanization of a VHH with an amino acid sequence shown in SEQ ID NO: 356.
In yet another preferred embodiment, the invention relates to an immunoglobulin single variable domain that has been obtained by affinity maturation of a VHH with an amino acid sequence shown in SEQ ID NO: 224. <_/—
-2916772
In yet another embodiment, said immunoglobulin single variable domain derived from a VHH with the amino acid sequence shown in SEQ ID NO: 224 is selected from immunoglobulin single variable domains with amino acid sequences shown in SEQ
ID NOs: 396 to 416.
In another preferred embodiment, the immunoglobulin single variable domain is a VHH with an amino acid sequence shown in SEQ ID NO: 402.
In an even more preferred embodiment, the immunoglobulin single variable domain has been obtained by humanization of the VHH with the amino acid sequence shown in SEQ ID NO: 402.
In another preferred embodiment, the immunoglobulin single variable domain is a VHH with an amino acid sequence shown in SEQ ID NO: 416.
In an even more preferred embodiment, the immunoglobulin single variable domain has been obtained by humanization ofthe immunoglobulin single variable domain with the amino acid sequence shown in SEQ ID NO: 416
In another preferred embodiment, the immunoglobulin single variable domain is a VHH with an amino acid sequence shown in SEQ ID NO:407.
In an even more preferred embodiment, the immunoglobulin single variable domain has been obtained by humanization of the immunoglobulin single variable domain with the amino acid sequence shown in SEQ ID NO: 413.
According to another embodiment, the immunoglobulin single variable domain is a VH domain, as defined herein.
In yet another embodiment, the représentatives of the class of DII4-and/or Ang2binding immunoglobulin single variable domains of the invention or présent in the polypeptides ofthe invention hâve amino acid sequences that correspond to the amino acid sequence of a naturally occurring VH domain that has been camelized, Le. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring variable heavy chain from a conventional 4-chain antibody by one or more amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, and reference is additionally be made to
-3016772
WO 1994/04678. Such camelization may preferentially occur at amino acid positions which are présent at the VH-VL interface and at the so-called Camelidae Hallmark residues (see for example also WO 1994/04678). A detailled description of such humanization and camelization techniques and preferred framework région sequences consistent therewith can additionally be taken from e.g. pp. 46 and pp. 98 of WO 2006/040153 and pp. 107 ofWO 2006/122786.
The DII4-or Ang2-binding components of the invention, e.g. immunoglobulin single variable domains and or polypeptides containing them, hâve specificity for DII4 or Ang2, respectively, in that they comprise one or more immunoglobulin single variable domains specifically binding to one or more epitopes within the DII4 or Ang2 molécule, respectively.
Spécifie binding of an DII4-and/or Ang2 binding component to its antigen DII4 or Ang2, respectively, can be determined in any suitable manner known per se, including, for example, the assays described herein, Scatchard analysis and/or compétitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA and ELISA) and sandwich compétition assays, and the different variants thereof known per se in the art.
With regard to the antigen DII4, a DII4-binding component of the invention, e.g. an immunoglobulin single variable domain, is not limited with regard to the species. Thus, the immunoglobulin single variable domains of the invention or polypeptides containing them preferably bind to human DII4, if intended for therapeutic purposes in humans. However, immunoglobulin single variable domains that bind to DII4 from another mammalian species, or polypeptides containing them, are also within the scope of the invention. An immunoglobulin single variable domain of the invention binding to one species form of DII4 may cross-react with DII4 from one or more other species. For example, immunoglobulin single variable domains of the invention binding to human DII4 may exhibit cross reactivity with DII4 from one or more other species of primates and/or with DII4 from one or more species of animais that are used in animal models for diseases, for example monkey (in partîcular Cynomolgus or Rhésus), mouse, rat, rabbit, pig, dog or) and in partîcular in animal models for diseases and disorders associated with DII4-mediated effects on angiogenesis (such as the species and animal models mentioned herein). Immunoglobulin single variable domains of the invention that show such cross-reactivity are advantageous in a
-3116772 research and/or drug development, since it allows the immunoglobulin single variable domains of the invention to be tested in acknowledged disease models such as monkeys, in particular Cynomolgus or Rhésus, or mice and rats, The same is true for
Ang2.
Also, the DII4-binding components of the invention are not limited to or defined by a spécifie domain or an antigenic déterminant of DH4 against which they are directed. Preferably, in view of cross-reactivity with one or more DII4 molécules from species other than human that is/are intended for use as an animal model during development of a therapeutic DII4 antagonist, a DII4-binding component recognizes an epitope in a région of the DII4 of interest that has a high degree of identity with human DII4. By way of example, in view of using a mouse model, an immunoglobulin single variable domain of the invention recognizes an epitope which is, totally or in part, located within the EGF-2 domain, which shows a high identity between human and mouse. The same is true for Ang2.
Therefore, according to a preferred embodiment, the invention relates to a DII4binding component, in particular an immunoglobulin single variable domain or a polypeptide containing same, wherein said immunoglobulin single variable domain is selected from the group that binds to an epitope that is totally or partially contained within the EGF-2 domain that corresponds to amino acid residues 252-282 of SEQ ID NO: 417.
If a polypeptide of the invention is a biparatopic molécule as defined herein, which contains more than one immunoglobulin single variable domain of the invention, at least one of the immunoglobulin single variable domain components binds to the epitope within the EGF-2 domain, as defined above.
Preferably, an immunoglobulin single variable domain of the invention binds to DII4 and/or Ang2 with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM (as determined by Surface Plasmon Résonance analysis, as described in Example 5.7).
Preferably, the immunoglobulin single variable domains of the invention hâve IC50 values, as measured in a compétition ELISA assay as described in Example 5.1. in the range of 10'6 to 10‘1° moles/litre or less, more preferably in the range of 10'8 to
-3216772
1O’10 moles/litre or less and even more preferably in the range of 10'9 to 10‘1° moles/litre or less.
According to a non-lîmiting but preferred embodiment of the invention, DII4-bindrng immunoglobulin single variable domains of the invention or polypeptides containing them bind to DII4 with an dissociation constant (Ko) of 10’5 to 10'12 moles/liter (M) or less, and preferably 10'7 to 10'12 moles/liter (M) or less and more preferably 10'8 to W12 moles/liter (M), and/or with an association constant (Ka) of at least 107 M'1, preferably at least 108 M’1, more preferably at least 109 M'1, such as at least 1012 M'1; and in particular with a KD less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The Ko and Ka values of the immunoglobulin single variable domain of the invention against DII4 can be determined. The same is true for Ang2.
In a further embodiment, the invention relates to DII4-binding components comprising two or more immunoglobulin single variable domains that bind to the antigen DII4 or Ang2, respectively, at different non-overlapping epitopes. More specifically, such polypeptide of the invention essentially consists of or comprises (i) a first immunoglobulin single variable domain specifically binding to a first epitope of DII4 or Ang2, respectively, and (li) a second immunoglobulin single variable domain specifically binding to a second epitope of DII4 or Ang2, respectively, wherein the first epitope of DII4/Ang2 and the second epitope of DII4/Ang2 are not îdentical epitopes. In other words, such polypeptide of the invention comprises or essentially consists of two or more immunoglobulin single variable domains that are directed against at least two different epitopes présent in DII4/Ang2, wherein said immunoglobulin single variable domains are linked to each other in such a way that they are capable of simultaneously binding DII4/Ang2. In this sense, the polypeptide of the invention can also be regarded as a bivalent or multivalent immunoglobulin construct, and especially as a multivalent immunoglobulin single variable domain construct, in that the polypeptide contains at least two binding sites for DII4/Ang2.
Such DII4-binding component of the invention includes (at least) two anti-DII4 immunoglobulin single variable domains, wherein (the) two immunoglobulin single variable domains are directed against different epitopes within the DII4 molécule.
Thus, these two immunoglobulin single variable domains will hâve a different antigen specificity and therefore different CDR sequences. For this reason, such polypeptides
-3316772 of the invention will herein also be named biparatopic polypeptides, or biparatopic single domain antibody constructs (if the immunoglobulin single variable domains consist or essentially consist of single domain antibodies), or biparatopic VHH constructs (if the immunoglobulin single variable domains consist or essentially consist of VHHs), respectively, as the two immunoglobulin single variable domains will include two different paratopes. The same is true for Ang2, mutatis mutandis.
According to a spécifie embodiment of the invention, in case that the polypeptide of the invention includes more than two anti-DII4 immunoglobulin single variable domains, i.e. three, four or even more anti-DII4 immunoglobulin single variable io domains, at Ieast two of the anti-DII4 immunoglobulin single variable domains are directed against different epitopes within the DII4 molécule, wherein any further immunoglobulin single variable domain may bind to any of these two different epitopes and/or a further epitope présent in the DII4 molécule. The same is true for Ang2, mutatis mutandis.
According to the invention, the two or more immunoglobulin single variable domains can be, independently of each other, VHs or VHHs, and/or any other sort of immunoglobulin single variable domains, such as VL domains, as defined herein, provided that these immunoglobulin single variable domains will bind the antigen, i.e. DII4 or Ang2, respectively.
The detailed description of the binding components is primarily provided for the DII4binding component. However, ali features and options outlined herein for the DII4binding component also apply equivalently for the Ang2-binding component, mutatis mutandis.
According to preferred embodiments, the binding molécules présent in the bispecific 25 binding molécules (the Ang2-binding molécules within the Ang2-binding component or the DII4-binding molécules within the DII4-binding component or the two adjacent Ang2- and DII4-binding components) may be connected with each other directly (i.e. without use of a linker) or via a linker. The linker is preferably a linker peptide and will be selected so as to allow binding of the two different binding molécules to each of 30 non-overlapping epitopes of the targets, either within one and the same target molécule, or within two different molécules.
-3416772
In the case of biparatopic binding molécules, sélection of linkers within the Ang2- or the DII4-binding component will inter alia dépend on the epitopes and, specifically, the distance between the epitopes on the target to which the immunoglobulin single variable domains bind, and will be clear to the skilled person based on the disclosure herein, optionally after some limited degree of routine expérimentation.
Two binding molécules (two VHHs or domain antibodies or VHH and a domain antibody), or two binding components, may be linked to each other via an additional VHH or domain antibody, respectîvely (in such binding molécules, the two or more immunoglobulin single variable domains may be linked directly to said additional immunoglobulin single variable domain or via suitable linkers). Such an additional VHH or domain antibody may for example be a VHH or domain antibody that provides for an increased half-life. For example, the latter VHH or domain antibody may be one that is capable of binding to a (human) sérum protein such as (human) sérum albumin or (human) transferrin.
Alternatively, the two or more immunoglobulin single variable domains that bind to the respective target may be linked in sériés (either directly or via a suitable linker) and the additional VHH or domain antibody (which may provide for increased half-life) may be connected directly or via a linker to one of these two or more aforementioned immunoglobulin sequences.
Suitable linkers are described herein in connection with spécifie polypeptides of the invention and may - for example and without limitation - comprise an amino acid sequence, which amino acid sequence preferably has a length of 9 or more amino acids, more preferably at least 17 amino acids, such as about 20 to 40 amino acids. However, the upper limit is not critical but is chosen for reasons of convenience regarding e.g. biopharmaceutical production of such polypeptides.
The linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutic purposes, the linker is preferably nonimmunogenic in the subject to which the bispecific binding molécule of the invention is administered.
One useful group of linker sequences are linkers derived from the hinge région of heavy chain antibodies as described in WO 1996/34103 and WO 1994/04678.
-3516772
Other examples are poly-alanine linker sequences such as Ala- Ala- Ala.
Further preferred examples of linker sequences are Gly/Ser linkers of different length such as (glyxsery)z linkers, induding (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3.
Some non-limiting examples of linkers are shown in Figures 40 and 48, e.g. the linkers
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (35GS; SEQ ID NO: 90);
GGGGSGGGS (9GS; SEQ ID NO: 91);
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (40GS; SEQ ID NO: 92).
If a bispecific binding molécule is modified by the attachment of a polymer, for example of a polyethylene glycol PEG (polyethylene glycol) moiety, the linker sequence preferably includes an amino acid residue, such as a cysteine or a lysine, allowing such modification, e.g. PEGylation, in the linker région.
Examples of linkers useful for PEGylation are:
GGGGCGGGS (“GS9.C5, SEQ ID NO:93);
GGGGCGGGGSGGGGSGGGGSGGGGS C'GS25,C5, SEQ ID NO:94)
GGGSGGGGSGGGGCGGGGSGGGGSGGG (GS27.C14, SEQ ID NO:95),
GGGGSGGGGSGGGGCGGGGSGGGGSGGGGSGGGGS ('‘GS35.C15, SEQ ID NO:96), and
GGGGCGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (“GS35,C5, SEQ ID NO:97).
Furthermore, the linker may also be a poly(ethylene glycol) moiety, as shown in e.g. WO 2004/081026.
In another embodiment, the immunoglobulin single variable domains are linked to each other via another moiety (optionally via one or two linkers), such as another ..
-3616772 polypeptide which, in a preferred but non-limiting embodiment, may be a further immunoglobulin single variable domain as described above. Such moiety may either be essentlally inactive or may hâve a biological effect such as improving the desired properties of the polypeptide or may confer one or more additional desired properties 5 to the polypeptide. For example, and without limitation, the moiety may tmprove the half-life ofthe protein or polypeptide, and/or may reduce its immunogenicity or improve any other desired property.
According to a preferred embodiment, a bispecific binding moiecule ofthe invention includes, especially when intended for use or used as a therapeutic agent, a moiety io which extends the half-life of the polypeptide of the invention in sérum or other body fluids of a patient. The term half-life is defined as the time it takes for the sérum concentration ofthe (modified) polypeptide to reduce by 50%, in vivo, for example due to dégradation of the polypeptide and/or clearance and/or séquestration by natural mechanisms.
More specifically, such half-life extending moiety can be covalently linked to orfused to an immunoglobulin single variable domain and may be, without limitation, an Fc portion, an albumin moiety, a fragment of an albumin moiety, an albumin binding moiety, such as an anti-albumin immunoglobulin single variable domain, a transferrin binding moiety, such as an anti-transferrin immunoglobulin single variable domain, a polyoxyalkylene moiecule, such as a polyethylene glycol molecuie, an albumin binding peptide or a hydroxyethyl starch (HES) derivatlve.
In another embodiment, the bispecific binding moiecule of the invention comprises a moiety which binds to an antigen found in blood, such as sérum albumin, sérum immunoglobulins, thyroxine-binding protein, fibrinogen or transferrin, thereby conferring an increased half-life in vivo to the resulting polypeptide of the invention. According to a specifically preferred embodiment, such moiety is an albumin-binding immunoglobulin and, especially preferred, an albumin-binding immunoglobulin single variable domain such as an albumin-binding VHH domain.
If intended for use in humans, such albumin-binding immunoglobulin single variable 30 domain preferably binds to human sérum albumin and preferably is a humanized albumin-binding VHH domain,
-3716772
Immunoglobulin single variable domains binding to human sérum albumin are known in the art and are described in further detail in e.g. WO 2006/122786. Specifically, useful albumin binding VHHs are ALB 1 and its humanized counterpart, ALB 8 (WO 2009/095489). Other albumin binding VHH domains mentioned in the above patent publication may, however, be used as well.
A specifically useful albumin binding VHH domain is ALB8 which consists of or contains the amino acid sequence shown in SEQ ID NO: 98 or 519.
According to a further embodiment ofthe invention, the two immunoglobulin single variable domains, in preferably VHHs, may be fused to a sérum albumin molécule, such as described e.g. in WO01/79271 and WO03/59934. As e.g. described in WO 2001/79271, the fusion protein may be obtained by conventional recombinant technology: a DNA molécule coding for sérum albumin, or a fragment thereof, is joined to the DNA coding for the bispecific binding molécule, the obtained construct is Inserted into a plasmid suitable for expression in the selected host cell, e.g. a yeast cell like Pichia pastoris or a bacterial cell, and the host cell is then transfected with the fused nucléotide sequence and grown under suitable conditions. The sequence of a useful HSA is shown in SEQ ID NO: 99.
According to another embodiment, a half-life extending modification of a polypeptide of the invention (such modification also reducing immunogenicity of the polypeptide) comprises attachment of a suitable pharmacologically acceptable polymer, such as straight or branched chain poly(ethylene glycol) (PEG) or dérivatives thereof (such as methoxypoly(ethylene glycol) or mPEG). Generally, any suitable form of PEGylation can be used, such as the PEGylation used in the art for antibodies and antibody fragments (including but not limited to domain antibodies and scFv's); reference is made, for example, to: Chapman, Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003); Harris and Chess, Nat. Rev. Drug. Discov. 2 (2003); and WO 2004/060965.
Various reagents for PEGylation of polypeptides are also commercially available, for example from Nektar Therapeutics, USA, or NOF Corporation, Japan, such as the
Sunbright® EA Sériés, SH Sériés, MA Sériés, CA Sériés, and ME Sériés, such as
Sunbright® ME-100MA, Sunbright® ME-200MA, and Sunbright® ME-400MA. .
-3816772
Preferably, site-directed PEGylation is used, in particular via a cysteine-residue (see for example Yang étal., Protein Engineering 16, 761-770 (2003)). For example, for this purpose, PEG may be attached to a cysteine residue that naturally occurs in a polypeptide ofthe invention, a polypeptide ofthe invention may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus of a polypeptide of the invention, ail using techniques of protein engineering known per se to the skilled person.
Preferably, for the polypeptides of the invention, a PEG is used with a molecular weight of more than 5 kDa, such as more than 10 kDa and less than 200 kDa, such as less than 100 kDa; for example in the range of 20 kDa to 80 kDa.
With regard to PEGylation, its should be noted that generally, the invention also encompasses any bispecific binding molécule that has been PEGylated at one or more amino acid positions, preferably in such a way that said PEGylation either (1 ) increases the half-life in vivo; (2) reduces îmmunogenicity; (3) provides one or more further bénéficiai properties known per se for PEGylation; (4) does not essentially affect the affinity of the polypeptide for its target (e.g. does not reduce said affinity by more than 50 %, and more preferably not by more than 10%, as determined by a suitable assay described in the art); and/or (4) does not affect any of the other desired properties of the bispecific binding molécules of the invention. Suitable PEGgroups and methods for attaching them, either specifically or non-specifically, will be clear to the skilled person. Various reagents for PEGylation of polypeptides are also commercially available, for example from Nektar Therapeutics, USA, or NOF Corporation, Japan, such as the Sunbright® EA Sériés, SH Sériés, MA Sériés, CA Sériés, and ME Sériés, such as Sunbright® ME-100MA, Sunbright® ME-200MA, and Sunbright® ME-400MA.
According to an especially preferred embodiment of the invention, a PEGylated polypeptide of the invention includes one PEG moiety of linear PEG having a molecular weight of 40 kDa or 60 kDa, wherein the PEG moiety is attached to the polypeptide in a linker région and, specifially, at a Cys residue at position 5 of a GS9linker peptide as shown in SEQ ID NO:93, at position 14 of a GS27-linker peptide as shown in SEQ ID NO:95, or at position 15 of a GS35-linker peptide as shown in SEQ
ID NO:96, or at position 5 of a 35GS-linker peptide as shown in SEQ ID NO:97.
-3916772
A bispecific binding molécule of the invention may be PEGylated with one of the PEG reagents as mentioned above, such as Sunbright® ME-400MA, as shown in the following chemical formula:
o II
O,
CHjO—(CH2CH2O)n-CH2CH2CH2NHC(CHa)2-N
Bispecific binding molécules that contain linkers and/or half-life extending functional groups are shown in SEQ ID NO: 81 and in Figure 48.
According to another embodiment, the immunoglobulin single variable domains are domain antibodies, as defined herein.
Immunoglobulin single variable domains présent in the bispecific binding molécules of the invention may also hâve sequences that correspond to the amino acid sequence of a naturally occurring VH domain that has been camelized, i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring variable heavy chain from a conventional 4-chain antibody by one or more amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, and reference is additionally be made to WO 94/04678. Such camelization may preferentially occur at amino acid positions which are présent at the VH-VL interface and at the so-called Camelidae Hallmark residues (see for example also WO 94/04678). A detailled description of such humanization and camelization techniques and preferred framework région sequences consistent therewith can additionally be taken from e.g. pp. 46 and pp. 98 of WO 2006/040153 and pp. 107 of WO 2006/122786.
The binding components hâve specificity for Ang2 or DII4, respectively, in that they comprise in a preferred embodiment one or more immunoglobulin single variable domains specifically binding to one or more epitopes within the Ang2 molécule or within the DII4 molécule, respectively.
Spécifie binding of a binding component to its antigen Ang2 or DII4 can be determined in any suitable manner known per se, including, for example, the assays described herein, Scatchard analysis and/or compétitive binding assays, such as
-4016772 radioimmunoassays (RIA), enzyme immunoassays (EIA and ELISA) and sandwich compétition assays, and the different variants thereof known per se in the art.
With regard to the antigen Ang2 or DII4, respectively, an immunoglobulin single variable domain is not limited with regard to the species. Thus, the immunoglobulin single variable domains preferably bind to human Ang2 or to human DII4, respectively, if intended for therapeutic purposes in humans. However, immunoglobulin single variable domains that bind to Ang2 or DII4, respectively, from another mammalian species, or polypeptides containing them, are also within the scope ofthe invention. An immunoglobulin single variable domain binding to one species form of Ang2 or DII4 may cross-react with the respective antigen from one or more other species. For example, immunoglobulin single variable domains binding to the human antigen may exhibit cross reactivity with the respective antigen from one or more other species of primates and/or with the antigen from one or more species of animais that are used in animal models for diseases, for example monkey (in particular Cynomolgus or Rhésus), mouse, rat, rabbit, pig, dog or) and in particular in animal models for diseases and disorders that can be modulated by inhibition of Ang2 (such as the species and animal models mentioned herein). Immunoglobulin single variable domains of the invention that show such cross-reactivity are advantageous in a research and/or drug development, since it allows the immunoglobulin single variable domains of the invention to be tested in acknowledged disease models such as monkeys, in particular Cynomolgus or Rhésus, or mice and rats.
Also, the binding components are not limited to or defined by a spécifie domain or an antigenic déterminant ofthe antigen against which they are directed. Preferably, in view of cross-reactivity with one or more antigen molécules from species other than human that is/are intended for use as an animal model during development of a therapeutic Ang2/DII4 antagonist, a binding component recognizes an epitope in a région of the the respective antigen that has a high degree of identity with the human antigen. By way of example, in view of using a mouse modei, an anti-Ang2 immunoglobulin single variable domain contained in the bispecific binding molécules ofthe invention recognizes an epitope which is, totally or in part, located within the EGF-2 domain of Ang2, which shows a high identity between human and mouse. v--4l16772
Therefore, according to a preferred embodiment, the bispecific binding molécule of the invention comprises a DII4-binding molécule which is an immunoglobulin single variable domain that is selected from the group that binds to an epitope that is totally or partially contained within the EGF-2 domain that corresponds to amino acid residues 252-282 ofSEQ IDNO:101.
In another aspect, the invention relates to nucleic acid molécules that encode bispecific binding molécules ofthe invention. Such nucleic acid molécules will also be referred to herein as nucleic acids of the invention and may also be in the form of a genetic construct, as defined herein. A nucleic acid of the invention may be genomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage that has been specifically adapted for expression in the intended host cell or host organism). According to one embodiment of the invention, the nucleic acid ofthe invention is in essentially isolated form, as defined hereabove.
The nucleic acid of the invention may also be in the form of, may be présent in and/or may be part of a vector, such as for example a plasmid, cosmid or YAC. The vector may especially be an expression vector, i.e. a vector that can provide for expression of the bispecific binding molécule in vitro and/or in vivo (i.e. in a suitable host cell, host organism and/or expression system). Such expression vector generally comprises at least one nucleic acid of the invention that is operably linked to one or more suitable regulatory éléments, such as promoter(s), enhancer(s), terminator(s), and the like. Such éléments and their sélection in view of expression of a spécifie sequence in a spécifie host are common knowledge ofthe skilled person. Spécifie examples of regulatory éléments and other éléments useful or necessary for expressing bispecific binding molécules of the invention, such as promoters, enhancers, terminators, intégration factors, sélection markers, leader sequences, reporter genes, and the like, are disclosed e.g. on pp. 131 to 133 of
WO 2006/040153.
The nucleic acids ofthe invention may be prepared or obtained in a manner known perse (e.g. by automated DNA synthesis and/or recombinant DNA technology), based on the information on the amino acid sequences for the polypeptides of the invention given herein, and/or can be isolated from a suitable natural source.
-4216772
In another aspect, the invention relates to host cells that express or that are capable of expressîng one or more bispecific binding molécules of the invention; and/or that contain a nucleic acid of the invention. According to a particularly preferred embodiment, said host cells are bacterial cells; other useful cells are yeast cells, fungal cells or mammalian cells.
Suitable bacterial cells include cells from gram-negative bacterial strains such as strains of Escherichia coli, Proteus, and Pseudomonas, and gram-positive bacterial strains such as strains of Bacillus, Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cell include cells from species of Trichoderma, Neurospora, and Aspergillus. Suitable yeast cells include cells from species of Saccharomyces ffor example Saccharomyces cerevisiae), Schizosaccharomyces (for example Schizosaccharomyces pombe), Pichia (for example Pichia pastoris and Pichia methanolica), and Hansenula.
