OA21935A - ‘‘Anti-IL-13RA2 Monoclonal Antibodies And Uses Thereof’’ - Google Patents

Abstract

Provided are antibodies or fragment thereof having binding speeificity to the human interleukin 13 receptor subunit alpha 2 (IL-13RA2) protein. These antibodies are capable of binding to IL13RA2 at high affinity and can mediate antibodydependent cellular cytotoxicity (ADCC) and effectively induce endocytosis. Also provided are methods and uses for treating cancers and inflammatory diseases.

Description

[00011 Interleukin 13 receptor subunit alpha 2 (IL-13RA2, or IL-13Ra2), also known as cluster of différentiation 213A2 (CD213A2), is a membrane bound protein that binds IL 13. Unlike the other receptor proteins, IL-13RA2 has a short cytoplasmic domain, and does not appear to fonction as a signal mediator. This protein is, however, able to regulate the effects of both IL-13 and IL-4, even though it does not bind directly to them.

[0002] The IL-13 signaling is regulated via a complex receptor system. In non-hematopoietic cells, IL-13 engages a heterodimerîc receptor composed of IL-4Ra and IL-13 Ral. IL-13 Ral binds IL-13 with low affinity, When it forms a complcx with IL-4Ra, however, it binds with much higher affinity, inducing the effector fonctions of IL-13. IL-13RA2 is closcly related to IL-13 Ral.

|0003] IL-13RA2 has been found to be over-expressed in a variety of cancers, including pancreatic, ovarian, melanomas, and malignant gliomas. Cells with high IL-13RA2 expression can rapidly and efficiently deplete extracellular IL-13. Likewise, IL-13 responses are enhanced in mice lacking IL13RA2. Therefore, IL-13RA2 can acl as a decoy receptor for IL-13 and elicits antagonistic activity against IL-13.

|0004] IL-1 3RA2 exists in both membrane-bound and soluble forms. The human soluble form of IL-13RA2 is generated by enzymatic cleavage of the membrane-bound form by matrix metalloproteinase 8.

[0005] Overexpression of IL-13RA2 has also been linked to inflammation. For instance, in patients with nasal polyps (NP), IL-13RA2 may contribute to airway inflammation and aberrant remodeling which are the main pathological fcaturcs.

SUMMARY |0006| Anti-IL-13RA2 antibodies arc discovered herein that hâve high binding affinity to the human IL-13RA2 protein and are efficient in mediating antibody-dependenl cellular cytotoxicity (ADCC), inducing cytoloxicity, and promoling cellular update. These antibodies, l

therefore, can be suitably used for treating diseases such as cancer and inilammatory diseases, and serve as targeting carrier of antibody-drug conjugatcs.

[0007] In one embodiment. an antibody or fragment thereof is provided that has specificity lo the human IL-13RA2 protein. In some embodiments, the antibody or fragment thereof comprises a heavy chain variable région comprising heavy chain complementarity delermining régions CDRHl, CDRH2, and CDRH3 and a light chain variable région light chain comprising complementarity determining régions CDRLl, CDRL2, and CDRL3.

[0008] In some embodiments, the CDRHl comprises the amîno acid sequence of SEQ ID NO: 25, the CDRII2 comprises the amino acid sequence of SEQ ID NO: 26, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 27, the CDRLl comprises the amino acid sequence of SEQ ID NO: 28, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 29. and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 30.

|0009| In some embodiments, the heavy chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13 and 54-57, and the light chain variable région comprises an amino acid sequence selected from the group consisting oi SEQ ID NO: 14 and 58-61. In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:54, and the light chain variable région comprises the amino acid sequence of SEQ ID NO:58.

|0(H0| In some embodiments, the CDRII1 comprises the amino acid sequence of SEQ ID NO: 31, the CDRII2 comprises the amino acid sequence of SEQ ID NO: 32 or 49, the CDR113 comprises the amino acid sequence of SEQ ID NO: 33, the CDRLl comprises the amino acid sequence of SEQ ID NO: 34, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 35, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 32.

|()0111 In some embodiments, the heavy chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11 and 62-65, and the light chain variable région comprises an amino acid sequence selected from the group consisting ol SEQ ID NO: 12 and 66-69. In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:62, and the light chain variable région comprises the amino acid sequence of SEQ ID NO:66.