Suitable mammalian cells include for example CHO cells, BHK cells, HeLa cells, COS cells, and the like. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for the expression of heterologous proteins can be used as well.
The invention further provides methods of manufacturing a bispecific binding molécule of the invention, such methods generally comprising the steps of:
- culturing host cells comprising a nucleic acid capable of encoding a bispecific binding molécule under conditions that allow expression of the bispecific binding molécule of the invention; and
- recovering or isolating the polypeptide expressed by the host cells from the culture; and
- optionally further purifying and/or modifying and/or formulating the bispecific binding molécule of the invention.
For production on an industrial scale, preferred host organisms include strains of
E. coli, Pichia pastoris, and S. cerevisiae that are suitable for large scale expression, production and fermentation, and in particular for large scale pharmaceutical expression, production and fermentation.
-4316772
The choice of the spécifie expression System dépends in part on the requirement for certain post-translational modifications, more specifically glycosylation. The production of a bispecific binding molécule of the invention for which glycosylation is desired or required would necessitate the use of mammalian expression hosts that have the ability to glycosylate the expressed protein. In this respect, it will be clear to the skilled person that the glycosylation pattern obtained (i.e. the kind, number and position of residues attached) will dépend on the cell or cell line that is used for the expression.
Bispecific binding molécules ofthe invention may be produced either in a cell as set out above intracellullarly (e.g. in the cytosol, in the periplasma or in inclusion bodies) and then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g. in the medium in which the host cells are cultured) and then isolated from the culture medium and optionally further purified.
Methods and reagents used for the recombinant production of polypeptides, such as spécifie suitable expression vectors, transformation or transfection methods, sélection markers, methods of induction of protein expression, culture conditions, and the like, are known in the art. Similarly, protein isolation and purification techniques useful in a method of manufacture of a polypeptide of the invention are well known to the skilled person.
In a further aspect, the invention relates to a peptide having an amino acid sequence of a CDR3 contained in an anti-DII4-VHH having an amino acid sequence selected from sequences shown in SEQ ID NOs: 1 to 166 and 458, SEQ ID NOs: 333 to 353, or SEQ ID NOs: 375 to 395, respectively, and a nucleic acid molécule encoding same.
These peptides correspond to CDR3s derived from the VHHs ofthe invention. They, in particular the nucleic acid molécules encoding them, are useful for CDR grafting in order to replace a CDR3 in an immunoglobulin chain, or for insertion into a nonimmunoglobulin scaffold, e.g. a protease inhibitor, DNA-binding protein, cytochrome b562, a helix-bundle protein, a disulfide-bridged peptide, a lipocalin or an anticalin, thus conferring target-binding properties to such scaffold. The method of CDRgrafting is well known in the art and has been widely used, e.g. for humanizing ν'—
-4416772 antîbodies (which usually comprises grafting the CDRs from a rodent antibody onto the Fv frameworks of a human antibody).
In order to obtain an immunoglobulin or a non-immunoglobulin scaffold containing a CDR3 of the invention, the DNA encoding such molécule may be obtained according to standard methods of molecular biology, e.g. by gene synthesis, by oligonucleotide annealing or by means of overlapping PCR fragments, as e.g. described by Daugherty étal., 1991, Nucleic Acids Research, Vol. 19, 9, 2471-2476. A method for inserting a VHH CDR3 into a non-immunoglobulin scaffold has been described by Nicaise étal., 2004, Protein Science, 13, 1882-1891.
The invention further relates to a product or composition containing or comprising at least one bispecific binding molécule of the invention and optionally one or more further components of such compositions known per se, i.e. depending on the intended use of the composition.
For pharmaceutical use, a bispecific binding molécule of the invention or a polypeptide containing same may be formulated as a pharmaceutical préparation or composition comprising at least one bispecific binding molécule of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active polypeptides and/or compounds. By means of non-limiting examples, such a formulation may be in a form suitable for oral administration, for parentéral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc. Such suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers for use in the préparation thereof, will be clearto the skilled person, and are further described herein.
Thus, in a further aspect, the invention relates to a pharmaceutical composition that contains at least one bispecific binding molécule, in particular one immunoglobulin single variable domain of the invention or a polypeptide containing same and at least one suitable carrier, diluent or excipient (i.e. suitable for pharmaceutical use), and optionally one or more further active substances.
-4516772
The bispecific binding molécules of the invention may be formulated and administered in any suitable manner known per se: Reference, in particular for the immunoglobulin single variable domains, is for example made to WO 2004/041862, WO 2004/041863, WO 2004/041865, WO 2004/041867 and WO 2008/020079, as well as to the standard handbooks, such as Remington’s Pharmaceutical Sciences, 18lh Ed., Mack Publishing Company, USA (1990), Remington, the Science and Practice of Pharmacy, 21,h Edition, Lippincott Williams and Wilkins (2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see for example pages 252-255).
For example, an immunoglobulin single variable domain of the invention may be formulated and administered in any manner known per se for conventional antibodies and antibody fragments (including ScFv's and diabodies) and other pharmaceutically active proteins. Such formulations and methods for preparing the same will be clear to the skilled person, and for example include préparations suitable for parentéral administration (for example intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecal administration) or for topical (i.e. transdermal or intradermal) administration.
Préparations for parentéral administration may for example be stérile solutions, suspensions, dispersions or émulsions that are suitable for infusion or injection. Suitable carriers or diluents for such préparations for example include, without limitation, stérile water and pharmaceutically acceptable aqueous buffers and solutions such as physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank’s solution; water oils; glycerol; éthanol; glycols such as propylene glycol or as well as minerai oils, animal oils and vegetable oils, for example peanut oil, soybean oil, as well as suitable mixtures thereof. Usually, aqueous solutions or suspensions will be preferred.
Thus, the bispecific binding molécule of the invention may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. For oral therapeutic administration, the bispecific binding molécule of the invention may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, élixirs, suspensions, syrups, wafers, and the like. Such compositions and préparations should contain at least 0.1% of the DII4-binding
-4616772 molécule of the invention, Their percentage in the compositions and préparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of the bispecific binding molécule of the invention in such therapeutically useful compositions is such that an effective dosage level will be obtained.
The tablets, pills, capsules, and the like may also contain binders, excipients, disintegrating agents, lubricants and sweetening or flavouring agents, for example those mentioned on pages 143-144 of WO 08/020079. When the unit dosage form is a capsule, it may contain, in addition to materials ofthe above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be présent as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or élixir may contain the bispecific binding molécules of the invention, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the bispecific binding molécules of the invention may be incorporated into sustainedrelease préparations and devices.
Préparations and formulations for oral administration may also be provided with an enteric coating that will allow the constructs of the invention to resist the gastric environment and pass into the intestines. More generally, préparations and formulations for oral administration may be suitably formulated for delivery into any desired part of the gastrointestinal tract. In addition, suitable suppositories may be used for delivery into the gastrointestinal tract.
The bispecific binding molécules of the invention may also be administered intravenously or intraperitoneally by infusion or injection, as further described on pages 144 and 145 of WO 2008/020079.
Fortopical administration ofthe bispecific binding molécules ofthe invention, it will generally be désirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid, as further described on page 145 of WO 2008/020079. vs/
-4716772
Generally, the concentration of the bispecific binding molécules of the invention in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
The amount of the bispecific binding molécules of the invention required for use in treatment will vary not only with the particular bispecific binding molécule selected, but also with the route of administration, the nature of the condition being treated and the âge and condition of the patient and will be ultimately at the discrétion of the attendant physician or clinician. Also, the dosage of the bispecific binding molécules of the invention varies depending on the target cell, tumor, tissue, graft, or organ. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more subdoses per day. The sub-dose itself may be further divided, e.g., into a number of discrète loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
An administration regimen may include long-term, daily treatment. By long-term” is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill in the art using only routine expérimentation given the teachings herein. See Remington’s Pharmaceutical Sciences (Martin, E.W., ed. 4), Mack Publishing Co., Easton, PA. The dosage can also be adjusted by the individual physician in the event of any complication.
According to a further embodiment, the invention relates to the use of bispecific binding molécules of the invention, e.g. immunoglobulin single variable domains or polypeptides containing them, for therapeutic purposes, such as
- for the prévention, treatment and/or alleviation of a disorder, disease or condition, especially in a human being, that is associated with DII4-mediated and/or Ang2related effects on angiogenesis or that can be prevented, treated or alleviated by modulating the Notch signaling pathway and/or the Tie2 signalling pathway with a bispecific binding molécule according to the invention,
- in a method of treatment of a patient in need of such therapy, such method comprising administering, to a subject in need thereof, a pharmaceutically active
-4816772 amount of at least one bispecific binding molécule ofthe invention, e.g. an immunoglobulin single variable domain, or a pharmaceutical composition containing same;
- for the préparation of a médicament for the prévention, treatment or alleviation of disorders, diseases or conditions associated with DII4-mediated and/or Ang2mediated effects on angiogenesis;
- as an active ingrédient in a pharmaceutical composition or médicament used for the above purposes.
According to a spécifie aspect, said disorder disorder, disease or condition is a îo cancer or cancerous disease, as defined herein.
According to another aspect, the disease is an eye disease associated with associated with DII4-mediated and/or Ang2-mediated effects on angiogenesis or which can be treated or alleviated by modulating the Notch signaling pathway and/or the Tie2 signalling pathway with a bispecific binding molécule.
is Depending on the cancerous disease to be treated, a bispecific binding molécule of the invention may be used on its own or in combination with one or more additional therapeutic agents, in particular selected from chemotherapeutic agents like DNA damaging agents or therapeutically active compounds that inhibit angiogenesis, signal transduction pathways or mitotic checkpoints in cancer cells.
The additional therapeutic agent may be administered simultaneously with, optionally as a component of the same pharmaceutical préparation, or before or after administration of the bispecific binding molécule.
In certain embodiments, the additional therapeutic agent may be, without limitation, one or more inhibitors selected from the group of inhibitors of EGFR, VEGFR, HER225 neu, Her3, AuroraA, AuroraB, PLK and PI3 kinase, FGFR, PDGFR, Raf, Ras, KSP, PDK1, PTK2, IGF-R or IR.
Further examples of additional therapeutic agents are inhibitors of CDK, Akt, sre/ber abl, cKit, cMet/HGF, c-Myc, Flt3, HSP90, hedgehog antagonists, inhibitors of JAK/STAT, Mek, mTor, NFkappaB, the protéasome, Rho, an inhibitor of wnt signaling
-4916772 or an inhibitor of the ubiquitination pathway or another inhibitor of the Notch signaling pathway.
Examples for Aurora inhibitors are, without limitation, PHA-739358, AZD-1152,
AT 9283, CYC-116, R-763, VX-680, VX-667, MLN-8045, PF-3814735.
An example for a PLK inhibitor is GSK-461364.
Examples for raf inhibitors are BAY-73-4506 (also a VEGFR inhibitor), PLX 4032, RAF-265 (also in addition a VEGFR inhibitor), sorafenib (also in addition a VEGFR inhibitor), and XL 281.
Examples for KSP inhibitors are ispinesib, ARRY-520, AZD-4877, CK-1122697, GSK 246053A, GSK-923295, MK-0731, and SB-743921.
Examples for a src and/or bcr-abl inhibitors are dasatinib, AZD-0530, bosutinib, XL 228 (also an IGF-1R inhibitor), nilotinib (also a PDGFR and cKit inhibitor), imatinib (also a cKit inhibitor), and NS-187.
An example for a PDK1 inhibitor is BX-517.
An example for a Rho inhibitor is BA-210.
Examples for PI3 kinase inhibitors are PX-866, BEZ-235 (also an mTor inhibitor), XL 418 (also an Akt inhibitor), XL-147, and XL 765 (also an mTor inhibitor).
Examples for inhibitors of cMet or HGF are XL-184 (also an inhibitor of VEGFR, cKit, Flt3), PF-2341066, MK-2461, XL-880 (also an inhibitor of VEGFR), MGCD-265 (also an inhibitor of VEGFR, Ron, Tie2), SU-11274, PHA-665752, AMG-102, and AV-299.
An example for a c-Myc inhibitor is CX-3543.
Examples for Flt3 inhibitors are AC-220 (also an inhibitor of cKit and PDGFR), KW 2449, lestaurtinib (also an inhibitor of VEGFR, PDGFR, PKC), TG-101348 (also an inhibitor of JAK2), XL-999 (also an inhibitor of cKit, FGFR, PDGFR and VEGFR), sunitinib (also an inhibitor of PDGFR, VEGFR and cKit), and tandutinib (also an inhibitor of PDGFR, and cKit).
Examples for HSP90 inhibitors are tanespimycin, alvespimycin, IPI-504 and CNF 2024.
-5016772
Examples for JAK/STAT inhibitors are CYT-997 (also interacting with tubulin),
TG 101348 (also an inhibitor of Flt3), and XL-019.
Examples for Mek inhibitors are ARRY-142886, PD-325901, AZD-8330, and XL 518.
Examples for mTor inhibitors are temsirolimus, AP-23573 (which also acts as a VEGF inhibitor), everolimus (a VEGF inhibitor in addition). XL-765 (also a PI3 kinase inhibitor), and BEZ-235 (also a PI3 kinase inhibitor).
Examples for Akt inhibitors are perifosine, GSK-690693, RX-0201, and triciribine.
Examples for cKit inhibitors are AB-1010, OSI-930 (also acts as a VEGFR inhibitor), AC-220 (also an inhibitor of Flt3 and PDGFR), tandutinib (also an inhibitor of Flt3 and PDGFR), axitinib (also an inhibitor of VEGFR and PDGFR), XL-999 (also an inhibitor of Flt3, PDGFR, VEGFR, FGFR), sunitinib (also an inhibitor of Flt3, PDGFR, VEGFR), and XL-820 (also acts as a VEGFR- and PDGFR inhibitor), imatinib (also a bcr-abl inhibitor), nilotinib (also an inhibitor of bcr-abl and PDGFR).
Examples for hedgehog antagonists are IPI-609 and CUR-61414,
Examples for CDK inhibitors are seliciclib, AT-7519, P-276, ZK-CDK (also inhibiting VEGFR2 and PDGFR), PD-332991, R-547, SNS-032, PHA-690509, and AG 024322.
Examples for protéasome inhibitors are bortezomib, carfilzomib, and NPI-0052 (also an inhibitor of NFkappaB).
An example for an NFkappaB pathway inhibitor is NPI-0052.
An example for an ubiquitination pathway inhibitor is HBX-41108.
In preferred embodiments, the additional therapeutic agent is an anti-angiogenic agent.
Examples for anti-angiogenic agents are inhibitors of the FGFR, PDGFR and VEGFR or the respective ligands (e.g VEGF inhibitors like pegaptanib or the anti-VEGF antibody bevacizumab), and thalidomides, such agents being selected from, without limitation, bevacizumab, motesanib, CDP-791, SU-14813, telatinib, KRN-951,
ZK-CDK (also an inhibitor of CDK), ABT-869, BMS-690514, RAF-265, IMC-KDR,
IMC-18F1, IMiDs (immunomodulatory drugs), thalidomide dérivative CC-4047, lenalidomide, ENMD 0995, IMC-D11, Ki 23057, brivanib, cediranib, XL-999 (also an
-5l16772 inhibitor of cKit and Flt3), 1B3, CP 868596, IMC 3G3, R-1530 (also an inhibitor of Flt3), sunitinib (also an inhibitor of cKit and Flt3), axitinib (also an inhibitor of cKit), lestaurtinib (also an inhibitor of Flt3 and PKC), vatalanib, tandutinib (also an inhibitor of Flt3 and cKit), pazopanib, GW 786034, PF-337210, IMC-1121 B, AVE-0005, AG-13736, E-7080, CHIR 258, sorafenib tosylate (also an inhibitor of Raf), RAF-265 (also an inhibitor of Raf), vandetanib, CP-547632, OSI-930, AEE-788 (also an inhibitor of EGFR and Her2), BAY-57-9352 (also an inhibitor of Raf), BAY-73-4506 (also an inhibitor of Raf), XL 880 (also an inhibitor of cMet), XL-647 (also an inhibitor of EGFR and EphB4), XL 820 (also an inhibitor of cKit), and nilotinib (also an inhibitor of cKit and brc-abl).
The additional therapeutic agent may also be selected from EGFR inhibitors, it may be a small molécule EGFR inhibitor or an anti-EGFR antibody. Examples for antiEGFR antibodies, without limitation, are cetuximab, panitumumab, matuzumab; an example for a small molécule EGFR inhibitor is gefitinib. Another example for an EGFR modulator is the EGF fusion toxin.
Among the EGFR and Her2 inhibitors useful for combination with the bispecific binding molécule of the invention are lapatinib, gefitinib, erlotinib, cetuximab, trastuzumab, nimotuzumab, zalutumumab, vandetanib (also an inhibitor of VEGFR), pertuzumab, XL-647, HKI-272, BMS-599626 ARRY-334543, AV 412, mAB-806, BMS-690514, JNJ-26483327, AEE-788 (also an inhibitor of VEGFR), ARRY-333786, IMC-11F8, Zemab.
Other agents that may be advantageously combined in a therapy with the bispecific binding molécule of the invention are tositumumab and ibritumomab tiuxetan (two radiolabelled anti-CD20 antibodies), alemtuzumab (an anti-CD52 antibody), denosumab, (an osteoclast différentiation factor ligand inhibitor), galiximab (a CD80 antagonist), ofatumumab (a CD20 inhibitor), zanolimumab (a CD4 antagonist), SGN40 (a CD40 ligand receptor modulator), rituximab (a CD20 inhibitor) or mapatumumab (a TRAIL-1 receptor agonist).
Other chemotherapeutic drugs that may be used in combination with the bispecific binding molécule s of the présent invention are selected from, but not limited to hormones, hormonal analogues and antihormonals (e.g. tamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate, flutamide, nilutamide, bicalutamide,
-5216772 cyproterone acetate, finasteride, buserelin acetate, fludrocortisone, fluoxymesterone, medroxyprogesterone, octreotide, arzoxifene, pasireotide, vapreotide), aromatase inhibitors (e.g. anastrozole, letrozole, liarozole, exemestane, atamestane, formestane), LHRH agonists and antagoniste (e.g. goserelin acetate, leuprolide, abarelix, cetrorelix, deslorelin, histrelin, triptorelin), antimetabolites (e.g. antifolates like methotrexate, pemetrexed, pyrimidine analogues like 5 fluorouracil, capecitabine, decitabine, nelarabine, and gemcitabine, purine and adenosine analogues such as mercaptopurine thioguanine, cladribine and pentostatin, cytarabine, fludarabine); antitumor antibiotics (e.g. anthracyclines like doxorubicin, daunorubicin, epirubicin ίο and idarubicin, mitomycin-C, bleomycin dactinomycin, plicamycin, mitoxantrone, pixantrone, streptozocin); platinum dérivatives (e.g. cisplatin, oxaliplatin, carboplatin, lobaplatin, satraplatin); alkylating agents (e.g. estramustine, meclorethamine, melphalan, chlorambucil, busulphan, dacarbazine, cyclophosphamide, ifosfamide, hydroxyurea, temozolomide, nitrosoureas such as carmustine and lomustine, thiotepa); antimitotic agents (e.g. vinca alkaloids like Vinblastine, vindesine, vinorelbine, vinflunine and vincristine; and taxanes like paclitaxel, docetaxel and their formulations, larotaxel; simotaxel, and epothilones like ixabepilone, patupilone, ZK-EPO); topoisomerase inhibitors (e.g. epipodophyllotoxins like etoposide and etopophos, teniposide, amsacrine, topotecan, irinotecan) and miscellaneous chemotherapeutics such as amifostine, anagrelide, interferone alpha, procarbazine, mitotane, and porfimer, bexarotene, celecoxib.
Particularly preferred combination partners of the bispecific binding molécules of the présent invention are VEGF antagonists, like bevacizumab (Avastin®), Vargatef®, Sorafenib and Sunitinib.
The efficacy of bispecific binding molécules of the invention or polypeptides containing them, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell- based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the spécifie disease or disorder of interest. Suitable assays and animal models will be clear to the skilled person, and for example include the assays described herein and used in the Examples below, e.g. a prolifération assay.
The data obtained in the experiments of the invention confirm that DII4-binding components of the invention hâve properties that are superior to those of DII4-binding
-5316772 molécules of the prior art, as can e.g. be taken from the ELISA data of Figure 10, showing that affinity-matured VHHs block hDLL4/hNotch1-Fc interaction in a complété manner, as well as the IC50 (nM) values for affinity matured VHHs in hDLL4/hNotch1-Fc compétition ELISA; and the affinity Ko (nM) of purified affinity matured VHHs on recombinant human DLL4 and mouse DLL4. This indicates that DII4-binding components of the invention are promising candidates to have therapeutic efficacy in diseases and disorders associated with DII4-mediated effects on angiogenesis, such as cancer.
According to another embodiment of the invention, there is provided a method of diagnosing a disease by
a) contacting a sample with a DII4-and/or Ang2 binding component of the invention as defined above, and
b) detecting binding of said DII4-and/or Ang2-binding component to said sample, and
c) comparing the binding detected in step (b) with a standard, wherein a différence in binding relative to said sample is diagnostic of a disease or disorder associated with DII4-mediated effects on angiogenesis.
For this and other uses, it may be useful to further modify a bispecific binding component of the invention, such as by introduction of a functional group that is one part of a spécifie binding pair, such as the biotin-(strept)avidin binding pair. Such a functional group may be used to link the bispecific binding molécule of the invention to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair, i.e. through formation of the binding pair. For example, a bispecific binding molécule of the invention may be conjugated to biotin, and linked to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such a conjugated bispecific binding molécule of the invention may be used as a reporter, for example in a diagnostic system where a détectable signal- l producing agent is conjugated to avidin or streptavidin. 1
-5416772
Brief description ofthe Figures:
Figure 1: Amino acid sequence alignment of human, rhésus and cynomolgus DLL4.
Figure 2: Human and mouse DLL4 délétion mutants (amino acid domain boundaries in superscript).
Figure 3: Purified VHHs blocking hDLL4/hNotch1-Fc interaction (ELISA).
Figure 4: Purified VHHs blocking hDLL4/hNotch1-Fc interaction (AlphaScreen).
Figure 5: Purified VHHs blocking CHO-hDLL4/hNotch1-Fc and CHO-mDLL4/hNotch1-Fc interaction (FMAT).
Figure 6: Purified VHHs blocking DLL4 mediated Notchl cleavage (reporter).
Figure 7: Binding of purified VHHs to recombinant human and mouse DLL4 (ELISA).
Figure 8: Binding of purified VHHs to recombinant human DLL1 and human Jagged-1 (ELISA).
Figure 9: Binding of purified VHHs to human/mouse/cynomolgus DLL4 (FACS).
Figure 10: Affinity matured VHHs blocking hDLL4/hNotch1-Fc interaction (ELISA).
Figure 11: Purified affinity matured VHHs blocking CHO-hDLL4/hNotch1-Fc and CHO-mDLL4/hNotch1-Fc interaction (FMAT).
Figure 12: Binding of purified VHHs to human/mouse DLL4 (ELISA).
Figure 13: Binding of purified affinity matured VHHs to recombinant human DLL1 and human Jagged-1 (ELISA).
Figure 14: Binding of purified VHHs to human/mouse/cynomolgus DLL4 (FACS).
Figure 15: Evaluation of VHH effects on DII4-mediated inhibition of HUVEC prolifération.
Figure 16: Description cycle 1 DLL4xAng2 VHHs
-5516772
Figure 17: Purified cycle 1 DLL4xAng2 VHHs blocking hDLL4-hNotch1 interaction (ELISA)
Figure 18: Purified cycle 1 DLL4xAng2 VHHs blocking CHO-hDLL4/Notch1 (44-1) and CHO-mDLL4/Notch1 (44-2) interaction (FMAT)
Figure 19: Purified cycle 1 DLL4xAng2 VHHs binding to human, mouse and cynomolgus DLL4 overexpressing CHO cells (FACS)
Figure 20: Purified cycle 1 DLL4xAng2 VHHs binding to human, mouse and rat DLL4 (ELISA)
Figure 21: Purified cycle 1 DLL4xAng2 VHHs binding to human DLL1 and Jagged-1 (ELISA)
Figure 22: Purified cycle 1 DLL4xAng2 VHHs blocking hAng2-hTie2 (48-1 ), mAng2mTie2 (48-2) and cAng2/cTie2 (48-3) interaction (ELISA)
Figure 23: Description cycle 2 DLL4xAng2 bispecific VHHs
Figure 24: Purified cycle 2 DLL4xAng2 VHHs blocking hDLL4-hNotch1 interaction (ELISA)
Figure 25: Purified cycle 2 DLL4xAng2 VHHs blocking CHO-hDLL4/Notch1 (51-1) and CHO-mDLL4/Notch1 (51-2) interaction (FMAT)
Figure 26: Purified cycle 2 DLL4xAng2 VHHs blocking hDLL4 mediated Notchl activation (reporter gene assay)
Figure 27: Purified cycle 2 DLL4xAng2 VHHs binding to human, mouse and cynomolgus DLL4 overexpressing CHO cells (FACS)
Figure 28: Purified cycle 2 DLL4xAng2 VHHs binding to human, mouse and rat DLL4 (ELISA)
Figure 29: Purified cycle 2 DLL4xAng2 VHHs binding to human DLL1 and Jagged-1 (ELISA)
Figure 30: Purified cycle 2 DLL4xAng2 VHHs blocking hAng2-hTie2 (56-1), mAng2mTie2 (56-2) and cAng2/cTie2 (56-3) interaction (ELISA)
-5616772
Figure 31: Purified cycle 2 DLL4xAng2 VHHs blocking hAng1-hTie2 interaction (ELISA)
Figure 32 : Purified cycle 2 DLL4xAng2 VHHs blocking hAng2 mediated HUVEC survival.