[0012] In sonie embodiments, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 37, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 38 or 50, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 39 or 51, thc CDRL1 comprises the amino acid sequence of SEQ ID NO: 40, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 41, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 42. in some embodiments, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 38, and the CDRH3 comprises the amino acid sequence of SEQ ID NO: 39.

|0(H3] In some embodiments, the heavy chain variable région comprises an amino acid sequence selected from thc group consisting of SEQ ID NO: 19 and 70-73, and the light chain variable région comprises an amino acid sequence selected from the group consisting ol SEQ ID NO: 20 and 74-77. In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:70, and the light chain variable région comprises the amino acid sequence of SEQ ID NO:74.

|0014| In some embodiments, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 43, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 44 or 52, the CDRII3 comprises thc amino acid sequence of SEQ ID NO: 45, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 46, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 47, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 48 or 53. In some embodiments, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 44, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 48.

[0015| In some embodiments, the heavy chain variable région comprises an amino acid sequence selected from thc group consisting of SEQ ID NO: 21 and 78-81, and the light chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22 and 82-85. In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:79, and the light chain variable région comprises the amino acid sequence of SEQ ID NO:83.

|0016| Also provided, in some embodiments, are polynucleotides encoding the antibody or fragment, antibody-drug conjugales based on the antibody or fragment, and compositions comprising the antibody or fragment thereof and a pharmaceutically acceptable carrier.

[0017] Methods and uses for thc treatment of diseases and conditions arc also provided. In one embodiment, provided is a method of trealing cancer or inflammatory in a patient in need thereof, comprising administering to the patient the antibody or fragment thereof of the présent disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS |00l8] FIG. lA-B show the binding affinity of mouse antibodics to IL-13RA2 on A375 cells.

[0019] FIG. 2A-B show the binding affïnity of rat antibodics to IL-I3RA2 on A375 cells.

|0020] FIG, 3 shows the binding affïnity of certain chimeric antibodies to IL-13RA2 on Λ375 cells.

[00211 FIG. 4 shows the Antîbody-Dependent Cellular Cytotoxicity (ADCC) of the chimeric antibodies.

[0022] FIG. 5 shows the cell killing activity of the chimeric antibodies.

|0023| FIG. 6A-C show the intemationization of lhe chimeric antibodies.

|0024] FIG. 7A-.I show the binding affïnity of the humanized antibodies.

|0025] FIG. 8A-B show the ADCC of the humanized antibodies, in IL-13RA2 overexpression cell Unes (A) and Endogenous Gliomas cell Unes (B).

[0026] FIG. 9A-C show the intemationization of selecled humanized antibodies.

|0027] FIG. 10A-C show the in vitro cytoxities of antibody-drug conjugales using lhe humanized antibodies.

|0028] FIG. 11A-B show lhe in vivo cytoxities of antibody-drug conjugales using the humanized antibodies.

DETAILED DESCRIPTION

Définitions

[0029| It is to be noted that the terni “a” or “an” entity refers to one or more of that entity; for example, “an antibody,” is understood to represent one or more antibodies. As such, lhe terms “a” (or an”), “one or more,” and “at least one” can be used interchangcably hercin.

[0030] As used herein, the terni polypeptide” is intendcd to encompass a singular polypeptide” as well as plural “polypeptides,” and refers to a molécule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The terni “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a spécifie length of the product. Thus, peptides, dipeptides, tri peptides, oligopeplides, “protein,” “amino acid chain,” or any other terni used to refer to a chain or chains of two or more amino acids, are included within the définition of “polypeptide,” and the terni polypeptide” may be used instead of, or interchangeably with any of thèse ternis. The terni “polypeptide” is also intendcd to refer to the producls of post-expression modifications of the polypeptide, including without limitation glycosylation, acétylation, phosphorylation, amidation, derivatization by known prolecting/blocking groups, protcolytic cleavage, or modification by non- naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant tcchnology. but is not nccessarily translated from a designated nucleic acid sequence. It may be gencratcd in any manner, including by Chemical synthesis.

[00311 “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molécules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, lhen the molécules arc homologous al thaï position. A degree of homology between séquences is a function of the number of matching or homologous positions shared by the séquences. An “unrclatcd” or “non-homologous” sequence shares less than 40% identity, though prcfcrably less than 25% identity, with one of the scquences of the présent disclosurc.

|0032| A polynucleotide or polynucleotide région (or a polypeptide or polypeptide région) has a certain percentage (for example, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 %) of “sequence identity” to another sequence means that, when aligned. that percentage of bases (or amino acids) are the same in comparing the two séquences.