Materials and methods
a) Génération CHO and HEK293 cell lines overexpressing human, mouse and cynomolgus DII4
The cDNAs encoding human (SEQ ID NO: 417; NM_019074.2) and mouse DII4 (NM_019454.3) are amplified from a Human Adult Normal Tissue Heart cDNA library (BioChain, Hayward, CA, USA) and a Mouse Heart Tissue cDNA library (isolated from C57/BI6 strain), respectively, using oligonucleotides designed in the 5’ and 3' UTR of the corresponding sequence (see Table 1 ; SEQ ID NO:421 to 426). Amplicons are cloned into the mammalian expression vector pCDNA3.1(+)-neo (Invitrogen, Carlsbad, CA, USA).
Table 1 : Oligonucleotide sequences used for amplification of DLL4 gene full length orthologues.
Human DLL4 Mouse DLL4 Cynomolgus DLL4
>Fwd_hDLL4 >Fwd_mDLL4 >Fwd_cDLL4
GCGAACAGAGCCAG ATTGAGG (SEQ ID NO:421) GAGCGACATCCCTA ACAAGC (SEQ ID NO:423) GCGAACAGAGCCAG ATTCAGG (SEQ ID NO:425)
>Rev_hDLL4 >Rev_mDLL4 >Rev_cDLL4
GGATGTCCAGGTAGG CTCCTG (SEQ ID NO:422) CCTCAACTCTGTTCC CTTGG (SEQ ID NO:424) CCAGACAGACACCC AAAGGT (SEQ ID NO:426)
-5716772
Cynomolgus DII4 cDNA is amplified from a Cynomolgus Normal Tissue Heart cDNA library (BioChain, Hayward, CA, USA), using primers designed on the 5’ and 3' UTR of the DII4 encoding sequence of the closely related species rhésus (Macaca mulatta DII4, SEQ ID NO:418; XM_001099250.1) (see Table 1). The final amplicon is cloned in the mammaiian expression vector pCDNA3.1(+)-neo (Invitrogen, Carlsbad, CA, USA). The amino acid sequence of cynomolgus DII4 was shown to be 100% identical to rhésus, and 99% identical to human (see Figure 1 ; différences from the human sequence are indicated as bold-underlined).
To establish Chinese Hamster Ovary (CHO) cells overexpressing human DII4, mouse DII4 or cynomolgus DII4, parental CHO cells are electroporated with pCDNA3.1(+)neo-hDII4, pcDNA3.1(+)-neo-mDII4 or pcDNA3.1(+)-neo-cDII4, respectively. Human Embyonic Kidney (HEK293) cells overexpressing human DII4 and mouse DII4 are generated by lipid-mediated transfection with Fugene (Roche) of pCDNA3.1(+)-neohDII4 or mDII4 plasmids, respectively, in the HEK293 parental cell line. For ail conditions, transfectants are selected by adding 1 mg/mL geneticin (Invitrogen, Carlsbad, CA, USA).
b) Génération of monoclonal anti-DII4 IgG and Fab fragment
In US 2008/0014196 (Genentech) a human/mouse cross-reactive DII4 mAb is described that was used by Ridgway et al. (2006) to show additive effects of VEGF mAb and DII4 mAb on tumor growth in a number of xenograft models. This anti-DII4 mAb and its corresponding Fab are purified to assess the properties of this antibody (fragment) in biochemical/cellular assays and xenograft models and for spécifie elutions during phage sélections. The published variable heavy and light chain sequences of DII4 mAb are cloned into a hlgG2aK framework, transiently expressed in HEK293 cells and purified from supernatants using protein A chromatography. Purified DII4 mAb shows binding to human DII4 and mouse DII4 in ELISA and FACS (using CHO-mDII4 and CHO-hDII4 cells), sub-nanomolar affinities to both growth factor orthologues in Biacore.
The corresponding DII4 Fab fragment is constructed via gene assembly based on back-translation and codon optimization for expression in E. coli using Leto’s Gene Optimization software (www.entechelon.com). Oligonucleotide primers for the assembly of the variable light chain (VJ, variable heavy chain (Vh), constant light
-5816772 chain (Cl) and constant domain 1 of the heavy chain (Cm) are designed and an assembly PCR is performed. The cDNA seqments encoding Vl+Cl and Vh+Chi are cloned into a pUC119-derived vector, which contains the LacZ promotor, a résistance gene for kanamycin, a multiple cloning site and a hybrid glII-pelB leader sequence, using the restriction sites Sfil and Asc/ and the restriction sites Kpn/ and Notl, respectively. In frame with the Fab coding sequence, the expression vector encodes a C-terminal HA and His6-tag. The Fab fragment is expressed in E. coli as His6tagged protein and subsequently purified from the culture medium by immobilized métal affinity chromatography (IMAC) and size exclusion chromatography (SEC).
io Relevant amino acid sequences of the variable heavy and variable light chain are depicted (SEQ ID NO: 1 and SEQ ID NO: 2; respectively, of US 2008/0014196); the amino acid sequences of the complété heavy and light chain are shown in SEQ ID NOs: 419 and 420, respectively.
c) Génération of DII4 mutants for epitope mapping
To identify the région in the extracellular domain (ECD) of DII4 that comprises the epitope recognized by the anti-DII4 VHHs, progressive délétion mutants ofthe D1I4 ECD are generated. The mammalian expression vector pSecTag2/Hygro (Invitrogen, Carlsbad, CA, USA) comprising a CMV promotor upstream of polynucleotides encoding a nested sériés of délétion fragments of the DII4 ECD fused to a polyHis20 tag are generated using standard recombinant DNA technology (see Figure 2; amino acid domain boundaries in superscript).). These recombinant proteins are expressed in transiently transfected HEK293 cells using the Freestyle 293 Expression System (Invitrogen, Carlsbad, CA, USA) from which conditioned medium is collected and purified via IMAC. Only DII4 mutants lacking the EGF2-like domain showed impaired binding to the humanized human/mouse cross-reactive anti-DII4 mAb described above (immobilized via a capturing anti-human IgG coated Biacore sensor chip). This IgG is known to have a spécifie binding epitope in this DII4 domain (patent application Genentech, US 2008/0014196A1 ).
d) Génération of DII4 reporter assay plasmids
A reporter assay is developed based on the γ-secretase mediated cleavage of Notchl and nucleartranslocation ofthe intracellular domain of Notchl (NICD) upon stimulation with DII4, essentially as described (Struhl and Adachi, Cell. 1998 May 15;
-5916772
93(4):649-60). Gal4/VP16 coding sequences are inserted into the NlCD-coding sequence. The potent hybrid transcriptional activator GAL4-VP16, which consists of a DNA binding fragment of yeast GAL4 fused to a Herpes simplex viral transcriptional activator domain VP16, is inserted carboxy-terminal to the transmembrane domain of 5 Notchl. Cleavage of this construct by γ-secretase results in the release of the
Gal4/VP16 NICD fusion protein which will translocate to the nucléus where it will bind to and transcriptionally activate a co-transfected luciferase reporter plasmid, containing a strong GAL4-UAS promoter sequence (Struhl, G. and Adachi, A., Cell, vol. 93, 649-660,1998). The human Notch1-Gal4/VP16 expression cassette is cloned in pcDNA3.1(+)-neo (Invitrogen, Carlsbad, CA, USA). The pGL4.31[Luc2P/Gal4UAS/Hygro] vector (Promega, Madison, Wl, USA) is used as luciferase reporter plasmid.
Example 1
Immunization with DII4 from different species induces a humoral immune response in llama
1.1. Immunizations
After approval of the Ethical Committee of the faculty of Veterinary Medicine (University Ghent, Belgium), 4 Hamas (designated No. 208, 209, 230, 231) are immunized with 6 intramuscular injections (100 or 50 pg/dose at weekly intervals) of recombinant human DII4 (R&D Systems, Minneapolis, MN, US). The DII4 antigen is formulated in Stimune (Cedi Diagnostics BV, Lelystad, The Netherlands). Three additional Hamas (designated No. 127b, 260, 261) are immunized according to standard protocols with 4 subcutaneous injections of alternating human DII4 and mouse DII4 overexpressing CHO cells which are established as described above. Cells are re-suspended in D-PBS and kept on ice priorto injection. Furthermore, three additional Hamas (designated No. 282, 283, 284) are immunized according to standard protocols with 4 intramuscular injections (100 or 50 pg/dose at biweekly intervals) of alternating recombinant human DII4 and mouse DII4 (R&D Systems,
Minneapolis, MN, US). The first injection at day 0 with human DII4 is formulated in
Complété Freund’s Adjuvant (Difco, Detroit, Ml, USA), while the subséquent
-6016772 injections with human and mouse DII4 are formulated in Incomplète Freund’s
Adjuvant (Difco, Detroit, Ml, USA).
7.2. Evaluation ofinduced immune responses in Marna
To evaluate the induction of an immune responses in the animais against human DII4 by ELISA, sera are collected from Hamas 208, 209, 230 and 231 at day 0 (preimmune), day 21 and day 43 (time of peripheral blood lymphocyte [PBL] collection), from Hamas 127b, 260 and 261 at day 0 and day 51, and from Hamas 282, 283 and 284 at day 0, day 28 and day 50. In short, 2 pg/mL of recombinant human DII4 or mouse DII4 (R&D Systems, Minneapolis, MN, USA) are immobilized overnight at 4°C in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1%). After addition of sérum dilutions, specifically bound immunoglobulins are detected using a horseradish peroxidase (HRP)-conjugated goat anti-llama immunoglobulin (Bethyl Laboratories Inc., Montgomery, TX, USA) and a subséquent enzymatic reaction in the presence of the substrate TMB (3,3^5,5tetramentylbenzidine) (Pierce, Rockford, IL, USA), showing that a significant antibody-dependend immune response against DII4 is induced. The antibody response is mounted both by conventîonal and heavy-chain only antibody expressing B-cell répertoires since specifically bound immunoglobulins can be detected with antibodies specifically recognizing the conventîonal llama lgG1 antibodies or the heavy chain only llama lgG2 or lgG3 antibodies (Table 2-A). In ail Hamas injected with mouse DII4, an antibody response is mounted by conventîonal and heavy chain only antibody expressing B-cells specifically against mouse DII4. Additionally, sérum titers of cell immunized animais are confirmed by FACS analysis on human and mouse DII4 overexpressing HEK293 cells (Table 2-B). The DII4 sérum titer responses for each llama are depicted in Table 2.
-6116772
Table 2: Antibody mediated spécifie sérum response against DLL4.
A) ELISA (recombinant protein solid phase coated)
Recombinant human DLL4 Recombinant mouse DLL4
Llam a Immunog en Tôt al IgG igGl IgG 2 IgG 3 Tôt al IgG IgG 1 IgG 2 IgG 3
208 rec. human DLL4 + + +/- +/- ND ND ND ND
209 rec. human DLL4 + + +/- +/- ND ND ND ND
230 rec. human DLL4 + + +/- +/- ND ND ND ND
231 rec. human DLL4 ++ ++ ++ ++ ND ND ND ND
127b CHOhDLL4 + CHO- mDLL4 ++ ++ +/- +/- + ++ +/- +/-
-6216772
260 CHOhDLL4 + CHO- mDLL4 ++ ++ + ++ ++ + ++
261 CHOhDLL4 + CHO- mDLL4 ++ ++ +/- +/- + 4“ +/- +/-
282 rec. human DLL4 + mouse DLL4 ++ ++ ++ ++ ++ ++ + +
283 rec. human DLL4 + mouse DLL4 ++ ++ ++ ++ ++ ++ ++
284 rec. human DLL4 + mouse DLL4 + + + + ++ + ++
ND: not determined
-6316772
B) FACS (natively expressed protein on HEK293 cells)
human DLL4 mouse DLL4
Llam a Immunog en Total IgG IgG 1 IgG 2 IgG 3 Tota I IgG igGi IgG 2 IgG 3
208 rec. human DLL4 ND ND ND ND ND ND ND ND
209 rec. human DLL4 ND ND ND ND ND ND ND ND
230 rec. human DLL4 ND ND ND ND ND ND ND ND
231 rec. human DLL4 ND ND ND ND ND ND ND ND
127b CHOhDLL4 + CHO- mDLL4 + ND ND ND + ND ND ND
-6416772
260 CHOhDLL4 + CHO- mDLL4 ++ ND ND ND ++ ND ND ND
261 CHOhDLL4 + CHO- mDLL4 F ND ND ND + ND ND ND
282 rec. human DLL4 + mouse DLL4 ND ND ND ND ND ND ND ND
283 rec. human DLL4 + mouse DLL4 ND ND ND ND ND ND ND ND
284 rec. human DLL4 + mouse DLL4 ND ND ND ND ND ND ND ND
ND: not determined
Example 2
Cloning of the heavy-chain only anti-DII4 antibody fragment répertoires and préparation of phage
-6516772
Following the final immunogen injection, immune tissues as the source of B-cells that produce the heavy-chain antibodies are collected from the immunized Hamas. Typically, two 150-ml blood samples, collected 4 and 8 days after the last antigen injection, and one lymph node biopsy, collected 4 days after the last antigen injection are collected per animal. From the blood samples, peripheral blood mononuclear cells (PBMCs) are prepared using Ficoll-Hypaque according to the manufacturées instructions (Amersham Biosciences, Piscataway, NJ, USA). From the PBMCs and the lymph node biopsy, total RNA is extracted, which is used as starting material for RT-PCR to amplify the VHH encoding DNA segments, as described in WO 05/044858. For each immunized llama, a library is constructed by pooling the total RNA isolated from ail collected immune tissues of that animal. In short, the PCRamplified VHH répertoire is cloned via spécifie restriction sites into a vector designed to facilitate phage display of the VHH library. The vector is derived from pUC119 and contains the LacZ promoter, a M13 phage glll protein coding sequence, a résistance gene for ampicillin or carbenicillin, a multiple cloning site and a hybrid glII-pelB leader sequence (pAX050). In frame with the VHH coding sequence, the vector encodes a C-terminal c-myc tag and a His6 tag. Phage are prepared according to standard protocols and stored after filter sterilization at 4°C for further use.
Example 3
Sélection of DII4 spécifie VHHs via phage display
VHH répertoires obtained from ail Hamas and cloned as phage library are used in different sélection strategies, applying a multiplicity of sélection conditions. Variables include i) the DII4 protein format (C-terminally His-tagged recombinantly expressed extracellular domain of human DII4 (Met1-Pro524) and mouse DII4 (Met1-Pro525) (R&D Systems, Minneapolis, MN, USA), orfull length human DII4 and mouse DII4 présent on DII4-overexpressing CHO or HEK293 cells, ii) the antigen présentation method (plates directly coated with DII4 or Neutravidin plates coated with DII4 via a biotin-tag; solution phase: incubation in solution followed by capturing on Neutravidincoated plates), iii) the antigen concentration and iv) different elution methods (nonspecific via trypsin or specfic via cognate receptor Notch1/Fc chimera or anti-DII4 IgG/Fab). AII sélections are done in Maxisorp 96-well plates (Nunc, Wiesbaden, Germany).
-6616772
Sélections are performed as follows: DII4 antigen préparations for solid and solution phase sélection formats are presented as described above at multiple concentrations. After 2h incubation with the phage libraries followed by extensive washing, bound phage are eluted with trypsin (1 mg/mL) for 30 minutes. In case trypsin is used for phage elution, the protease activity is immediately neutralized applying 0.8 mM protease inhibitor ABSF. As control, sélections w/o antigen are performed in parallel. Phage outputs that show enrichment over background (nonantigen control) are used to infect E. coli. Infected E. coli cells are either used to préparé phage for the next sélection round (phage rescue) or plated on agar plates (LB+amp+glucose2%) for analysis of individual VHH clones. In order to screen a sélection output for spécifie binders, single colonies are picked from the agar plates and grown in 1 mL 96-deep-well plates. LacZ-controlled VHH expression is induced by adding IPTG (0.1-1 mM final) in the absence of glucose. Periplasmic extracts (in a volume of - 80 uL) are prepared according to standard protocols
Example 4
Screening of periplasmic extracts in DII4-Notch1 AlphaScreen and FMAT compétition assay
Periplasmic extracts are screened in a human DII4/human Notchl AlphaScreen assay to assess the blocking capacity of the expressed VHHs. Human DII4 is biotinylated using biotin (Sigma, St Louis, MO, USA) and biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester sodium sait (Sigma, St Louis, MO, USA). Notchl/Fc chimera (R&D Systems, Minneapolis, MN, USA) is captured using an antiFc VHH which is coupied to acceptor beads according to the manufacturées instructions (Perkin Elmer, Waltham, MA, US). To evaluate the neutralizing capacity of the VHHs, dilution sériés of the periplasmic extracts are pre-incubated with biotinylated human DII4. To this mixture, the acceptor beads and the streptavidin donor beads are added and further incubated for 1 hour at room température. Fluorescence is measured by reading plates on the Envision Multilabel Plate reader (Perkin Elmer, Waltham, MA, USA) using an excitation wavelength of 680 nm and an émission wavelength of 520 nm. Decrease in fluorescence signal indicates that the binding of biotinylated human DII4 to the human Notchl/Fc receptor is blocked by the VHH expressed in the periplasmic extract.
-6716772
Alternatively, CH0-hDII4 and CH0-mDII4 cells are used in a human Notch1/Fc FMAT (Fluorometric Microvolume Assay Technology) compétition assay. Recombinant human Notch1/Fc chimera (R&D Systems, Minneapolis, MN, USA) is randomly labeled with Alexa-647 (Invitrogen, Carlsbad, CA, USA). In brief, 5 pL periplasmic material is added to 100 pM or 175 pM labeled human Notch1/Fc together with 7,500 CHO-hDII4 or CHO-mDII4 overexpressing cells, respectively, and readout is performed after 2 hours of incubation. To set the no-competition baseline, at least 30 replicates of cells with human Notch1/Fc~Alexa647 are included and the percentage of inhibition is calculated from this baseline. Ail calculations are based on îo the FL1_total signal which comprises the average of the fluorescence per well times the number of counts per well.
From this screening, inhibiting VHHs are selected and sequenced. Sequence analysis revealed 167 unique VHHs belonging to 40 different B-cell lineages. The total number of variants found for each B-cell lineage is depicted in Table 3. An 15 overview of periplasmic screening data is given in Table 4. The amino acid sequences of ail obtained unique VHHs are shown in the Sequence Listing (SEQ ID NO:167 - 332 and 459) and in Table 5 (CDRs and framework régions are indicated).
Table 3: Sélection parameters used for the identification of DLL4 spécifie VHH B-cell lineages.
B-cell lineag e VHH ID # variant s librar y sélection format phage elution selectio n rounds
1 DLLBII8A 09 31 231 rhDLL4 (3 nM) trypsin 1
2 DLLBII5B 11 1 231 rhDLL4 (3 nM) trypsin 1
-6816772
3 DLLBII7B 5 21 231 RI: biotrhDLL4 (3 nM) RII: bîotrhDLL4 (0.03 nM) trypsin 2
4 DLLBII6B 11 13 231 biot-rhDLL4 (3 M) trypsin 1
5 DLLBII8C 11 5 231 RI: biotrhDLL4 (3 nM) RII: biotrhDLL4 (3 nM) trypsin 2
6 DLLBII19 D10 1 231 biot-rhDLL4 (3 nM) trypsin 1
7 DLLBII33 C5 2 231 CHO-hDLL4 (2E6/mL) trypsin 1
8 DLLBII28 B6 2 231 rmDLL4 (0.5 ug/mL) trypsin 1
9 DLLBII17 G10 1 231 biot-rhDLL4 (3 nM) trypsin 1
10 DLLBII17 C1 8 231 biot-rhDLL4 (3 nM) trypsin 1
11 DLLBII19 F4 1 231 biot-rhDLL4 (3 nM) trypsin 1
12 DLLBII17 F10 1 231 biot-rhDLL4 (3 nM) trypsin 1
-6916772
13 DLLBII17 B3 5 231 biot-rhDLL4 (3 nM) trypsin 1
14 DLLBII19 F12 2 231 biot-rhDLL4 (3 nM) trypsîn 1
15 DLLBII42 B7 1 231 RI: biotrhDLL4 (3 nM) RII: biotrhDLL4 (3 nM) rhNotch 1/Fc 2
16 DLLBII47 D1 1 230 RI: biotrhDLL4 (3 nM) RII: biotrhDLL4 (3 nM) rhNotch 1/Fc 2
17 DLLBII56 A09 15 230 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) rhNotch 1/Fc 2
18 DLLBII95 F2 5 230 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) trypsin 2
-7016772
19 DLLBII96 C3 20 230 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) trypsin 2
20 DLLBII10 4G1 1 230 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) RIII: biotrhDLL4 (+rhDLL4) rhNotch 1/Fc (RI-RII) trypsin (RHI) 3
21 DLLBII10 2F8 3 230 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) RIII: biotrhDLL4 (0.01 nM) rhNotch 1/Fc (RI-RII) trypsin (RHI) 3
-7116772
22 DLLBII11 2A3 1 209 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) trypsin 2
23 DLLBII10 2G4 2 230 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) RIII: biotrhDLL4 (0.01 nM) rhNotch 1/Fc (RI-RII) trypsin (RI) 3
24 DLLBII10 1G8 1 230 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) RIII: biotrhDLL4 (0.1 nM) rhNotch 1/Fc (RI-RII) trypsin (RHi) 3
25 DLLBII11 2Α4 1 209 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) trypsin 2
26 DLLBII10 1Η9 1 230 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) RIII: biotrhDLL4 (0.1 nM) rhNotch 1/Fc (RI-RII) trypsin (RHI) 3
27 DLLBII10 1Η5 1 230 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) RIII: biotrhDLL4 (1 nM) rhNotch 1/Fc (RI-RII) trypsin (RIII) 3
28 DLLBII11 2E7 1 209 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) trypsin 2
29 DLLBII10 1F1 1 230 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) RI II: biotrhDLL4 (1 nM) rhNotch 1/Fc (RI-RII) trypsin (RIII) 3
30 DLLBII10 4A3 1 230 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) RI II: biotrhDLL4 (1 nM) + rhDLL4 rhNotch 1/Fc (RI-RII) trypsin (RHI) 3
-7416772
31 DLLBII10 4C4 1 230 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) RI II: biotrhDLL4 (1 nM) + rhDLL4 rhNotch 1/Fc (RI-RII) trypsin (RIII) 3
32 DLLBII10 4B5 1 230 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) RIII: biotrhDLL4 (1 nM) + rhDLL4 rhNotch 1/Fc (RI-RII) trypsin (RIII) 3
33 DLLBII10 7C3 1 208 RI: CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) rhNotch 1/Fc 2
-7516772
34 DLLBII58 A11 4 260 RI: biotrhDLL4 (3 nM) RII: biotrmDLL4 (3 nM) rh Notch 1/Fc 2
35 DLLBII61 F5 1 260 RI: HEK293HhDLL4 (2E6/mL) RII: HEK293HhDLL4 (2E6/mL) trypsin 2
36 DLLBII61 F7 1 260 RI: HEK293HhDLL4 (2E6/mL) RII: HEK293HhDLL4 (2E6/mL) trypsin 2
37 DLLBII62 C11 1 260 RI: HEK293HhDLL4 (2E6/mL) RII: HEK293HmDLL4 (2E6/mL) trypsin 2
-7616772
38 DLLBII11 5A5 1 230 RI; CHOmDLL4 (2E6/mL) RII: CHOmDLL4 (2E6/mL) RIII: biotrhDLL4 (1 nM) RIV:CHOmDLL4 (2E6/mL) rhNotch 1/Fc (RI-RII) trypsin (RHI) trypsin (RIV) 4
39 DLLBII83 G1 4 284 RI: CHOmDLL4 (2E6/mL) RI: CHOhDLL4 (2E6/mL) DLL4 IgG 2
40 DLLBII80 E8 1 283 RI; CHOhDLL4 (2E6/mL) RI: CHOhDLL4 (2E6/mL) DLL4 IgG 2
-7716772
Table 4: Screening of periplasmic extracts containing expressed anti-DLL4 VHH
Bcell line âge Représentative VHH ID # unique sequen ces ELISA Alpha Screen FM AT FMAT Biacore (a)
hDLL4 % inhibi tion hDLL4 % inhibi tion hDL L4 % inhi bit tion mDLL4 % inhibit tion kd(s'1)
1 DLLBII8A09 31 96 - - - (7,2e03- 2.4e'04)
2 DLLBII5B11 1 98 - - - -
3 DLLBII7B05 21 84 - - - (2.4e'04)
4 DLLB1I6B11 13 98 - - - (9.4e'04- 3.7e'04)
5 DLLBII8C11 5 57 - - - (7.3e'04- 6.0e'04)
6 DLLBII19D1 0 1 98 85 - - 1.3e-03
7 DLLBII33C0 5 2 86 75 - - 9.2e’04 (2 je-03)
8 DLLBII28B0 6 2 23 54 - - 7.5e'03 (1.6e'04)
9 DLLBII17G1 0 1 93 82 - - 1.5e'03
-7816772
10 DLLBII17C0 1 8 82 84 - - 5.6e'04 (5.6e'045.3e'04)
11 DLLBII19F0 4 1 98 95 - - 1.1e’03
12 DLLBII17F1 0 1 98 88 - - 1.1e-03 / 3 jE-04 (b)
13 DLLBII17B0 3 5 76 77 - - 1.2e*03 / 2 2e-°4 Φ)
14 DLLBII19F1 2 2 98 98 - - 4.9e'04 (1.0e'03)
15 DLLBII42B0 7 1 - - - - -
16 DLLBII47D0 1 1 - - 87 - -
17 DLLBII56A0 9 15 - - - - 1.1e-03 (9.5e’031.1e-03)
18 DLLBII95F0 2 5 - - 81 71 6.7e'04
19 DLLBII96C0 3 20 - - 75 83 -
20 DLLBII104G 01 1 - - 94 86 1 2e‘03 (1.4e-039.4e'04)
21 DLLBII102F 08 3 - - 85 75 -
-7916772
22 DLLBIH12A 03 1 - - 72 97 -
23 DLLBII102G 04 2 - - 86 82 -
24 DLLBII101G 08 1 - - 91 92 2 ήΕ-03
25 DLLBII112A 04 1 - - 75 90 -
26 DLLBII101H 09 1 - - 87 75 -
27 DLLBII101H 05 1 - - 85 83 -
28 DLLBII112E 07 1 - - 80 85 -
29 DLLBI1101F 01 1 - - 85 78 2.Oe‘02
30 DLLBII104A 03 1 - - 86 83 -
31 DLLBII104C 04 1 - - 87 83 1.0E‘°3
32 DLLBII104B 05 1 - - 86 78 -
33 DLLBII107C 03 1 - - 75 80 -
34 DLLBII58A1 1 4 - - 95 73 1 .6e-03 (1.7e'031.6e'03)
Μ-A
-8016772
35 DLLBII61F0 5 1 - - 74 76 -
36 DLLBII61F0 7 1 - - 79 77 -
37 DLLBII62C1 1 1 - - 74 71 -
38 DLLBII115A 05 1 - - 74 84 3.1e'03
39 DLLBII83G0 1 4 - - 87 93 4.1e'04
40 DLLBII80E0 8 1 - - 71 82 -
(a) if multiple unique variants within a B-cell lineage are identified, the range (max-min) in off-rate or the off-rate of a lineage member is given between brackets in italics).
<b) heterogeneous fit: fast and slow off-rate determined.
-8l16772
Table 5 Framework and CDR Sequences of anti-DLL4 VHH
VHH !D SEQ ID NO Framework 1 CDR1 Framework 2 CDR 2 Framework 3 CDR 3 Framework 4
DLLBIIO 5B06 167 EVQLVESGGG LVQPGGSLRL SCAASGFTLD TYNIG WFRQAPGKER EWVS CISSSD GSTNYA DSVKG RFTISRDNAKNTVY LQMNNLKPEDTAV YYCAA PFAYYSDLC GVNGVDY WGQGTQVTVSS
DLLBIIO 5B08 168 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CINSSD GSTYYT DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAIY YCAA PFSYYSHLC GVNGYDY WGQGTQVTVSS
DLLBIIO 5B09 169 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CISSSD GSTAYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFSYYSSLC GVNEYDY WGQGTQVTVSS
DLLBIIO 5B11 170 EVQLVESGGG LVQPGGSLRL SCAISGFTLD LHVIG WLRQAPGKER EWVS CISSSD GSTYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PWDSWYCGI GNDYDY WGQGTQVTVSS
DLLBIIO 5D11 171 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CIRGSN GSTGYT DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFIHYSDLCG VNGYDY WGQGTQVTVSS
DLLBIIO 6A02 172 EVQLVESGGG LVQAGGSLRL SCAASGFTLD KYAIG WFRQAPGKER EGVS CISSRG GSTYYV DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA DPIHNCYSG SSYYYSPEA VYDY WGQGTQVTVSS
DLLBIIO 6A05 173 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CITSSN GSTYYT DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFAHYSDLC GVNGYDY WGQGTQVTVSS
DLLBIIO 6B11 174 EVQLVESEGG LVQAGGSLRL SCAASGSTFS SYAM G WYRQAPGKQ RELVA VISNGG ITNYPN SVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCFY SGSYYYPTD VHEYDY WGQGTQVTVSS
DLLBIIO 6E02 175 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CINSSD GSTYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFEYYSDLC GVNGYDY WGQGTQVTVSS
DLLBIIO 6E04 176 EVQLVESGGG LVQAGGSLRL SCAASGFTLD YYAIG WFRQAPGKER EGIS CISSRG GSTFYV DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA DPIHNCYSG RYYYSPEAV YEY WGQGTQVTVSS
DLLBIIO 6E12 177 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YHNIG WFRQAPGKER EWVS CISSSG GSTAYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFSHYSDLC GVNAIDY WGQGTQVTVSS
DLLBIIO 6G09 178 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CINSSD GSTYYA DSVKG RFTVSRDNAKNTVY LQMNSLKPEDTAVY YCAA PFEYYSDLC GVNGYDY WGQGTQVTVSS
DLLBIIO 7A02 179 EVQLVESGGG LVQAGGSLRL SCAASGSTFN SYAM G WYRQAPGKQ REWVA AFSTGG STNYAD SVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCFY SGSYYYPTD VFEYDY WGQGTQVTVSS
DLLBIIO 7B05 180 EVQLVESGGG LVQAGGSLRL SCAASGFALD YYAV G WFRQAPGKER EGVS CISSRG GSTFYA DSVKG RFTTSRNNAKNTVY LQMNSLKPEDTAVY YCAA HPLQNCCGG SAYASPEAV YEY WGQGTQVTVSS
DLLBIIO 8A09 181 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CINSSD GSTYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFAYYSNLC GVNGYDY WGQGTQVTVSS
DLLBIIO 8B05 182 EVQLVESGGG LVQAGGSLRL SCAASGFALD YYAV G WFRQAPGKER EGVS CISSRG GSTYYV DSVKG RFTTSRDNAKNTVY LQMNSLKPEDTAVY YCAA DPIHNCYSG NYYASPEAV YDY WGQGTQVTVSS
DLLBIIO 8C11 183 EVQLVESGGG LVQAGGSLRL SCAASGFTFD DYAIG WFRQAPGKER EGVS CISSHD RTTYYA DSVKG RFTISSDNAKNTVY LQMNSLKPEDTAVY YCAA DPLVCGYND PRLADY WGQGTQVTVSS
DLLBIIO 8H06 184 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CITSSY GSTYYT DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFAHYSDLC GVNGYDY WGQGTQVTVSS
DLLBIIO 9C01 185 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YHNIG WFRQAPGKER EWVS CISSSD GRTAYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFTHYSDLC GVNEYDY WGQGTQVTVSS
DLLBII1 00G01 186 EVQLVESGGG LVQPGGSLRL SCAASGFTFD DYAIG WFRQAPGKEP EGIG CISSSG GSTYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAT PGIAACRGIH Y WGQGTQVTVSS
DLLBII1 01B12 187 KVQLVESGGG LVQPGGSLRL SCAASGFTFD DYAIG WFRQAPGKEP EGIS CISSSG GITYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAT PGIAACRGIH Y TGQGTQVTVSS
DLLBII1 01E04 188 EVQLVESGGG LVQPGGSLRL SCAASGFTFG NYDM S WVRQAPGKGP EWVS AINSGG GTTYYA DSVKG RFTISRDNAKNTLY LQMNSLKPEDTAVY YCAT PRGWGPTG PHEYGY WGQGTQVTVSS
DLLBII1 O1FO1 189 EVQLVESGGG LVQAGGSLRL SCAASGRTFS NYAM G WFRQAPGKER EFVA AISWSG GDTYYA DSVKG RFTISRDNAKNTVC LQMNSLKPEDTAVY YCAA SFQSGAAPG ANFYDY WGQGTQVTVSS
DLLBII1 01F03 190 EVQLVESEGG SVQAGGSLRL SCAASGRTFS SYAM G WFRQAPGKER EFVA AINWSG GYTYYA DSVRG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PAPGSSGYE YDY WGQGTQVTVSS
DLLBII1 01F06 191 EVQLVESGGG LVQAGGSLRL SCAASGRTFS SYAM G WFRQAPGKER EFVA AlFWSG GSTYYA DSVRG RFTISRDIAKNTVYL QMNSLKPEDTAVY YCAA PSPGSSGYE YDY WGQGTQVTVSS
DLLBII1 01F08 192 EVQLVESGGG LVQTGDSLRLS CAASGSTFS NYRM G WFRQGPGKER EFVA AIGRNG QNTYYT DSVKG RFTVTRDNAKNMM YLQMNSLKPEDSA VYTCAA SLRGWDTTR IDYEY WGQGTQVTVSS
DLLBII1 01F10 193 EVQLVESGGG LVQPGGSLRL SCTASGFTFD VYAIG WFRQAPGKEP EGIS CISSSG SITYYA DSVKG RFTTSRDSAKNTVY LQMNSLKPEDTAVY YCAT PGIAACRGIH Y WGQGTQVTVSS
DLLBII1 01G02 194 EVQLVESGGG LVQPGGSLRL SCAASGFTFG NYDM S WVRQAPGKGP EWVS AINSGG DTTYYA DSVKG RFTISRDNAKNTLY LQMNSLKPEDTAVY YCAT PRGWGPTG PHEYGY WGQGTQVTVSS
DLLBII1 01G03 195 EVQLVESRGG LVQAGGSLRL SCAASGRTFN SYAM G WFRQAPGKER EFVA TINWSG GSTYYA DSVKG RFTISRDNAKNTAY LQMNSLKPEDTAVY YCAA PAPGSSGYE YDY WGQGTQVTVSS
DLLBII1 01G05 196 EVQLVESGGG SVQAGGSLRL SCAASGRTFS SYAM G WFRQAPGKER EFVA AVYWS GGSTY YADSVR G RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PSPGSSGYE YDY WGQGTQVTVSS
DLLBII1 01G08 197 EVQLVESGGG LVQAGGSLRL SCAASGRTFS SYAM A WFRQAPGKER EFVA AIRWSG GTAYYA DSVQG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAN RAADTRLGP YEYDY WGQGTQVTVSS
DLLBII1 01H02 198 EVQLVESGGG LVQPGGSLRL SCAASGFTFG NYDM S WVRQAPGKGP EWVS AINSGG GITYYA DFVKG RFTISRDNAKNTLY LQMSSLKPEDTAVY YCAT PRGWGPTG PHEYGY WGQGTQVTVSS
DLLBII1 01H03 199 EVQLVESGGG LVQPGGSLRL SCAASGFTFD DYAIG WFRQAPGKEP EGIS CISSSG GITYYT DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAT PGIAACRGIH Y TGQGTQVTVSS
DLLB1I1 01H05 200 EVQLVESGGG SAQAGGSLRL SCAASGRTSS TYAM G WFRQAPGKEH EFVS AIGRGT GATSY GDSVK G RFTISRDNAKNTVY LQMNSLQLEDTGD YYCVA GRGFYHDYS SYEY RGQGTQVTVSS
DLLBII1 01H09 201 EVQLVESGGG LVQPGGSLRL SCAASGFTLG YYTIV WFRQAPGKER KGVS CISSRD GSRYY ADSVK G RFTISRDNAKNTVY LRMNSLKPEDTAVY YCAA GPDCSSYDY WGQGTQVTVSS
DLLBII1 02F08 202 EVQLVESGGG LVQTGDSLRLS CAASGSTFS NYRM G WFRQGPGKER EFVA AIGRNG QNTYYT DSVKG RFTVTRDNAKNMV YLQMNSLKPEDSA VYTCAA SLRGWDTTR IDYEY WGQGTQVTVSS
DLLBII1 02G04 203 EVQLMESGGG LVQPGGSLRL SCAASGFTFS SYAM S WVRQAPGKGL EWVS RITSGG RTTYRD SVKG RFTISRDNSKNTLY LQMNSLKPEDTALY YCAK ARGDIDVYTL SDS RGQGTQVTVSS
DLLBII1 02H07 204 EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYAM S WVRQAPGKGL EWVS RITSGG RATYRD SVKG RFTISRDNSKNTLY LQMNSLKPEDTALY YCAK ARGDIDVYTL SDS RGQGTQVTVSS
DLLBII1 02H09 205 EVQLVESGGG LVQPGGSLRL SCAASGFTFG NYDM S WVRRPPGKGP EWVS AINSGG GSTYYA DSVKG RFTISRDNAKNTLY LQMNSLKPEDTAVY YCAT PRGWGPTG PHEYGY WGQGTQVTVSS
DLLB1I1 03A04 206 EVQLVESGGG LVQAGGSLRL SCAASGFTFD DYAIG WFRQAPGKER EGVS CISSSD GSTYYA DSVKG RFTVSSNNADDTVY LQMNSLKPEDTAVY YCAV RLFSGGCAV VAGTSWADF GS SGQGTQVTVSS
DLLBII1 03B05 207 AVQLVESGGG LVQAGGSLRL SCAASGFTFD DYAIG WFRQAPGKER EGVS CISSSD GSTHYA DSVKG RFTISSDKVKNTVY LQMNSLKPEDTAVY YCAV RLFKGGCAV VAGTSWADF GS TGQGTQVTVSS
DLLBII1 04A03 208 EVQLVESGGG LVQAGGSLRL SCAASGDIPR IAAMG WYRQAPGKQ RELVA TVSNAA TTRYAD SAKG RFTISRDNAKTVSL QMDNLKPEDTGVY YCYS LATTVTPSW VNY WGQGTQVTVSS
DLLBII1 04A05 209 EVQLVESGGG LVQPGGSLRL SCAASGFAFG YYDM S WVRQAPGKGP EWVS AINSGG GSTYYA DSVKG RFTISRDNAKNMLY LQMNSLKPEDTAVY YCAT PRGWGPTG PHEYDY WGQGTQVTVSS
DLLBII1 04B02 210 EVQLVESGGG LVQAGGSLRL SCDASGRGFS YYRM G WFRQAPGKER EFVA AIGKSG RNTYY GDYVK G RFTVSRDNAKNTVY LQMTSLKPEDTAVY TCAA SLRGWDTTW IDYEY WGQGTQVTVSS
DLLBI11 04B05 211 EVQLVESGGG SVQAGGSLRL SCAASGSISR IDVMA WYRQAPGKER ELVA SISSGG STNYAD SVKG RFTISREYFKNMMY LQMNSLKFEDTAVY YCNA DSRRGGVGN FFRS WGQGTQVTVSS
DLLBII1 04B08 212 EVQLVESRGG LVQPGGSLRL SCAASGFTFG SYDM S WVRQAPGKGP EWVS AINSGG GSTYYA DSVKG RFTISRDNAKSTLYL QMNSLKPEDTAVY YCAI PRGWGPTG PIEYAY WGQGTQVTVSS
DLLBII1 04C04 213 EVQLVESGGG LVQAGGSLRL SCAAAGSTFS SYVM G WYRQAPGKQ RELVA HISTRGI TYYADS VKG RFTISRDNAKNTMY LQMNSLKPEDTAVY YCNT RRNFLSNY WGQGTQVTVSS —
DLLBII1 04C12 214 EVQLVESGGG LVQPGGSLRL SCAASGFTFD DYAIG WFRQAPGKEP EGIS CISSSG GITYYA DSVKG RFTISRDNAKSTVY LQMNSLKPEDTAVY YCAT PGIAACRGIH Y TGQGTQVTVSS
DLLBII1 04G01 215 EVQLVESGGG LVQAGGSLRL SCAASGFTFD DYAIG WFRQAPGKER EGVS CISSSD GSTYYA DSVKG RFTISSDNAKNTVY LQMNSLKPEDTAVY YCAT AWCDSSWY RSFVGY WGQGTQVTVSS
DLLBII1 05G01 216 EVQLVESGGG LVQAGGSLRL SCAASGFTFD DYAIG WFRQAPGKER EGVS CISSSD DSTYYA DSVKG RFTISSNNAKNTAY LQMNSLKPEDTAVY YCAV RLFSGGCAV VARTSWADF GS SGQGTQVTVSS
DLLBII1 06A01 217 EVQLVESGGG FVQPGGSLRL SCAASGFTFD DYAIG WFRQAPGKEP EEIS CISSSG GITYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAT PGIAACRGIH Y WGQGTQVTVSS
DLLB1I1 06F01 218 EVQLVESGGG LVQSGGSLRL SCAASGFTFG SYDM S WVRQAPGKGP EWVS AINSGG GITYYA DSVNG RFTISRDNTKNTLYL QMNSLKPEDTAVY YCAT PRGWGPTG PHEYGY WGQGTQVTVSS
DLLBII1 06H01 219 EVQLVESGGG LVQAGGSLRL SCAASGSTFS NYAM G WYRQAPGKQ RELW GISSDG STHYAD SAKG RFTISRDDAKNTVY LQMNSLKTEDTAVY YCYV PVKVAGLEY AY WGQGTQVTVSS
DLLBII1 07C03 220 EVQLVESGGG LVQPGGSLRL SCEVSGSIGS VSDM R WYRQAPGLQY ELVA RITSGSI TDYSDS VKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCNA DVQHSAWLK PLTY WGQGTQVTVSS
DLLBI11 12A03 221 EVQLVESGGG LVQPGGSLRL SCAASGIRFS INGM G WYRQAPGKQ REAVA TITRGGI RDYTDS VKG RFTISRDIARNTVYL QMNNLKPEDSAVY YCNI DIY WGRGTQVTVSS
DLLBII1 12A04 222 EVQLVESGGG LVQAGGSLRL SCAAFGRTPY GMG WFRQAPGKER EFVA AITSDG STNYAD SVKG RFTISRDNAKNAVS LQMNSLKPEDTAVY YCTA PYYSDFEGT TTEYDY WGQGTQVTVSS
DLLBII1 12E07 223 EVQLVESGGG LVQAGGSLRL SCAASGRTVR SYAT G WFRQAPGKER EFVA ALRWSI GSIASV YYDDSV KG RFTISGDNAENTVY LQMNALKPEDTAIY YCAS TTRGRYSAL SASAYDY WGQGTQVTVSS
DLLBII1 15A05 224 EVQLVESGGG LVQPGGSLRL SCAASGFTFG SYDM S WVRRSPGKGP EWVS SINSGG GSTYYA DFVKG RFTISRDNAKNTLY LQMNSLKPEDTAVY YCAA DRYIRARQG DYWGAYEYD Y WGQGTQVTVSS
DLLBII1 6A03 225 EVQLVESGGG LVQAGGSLRL SCAASGFTLD YYAIG WFRQAPGKER EGVS CISSRG GSTYYE DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA DPIHNCYSG RYYASPDAV YDY WGQGTQVTVSS
DLLBII1 6A07 226 KVQLVESGGG LVQAGGSLRL SCAASGFTFD DYAIG WFRQAPGKER EGVS CISSHD GTTYYA DSVKG RFTISSDNAKNTVY LQMNSLKPEDTAVY YCAA DPLVCGYND PRLADY WGQGTQVTVSS
DLLBII1 6A09 227 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YHNIG WFRQAPGKER EWVS CISSSG GSTAYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFNHYSDLC GVNAIDY WGQGTQVTVSS
DLLBII1 6C11 228 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CISSSD GSTAYA DSVKG RFTISRDNGKNTVY LQMNSLKPEDTAVY YCAA PFSYYSSLC GVNEYDY WGQGTQVTVSS
DLLBII1 6D11 229 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CISSTN GNTYYA DSVKG RFSISRDNARNTVY LQMNSLKPEDTAVY YCAA PFSYYNNLC GVNGVDY WGQGTQVTVSS
DLLBII1 6E02 230 EVQLVESGGG LVQAGGSLRL SCAASGFTFD DYAIG WFRQAPGKER EGVS CISSSD DSTYYA DSVKG RFTISSNNAKNTVY LQMNSLKPEDTAVY YCAV RLFSGGCAV VAGTSWADF GS SGQGTQVTVSS
DLLBII1 6E08 231 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EGVS CITSSN GSTYYT DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFAHYSDLC GVNGYDY WGQGTQVTVSS
DLLBII1 6H02 232 EVQLVESGGG LVQPGGSLRL SCAASGFALD YYNIG WFRQAPGKER EWVS CISSSD GSTGY ADSVK G RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFAYYSDLC GVNEYDY WGQGTQVTVSS
DLLBII1 6H09 233 EVQLVESGGG LVQAGGSLRL SCAASGSTFT SYAM G WYRQAPGKQ RELVA AISSDD STYYAD CVKG RFTISRDYAKNTVY LQMNSLKPEDTAVY YCNA PHSDYDEEA PSDFGS WGQGTQVTVSS
DLLBII1 7A12 234 EVQLVESGGG LVQAGGSLRL SCAASGFTFD DYAIG WFRQAPGKER EGVS CISSSD DSTYYA DSVKG RFTISRNNAKNTVY LQMNSLKPEDTAVY YCAV RLFSGGCAV WGTSWADF GS SGQGTQVTVSS
DLLBII1 7B03 235 EVQLVESGGG LVQPGGSLRL SCAASGFTFE NYAL G WFRQAPGKER EWVS CISSSD GTTYYA DSVKG RFTISRDNVKNTVY LQMNRLKPEDTAIY YCAL SLGSSWCAY DY WGQGTQVTVSS
DLLBII1 7B09 236 EVQLMESGGG LVQAGGSLRL SCAASGFTFD DYAIG WFRQAPGKER EGVS CISSYD GTTYYA DSVKG RFTISSDNAKNTVY LQMNSLKPEDTAVY YCAA DPLVCGYND PRLADY WGQGTQVTVSS
DLLBII1 7C01 237 EVQLVESGGG LVQAGGSLRL SCAASGFTFD DYPIG WLRQAPGKER EGVS CISSSD DSTYYA DSVKG RFTISSNNAKNTVY LQMNSLKPEDTAVY YCAV RLFSGGCAV VAGTSWADF GS SGQGTQVTVSS
DLLBII1 7008 238 EVQLVESGGG LVQAGGSLRL SCAASGSTFS SYAM G WYRQAPGKQ RELVA VISSGD RTNYLD SVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCFY SGSYYYPTD VHEYAY WGQGTQVTVSS
-Z.6-
DLLBII1 7E04 239 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CISSGD GSTYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFEYYSAYC GVNRYDY WGQGTQVTVSS
DLLBII1 7F10 240 EVQLVESGGG LVQAGGSLRL SCASSGRTLL NYAM G WFRQAPGKER EFVS GINWS GGSTY YADSVK G RFTISRDNAENTVY LHMNSLKPEDTAVY YCAA AHDNYWFTD DSLGRGLKY WGQGTQVTVSS
DLLBII1 7G10 241 EVQLVESGGG LVQAGGSLRL SCAASGSTFS SYAM G WYRHQAPGK QRELV AAISSD GSTHYA DSVKG RFTISRDNAKNTMY LQMNSLKPEDTAVY YCNT KTFGSNWYD DY WGQGTQVTVSS
DLLBII1 8D02 242 EVQLVESGGG LVQAGGSLRL SCAASGFTLD YYAIG WFRQAPGKER EGVS CISSRG GSTYYT DSVKG RFTISRDNAKNTVY LQMNSLKPEDTGV YYCAA DPIHNCYSG RYYASPEAV YDY WGQGTQVTVSS
DLLB1I1 8F05 243 EVQLVESGGG LVQAGGSLRL SCAASAFTLD YYAV G WFRQAPGKER EGVS CISSSG GSTYYE DSVKG RFTISRDNAKNTVY LQMNNLKPEDTAV YYCAA DPFHNCYSG SHYSSPEAV YEY WGQGTQVTVSS
DLLBII1 8H08 244 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKGR EWVS CINSSD GSTYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFEYYSDLC GVNGYDY WGQGTQVTVSS
DLLBII1 9B09 245 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CISSSD GRTNY VDSVK G RFTMSRDNAKNTV YLQMNSLKPEDTAV YYCAA PFNYYSDLC GVNGVDY WGQGTQVTVSS
DLLBII1 9D04 246 EVQLVESGGG LVQAGGSLRL SCAASGSTFS SYAM G WYRQAPGNQ RELVA VISSGG STNYAD SVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY CCFY SGSYYYPTD VHEYAY WGQGTQVTVSS
DLLBII1 9D07 247 EVQLVESGGG LVQPGGSLRL SCAASGFTLD NYNIG WFRQAPGKER EWVS CITSSN GSTYYT DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFAHYSDLC GVNGYDY WGQGTQVTVSS
DLLBII1 9D10 248 EVQLVESGGG LVQAGDSLRLS CAASGRTVG SYDM S WVRQGPGKE REWVS SINSGV GKTYYA DSVKG RFTIFRDNAKNMVY LQMNNLKPEDTAV YYCAT EMDGSRYV EGQGTQVTVSS
DLLBII1 9F04 249 EVQLVESGGG LVQAEGSLRLS CAASGSTFS TYAM A WYRQAPGKQ RELVA GISFDG STHYAE SVKG RFTISRDDAKNTVS LQMNSLKPEDAAV YYCYS VHPSTGFGS WGQGTQVTVSS
DLLBII1 9F12 250 EVQLVESGGG LVQAGGSLRL SCTASGSTFT SYAM G WYRQAPGKQ RELVA AISSDD STYYAD CVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCNA PHSDYDEEA PSDFGS WGQGTQVTVSS
DLLBII2 4C07 251 EVQLVESGGG LVQAGGSLRL SCAASGFTFD DYAIG WFRQAPGKER EGVS CISSSD DSTYYA DSVKG RFTISSNNAKNTVY LTMNSLKPEDTAVY YCAV RLFSGGCAV VASTSWADF GS SGQGTQVTVSS
DLLBII2 4D07 252 EVQLVESGGG LVQAGGSLRL SCAASGFTFD DYAIG WFRQAPGKER EGVS CISSSD GSTYYA DSVKG RFTISSNNAKNRAY LQMNSLKPEDTAVY YCAV RLFRGGCAV VAGTSWADF GS SGQGTQVTVSS
DLLBII2 5G05 253 EVQLVESGGG LVQAGGSLRL SCAASGFTFD DYAIG WFRQAPGKER EGVS CISSYD GTTYYA DSVKG RFTISSDNAKNTVY LQMNSLKPEDTAVY YCAA DPLVCGYND PRLADY WGQGTQVTVSS
(Λ
- 100-
DLLBII2 5H07 254 EVQLVESGGG LVQAGGSLRL SCAASGFTLD YYAIG WFRQAPGKER EGVS CISSRG GSTYYE DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA DPIHNCYSG RYYASPDAV YEY WGQGTQVTVSS
DLLBII2 6C02 255 EVQLVESGGG LVQAGGSLRL SCAASGFTLD YYAIG WFRQAPGKER EGVS CISSRG SSTYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA DPIHNCYSG NGYDSPEAV YDY WGQGTQVTVSS
DLLBII2 6H11 256 EVQLVESGGG LVQAGGSLRL SCTASGFTLD YYAIG WFRQAPGKER EGVS CISSSG GSTYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA DPFHNCYSG SAYSSPEAV YEY WGQGTQVTVSS
DLLBII2 8B06 257 EVQLVESGGG LVQAGGSLRL SCAASGSTFS TYAM G WYRQDPGNQ RELVA AISSDG STHYAD SVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCYA PVKVAGLEY DY WGQGTQVTVSS
DLLBII3 3A06 258 EVQLVESGGG LVQPGGSLRL SCAASGFTLD NYAIG WFRQAPGKER EWVS CISGFD GSTYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA SVGSSWCAY DY WGQGTQVTVSS
101 -
DLLBII3 3A10 259 EVQLVESGGG LVQPGGSLRL SCAASGFTFE NYAL G WFRQAPGKER EWVS CISSSD GTTYYA DSVRG RFTISRDNAKNTVY LQMNRLKPEDTAIY YCAL SLGSSWCAY DY WGQGTQVTVSS
DLLBI13 3C05 260 EVQLVESGGG LVQAGGSLRL SCAASGFTLD NYVIG WFRQAPGKER EEVS CISSSG GSTDYL DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA DSLPCYYDK MVYDY WGQGTQVTVSS
DLLBII3 3C09 261 EVQLVESGGG LVQAGGSLRL SCTASGFKLD YYVIG WFRQAPGKER EGVS CTSSSG GSTYYA DSVKG RFTISRDNAKNTVY LQMHSLKPEDTAVY YCAA DSFACDYGK MIYDY WGQGTQVTVSS
DLLBII3 3C10 262 EVQLVESGGG LVQPGGSLRL SCAASGFGFD NYAM G WFRQAPGKER EWVS CISGSD GSTYYA DSVKG RFTISRDNAKNTVY LQMHSLKPEDTAVY YCAA SLGSSWCAY DY WGQGTQVTVSS
DLLBII3 3D02 263 EVQLVESGGG LVQAGGSLRL SCSASGFTFD DYAIG WFRQAPGKER EGVS CISSHD GTTYYA DSVKG RFTISSDNAKNTVY LQMNSLKPEDTAVY YCAA DPLVCGYND PRLADY WGQGTQVTVSS
- 102-
DLLBII3 3E01 264 EVQLVESGGG LVQAGGSLRL SCAASGSTFS SYAM G WYRQAPGKQ RELVA AISNGG STNYVD SVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCFY SGSYYYPTD VHEYDY WGQGTQVTVSS
DLLBII3 3E03 265 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CISSSD GRTNY VDSVK G RFTMSRDNAKNTV YLQMNSLKPEDTAV YYCAA PFNYYSNLC GVNGVDY WGQGTQVTVSS
DLLBII3 3F01 266 EVQLVESGGG LVQAGGSLRL SCAASGFSLD YYAIG WFRQAPGKER EGIS CISGRG GSTYYI DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA DPIHNCYSG SHYYSPEAV YEY WGQGTQVTVSS
DLLB1I3 3F04 267 EVQLVESGGG LVQAGDSLRLA CAASGFALD YYAV G WFRQAPGKER EGVS CISSRG GSTFYA DSLKG RFTTSRDNAKNTVY LQMNSLKPEDTAVY YCAA DPIHNCYSG SDYASPEAV YEY WGQGTQVTVSS
DLLBII3 3H04 268 EVQLVESGGG LVQPGDSLRLS CAASGFTLD YYNIG WFRQAPGKER EWVA CIRSSD GSTYYT DSVKG RFTISRNNAKNTVY LQMNSLKPEDTAVY YCAA PFIHYSDLCG VNGNDY WGQGTQVTVSS
- 103 -
DLLBII4 2A08 269 EVQLVESGGG LVQAGGSLRL SCAASGSTFN SYAM G WYRQAPGKQ RELVA VISSGS VTNYAD SVKG RFTISRDNAKNTVS LQMNSLKPEDTAVY YCFY SGSYYYPTD VHEYDY WGQGTQVTVSS
DLLBII4 2B07 270 EVQLVESGGG LVQPGGSLRL SCAASGFRLD YYAIG WFRQAPGKER EWVS CMGSS VRSTYY ADSVK G RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA APIFECPSGE IYDY WGQGTQVTVSS
DLLB1I4 2B10 271 EVQLVESGGG LVQAGGSLRL SCAASGFTLD YYAIG WFRQAPGKER EGVS CISSRG GSTYYT DSVKG RFTISRDNAKNTVY LQMNSLKPEDTGV YYCAA DPIHNCYSGT YYASPEAVY EY WGQGTQVTVSS
DLLBI14 2F08 272 EVQLVESGGG LVQAGGSLRL SCAASGFALD YYAV G WFRQAPGKER EGVS CISSRG GSTYYV DSVKG RFTTSRDNAKNTVY LEMNSLKPEDTAVY YCAA DPIHNCYSG SYYASPEAV YDY WGQGTQVTVSS
DLLBII4 2G04 273 EVQLVESGGG LVQAGGSLRL SCAASGSTFS SYAM G WYRQAPGKQ RELVA VISSGD STNYSD SVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCFY SGSYYYPTD VHEYAY WGQGTQVTVSS
104-
DLLBII4 3A05 274 EVQLVESGGG LVQAGGSLRL SCAASGSSFS SYAM G WYRQAPGKQ RELVA VISSGD RTNYLD SVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCFY SGSYYYPTD VHEYAY WGQGTQVTVSS
DLLBII4 3A10 275 EVQLVESGGG LVQPGGSLRL SCAASGFALD YYNIG WFRQAPGKER EWVS CISGSD GSTGY ADSVK G RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFAYYSDLC GVNEYDY WGQGTQVTVSS
DLLBII4 3A12 276 EVQLVESGGG LVQPGGSLRL SCAASGFALD GHNl G WFRQAPGKER EWVS CINSGD GSTGY ADSVK G RFTISRDNAKNTVY LQMNRLKPEDTAV YYCAA PFNHYSFLC GVNEYDY WGQGTQVTVSS
DLLBII4 3B04 277 EVQLVESGGG LVQAGGSLRL SCAASGSTFS SYAM G WYRQAPGKQ RELAA VISTGD NTNYAD SVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY HCFY SGSYYYPTE VYEYDY WGQGTQVTVSS
- 105 -
DLLBII4 3B11 278 EVQLVESGGG LVQAGGSLRL SCAASGSTFR SYAM G WYRQVPGNQ RELVA VISSGD SANYAD SVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCFY SGSYYYPTD VHEYDY WGQGTQVTVSS
DLLBII4 3C12 279 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CINSSD GTTYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFEYYSDLC GVNGYDY WGQGTQVTVSS
DLLBII4 7A05 280 EVQLVESGGG LVQAGGSLRL SCGASGFSLD YYAIG WFRQAPGKER EGVS CISGRG SNTYYL DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA DPIHNCYGG SYYASPEAV YEY WGQGTQVTVSS
DLLBII4 7D01 281 EVQLVESGGG LVQPGGSLRL SCAASGFTFD DYAIG WFRQAPGKER EGVS CISSSG STYYAD SVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAI AGASSWCFP PGY WGQGTQVTVSS
DLLBH4 7E03 282 EVQLVESGGG LVQAGGSLRL SCAASGFALD YYAV G WFRQAPGKER EGVS CISSRG GSTYYA DSVKG RFTTSRDNAKNTVY LQMNSLKPEDTAVY YCAA DPIHNCYSGI YYASPEAVY DY WGQGTQVTVSS
- 106-
DLLB1I4 7E12 283 EVQLVESGGG LVQAGGSLRL SCAASGSTFS SYAM G WYRQAPVKQR ELVA VISNGG STNYAD SVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCFY SGSYYYPTD VHEYDY WGQGTQVTVSS
DLLBII4 7F06 284 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYAIG WFRQAPGKER EGVS CISSRG GSTYYE DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA DPIHNCYSG RYYASPDAV YDY WGQGTQVTVSS
DLLBII4 7G11 285 EVQLVESGGG LVQPGGSLRL SCAASEFTLD HYNIG WFRQAPGKER EWVS CISSSD GSTGY ADSVK G RFTISRDKAKNTVY LQMNSLKPEDTAVY YCAA PFSYYSDLC GVNGYDY WGQGTQVTVSS
DLLBII4 7H02 286 EVQLVESGGG LVQAGGSLRL SCAASGSTFS SYAM G WYRQAPGKQ RELVA VISSGD STNYAD SVKG RFTMSRDNAKNTV YLQMNSLRPEDTA VYYCFY SGSYYYPSD VHEYDY WGQGTQVTVSS
DLLBII4 8A01 287 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CISSSD GSTDYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFSYYSGLC GVNGVDY WGQGTQVTVSS
- 107-
DLLBII4 8A08 288 EVQLVESGGG LVQPGGSLRL SCAASGFTLG VYAT G WFRQAPGKER EWVS CISGSD GSTWY ADSVK G RFTISRDNAKNTVY LQMNSPKSEDTAV YYCAL SLGSSWCAY DY WGQGTQVTVSS
DLLBII4 8E03 289 EVQLVESGGG LVQAGGSLRL SCAASGFTLD YYAIG WFRQAPGKER EGVS CISGRG GSTYYT DSVKG RFTISRDNAKNTVY LQMNSLKPEDTGV YYCAA DPVHNCYSG RYYASPDAV YEY WGQGTQVTVSS
DLLBII4 8F05 290 EVQLVESGGG LVQPGGSLRL SCAASGSTFS SYAM G WYRQAPGKQ RELVA VISNGG STNYAD SVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY ICFY SGSYYYPTD VHEYAY WGQGTQVTVSS
DLLBII4 9A12 291 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YHNIG WFRQAPGKER EWVS CISSSG GSTAYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFSHYNDLC GVNAIDY WGQGTQVTVSS
DLLBII4 9B05 292 EVQLVESGGG LVQAGGSLRL SCAASGFTLD YYAIG WFRQAPGKER EGVS CISSRG ASTYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA DPIHNCYSG NGYDSPEAV YDY WGQGTQVTVSS
-801
DLLBII4 9E01 293 EVQLVESGGG LVQPGGSLRL SCAASGFTLH YYNIG WFRQAPGKER EWVS CINSSD GSTHYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFEYYSDLC GVNGYDY WGQGTQVTVSS
DLLBII4 9F05 294 KVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CINSSD GSTYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFEYYSNLC GVNGYDY WGQGTQVTVSS
DLLBII4 9G02 295 EVQLVESGGG LVQAGGSLRL SCAASGFTLD KYSIG WFRQAPGKER EGVS CISSSG GSTYYV DSVKG RFTISRDNAKNTVY LQMNNLKPEDTAV YYCAA DPLHNCYSG RGYYSPEAV YEY WGQGTQVTVSS
DLLBI14 9G05 296 EVQLVESGGG LVQAGGSLRL SCAASGFTLD YYAIG WFRQAPGKER EGVS CISSRG GSTYYT DSVKG RFTISRDNAKNTVY LQMNSLKPEDTGV YYCAA DPIHNCYSG SYYASPEAV YEY WGQGTQVTVSS
DLLBII4 9H05 297 EVQLVESGGG LVQPGGSLRL SCAASGFTLD YYNIG WFRQAPGKER EWVS CINSSD GSTYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PFEYYSNLC GVNGYDY WGQGTQVTVSS
- 109-
DLLBII5 5A07 298 EVQLVESGGG LVQPGGSLRL SCTASGFTFD DYAIG WFRQAPGKEP EGIS CISSSG SITYDA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAT PGIAACRGIH Y WGQGTQVTVSS
DLLBII5 5D12 299 EVQLVESGGG LVQPGGSLRL SCAASGFTFD DYAIG WFRQAPGKEP EGIS CISSSG GITYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAT PGIAACRGIH Y TGQGTQVTVSS
DLLBII5 6A09 300 EVQLVESGGG LVQPGGSLRL SCAASGFTFD DYAIG WFRQAPGKEP EGIS CISSSG GITYYA DSVKG RFTTSRDNAKNTVY LQMNSLKPEDTAVY YCAT PGIAACRGIH Y TGQGTQVTVSS
DLLBII5 6C04 301 EVQLVESGGG LVQPGGSLRL SCTASGFTFD VYAIG WFRQAPGKEP EGIS CISSSG SITYYA DSVKG RFTTSRDSAKNTVY LQMNSLKPEDTAVY YCAT PGIAACRGIH Y WGQGTQVTVSS
DLLBII5 6H08 302 EVQLVESGGG LVQPGGSLRL SCAASGFTFD DYAIG WFRQAPGKEP EEIS CISSSG GITYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAT PGIAACRGIH Y TGQGTQVTVSS
-110-
DLLBII5 8A11 303 EVQLVESGGG LVQAGGSLRL SCTTSERTVS RYSM G WFRQAPGKER EAVA TISWSG DSTYYA DSVKG RFTISRDNTKNTLYL QIDSLKPEDTAVYY CVA KPNLKYGSY WPPRGYDY WGQGTQVTVSS
DLLB1I5 8B01 304 EVQLVESGGG LVQAGGSLRL SCTTSERTVS RYSM G WFRQAPGKER EAVA TISWSG DSTYYA DSVKG RFTISRDNTKNMLY LQMNSLKPEDTAVY YCVA KPNLKYGST WPPRGYDY WGQGTQVTVSS
DLLBII5 9B01 305 EVQLVESGGG LVQAGGSLRL SCTTSERAVS RYTM G WLRQAPGKER EAVA TISWSG DSTYYA DSVKG RFTISRDNTKNTLYL QMNSLKPEDTADY YCAA KPNLKYGSY WPPRGYDY WGQGTQVTVSS
DLLBII5 9B11 306 EVQLVKSGGG LVQAGGSLRL SCTTSERTVS RYGM G WFRQAPGKER EAVA TISWSG DSTYYA DSVKG RFTISRDNTKNTLYL QMNSLKPEDTAVY YCAA KPNLKYGSD WPPRGYDY WGQGTQVTVSS
DLLBII6 1F05 307 EVQLVESGGG LVQPGGSLRL SCTASGFTFS SYAM S WVRQAPGKGL EWVS FINKDG SDTGYA DSVKG RFTISRDNAKNTMY LQMNSLKPEDTAVY FCET RTSRSPRP RGQGTQVTVSS
DLLBII6 1F07 308 EVQLVESGGG LVQAGGSLRL SCAASGRTFS RYAM G WFRQAPGKER EFVA AINWSG GSTYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY DCAA SNYYSVYDD RPVMDY WGQGTQVTVSS
DLLBII6 2C11 309 EVQLMESGGG LVQPGGSLRL SCVAAGFTFS NYYM S WVRQAPGKGL EWVS VISPDG SNTYYA DTVKG RFTISRGNAKNTLF LQMTGLKSEDAAV YYCAR GSGSWGV HGQGTQVTVSS
DLLBII7 8B03 310 EVQLVESGGG LVQAGGSLRL SCAASGRTFS NYIM G WFRQAPGKER EFVA GISRYG DYTAYA DSVKG RFTISRDNVKNTVY LRMNSLKPDDTAVY YCAA NEGYCSGYG CYEDSGQYD Y WGQGTQVTVSS
DLLBII7 8B04 311 EVQLVESGGG LVQAGGSLRL SCAASGRTFS NYIM G WFRQAPGKER EFVA GISRYG DYTYYA DSVKG RFTISRDNVKNTVY LRMNSLKPDDTAVY YCAA NEGYCSGYG CYEDSGQYD Y WGQGTQVTVSS
DLLBII8 0E08 312 EVQLVESGGG LVQAGGSLTLS CAASGGTFT TYAM G WFRQAPGKER EFVA AVSRFG VSWDY ADSVK G RFTISRDNTANTLKL RMNSLKADDTAVY YCAA GGRSFLPFV PAY WGQGTQVTVSS
112-
DLLBII8 3G01 313 EVQLVESGGG LVQAGGSLRL SCAASGRTFS NYIM G WFRQAPGKER EFVA GISRYA DYTGYA DSVKG RFTISRDNVKNTVY LRMNSLKPDDTAVY YCAA NEGYCSGYG CYEDSGQYD Y WGQGTQVTVSS
DLLBII8 3G04 314 EVQLVESGGG LVKPGGSLRLS CAASGRTLY IMG WYRQAPGKER EFVA GISRYG DITYAA DSVKG RFTISRDSVKNTVY LRMNSLKPDDTAVY YCAA NGGYCSGYG CYEDSGQYD Y WGQGTQVTVSS
DLLBII8 7B06 315 EVQLVESGGG LVQAGGSLRL SCAASGFTFD DYAIG WFRQAPGKEP EGIS CISSSG GITYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAT PGIAACRGIH Y TGQGTQVTVSS
DLLBII8 9B04 316 EVQLVESGGG LVQPGGSLRL SCAASGFTFD DYAIG WFRQAPGKEP EEIS CISSSG GITYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAT PGIAACRGIH Y TGQGTQVTVSS
DLLBII9 0E10 317 EVQLVESGGG LVQAGGSLRL SCAVSGFSFD DYAIG WFRQAPGKEP EGIS CISSSG GITYYA DSVKG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAT PGIAACRGIH Y TGQGTQVTVSS
-113-
DLLBII9 5A01 318 EVQLVESGGG LVQPGGSLRL SCAASGFTFG SYDM S WVRQAPGKGP EWVS AINSGG GSTYYA DSVKG RFTISRDNAKNTLY LQMNSLKPEDTAVY YCAI PRGWGPTG PIEYAY WGQGTQVTVSS
DLLBII9 5B03 319 EVQLVESGGG LVQPGGSLRL SCAASGFTFG SYDM S WVRQAPGKGP EWVS AINSGG GSTYYA DSVKG RFTISRDNAKNTLY LQMNSLKPEDTAVY YCAI PRGWGPTG PIEYGY WGQGTQVTVSS
DLLBII9 5C03 320 EVQLVESGGG LVQPGGSLRL SCAASGFTFG SYDM S WVRQAPGKGP EWVS AINSGG GSTYYA DSVKG RFTISRDNAKNTLY LQMNSLKPEDTGV YSCAI PRGWGPTG PHEYGY WGQGTQVTVSS
DLLBII9 5D02 321 EVQLVESGGG LVQSGGSLRL SCAASGFTFG SYDM S WVRQAPGKGP EWVS AINSGG GSTYYA DSVKG RFTISRDNAKNTLY LQMNSLKPEDTAVY YCAI PRGWGPTG PHEYAY WGQGTQVTVSS
DLLBII9 5F02 322 EVQLVESGGG SVQAGGSLRL SCAASGRTFS SYAM G WFRQAPGKER EFVA AINWSG GYTYYA DSVRG RFTISRDNAKNTVY LQMNSLKPEDTAVY YCAA PAPGSSGYE YDY WGQGTQVTVSS
-114-
DLLBII9 5F03 323 EVQLVESGGG LVQPGGSLRL SCTASGFTFD VYAIG WFRQAPGKEP EGIS CISSSG SITYYA DSVKG RFTISRDSAKNTVY LQMNSLKPEDTAVY YCAT PGIAACRGIH Y WGQGTQVTVSS
DLLBII9 5H02 324 EVQLVESGGG LVQPGGSLRL SCAASGFTFG NYDM S WVRHAPGKGP EWVS AINSGG GSTYYT DSVKG RFTISRDNAKNTLY LQMNSLKPEDTAVY YCAI PRGWGPTG PHEYAY WGQGTQVTVSS
DLLBII9 6C02 325 EVQLVESGGG LVQPGGSLRL SCAASGFTFG SYDM S WVRQAPGKGP EWVS AINSGG GTTYYA DSVKG RFTISRDNAKNTLFL QMNSLKPEDTAVY YCAI PRGWGPTG PLEYGY WGQGTQVTVSS
DLLBII9 6C03 326 EVQLVESGGG LVQPGGSLRL SCAASGFTFG NYDM S WVRQAPGKGP EWVS AINSGG GDTYYA DSVKG RFTISRDNAKNTLY LQMNSLKPEDTAVY YCAT PRGWGPTG PHEYGY WGQGTQVTVSS
DLLBI19 6F02 327 EVQLVESGGG LVQPGGSLRL SCAASGFTFG NYDM S WVRQAPGKGP EWVS AINSGG GITYYA DSVKG RFAISRDNAKTTLYL QMNNLQPEDTAVY YCAT PRGWGPTG PHEYGY WGQGTQVTVSS
115 -
DLLBII9 6H02 328 EVQLVESGGG LVQAGGSLRL SCAASGFTFG SYDM S WVRQAPGKGP EWVS AINSGG GITYYA DLVKG RFTISRDNAKNTLY LQMNSLKPEDTAVY YCAI PRGWGPTG PHEYGY WGQGTQVTVSS
DLLBI19 7B02 329 EVQLVESGGG LVQPGGSLRL SCAASGFTFG NYDM S WVRQAPGKGP EWVS AINSGG GITYYA DSVKG RFTISRDNAKNTLY LQMNSLKPEDTAVY YCAT PRGWGPTG PHEYGY WGQGTQVTVSS
DLLBII9 7D01 330 EVQLVESGGG LVQPGGSLRL SCAASGFTFG SYDM S WVRQAPGKGP EWVS AINSGG GSTYYA DSVKG RFTISRDNAKNTLY LQMNSLTPEDTAVY YCAI PRGWGPTG PHEYAY WGQGTQVTVSS
DLLBI19 7E01 331 EVQLVESGGG LVQPGGSLRL SCTASGFTFG NYDM S WVRRPPGKGP EWVS AINSGG GSTYYA DSVKG RFTISRDNAKNTLY LQMNSLKPEDTAVY YCAT PRGWGPTG PHEYGY WGQGTQVTVSS
DLLBII9 7E02 332 EVQLVESGGG LVQPGGSLRL SCAASGFTFG SYDM S WVRQAPGKGP EWVS AINSGG GSTYYA DSVKG RFTISRDNAKNTLY LQMNNLKPEDTAV YSCAI PRGWGPTG PHEYAY WGQGTQVTVSS
Example 5
Characterization of purified anti-DII4 VHHs
Inhibitory anti-DII4 VHHs selected from the screening described in Example 4 are further purified and characterized. Selected VHHs are expressed in E. coli TGI as c-myc, His6-tagged proteins. Expression is induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 37’C. After spinning the cell cultures, periplasmic extracts are prepared by freeze-thawing the pellets. These extracts are used as starting material and VHHs are purified via IMAC and size exclusion chromatography (SEC) resulting in 95% purity as assessed via SDS-PAGE.
5.1. Evaluation of DII4 blocking VHHs in ELISA
The blocking capacity of the VHHs is evaluated in a human DII4 - human Notch1/Fc blocking ELISA. In brief, 1 pg/mL of human Notch1/Fc chimera (R&D Systems, Minneapolis, MN, USA) is coated in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). A fixed concentration of 15 nM biotinylated human DII4 is preincubated with a dilution sériés ofthe VHH for 1 hour, afterwhich the mixture is incubated on the coated Notchl receptor for an additional hour. Residual binding of biotinylated human DII4 is detected using horseradish peroxidase (HRP) conjugated extravidin (Sigma, St. Louis, MO, USA) (Figure 3). Human DII4 is biotinylated as described above. The IC50 values for VHHs blocking the human DII4 - human Notchl/Fc interaction are depicted in Table 6, v-·
-11616772
Table 6: IC50 (nM) values for VHHs in hDLL4/hNotch1-Fc compétition ELISA
VHH ID ICS0 (nM)
6B11 1.5
55D12 12.3
56A09 4.9
56C04 33.9
56H08 6.9
57C11 17.3
62C11 72.0
96C03 38.4
101G08 9.5
104G01 1.1
115A05 9.1
antiDLL4 Fab 0.7
5.2. Evaluation ofDII4 blocking VHHs in AlphaScreen
In brief, 1 nM biotinylated human DII4 is captured on streptavidin-coated donor beads (20 pg/mL), while 0.4 nM of the receptor human Notchl (as a Fc fusion 5 protein) is captured on anti-human Fc VHH-coated acceptor beads (20 pg/mL).
Both loaded beads are incubated together with a dilution range of the competing VHH (Figure 4). The IC50 values for VHHs blocking the human DII4 - human Notchl/Fc interaction are depicted in Table 7. v-11716772
Table 7: IC50 (nM) values for VHHs in hDLL4/hNotch1 compétition AlphaScreen
VHH ID IC50 (nM)
5B11 0.7
6B11 0.3
7A02 0.4
7B05 1.1
8A09 0.4
8C11 0.7 (a)
19F04 0.05 (a)
55D12 2.3
56A09 1.2
56C04 5.4
56H08 1.6
57C11 2.2
62C11 24.1
115A05 5.0
antiDLL4 Fab 0.3
(a) partial inhibitor
-11816772
5.3. Inhibition by anti-DII4 VHHs of human Notch1/Fc binding to human or mouse
DII4 expressed on the CHO cells
The blocking capacity of the VHHs is evaluated in a human and mouse DII4 human Notch1/Fc compétitive FMAT assay (Figure 5) as outlined in Example 4.
The IC50 values for VHHs blocking the interaction of human Notch1/Fc to human or mouse DII4 expressed on CHO cells are depicted in Table 8.
Table 8: (Mean) IC50 values (nM) of purified VHHs blocking the interaction of human Notch1/Fc to human or mouse DLL4 expressed on CHO cells (FMAT)
hDLL4 mDLL4
VHH ID IC50 (nM) IC50 (nM)
6B11 8.9 -
8A09 5.5 -
19F04 33.0 -
55D12 39.1 41.0
56A09 10.6 15.0
56C04 28.7 49.6
56H08 22.0 33.7
57C11 53.9 49.5
62C11 172.2 106.3
96C03 160.8 28.8
101G08 24.6 92.1
104G01 2.5 -
115A05 22.0 43.0
antiDLL4 Fab 5.4 2.3
- 119 16772
5.4. Evaluation of DII4 blocking VHHs in reporter assay
To evaluate the potency of the selected VHHs, a reporter assay is set up which is based on the γ-secretase mediated cleavage of Notchl and release of the intracellular domain of Notchl (NICD) upon stimulation with DII4. The Notchl 5 GAL4/VP16 construct is cotransfected with the pGL4.31 [Luc2P/Gal4UAS/Hygro] reporter plasmid in HEK cells resulting in a transient expression of the fusion protein. These transiently transfected cells are stimulated for 24 hours by coculture with a HEK293-hDII4 stable cell line. Forty-eight hours post-transfection, the readout is performed. The VHHs are preincubated with the HEK293-hDII4 cells îo 1 hour before the start of the co-culture and are included during the co-culture (Figure 6). The IC50 values of the VHHs for blocking the DII4-mediated cleavage of Notchl and subséquent translocation of its NICD to the nucléus of the receptor cell are depicted in Table 9.
Table 9: (Mean) IC50 values (nM) of purified anti-DII4 VHHs in a DLL4/Notch1 15 reporter assay
VHH ID IC50
56A09 540
62C11 4663
96C03 5156
101G08 2760
104G01 964
115A05 1740
anti-DLL4 Fab 133
- 120 16772
5.5. Epitope binning
In order to détermine whether VHHs can bind simultaneously to DII4 when e.g. a benchmark antibody is bound, epitope binning experiments are carried out (via Surface Plasmon Résonance (SPR) on a Biacore T100 instrument). Anti-DII4 Fab fragment is irreversibly immobilized on the reference and on the active flow cell of a CM5 sensor chip. For each sample (cycle), human DII4 is injected on the active and reference flow cell and reversibly captured by anti-DII4 Fab. Additional binding of VHHs is evaluated by injection over the immobilized surface. Ail VHHs and antiDII4 Fab are injected at 100 nM with a surface contact time of 120 seconds and a flow rate of 10 uL/minute. Surface is regenerated using 10 mM glycine (pH1.5). Processed curves are evaluated with Biacore T100 Evaluation software.
Table 10-A représente the sequential injection/regeneration path of analysed VHHs and controls. VHHs DLLBII56A09 (SEQ ID NO: 300), DLLBII96C03 (SEQ ID NO: 326), DLLBII101G08 (SEQ ID NO: 197) and DLLBII115A05 (SEQ ID NO: 224) are shown not to additionally bind to human DII4 captured by DII4 Fab. Injection of DII4 Fab also failed to additionally bind human DII4 indicating that ail epitopes are saturated. Therefore, it can be concluded that these VHHs recognize an epitope overlapping with DII4 Fab for binding human DII4. Human-only VHHs DLLBII6B11 (SEQ ID NO: 174) and DLLBII104G01 (SEQ ID NO: 215) show additional binding on DII4 Fab captured human DII4, indicating that these VHHs that are spécifie for human DII4 recognize a different epitope than the human/mouse cross-reactive VHHs.
- 121 16772
Table 10-A: Epitope binning of anti-DLL4 VHHs - simultaneous binding with
DLL4 Fab
Injection step Binding/ Régénération [sample ] Binding level (RU)
1 hDLL4 100 nM 1727
2 DLL4 Fab 100 nM no binding
3 59A9 100 nM no binding
4 6B11 100 nM 405
5 Glycine pH1.5 10 mM 90
6 hDLL4 100 nM 1349
7 104G1 100 nM 276
8 Glycine pH1.5 10 mM 87
9 hDLL4 100 nM 1336
10 Glycine pH1.5 10 mM 70
11 hDLL4 100 nM 1333
12 96C3 100 nM no binding
13 101G8 100 nM no binding
14 115A05 100 nM no binding
15 Glycine pH1,5 10 mM 70
- 122 16772
5.6. Epitope mappîng using DII4 délétion mutants
Binding ofthe VHHs to these DII4 mutants is assessed in Biacore. In brief, VHHs DLLBII101G08 (SEQ ID NO:197) and DLLBII115A5 (SEQ ID NO: 224) are coated on a CM4 Sensorchip and 200 nM of each délétion mutant is injected across the chip. Binding is qualitatively assessed. No binding of DLLBII56A09 (SEQ ID NO: 300), DLLBII101G08 (SEQ ID NO: 197) and DLLBII115A05 (SEQ ID NO: 224) is observed to human and mouse DII4 mutants hDII4.1 and mDH4.8, respectively, lacking EGF-like 2 domain (Table 10-B). Indirect evidence using a hDII4/DII4 IgG compétitive ELISA already pointed to this observation. In brief, 1 pg/mL of DII4 IgG is coated in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). A fixed concentration of 6 nM biotinylated human DII4 is preincubated with a dilution sériés of the VHH for 1 hour, after which the mixture is incubated on the coated IgG for an additional hour. Residual binding of biotinylated human DII4 is detected using horseradish peroxidase conjugated extravidin (Sigma, St. Louis, MO, USA) (data not shown). Human DII4 is biotinylated as described above. It is known from patent literature that the monoclonal anti-DII4 IgG (Genentech,
US 2008/0014196A1 ) binds to an epitope within the EGF-like 2 domain of DII4.
Table 10-B: Epitope mapping of anti-DLL4 VHHs - binding to DLL4 délétion mutants
DLLBII56A9 DLLBII101G8 DLLBII115A5
sample Binding (RU) kd (1/s) Binding (RU) kd (1/s) Binding (RU) kd (1/s)
hDLL4 281 9.5E-04 373 2.0E-03 324 3.5E-03
mDLL4 389 1.9E-03 502 6.0E-03 344 6.5E-03
hDLL4.1 no binding no binding no binding
- 123 16772
hDLL4.3 125 7.4E-04 198 4.65E-03 137 3.5E-03
hDLL4.5 143 1.2E-03 266 2.19E-03 162 4.2E-03
hDLL4.6 136 1.1E-03 229 2.20E-03 152 4.1E-03
mDLL4.8 no binding no binding no binding
mDLL4.10 141 1.1E-03 189 5.14E-03 121 3.8E-03
mDLL4.11 132 1.6E-03 210 6.16E-03 121 6.6E-03
mDLL4.12 161 1.3E-03 244 4.52E-03 152 3.1E-03
5.7. Determining the affinity ofthe hDII4 - VHH interaction
Kinetic analysis to détermine the affinity ofthe DII4 - VHH interaction is performed by Surface Plasmon Résonance (SPR) on a Biacore T100 instrument.
Recombinant human DII4 is immobilized onto a CM5 qhip via amine coupling using EDC and NHS) or biotinylated human DII4 is captured on a SA chip (streptavidin surface). Purified VHHs or Fab fragment are injected for 2 minutes at different concentrations (between 10 and 300 nM) and allowed to dissociate for 20 min at a flow rate of 45 μΙ/min. Between sample injections, the surfaces are regenerated îo with 10 mM glycine pH1.5 and 100 mM HCl. HBS-N (Hepes buffer pH7.4) is used as running buffer. If possible, data are evaluated by fitting a 1:1 interaction model (Langmuir binding) onto the binding curves. The affinity constant Ko is calculated from resulting association and dissociation rate constants (ka) and (kq). The affinities of the anti-DII4 VHHs are depicted in Table 11. v—
- I2416772
Table 11: Affinity KD (nM) of purified VHHs for recombinant human DLL4
rhDLL4
VHH ID ka (M'1.s’1) kd (s‘1) Ko(nM)
56A09 1.7E+05 9.3E-04 5.6
56C04 1.1E+05 4.9E-03 45
56H08 1.2E+05 1.1E-03 9.4
62C11 1.2E+06 1.3E-01 120
96C03 1.6E+05 4.8E-02 310
101G08 4.3E+04 2.2E-03 52
104G01(a) 1.2E+05- 1.5E+05 3E-03 - 6E-04 4-24
115A05 1.5E+05 3.9E-03 25
antiDLL4 Fab 2.3E+05 3.4E-04 1.5
(a) heterogeneous binding curve resulting in no 1:1 fit
5.8. Binding to orthologues (mDII4, cDII4) and family members (hJagged-1,hDLL1)
In order to détermine cross-reactivity to mouse DII4 a binding ELISA is performed.
In brief, recombinant mouse DII4 (R&D Systems, Minneapolis, MS, USA) is coated overnight at 4°C at 1 pg/mL in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1% in PBS). VHHs are applied as dilution sériés and binding is detected using a mouse anti-myc (Roche) and an anti-mouse-AP conjugate (Sigma, St Louis, MO, USA) (Figure 7). As reference, binding to human DII4 is measured. EC50 values are summarized in Table 12.
- 12516772
Table 12: ECgo (nM) values for VHHs in a recombinant human DLL4 and mouse DLL4 binding ELISA
rhDLL4 rmDLL4
VHHID ECso (nM) EC50 (nM)
5B11 1.8 -
6B11 1.4 -
7A02 1.4 -
7B05 7.2 -
8A09 0.9 -
8C11 1.1 -
17F10 0.9 -
19F04 0.9 0.8
55D12 13.1 30.0
56A09 3.6 6.3
56C04 44.3 244.0
56H08 4.1 8.7
57C11 7.9 83.4
62C11 137.0 13.1
96C03 86.5 8.7
101G08 8.9 53.9
104G01 8.4 -
- 126 16772
115A05 5.0 33.4
antiDLL4 Fab 3.0 3.0
In order to détermine the cynomologus cross-reactivity of the VHHs, a FACS binding experiment is performed. Cynomolgus DII4 expressing HEK293 cells (transient or stable transfection) are used for a titration binding experiment of the VHHs. After a 30 minutes incubation on ice, ail samples are washed and détection is performed by applying anti-c-myc~Alexa647 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Human and mouse DII4 overexpressing HEK293 cells are taken as reference. The mean MCF value is determined on the FACS Array and used for calculation of the EC50 value (see Figure 9).
Absence of binding to homologous ligands human DLL1 and human Jagged-1 is assessed via solid phase binding assay (ELISA). In brief, human DLL1 (Alexis, San Diego, CA, USA) and human Jagged-1 (Alexis, San Diego, CA, USA) are coated overnight at 4’C at 1 pg/mL in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1% in PBS). VHHs are applied as dilution sériés and binding is detected using a mouse anti-myc (Roche) and an anti-mouse-AP conjugale (Sigma, St. Louis, MO, USA). Ail anti-DII4 VHHs are considered as being non-cross reactive to these homologous ligands (Figure 8).
5.9. Evaluation of VHHs in blocking DII4- mediated HUVEC prolifération
The potency of the selected VHHs is evaluated in a prolifération assay, as described by Ridgway étal., Nature. 2006 Dec 21 ;444(7122): 1083-7), in modified form. In brief, 96-well tissue culture plates are coated with purified DII4-HÎS (RnD Systems; C-terminal His-tagged human DII4, amino acid 27-524, 0.75ml/well, 10 ng/ml) in coating buffer (PBS, 0.1% BSA). Wells are washed in PBS before 4000 HUVE cells/well are seeded in quadruplicate. Cell prolifération is measured /
- 127 by [3H]-Thymidine incorporation on day 4. The results, shown in Figure 15, demonstrate that the DLL4 VHHs DLLBII101G08, DLLBII104G01, DLLBII115A05, DLLBII56A09 and the DLL4 Fab inhibit the DLL4-dependent effect on HUVEC prolifération in a dose-dependent manner, the IC50 values are summarized in Table 13. The tested VHHs achieve a complété inhibition of the DLL4-dependent effect at 10μΜ.
Table 13 IC50 values obtained in the DLL4 prolifération assay
Fab 56A9 104G1 101G8 115A5
IC50 (nM) (experiment 1) 4.9 11.0 103 401 10002
IC50 (nM) (experiment 2) 5.6 6.8 32 112 N.D.
n 2 2 2 2 1
Example 6
Affinity maturation of selected anti-DII4 VHHs
VHHs DLLBII101G08 and DLLBII115A05 are subjected to two cycles of affinity maturation.
In a first cycle, amino acid substitutions are introduced randomly in both framework (FW) and complementary determining régions (CDR) using the errorprone PCR method. Mutagenesis is performed in a two-round PCR-based approach (Genemorph II Random Mutagenesis kit obtained from Stratagene, La Jolla, CA, USA) using 1 ng of the DLLBII101G08 or DLLBII115A05 cDNA template, followed by a second error-prone PCR using 0.1 ng of product of round 1. After a polish step, PCR products are inserted via unique restriction sites into a vector designed to facilitate phage display of the VHH library. Consecutive
- 12816772 rounds of in-solution sélections are performed using decreasing concentrations of biotinylated recombinant human DLL4 (biot-rhDLL4) and trypsin elutions. Affinitydriven sélections in a third round using cold rhDLL4 (at least 100x excess over biot-rhDLL4) are also performed. No sélections on murine DLL4 are included as (conservation of) cross-reactivity is assessed at the screening level. Individual mutants are produced as recombinant protein using an expression vector derived from pUC119, which contains the LacZ promoter, a résistance gene for ampicillin, a multiple cloning site and an ompA leader sequence (pAX50). E. coliTGî cells are transformed with the expression vector library and plated on agar plates io (LB + Amp + 2% glucose). Single colonies are picked from the agar plates and grown in 1 mL 96-deep-well plates. VHH expression is induced by adding IPTG (1 mM). Periplasmic extracts (in a volume of ~ 80 uL) are prepared according to standard methods and screened for binding to recombinant human and mouse DII4 in a ProteOn (BioRad, Hercules, CA, USA) off-rate assay. In brief, a GLC
ProteOn Sensor chip is coated with recombinant human DII4 on the “ligand channels” L2 and L4 (with L1/L3 as reference channel), while “ligand channels L3 and L6 is coated with mouse DII4. Periplasmic extract of affinity matured clones is diluted 1/10 and injected across the analyte channels A1-A6. An average off-rate is calculated of the wild type clones présent in the plate and served as a reference to calculate off-rate improvements.
In a second cycle, a combinatorial library is created by simultaneously randomising the susceptible positions identified in cycle one. For this, the full length DLLBII101G8 or DLLBII115A05 cDNA is synthesized by overlap PCR using oligonucleotides degenerated (NNS) at the randomisation positions and a rescue
PCR is performed. A list of the primers used for generating the combinatorial library can be found in Table 14 and SEQ ID NOs: 427 to 457. The randomised
VHH genes are inserted into a phage display vector (pAX50) using spécifie restriction sites as described above (Example 2). Préparation of periplasmic extracts of individual VHH clones is performed as described before. __
- 12916772
Table 14: Oligonucleotides affinity maturation libraries
101G08 combinatorial library oligonucleotides 115A5 combinatorial library oligonucleotides
>101G08CL_fwd1-bis gaggtgcaattggtggagtctgggGGTGG TCTGGTTCAGGCTGGT (SEQ ID NO:427) >101G08CL_fwd_2 TCCTGCGCAGCTTCTGGTCGTA CCTTCTCCAGCTACGCGATGG CT (SEQ ID NO:428) >101 G08CL_fwd_3 CCAGGCAAAGAACGCGAGTWC GTAGCCGCAATCCGTTGGAGC GGT (SEQ ID NO:429) >101G08CL_fwd_4 CTGATTCCGTTCAGGGTCGTTT CACCATCTCTCGTGACAACGC G (SEQ ID NO:430) >101G08CL_fwd_5 CTGCAGATGAACTCTCTGAAAC CGGAAGATACGGCAGTCTACT AC (SEQ ID NO:431) >101G08CL_fwd_6-4 >115A05CL_fwd_1 gaggtgcaattggtggagtctgggGGTGGTCT GGTTCAGCCAGGT (SEQ ID NO:443) >115A5CL_rev1 -bis TGAGGAGACGGTGACCTGGGTCCC CTGACCCC (SEQ ID NO:444) >115A05CL_fwd_2 GTGCAGCTTCCGGCTTTACGWTCGG CTCCTACGACATGTCTTGGG (SEQ ID NO:445) >115A05CL1_rev_2 ACGCACCCCAGTATTCACCCTGACG CGCCCAAATGTAGCGATCTGCAGC (SEQ ID NO:446) >115A05CL_fwd_3 AGGTCCGGAATGGGTGTCCKCTATC AACTCTGGTGGTGGTAGCAC (SEQ ID NO:447) >115A05CL_rev_3 TCTTCCGGTTTCAGGCTGTTCATCTG CAGGTACAGCGTGTTTTTG (SEQ ID
- 130 16772
GACACTCGTCTGcgtCCGTACctg TACGACYATTGGGGTCAGGGT A (SEQ ID NO:432) >101G08CL_fwd_6-3 GACACTCGTCTGGvACCGTACct gTACGACYATTGGGGTCAGGGT A (SEQ ID NO:433) >101G08CL_fwd_6-2 GACACTCGTCTGcgtCCGTACG AGTACGACYATTGGGGTCAGG GTA(SEQ ID NO:434) >101G08CL_fwd_6-1 GACACTCGTCTGGVACCGTAC GAGTACGACYATTGGGGTCAG GGTA (SEQ ID NO:435) >101G08CL_rev_2-2 CAGACGAGTGTCcggCGCACGG TTTGCACAGTAGTAGACTGCCG T (SEQ ID NO:436) >101G08CL_rev_2-1 CAGACGAGTGTCTRCCGCACG GTTTGCACAGTAGTAGACTGCC GT (SEQ ID NO:437) >101G08CL_rev_3 NO;448) >115A05CLJwd_4 AAAGGTCGTTTCACCATCTCTCGTGA CAACGCCAAAAACACGCTG (SEQ ID NO:449) >115A05CL_rev_4 TGAAACGACCTTTTWCGWAGTCGGY GTAGWAGGTGCTACCACCAC (SEQ ID NO:450) >115A05CL_fwd_5 TGAAACCGGAAGATACCGCGGTATA CTACTGCGCTGCAGATCGCT (SEQ ID NO:451) >115A05CL_rev_5 CCATTCCGGACCTTTACCCGGAGAA CGACGAACCCAAGACATGTC (SEQ ID NO:452) >115A05CL_fwd_6-1 TACTGGGGTGCGTACGHATACGACT ACTGGGGTCAGGGTAC (SEQ ID NO:453) >115A05CL_fwd_6-2 TACTGGGGTGCGTACcagTACGACTA CTGGGGTCAGGGTAC (SEQ ID NO:454)
- I3I 16772
AGAGTTCATCTGCAGATAGACG GTGTTTTTCGCGTTGTCACGAG A (SEQ ID NO:438) >101G08CL_rev_4
CTGAACGGAATCAGSGTAATAC GCAGTTYCACCGCTCCAACGG AT (SEQ ID NO:439) >101G08CL_rev_5
GCGTTCTTTGCCTGGAGCCTG ACGAWACCAAGCCATCGCGTA GCT (SEQ ID NO:440) >101G08CL_rev_6
AGAAGCTGCGCAGGACAGACG GAGAGAGCCACCAGCCTGAAC CAG (SEQ ID NO:441) >101G08CL_rev1-bis
TGAGGAGACGGTGACCTGGGT CCCCTGACCCCAAT (SEQ ID NO:442) >115A05CL_rev_6
CCGGAAGCTGCACAGCTCAGACGCA
GAGAACCACCTGGCTGAACC(SEQ
ID NO:455) >115A05CL2_rev_2-2
ACGCACCCCAGTAGTAACCCTGACG CGCCCRAATGTAGCGATCTGCAGC (SEQ ID NO:456) >115A05CL2_rev_2-1
ACGCACCCCAGTAKTCACCCTGACG CGCCCRAATGTAGCGATCTGCAGC (SEQ ID NO:457)
Screening for binding to recombinant human DII4 in a ProteOn off-rate assay identifies clones with up to 38-fold (DLLBII101G08) and 11-fold (DLLBII115A05) improved off-rates (Table 15). v—
- 13216772
Table 15: Off-rate screening of DLLBII101G08 and DLLBII115A05 affinity-matured clones.
hDLL4 mDLL4
kd (s1) fold kd (s-1) fold
DLLBII101G08 2.2E-03 1 6.7E-03 1
DLLB1I129D08 5.9E-05 38 1.9E-04 35
DLLBII129H04 6.ΘΕ-05 33 2.5E-04 27
DLLBII129G10 7.3E-05 31 2.6E-04 26
DLLBII129H07 7.4E-05 30 2.5E-04 27
DLLBII129B02 7.6E-05 30 2.6E-04 26
DLLBII129E11 8ΌΕ-05 28 2.5E-04 26
DLLBII130F06 6.5E-05 27 2.6E-04 19
DLLBII130B03 6.7E-05 27 2.4E-04 20
DLLBII129D01 8.5E-05 26 2.6E-04 26
DLLBII130D06 6.9E-05 26 3.1E-04 16
DLLBII129G09 8.8E-05 26 3.4E-04 20
DLLBII129B05 9.3E-05 24 3.4E-04 20
DLLBII130E03 7.5E-05 24 2.7E-04 18
DLLBII129H05 9.4E-05 24 3.5E-04 19
DLLBII130A05 7.5E-05 24 3.0E-04 17
- 133 16772
DLLBII130B02 7.8E-05 23 2.9E-04 17
DLLBII129H02 9.9E-05 23 3.4E-04 19
DLLBII130B04 8.3E-05 22 2.9E-04 17
DLLBII129E07 1.1E-04 21 2.8E-04 24
DLLBII129E03 1.1E-04 20 3.6E-04 18
DLLBII129A03 1.2E-04 19 3.8E-04 18
The best top DLLBII101G08 variants and DLLBII115A05 variants are cloned into expression vector pAXIOO in frame with a C-terminal c-myc tag and a (His)6 tag. Off-rates on recombinant mouse DII4 are also improved. VHHs are produced in
E. coli as His6-tagged proteins and purified by IMAC and SEC. Sequences are represented in Tables 16-A (LLBII101G08) and 16-B (DLLBII115A05), respectively.
- 13416772
135Table 16-A Framework and CDR région sequences of DLLBII101G08 variants
VHH ID SEQ ID NO FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4
DLLBII12 9A03 354 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WFRQAPGK EREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMNSLR PEDTAVYYCA N RAPDTRLR PYLYDY WGQGTQV TVSS
DLLBII12 9B02 355 EVQLVESGGGLV QAGGSLSLSCAA SGRTFS SYAM A WYRQAPGK EREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLE PYEYDH WGQGTQV TVSS
DLLBI112 9B05 356 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WYRQAPGK EREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLA PYEYDH WGQGTQV TVSS
- 136-
DLLBII12 9D01 357 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WFRQAPGK EREYVA AIRWSGGT AYYADSVQ S RFTITRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLE PYLYDH WGQGTQV TVSS
DLLBI112 9D08 358 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WYRQAPGK EREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLE PYLYDY WGQGTQV TVSS
DLLBII12 9E03 359 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WFRQAPGK DREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLA PYLYDY WGQGTQV TVSS
DLLBII12 9E07 360 EVQLVESGGGLV QAGGSLRLSCSA SGRTFS SYAM A WYRQAPGK EREYVA AIRWSGGT AYYPDSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLA PYEYDH WGQGTQV TVSS
DLLBI112 9E11 361 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WYRQAPGK EREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLR PYLYDY WGQGTQV TVSS
- 137-
DLLBII12 9G09 362 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WYRQAPGK EREYVA AIRWSGET AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLE PYLYDH WGQGTQV TVSS
DLLBII12 9G10 363 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WYRQAPGK EREYVA AIRWSGGT AYYADSVQ S RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLE PYEYDH WGQGTQV TVSS
DLLBII12 9H02 364 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WYRQAPGK EREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLR PYEYDY WGQGTQV TVSS
DLLBII12 9H04 365 EVQLVESRGGLV QAGGSLRLSCAA SGRTFS SYAM A WFRQAPGK EREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLE PYLYDH WGQGTQV TVSS
DLLBII12 9H05 366 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WYRLAPGK EREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLG PYLYDY WGQGTQV TVSS
- 138-
DLLBII12 9H07 367 EVQLVESGGGLV QAGGSLRLSCAA SG RTFS SYAM A WYRQAPGK EREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLE PYEYDY WGQGTQV TVSS
DLLBII13 0A05 368 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WYRQAPGK EREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLG PYLYDH WGQGTQV TVSS
DLLBII13 0B02 369 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WFRQAPGK EREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMYSLK PEDTAVYYCA N RAPDTRLA PYLYDH WGQGTQV TVSS
DLLBII13 0B03 370 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WYRQAPGK EREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLA PYLYDY WGQGTQV TVSS
DLLBII13 0B04 371 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WFRQAPGK EREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLR PYLYDH WGQGTQV TVSS
139-
DLLBII13 0D06 372 EVQLVESGGGLV QAGGSLRLSCSA SGRTFS SYAM A WYRQAPGK EREYVA AIRWSGET AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLE PYLYDH WGQGTQV TVSS
DLLBII13 0E03 373 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WYRQAPGK EREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLE PYEYDH WGQGTQV TVSS
DLLBII13 0F06 374 EVQLVESGGGLV QAGGSLRLSCAA SGRTFS SYAM A WYRQAPGK EREYVA AIRWSGGT AYYADSVQ G RFTISRDNAKN TVYLQMNSLK PEDTAVYYCA N RAPDTRLA PYEYDY WGQGTQV TVSS
- 140Table 16-B Framework and CDR région sequences of DLLBII115A05 variants
VHH ID SEQ ID NO FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4
DLLBI11 33A05 396 EVQLVESGGGLV QPGGSLRLSCAA SGFTFG SYDM S WVRRSPGK GPEWVS AINSGGGST FYTDYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY QYDY WGQGTQV TVSS
DLLBII1 33A09 397 EVQLVESGGGLV QPGGSLRLSCAA SGFTFG SYDM S WVRRSPGK GPEWVS AINSGGGST YYADYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY AYDY WGQGTQV TVSS
DLLBII1 33A12 398 EVQLVESGGGLV QPGGSLRLSCAA SGFTFG SYDM S WVRRSPGK GPEWVS AINSGGGST YYTDYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY VYDY WGQGTQV TVSS
- 141 -
DLLBII1 33D06 399 EVQLVESGGGLV QPGGSLRLSCAA SGFTIG SYDM S WVRRSPGK GPEWVS AINSGGGST YYADYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY QYDY WGQGTQV TVSS
DLLBII1 33F01 400 EVQLVESGGGLV QPGGSLRLSCAA SGFTIG SYDM S WVRRSPGK GPEWVS AINSGGGST YYTDYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY VYDY WGQGTQV TVSS
DLLBII1 33F06 401 EVQLVESGGGLV QPGGSLRLSCAA SGFTFG SYDM S WVRRSPGK GPEWVS AINSGGGST YYTDYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY QYDY WGQGTQV TVSS
DLLBII1 33G05 402 EVQLVESGGGLV QPGGSLRLSCAA SGFTFG SYDM S WVRRSPGK GPEWVS AINSGGGST YYTDYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY AYDY WGQGTQV TVSS
DLLBII1 33H03 403 EVQLVESGGGLV QPGGSLRLSCAA SGFTIG SYDM S WVRRSPGK GPEWVS AINSGGGST YYTDYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY AYDY WGQGTQV TVSS
142-
DLLBII1 34B11 404 EVQLVESGGG LV QPGGSLRLSCAA SGFTIG SYDM S WVRRSPGK GPEWVS AINSGGGST YYTDFVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY AYDY WGQGTQV TVSS
DLLBII1 34D10 405 EVQLVESGGGLV QPGGSLRLSCAA SGFTIG SYDM S WVRRSPGK GPEWVS SINSGGGST YYTDYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY AYDY WGQGTQV TVSS
DLLBII1 35H04 406 EVQLVESGGGLV QPGGSLRLSCAA SGFTIG SYDM S WVRRSPGK GPEWVS AINSGGGST YYADYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY QYDY WGQGTQV TVSS
DLLBII1 36C07 407 EVQLVESGGGLV QPGGSLRLSCAA SGFTFG SYDM S WVRRSPGK GPEWVS SINSGGGST YYADYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY EYDY WGQGTQV TVSS
DLLBII1 36D01 408 EVQLVESGGGLV QPGGSLRLSCAA SGFTIG SYDM S WVRRSPGK GPEWVS AINSGGDST FYADYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY AYDY WGQGTQV TVSS
- 143-
DLLBI11 36H03 409 EVQLVESGGGLV QPGGSLRLSCAA SGFTFG SYDM S WLRRSPGK GPEWVS AINSGGGST YYADYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GDYWGAY VYDY WGQGTQV TVSS
DLLBII1 37A04 410 EVQLVESGGGLV QPGGSLRLSCAA SGFTFG SYDM S WVRRSPGK GPEWVS AINSGGGST YYTDYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GDYWGAY AYDY WGQGTQV TVSS
DLLBII1 37A06 411 EVQLVESGGGLV QPGGSLRLSCAA SGFTFG SYDM S WVRRSPGK GPEWVS AINSGGGST YYTDYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIRARQ GEYWGAY AYDY WGQGTQV TVSS
DLLBII1 37B06 412 EVQLVESGGGLV QPGGSLRLSCAA SGFTIG SYDM S WVRRSPGK GPEWVS SINSGGGST YYTDFVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY AYDY WGQGTQV TVSS
DLLBII1 37C04 413 EVQLVESGGGLV QPGGSLRLSCAA SGFTIG SYDM S WVRRSPGK GPEWVS AINSGGGST YYTDYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY EYDY WGQGTQV TVSS
144-
DLLB1I1 37F04 414 EVQLVESGGGLV QPGGSLRLSCAA SGFTIG SYDM S WVRRSPGK GPEWVS SINSGGGST FYTDFVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY AYDY WGQGTQV TVSS
DLLBI11 38F12 415 EVQLVESGGGLV QPGGSLRLSCAA SGFTFG SYDM S WVRRSPGK GPEWVS AINSGGGST YYTDYVKG RFTISRNNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY QYDY WGQGTQV TVSS
DLLBIIO 15 416 EVQLVESGGGLV QPGGSLRLSCAA SGFTIG SYDM S WVRRSPGK GPEWVS AINSGGGST YYADYVKG RFTISRDNAKN TLYLQMNSLK PEDTAVYYCA A DRYIWARQ GEYWGAY AYDY WGQGTQV TVSS
Example 7
Characterization of affinity matured purified anti-DII4 VHHs
Affinity-matured variants of VHHs DLLBII101G08 and DLLBII115A05 are expressed and purified as described above (Example 6). VHHs are characterized in the rhDLLI/ rhJAGI binding ELISA and hDII4/ mDII4/ cynoDII4 FACS (Example 5.8; Table 20; Figure 12 and 13), the rhDII4 - rhNotchl compétition ELISA (Example 5.1; Table 17; Figure 10), the compétition rhNotchl - CHO-hDII4 FMAT (Example 5.3; Table 18;
Figure 11).
Characterization data are summarized in Table 21. Overall, the affinity matured VHHs show clear improvements in affinity and potency, while their binding to mDII4 and cyno DII4 is maintained and no binding to hDLL1 or hJAG1 is observed
Table 17: IC50 (nM) values for affinity matured VHHs in hDLL4/hNotch1-Fc compétition ELISA
VHH ID IC50 (nM)
101G08 10.0
129A03 1.8
129B05 0.9
129D08 1.2
129E11 1.3
129H07 1.0
130B03 1.5
130F06 1.3
anti-DLL4 Fab 1.5
145
VHH ID IC50 (nM)
115A05 7.5
133A05 2.1
133A09 1.5
133G05 2.0
134D10 1.3
136C07 1.4
015 0.9
anti-DLL4 Fab 1.2
Table 18: IC50 values (nM) of purified affinity matured VHHs blocking the interaction of human Notch1/Fc to human or mouse DLL4 expressed on CHO cells (FMAT)
hDLL4 mDLL4
VHH ID IC50 (nM) IC5o{nM)
101G08 69.3 140.5
129B05 7.4 14.4
129D08 7.8 11.0
129E11 8.1 12.3
anti-DLL4 Fab 5.5 3.0
I46
hDLL4 mDLL4
VHH ID IC50 (nM) IC50 (nM)
115A05 106.7 348.9
133A09 6.6 18.6
133G05 5.9 12.0
136C07 8.0 31.2
015 5.7 21.2
anti-DLL4 Fab 3.4 1.6
Table 19: Affrnity KD (nM) of purified affinity matured VHHs on recombinant human DLL4 and mouse DLL4
rhDLL4 rmDLL4
VHH ID ka kd (s1) KD (nM) ka (NT1 s*1) kd (s-1) KD(nlVI)
101G08 (wt) 4.8E+04 2.3E-03 48.0 9.4E+04 5.6E- 03 60.0
129A03 2.1E+05 1.2E-04 0.5
129B05 2.3E+05 7.9E-05 0.3 2.7E+05 3.1 E- 04 1.1
129D08 1.8E+05 6.4E-05 0.4 2.7E+05 2.0E- 04 0.8
129E11 1.9E+05 9.0E-05 0.5 2.5E+05 2.9E- 04 1.2
129H07 1.6E+05 7.3E-05 0.5
130B03 2.2E+05 6.8E-05 0.3
I47
130F06 2.0E+05 8.0E-05 0.4
antiDLL4 Fab 2.3E+05 3.4E-04 1.5
rhDLL4 rmDLL4
VHH ID ka (M‘1s‘ 1) kd (s’1) Kd (nM) ka (M‘1s'1) kd (s1) KD(nM)
115A05 (wt) 2.5E+05 4.0E- 03 16.0 1.7E+05 9.1E-03 53.0
133A09 4.4E+05 9.0E- 04 2.1 3.5E+05 2.7E-03 7.8
133G05 5.9E+05 4.7E- 04 0.8 4.7E+05 1.6E-03 3.4
136C07 6.2E+05 3.9E- 04 0.6 5.0E+05 1.3E-03 2.6
015 4.5E+05 O A m 1 1.0 3.5E+05 1.5E-03 4.3
anti- DLL4 Fab 2.3E+05 3.4E- 04 1.5
148
Table 20: EC50 (nM) values of affinity matured VHHs for binding on CHO-hDLL4,
CHO-mDLL4 and CHO-cDLL4 (FACS)
hDLL4 mDLL4 cDLL4
VHH ID EC50 (nM) ECS0 (nM) EC50 (nM)
101G08(wt) 17.5 11.2
129B05 9.7 3.9 3.9
129D08 9.6 3.7 3.8
129E11 1.4 4.1 4.2
anti-DLL4 Fab 5.6 2.1 2.5
hDLL4 mDLL4 CDLL4
VHH ID ECS0 (nM) EC50 (nM) EC5o (nM)
115A05(wt) 11.3 13.8
133A09 7.2 1.7 2.3
133G05 8.5 2.8 2.7
136C07 10.9 8.3 3.5
015 14.8 7.0 5.1
anti-DLL4 Fab 5.6 2.1 2.5
149
Table 21: Characteristics of affinity-matured VHHs derived from DLLBII101G08 and
DLLBII115A05
ELI SA FM AT hD LL4 FMA T mDL L4 FA CS FA CS FA CS ELI SA ELI SA
KD (nM) hDL L4 KD (nM ) mD LL4 IC50 (nM ) IC50 (nM ) IC50 (nM) EC 50 (nM ) EC 50 (nM ) EC 50 (nM ) hD LL1 hJa 9-1
101G08 48.0 60.0 10.0 69.3 140. 5 17.5 NF 11. 2 nb nb
129A03 0.5 1.8
129B05 0.3 1.1 0.9 7.4 14.4 9.7 3.9 3.9 nb nb
129D08 0.4 0.8 1.2 7.8 11.0 9.6 3.7 3.8 nb nb
129E11 0.5 1.2 1.3 8.1 12.3 10.4 4.1 4.2 nb nb
129H07 0.5 1.0
130B03 0.3 1.5
130F06 0.4 1.3
DLL4 Fab 1.5 1.5 5.5 3.0 5.6 2.1 2.5
150
ELI SA FMAT hDLL 4 FMAT mDLL 4 FA CS FA CS FA CS ELI SA ELI SA
Kd (nM) hDL L4 Kd (nM) mDL L4 IC50 (nM) IC50 (nM) IC50 (nM) EC 50 (nM ) EC 50 (nM ) EC 50 (nM ) hD LL1 hJa g-1
115AG 5 16.0 53.0 7.5 106.7 348.9 11.3 NF 13.8 nb nb
133A0 5 2.1
133A0 9 2.1 7.8 1.5 6.6 18.6 7.2 1.7 2.3 nb nb
133G 05 0.8 3.4 2.0 5.9 12.0 8.5 2.8 2.7 nb nb
134D 10 1.3
136C 07 0.6 2.6 1.4 8.0 31.2 10.9 8.3 3.5 nb nb
015 1.0 4.3 0.9 5.7 21.2 14.8 7.0 5.1 nb nb
DLL4 Fab 1.5 1.2 3.4 1.6 5.6 2.1 2.5
nb: no binding
I5l
Example 8
Construction, production and characterization of bispecific VHHs targeting DLL4 and Ang2 using anti-serum albumin binding as half-life extension
In a first cycle, the anti-DLL4 VHH DLLBII00018 (US 2011/0172398 A1) and the cycle 1 sequence optimized anti-Ang2 VHHs 00042 (SEQ ID NO: 482), 00045 (SEQ ID NO: 484) and 00050 (SEQ ID NO:483) are used as building blocks to generate bispecific VHHs DLLANGBII00001-00016. A genetic fusion to a sérum albumin binding VHH is used as half-life extension methodology. Building blocks are linked via a 9 Gly-Ser flexible linker. VHHs are produced and purified as described in Example 5. An overview of the format and sequence of ail bispecific VHHs is depicted in Figure 16 and Table 22-A (linker sequences underlined), SEQ ID Nos 460-475. Expression levels are indicated in Table 22-B.
Table 22-A
Sequences of bispecific VHH targeting DLL4 and Ang2
VHH ID AA sequence
DLLANGBII00001 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGR FTISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFT ISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNSKNTV YLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQ PGGSLRLSCAASG FT FDDYALGW FRQAPGKEREGVSCIRCS DGSTYYADSVKGRFTISS DNSKNTVY LQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 460)
DLLANGBI100002 DVQLVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSDGSTYYADSVKGR FTISSDNSKNTVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSDGSTYYADSVKGRF TISSDNSKNTVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGNSLRLSCAASG FT FSS FGMSWVRQAPGKGLEWVS SISGSGS DT LYADSVKGR FT ISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV Q PGGSLR LSCAASGRTFS SYAMAWYRQA PGKEREYVAAIRWSGGTAYYADSVKGR FTIS RDNAKNT V YLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSS (SEQ ID NO: 461)
DLLANGBII00003 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGR FTISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFT ISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNSKNTV YLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 462)
DLLANGBII00004 DVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADS VKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVE SGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGRFTISRD NAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSS (SEQ ID NO: 463)
152
DLLANGBII00005 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGR FTISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGNSLRLSCAASGFT FS S FGMSWVRQAPGKGLEWVSSISGSGS DTLYADSVKGR FT ISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNSKNTV YLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESG GGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNS KNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 464)
DLLANGBI100006 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGR FTISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFT ISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGFALDYYAIGWFRQAPGKEREGVSCISSSDGITYYADSVKGRFTISRDNSKNTV YLQMNSLRPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSS (SEQ ID NO: 465)
DLLANGBII00007 DVQLVESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQAPGKEREGVSCISSSDGITYYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSSGGGGSGGG SEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKG RFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGG GLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGRFTISRDNAK NTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSS (SEQ ID NO: 466)
DLLANGBII00008 DVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADS VKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSG GGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGRFT ISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSS (SEQ ID NO: 467)
DLLANGBII00009 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGR FTISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSDGSTYYADSVKGRFT ISSDNSKNTVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQ LVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSDGSTYYADSVKGRFTI SSDNSKNTVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTIS RDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 468}
DLLANGBII00010 DVQLVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSDGSTYYADSVKGR FTISSDNSKNTVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSDGSTYYADSVKGRF TISSDNSKNTVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGRFT ISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSEVQL VESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTIS RDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 469)
DLLANGBÎI00011 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGR FTISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFT ISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGG GSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVK GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 470)
DLLANGBII00012 DVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGG S EVQLVES GGG LVQ PGGS LRLS CAASGRT FS SYAMAWYRQAPGKEREYVAAIRWSGGTAY YADS VKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGG GSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVK GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 471)
DLLANGBII00013 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGR FTISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFT ISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGG GSEVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVK GRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGG GSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYA
153
DSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 472)
DLLANGBII00014 DVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADS VKGRFTIS S DNSKNTVYLQMNS LR PEDTAVYYCAAV PAGRLR FGEQWY PLYEYDAWGQGTLVTVSSG GGGSGGGSEVQLVESGGGLVQPGGSLRL3CAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAY YADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGG GSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYA DSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 473)
DLLANGBII00015 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGR FTISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQAPGKEREGVSCISSSDGITYYADSVKGRFT ISRDNSKNTVYLQMNSLRPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 474)
DLLANGBII00016 DVQLVESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQAPGKEREGVSCISSSDGITYYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSSGGGGSGGG SEVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKG RFTISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 475)
Table 22-B
]54
n.e.: no expression performed
To explore the anti-DLL4 blocking properties in comparison with the monovalent building block DLLBII00018, ail purified bispecific VHHs are analyzed in the hDLL4/hNotch1 compétition ELISA (see Example 5.1 as described in patent US 2011/0172398 A1) (Figure 17) and the CHO-hDLL4 / CHO-mDLL4 compétition FMAT (see Example 5.3 as described in patent US 2011/0172398 A1) (Figure 18). Here, the ELISA compétition assay is performed with a fixed concentration of 8 nM biotinylated hDLL4. Both ELISA and the FMAT compétition assay are also performed after preincubation ofthe VHH with 12.5 μΜ and 25 μΜ human sérum albumin, respectively. A summary of IC5o values and % inhibition is shown in Table 23.
Table 23: IC50 values (nM) and % inhibition in hDLL4/hNotch1 compétition ELISA and CHO-hDLL4 and CHO-mDLL4 compétition FMAT.
I1DLL4 ELISA CHO-11DLL4 FMAT CHO-mDLL4 FMAT
VHH ID Format HSA icso (nM) % inli ICSO (nM) % inh ic50 (nM) % inh
DLLBII00018 0001B - 4.4 85 6.3 66 4.5 93
n.d. n.d 4.2 67 4.3 95
DLLANGBIIOOOOI 00018 - 4.8 91 8.1 76 6.5 95
+ 5.4 94 17.7 87 10.0 97
155
156
DLL4 Fab - 3.1 95 3.3 84 0.8 99
+ n.d. n.d. 3.0 91 1.0 101
n.d., not determined
Additionally, in order to détermine cross-reactivity of the bispecific VHHs to murine and cynomolgus DLL4, a FACS binding experiment is performed. Briefly, CHO cells overexpressing mouse and cynomolgus DLL4 are used for a titration binding experiment of the VHHs. After a 30 min incubation on ice, ail samples are washed and a
2-step détection using anti-c-myc followed by goat-anti mouse IgG-ΡΕ labeled is performed. CHO cells overexpressing human DLL4 are taken as reference. The mean MCF value is determined using a FACS Array and used for calculation of the EC50 value (Table 24; Figure 19).
Table 24: EC50 values of bispecific VHHs binding to human, mouse and cyno DLL4 overexpressed on CHO cells (FACS)
CHO-I1DLL4 ECJ0 (nM) CHO-niDLL4 ECÎO (nM) CHO-CDLL4 ECS0 (nM)
DLLBII00018 1.5 1.2 0.8
DLLANGBII00001 1.2 0.9 0.8
DLLANGB1100003 1.1 0.9 0.7
DLLANGBII00005 1.1 1.1 0.8
DLL ANGBI100007 1.5 i.2 0.9
DLLANGBII00009 1.4 1.1 0.8
DLLANGBII00012 0.8 0.8 0.7
DLLANGBII00014 1.8 1.4 1.2
DLL4Fab 7.5 2.3 1.1
157
In order to détermine cross-reactivity to mouse DLL4 and rat DLL4, a binding ELISA is performed. In brief, recombinant mouse DLL4 (R&D Systems, Minneapolis, Ml, USA) and rat DLL4 is coated overnight at 4°C in a 96-well MaxiSorp plate (Nunc, Wiesbaden,
Germany). Wells are blocked with a 1% casein solution. VHHs are applied as dilution sériés and binding is detected biotinylated anti-VHH 1A4 followed by extravidin-HRP.
1A4 is an anti-VHH VHH (generetad in-house by Ablynx NV). As reference binding to human DLL4 is measured. EC5o values are summarized in Table 25 and Figure 20.
Table 25: EC5o values of bispecific VHHs binding to human, mouse and rat DLL4 (ELISA)
11DLL4 ECjo (nM) mDLL4 ECj0 (nM) rDLL4 ECJ0 (nM)
DLLBII00018 2.5 3.3 2.6
DLLANGBII00001 2.5 3.6 3.4
DLLANGB1I00003 2.1 3.2 2.7
DLLANGBII00005 2.0 3.1 2.9
DLLANGBII00007 2.4 3.3 2.8
DLLANGBH00012 2.9 3.3 3.1
DLLANGBII00014 3.2 4.2 3.8
Absence of binding to the homologous human ligands DLL1 and Jagged-1 is assessed via a solid phase binding assay (ELISA). In brief, 1 pg/mL of recombinant human DLL1 (Alexis, San Diego, CA, USA) or recombinant human Jagged-1 (Alexis, San Diego, CA, USA) is coated overnight at 4’C in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a 1% casein solution. VHHs are applied as dilution sériés and binding is detected biotinylated anti-VHH 1A4 followed by extravidin-HRP. Ail bispecific VHH are considered as being non-cross reactive to these homologous ligands. Results are shown in Figure 21.
To explore the anti-Ang2 blocking properties in comparison with the monovalent antiAng2 building blocks 00042, 00045 and 00050, ail purified bispecific VHHs are analyzed in a human Ang2/hTie2-Fc (Figure 22-1), mouse Ang2/mTie2 (Figure 22-2) and cyno Ang2/cTie2 (Figure 22-3) compétition ELISA. This assay is also performed after incubation of the VHH with 0.5 μΜ human sérum albumin. A summary of IC5o and % inhibition values is shown in Table 26.
158
Table 26: IC50 values (pM) and % inhibition in human, mouse and cyno Ang2/Tie2 compétition ELISA
VHH ID
Format
IC50 (pM)
ntAng2 cAng2
00050
DLLANGBII00001
DLLANGBII00002 oooia
DLLANGBII00009
DLLANGBHOOOIO
00018
AMG386
00042
DLLANGBII00003
DLLANGB1I00004
00018
DLLANGBII00005
0001e
DLLANGB1I00006
00018
DLLANGBII000II
00018
DLLANGBII00012
DLLANGBII00013
1 00018
DLLANGBII00014
HSA
100
7,063
10,556
11,363
6,569
IC50 (pM)
ICS0 (pM)
159
00018
AMG386 - 4 100 5 100 17 100
+ 4 100 n.d. n d n.d. n.d.
00045 A - 106 100 146 100 164 100
+ n.d. n.d. n.d. n.d. n.d. n.d.
DLLANGBII00007 ÛÛO1È - 242 100 364 100 312 100
+ 337 100 428 100 452 100
DLLANGBII00008 - n.d. n.d. n.d. n.d. n.d. n.d.
+ n.d. n d n.d. n.d. n.d. n.d.
DLLANGBII00015 00016 - 201 100 n.d. n.d. n.d. n.d.
+ 207 100 n.d. n.d. n.d. n.d.
DLLANGBII00016 - n.d. n.d. n.d. n.d. n.d. n.d.
+ n.d. n.d. n.d. n.d. n.d. n.d.
AMG386 - 4 100 8 100 21 100
+ n.d. n.d. n.d. n.d. n.d. n.d.
n.d., not determined
Affinities of certain DLL4-Ang2 bispecific VHHs for human sérum albumin hâve been determined (see Example 5) and are shown in Table 27. The affinity constant KD is calculated from resulting association and dissociation rate constants ka and kd.
Table 27: Affinity Kd of purified VHHs for human sérum albumin (HSA)
160
In a second cycle, the anti-DLL4 VHH DLLBII00018 (US 2011/0172398 A1) and the final sequence optimized antî-Ang2 VHHs 00921 (SEQ ID NO: 485), 00938 (SEQ ID NO:486) and 00956 (SEQ ID NO:488) are used as building blocks to generate bispecific VHHs DLLANGBII00017-00019. A genetic fusion to a sérum albumin binding VHH is used as half-life extension methodology. Building blocks are linked via a 9 Gly-Ser flexible linker. An overview of the format and sequence of ali bispecific VHHs is depicted in Figure 23 and Table 28 (linker sequences underlined), SEQ ID Nos 476-478.
Table 28
Sequences of bispecific VHH targeting DLL4 and Ang2
VHH ID AA sequence
DLLANGBIIQ0017 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADSVKGR FTISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFT ISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLV QPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGRFTISSDNSKNTV YLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQ PGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGRFTISSDNSKNTVY LQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 476)
DLLANGBIIQ0018 DVQLVESGGGLVQPGGSLRLSCAVSGITLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGR FTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAYYADS VKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGGGSGG GSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVK GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 477)
DLLANGBII00019 DVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGR FTISSDNSKNTVY LQMNS LR PE DTAVY YCAAV PAGRLR FGEQWYPLYEY DAWGQGTLVTVS SGGGGS GGGSEVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADS VKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSG GGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMAWYRQAPGKEREYVAAIRWSGGTAY YADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCANRAPDTRLAPYEYDHWGQGTLVTVSSGGG GSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYA DSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 478)
To explore the anti-DLL4 blocking properties in comparison with the monovalent building block
DLLBII00018, ali purified bispecific VHHs are analyzed in the hDLL4/hNotch1 compétition ELISA (see Example 5.1 as described in patent US 2011/0172398 A1) (Figure 24), the CHOhDLL4 / CHO-mDLL4 compétition FMAT (see Example 5.3 as described in patent US 2011/0172398 A1) (Figure 25) and the hDLL4 mediated Notch 1 activation (reporter gene) assay (see Example 5.4 as described in patent US 2011/0172398 A1) (Figure 26). Here, the ELISA compétition assay is performed with a fixed concentration of 8 nM biotinylated hDLL4. The ELISA compétition assay, the FMAT compétition assays and the reporter gene assay are also performed after preincubation of the VHH with 12.5 μΜ, 25 μΜ and 162 μΜ human sérum albumin, respectively. A summary of ΙΟΜ values and % inhibition is shown in Table 29.
161
Table 29: IC50 values (nM) and % inhibition in hDLL4/hNotch1 compétition ELISA, CHO-hDLL4 and CHO-mDLL4 compétition
FMAT and hDLL4 mediated Notchl activation (reporter gene) assay σ' NJ
HDLL4 ELISA
CHO-hDLL4 FMAT(“
CH0-mDLL4 FMATW hDLL4 reporter
VHH ID
DLLBII00018
DLL4 Fab
Format
DLLANGBÜ00017
DLLANGBII00018
DLLANGBII00019
ICS0 (nM)
IC50 (nMl
ICjo (nM)
ICM (nM)
(a) tagged versions of VHH were used as these had a higher purity which avoided assay interférence at higher VHH concentration.
(b) maximum inhibition not reached at highest VHH concentration
Binding to human DLL4, mouse DLL4 and rat DLL4 is assessed in Biacore. Briefly, kinetic analysis of the bispecific VHHs is performed by SPR on a Biacore T100 instrument. Recombinant human DLL4 (R&D Systems, Minneapolis, Ml, USA) and mouse DLL4 (R&D Systems, Minneapolis, Ml, USA) are immobilized on a CM5 chip via amine coupling. VHHs are injected over these surfaces at different concentrations between 2.5 and 1,800 nM. Samples are injected for 2 min and allowed to dissociate for 20 min at a flow rate of 45 μΙ/min. Between sample injections, the surfaces were regenerated with a 100s puise of 10mM glycine pH 1.5. Association/dissociation data are evaluated by fitting a 1:1 interaction model (Langmuir binding). The affinity constant Kd is calculated from resulting association and dissociation rate constants ka and kd (Table 30).
Table 30: Binding kinetcs of bispecific VHHs for binding to human and mouse DLL4 (Biacore)
hDLL4 mDLL4
ka (1/Ms) ka (1/s) KD (nM) ka (1/Ms) ka (1/s) . KD (nM)
DLLANGBII00017 1.6E+05 9.5E-05 0.6 9.1E+05 2.6E-04 2.4
DLLANGBII00018 2.0E+05 9.3E-05 0.5 1.3E+05 2.9E-04 1.9
DLLANGBII00019 1.1E+05 7.8E-05 0.7 1.5E+05 2.8E-04 3.0
Additionally, in order to détermine cross-reactivity of the bispecific VHHs to murine and cynomolgus DLL4, a FACS binding experiment is performed. Briefly, CHO cells overexpressing mouse and cynomolgus DLL4 are used for a titration binding experiment of the VHHs. After a 30 min incubation on ice, ail samples are washed and a 2-step détection using biotinylated anti-VHH 1A4 followed by PE labeled streptavidin is performed. CHO cells overexpressing human DLL4 are taken as reference. The mean MCF value is determined using a FACS Array and used for calculation of the EC50 value (Table 31; Figure 27).
163
Table 31: EC50 values of bispecific VHHs binding to human, mouse and cyno DLL4 overexpressed on CHO cells (FACS)
CHO-hDLL4 EC50 (nM) CHO-mDLL4 ECS0 (nM) CHO-cDLL4 EC50 (nM)
DLLANGBII00017 6.0 6.2 4.7
DLLANGBH00018 7.9 6.7 5.6
DLLANGBII00019 6.7 6.3 5.3
DLL4Fab 7.0 6.0 5.3
In order to détermine cross-reactivity to mouse DLL4 and rat DLL4, a binding ELISA is performed. In brief, recombinant mouse DLL4 (R&D Systems, Minneapolis, Ml, USA) and rat DLL4 is coated overnight at 4’C in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a 1% casein solution. VHHs are applied as dilution sériés and binding is detected biotinylated anti-VHH 1A4 followed by extravidin-HRP. As reference binding to human DLL4 is measured. EC50 values are summarized in Table 32 and Figure 28.
Table 32: EC50 values of bispecific VHHs binding to human, mouse and rat DLL4 (ELISA)
hDLL4 EC50 (nM) mDLL4 EC50 (nM) cDLL4 EC50 (nM)
DLLANGB1I00017 1.0 1.6 1.7
DLLANGBII00018 1.2 1.6 1.7
DLLANGBII00019 1.1 1.5 1.9
164
Absence of binding to the homologous human ligands DLL1 and Jagged-1 is assessed via a solid phase binding assay (ELISA). In brief, 1 pg/mL of recombinant human DLL1 (Alexis, San Diego, CA, USA) or recombinant human Jagged-1 (Alexis, San Diego, CA,
USA) is coated overnight at 4’C in a 96-well MaxiSorp plate (Nunc, Wiesbaden,
Germany). Wells are blocked with a 1% casein solution. VHHs are applied as dilution sériés and binding is detected biotinylated anti-VHH 1A4 followed by extravidin-HRP. Ail bispecific VHH are considered as being non-cross reactive to these homologous ligands. Results are shown in Figure 29.
To explore the anti-Ang2 blocking properties in comparison with the final sequence optimized monovalent anti-Ang2 building blocks 00921, 00938 and 00956, ail purified bispecific VHHs are analyzed in a human Ang2/hTie2 (Figure 30-1), mouse Ang2/mTie2 (Figure 30-2), cyno Ang2/cTie2 (Figure 30-3), a hAng1/hTie2 (Figure 31) compétition ELISA and the hAng2 mediated HUVEC survival assay (Figure 32). A summary of IC50 and % inhibition values is shown in Table 33.
165
Table 33: IC50 values (pM) and % inhibition in human, mouse and cyno Ang2/Tie2 compétition ELISA, hAngl compétition
ELISA and hAng2 mediated HUVEC survival assay
hAng2 mAng2 cAng2 hAngl /hAng2 IC$o ratio HUVEC survival
VHH ID Format HSA ICJ0 (pM) % inh IC50 (pM) % inh ICM (pM) % inh IC» (nM) % inh
00921 - 20,400 100 27,200 100 43,400 100 >98 18.8 100
+ n.d n.d. n.d. n.d. n d ll.d. n.d. n.d. n.d.
DLLANGBII000017 0001S - 8 100 12 100 28 100 > 257,632 tbd tbd
13 100 20 100 35 100 > 158,855 n.d. n.d.
AMG386 • 3 100 3 100 15 100 14,421 tbd tbd
+ ll.d. n.d. n.d. n.d. n.d n.d. n.d n.d n.d.
00938 W - 40 100 62 100 105 100 > 50,234 4.3 100
+ n.d. n.d. n.d. n d. n.d n.d. n.d. n.d. n.d.
DLLANGBII00018 - 55 100 85 100 130 100 > 36,392 4.0 100
+ 61 100 91 100 131 100 > 32,684 n d. n d.
AMG386 - 3 100 3 100 19 100 17,452 1.7 100
+ n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
00956 -ÛWS6 - 1,180 100 2,230 100 2,030 100 > 1,698 6.8 100
+ n.d n.d. n.d n.d. n.d. n d. n.d. n.d. n.d
DLLANGBII00019 - 32 100 45 100 76 100 >61,802 3.6 100
+ 40 100 51 100 76 100 > 50,234 n.d. n.d.
AMG386 - 3 100 2 100 16 100 11,482 1.2 100
+ n.d. n d n d il d ll.d n.d. n.d n.d n.d.
n.d., not determined; tbd, to be determined t
Affinities of DLLANGBII00017-18-19 for human, mouse, cyno and rat Ang2 (see
Example 5) hâve been determined and are shown in Table 34.
Table 34: Binding kinetics of purified VHHs for recombinant human, cyno, mouse and rat Ang2
human Ang2-FLD cyno Ang2-FLD
k» (1/Ms) (1/s) KD (M) kn (1/Ms) Ιςι (1/s) Kd (M)
DLLANGBII00017 1.90E+06 1.30E-02 6.60E-09 2.50E+06 1.20E-02 4.70E-09
DLLANGBII00018 8.80E+05 3.30E-05 3.80E-11 1.30E+06 3.20E-05 2.40E-11
DLLANGBII00019 5.10E+05 1.60E-03 3.10E-09 6.30E+05 1.30E-03 2.10E-09
mouse Ang2-FLD rat Ang2-FLD
ka (1/Ms) kd (1/s) KD (M) k;> (1/Ms) kd (i/s) KD (M)
DLLANGBII00017 9.10E+05 1.50E-02 1.70E-08 6.70E+05 3.30Ê-02 4.90E-08
DLLANGBII00018 4.40E+05 6.90E-05 1.60E-10 3.30E+05 9.00E-05 2.70E-10
DLLANGBII00019 3.80E+05 3.80E-03 1.00E-08 2.80E+05 6.00E-03 2.10E-08
Affinities of DLLANGBII00017-18-19 for human, mouse and cyno sérum albumin hâve been determined (Example 5) and are shown in Table 35. The affinity constant Ko is calculated from resulting association and dissociation rate constants ka and kd.
Table 35: Binding kinetics of purified VHHs for recombinant human, mouse and cyno sérum albumin
USA CSA
ka (1/Ms) kj (1/s) K-D (nM) ka (1/Ms) Iq (1/s) Kd (nM)
ALB11 4.5E+05 1.7E-03 3.8E-09 4.2E+05 1.7E-03 3.9E-09
DLLANGBII000I7 1.4E+05 4.4E-03 3.1E-08 1.4E+05 4.2E-03 2.9E-08
DLLANGBI100018 1.6E+05 4.7E-03 2.9E-08 1.6E+05 4.6E-03 2.9E-08
DLLANGBII00019 8.1E+04 5.6E-03 6.9E-08 8.1E+04 5.5E-03 6.8E-08
167 (/—
MSA
(1/Ms) kd (1/s) KD (nM)
ALBll 5.5E+05 3.0E-02 5.5E-08
DLLANGBII00017 1.4E+05 1.1E-01 7.7E-07
DLLANGB11000I8 * * ★
DLLANGBI100019 * * *
* could not be properly fitted i 68
Ang2-binding components (Table 36) (1D01 (SEQ ID No: 479); 7G08 (SEQ ID No:480); 027 (SEQ ID No:481); 00042 (SEQ ID No:482); 00050 (SEQ DI No:483); 00045 (SEQ ID No:484); 00921 (SEQ ID No:485); 00928 (SEQ ID No:486); 00938 (SEQ ID No:487); 00956 (SEQ ID No:488)
FRI CDR1 FR2 CDR2
1D01 EVQLVE S GGGLVQAGGS LRLS CAASG FT FD DYALG WFRQAAGKEREGVS CIRCSDGSTYYADSVKG
7G08 EVQLVESGGGLVQPGGSLRLSCAASGFALD YYAIG WFRQVPGKEREGVS CISSSDGITYYVDSVKG
027 EVQLVESGGGLVQAGGSLRLSCAASGFTLD DYAIG WFRQAPGKEREGVS CIRDS DGSTYYADSVKG
FR3 CDR3 FR4
1D01 RFTISSDNAKNTVYLQMNSLKPEDTAVYYCAA SIVPRSKLEPYEYDA WGQGTQVTVSS
7G08 RFTIS RDNAKNTVYLQMNS LKPEDTAVYYCAT DSGGYIDYDCMGLGYDY WGQGTQVTVSS
027 RFTISSDNDKNTVYLQMNSLKPEDTAVYYCAA VPAGRLRFGEQWYPLYEYDA WGQGTQVTVSS
FRI CDR1 FR2 CDR2
00042 EVQLVESGGG LVQPGGSLRLSCAAS G FTL D DYAIG WFRQAPGKEREGVS SIRDNDGSTYYADSVKG
00050 EVQLVESGGGLVQPGGSLRLSCAASG FT FD DYALG WFRQAPGKEREGVS CIRCSDGSTYYADSVKG
00045 EVQLVESGGGLVQPGGSLRLSCAASGFALD YYAIG WFRQAPGKEREGVS CISSSDGITYYADSVKG
FR3 CDR3 FR4
00042 RFTISSDNSKNTVYLQMNSLRPEDTAVYYCAA VPAGRLRFGEQWYPLYEYDA WGQGTLVTVSS
00050 RFTISSDNSKNTVYLQMNSLRPEDTAVYYCAA SIVPRSKLEPYEYDA WGQGTLVTVSS
0004 5_______RFTISRDNSKNTVYLQMNSLRPEDTAVYYCAT DSGGYIDYDCMGLGYDY WGQGTLVTVSS
FRI
CDR1 FR2
CDR2
00921
00928
00938
00956
EVQLVESGGGLVQPGGSLRLSCAASGFTFD DYALG
EVQLVESGGGLVQPGGSLRLSCAASGFALD YYAIG
EVQLVESGGGLVQPGGSLRLSCAVSGITLD DYAIG
EVQLVESGGGLVQPGGSLRLSCAASGFTLD
WFRQAPGKEREGVS CIRCSGGSTYYADSVKG
WFRQAPGKEREGVS CISSSGGITYYADSVKG
WFRQAPGKEREGVS AIRSSGGSTYYADSVKG
DYAIG WFRQAPGKEREGVS AIRSSGGSTYYADSVKG
FR3
CDR3
FR4
00921
00928
00938
00956
RFTISSDNSKNTVYLQMNSLRPEDTAVYYCAA R FTISRDNSKNTVYLQMNSLRPEDTAVYYCAT RFTISSDNSKNTVYLQMNSLRPEDTAVYYCAA RFTISSDNSKNTVYLQMNSLRPEDTAVYYCAA
SIVPRSKLEPYEYDA WGQGTLVTVSS DSGGYIDYDCSGLGYDY WGQGTLVTVSS VPAGRLRYGEQWYPIYEYDA WGQGTLVTVSS
VPAGRLRFGEQWYPLYEYDA WGQGTLVTVSS

Claims (33)

  1. Claims
    1. A bispecific binding molécule comprising at least one Ang2-binding component and at least one DII4-binding component.
  2. 2. The bispecific binding molécule of claim 1, further comprising at least one sérum albumin binding component.
  3. 3. The bispecific binding molécule of claim 1 or 2, comprising a DII4-binding component comprising at least a variable domain with four framework régions and three complementarity determining régions CDR1, CDR2 and CDR3, respectively, wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in
    a. SEQ IDs NOs: 1 to 166 and 458,
    b. SEQ ID NOs: 333 to 353, or
    c. SEQ ID NOs: 375 to 395.
  4. 4. The bispecific binding molécule of claim 3, the DII4-binding component of which is an isolated immunoglobulin single variable domain or a polypeptide containing one or more of said immunoglobulin single variable domains, wherein said immunoglobulin single variable domain consists of four framework régions and three complementarity determining régions CDR1, CDR2 and CDR3, respectively, and wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in
    a. SEQ ID NOs: 1 to 166 and 458,
    b. SEQ ID NOs: 333 to 353, or
    c. SEQ ID NOs: 375 to 395.
  5. 5. The bispecific binding molécule of claim 4, wherein said one or more immunoglobulin single variable domain contain
    a. a CDR3 with an amino acid sequence selected from a first group of amino acid sequences shown in SEQ ID NOs: 1 to 166;
    b. a CDR1 and a CDR2 with an amino acid sequences that is contained, as indicated in Table 5, as partial sequence in a sequence selected from a second group of amino acid sequences shown SEQ ID NOs: 167 to 332 and 459;
    I70
    c. wherein a SEQ ID NO: x of said first group, for SEQ ID NOs:
    1-166 corresponds to SEQ ID NO: y of said second group in that y = x +166.
  6. 6. The bispecific binding molécule of claim 4, wherein said one or more immunoglobulin single variable domains contain
    a. a CDR3 with an amino acid sequence selected a said first group of amino acid sequences shown in SEQ ID NOs: 333 to 353;
    b. a CDR1 and a CDR2 with an amino acid sequence that is contained, as indicated in Table 16-A , as a partial sequence in a sequence selected from a second group of sequences shown SEQ ID NO: 354 to 374;
    c. wherein a SEQ ID NO: x of said first group corresponds with SEQ ID NO: y of said second group in that y = x +21.
  7. 7. The bispecific binding molécule of claim 4, wherein said one or more immunoglobulin single variable domains contain
    a. a CDR3 with an amino acid sequence selected a said first group of amino acid sequences shown in SEQ ID NOs:375 to 395;
    b. a CDR1 and a CDR2 with an amino acid sequence that is contained, as indicated in Table 16-B, as a partial sequence in a sequence selected from a second group of sequences shown in SEQ ID NOs: 396 to 416;
    c. wherein a SEQ ID NO: x of said first group corresponds to SEQ ID NO: y of said second group in that y = x +21.
  8. 8. The bispecific binding molécule of any one of claims 4 to 7, wherein said one or more immunoglobulin single variable domains are VHHs.
  9. 9. The bispecific binding molécule of claim 8, wherein said one or more VHHs hâve an amino acid sequence selected from amino acid sequences shown in SEQ ID NOs: 167 to 332 and 459.
  10. 10. The bispecific binding molécule of claim 8, said one or more VHHs hâve an amino acid sequence selected from amino acid sequences shown in SEQ ID NOs: 354 to 374.^
    171
  11. 11. The bispecific binding molécule of claim 8, wherein said one or more VHHs hâve an amino acid sequence selected from amino acid sequences shown in SEQ ID NOs:396 to 416.
  12. 12. A VHH which has been obtained by affinity maturation of a VHH as defined in claim 9.
  13. 13. A DII4-binding VHH with an amino acid sequence selected from acid sequences shown in SEQ ID NOs: 356 and 358.
  14. 14. A DII4-binding VHH with an amino acid sequence selected from sequences shown in SEQ ID NOs: 402, 407 and 416.
  15. 15. An immunoglobulin single variable domain which has been obtained by humanization of an immunoglobulin single variable domain as defined in claim 5.
  16. 16. The bispecific binding molécule of claim 1, which binds to an epitope of DII4 that is totally or partially contained within the EGF-2 domain that corresponds to amino acid residues 252-282 of SEQ ID NO: 417.
  17. 17. The bispecific binding molécule of claim 16, which is a immunoglobulin single variable domain or a polypeptide containing same.
  18. 18. The bispecific binding molécule of any one of claims 1 to 16, comprising an Ang2-binding component comprising at least a variable domain with four framework régions and three complementarity determining régions CDR1, CDR2 and CDR3, respectively, wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in SEQ IDs NOs: 491, 494, 497, 500, 503, 506, 509, 512, 515, or 518.
  19. 19. The bispecific binding molécule of claim 18, the Ang2-binding component of which is an isolated immunoglobulin single variable domain or a polypeptide containing one or more of said immunoglobulin single variable domains, wherein said immunoglobulin single variable domain consists of four framework régions and three complementarity determining régions CDR1, CDR2 and CDR3, respectively, and wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in SEQ IDs NOs: 491, 494, 497, 500, 503, 506, 509, 512, 515, or 518.
  20. 20. The bispecific binding molécule of claim 19, wherein said one or more immunoglobulin single variable domain contain
    172
    a. a CDR3 with an amino acid sequence selected from a first group of amino acid sequences shown in SEQ ID NOs: SEQ IDs NOs: 491, 494, 497, 500, 503, 506, 509, 512, 515, or 518 (Table 36);
    b. a CDR1 with an amino acid sequences that is contained, as indicated in Table 22-A or 28, as partial sequence in a sequence selected from a second group of amino acid sequences shown SEQ ID NOs: 489, 492, 495, 498, 501,504, 507, 510, 513, or 516 (Table 36);
    c. a CDR2 with an amino acid sequences that is contained, as indicated in Table 22-A or 28, as partial sequence in a sequence selected from a second group of amino acid sequences shown SEQ ID NOs: 490, 493, 496, 499, 502, 505, 508, 511, 514, or 517 (Table 36).
  21. 21. The bispecific binding molécule of any one of claimsl 8 to 20, wherein said one or more immunoglobulin single variable domains are VHHs.
  22. 22. The bispecific binding molécule of claim 21, wherein said one or more VHHs hâve an amino acid sequence selected from amino acid sequences shown in SEQ ID NOs: 479, 480, 481,482, 483, 484, 485, 486, 487, or 488.
  23. 23. An Ang2-binding VHH with an amino acid sequence selected from acid sequences shown in SEQ ID NOs: 479, 480, 481,482, 483, 484, 485, 486, 487, or 488.
  24. 24. The binding molécule of any one of claim 2 to 23, the sérum albumin binding component of which is an isolated immunoglobulin single variable domain or a polypeptide containing one or more of said immunoglobulin single variable domains, wherein said immunoglobulin single variable domain consists of four framework régions and three complementarity determining régions CDR1, CDR2 and CDR3, respectively, and wherein said CDR3 has an amino acid sequence selected from amino acid sequences shown in SEQ ID NOs: 522, 525, 528, 531,534, 537, or 540.
  25. 25. The binding molécule of claim 24, wherein said one or more immunoglobulin single variable domain contain la-—
    173
    a. a CDR3 with an amino acid sequence selected from a first group of amino acid sequences shown in SEQ ID NOs: SEQ IDs NOs: 522, 525, 528, 531, 534, 537, or 540;
    b. a CDR1 with an amino acid sequences selected from a second group of amino acid sequences shown SEQ ID NOs: 520, 523, 526;
    529, 532, 535, or 538;
    c. a CDR2 with an amino acid sequences selected from a second group of amino acid sequences shown SEQ ID NOs: 521, 524, 527,
    530, 533, 536, or 539.
  26. 26. The bispecific binding molecuie of claim 24 to 25, wherein said one or more immunoglobulin single variable domains are VHHs.
  27. 27. The bispecific binding moiecule of claim 26, wherein said one or more VHHs hâve an amino acid sequence shown in SEQ ID NOs: 98 or 519.
  28. 28. The bispecific binding molecuie of any one of daims 2 to 27 having the amino acid sequence selected from amino acid sequences shown in SEQ ID NOs: 460 to 478.
  29. 29. A nucleic acid moiecule encoding a binding molecuie of any one of daims 1 to 28 or a vector containing same.
  30. 30. A pharmaceutical composition containing at least one bispecific binding molecuie of any one of daims 1 to 28 as the active ingrédient.
  31. 31. The pharmaceutical composition of claim 30 for the treatment of a disease that is associated with DII4-mediated and/or Ang2-mediated effects on angiogenesis.
  32. 32. The pharmaceutical composition of daim 31 for the treatment of cancer and cancerous diseases.
  33. 33. The pharmaceutical composition of claim 30 for the treatment of eye diseases.
OA1201300406 2011-04-01 2012-03-30 Bispecific binding molecules binding to DII4 and Ang2. OA16772A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP11160920.2 2011-04-01

Publications (1)

Publication Number Publication Date
OA16772A true OA16772A (en) 2016-01-04

Family

ID=

Similar Documents

Publication Publication Date Title
CA2827817C (en) Bispecific binding molecules binding to vegf and ang2
US20110195494A1 (en) Dll4-binging molecules
US20200207845A1 (en) Ang2-binding molecules
CA2808696C (en) Vegf-binding molecules
JP5833009B2 (en) Bispecific binding molecules for antiangiogenic therapy
EP2694545B1 (en) Bispecific binding molecules binding to dll4 and ang2
NZ614243B2 (en) BISPECIFIC BINDING MOLECULES BINDING TO Dll4 AND Ang2
HK1171031A (en) Dll4-binding molecules
OA16601A (en) Bispecific molecules binging to VEGF and Ang2.
HK1190735A (en) Bispecific binding molecules binding to d114 and ang2