[0033] The terni “an équivalent nucleic acid or polynucleotide” refers to a nucleic acid having a nucléotide sequence having a certain degree of homology, or sequence identity. with the nucléotide sequence of the nucleic acid or complément thereof. A homolog of a double stranded nucleic acid is intendcd to include nucleic acids having a nucléotide sequence which has a certain degree of homology with or with the complément thereof. In one aspect, homologs of nucleic acids are capable of hybridizing to the nuclcic acid or complément thereof. Likewise, “an équivalent polypeptide” refers to a polypeptide having a certain dcgrce of homology, or sequence identity. with the amino acid sequence of a référencé polypeptide. In some aspects, the sequence identity is at least about 70%. 75%, 80%, 85%. 90%. 95%. 98%, or 99%. In some aspects, the équivalent polypeptide or polynucleotide has one. two, threc, four or five addition, délétion, substitution and their combinations thereof as compared to the reference polypeptide or polynucleotide. In some aspects, the équivalent sequence retains the activity (e.g., epitope-binding) or structure (e.g., sall-bridge) of the référence sequence.

|0034] As used hcrcin, an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically rccognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. Thus the term “antibody includes any protein or peptide containing molécule that comprises at least a portion ofan immunoglobulin molécule having biological activity of binding to the antigen. Examples of such include, but arc not limited to a complcmentarily determining région (CDR.) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable région, a heavy chain or light chain constant région, a framework (l’R) région, or any portion thereof, or at least one portion of a binding protein.

[0035] The tenus “antibody fragment” or “antigen-binding fragment”, as used herein, is a portion ofan antibody such as Iftab')?, F(ab)?, Fab¹, Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term “antibody fragment” includes aptamers, spicgelmers, and diabodies. The term “antibody fragment” also includes any synthetic or gcnclically cngincercd protein thaï acts like an antibody by binding to a spécifie antigen to form a complex.

|0036] A “single-ehain variable fragment” or scFv” refers to a fusion protein of the variable régions of the heavy (V_H) and light chains (Vl) of immunoglobulins. In some aspects, the régions are connected with a short linker peptide of ten to about 25 amino acids. The linker ean bc rich in glycine for flexibilily, as well as serine or thréonine for solubility, and can cither connect the N-terminus of the Vh with the C-terminus of the V_L, or vice versa, This protein retains the specificity of the original immunoglobulin, despile rcmoval of the constant régions and the introduction of the linker. ScFv molécules arc known in the art and arc described. e.g., in US patent 5,892,019.

|<)037] The lerm antibody cncompasses varions broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ. ε) with some subclasses among them (e.g., γ l- γ4). It is the nature of this chain that déterminés the “class” ofthe antibody as 5 IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isolypes) e.g., IgG|, IgG₂, IgG?, IgG₄, IgG₅, etc. are well characterized and are known to confer functional specîalization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view ofthe instant disclosure and, accordingly, are within the scope ofthe instant disclosure. Ail immunoglobulin classes arc clcarly within the scope 10 ofthe présent disclosure, the following discussion will generally be directcd to the IgG class of immunoglobulin molécules. With regard to IgG, a standard immunoglobulin molécule comprises two idenlical light chain polypeptides of molecular weight approximately 23,000 Dallons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are lypically joined by disulfide bonds in a “Y configuration wherein the light 15 chains brackct the heavy chains starting at the mouth of the “Y” and continuing through the variable région.

[0038] Antibodies, antigen-bînding polypeptides, variants, or dérivatives thereof of the disclosure include, but are not limiled to, polyclonal, monoclonal, multispccific, human, humanized, primatized, or chimcric antibodies, single chain antibodies, epitope-binding 20 fragments, e.g., Fab, Fab' and F(ab')2, Fd. Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VK or VH domain, fragments produced by a Fab expression library, and anti- idiotypie (anti-Id) antibodies (including, e.g., anti-Id antibodies to LIGHT antibodies discloscd hercin). Immunoglobulin or antibody molécules ofthe disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, 25 and IgY), class (e.g., IgG 1, lgG2, IgG3, IgG4, IgAl and lgA2) or subclass of immunoglobulin molécule.

|00391 Light chains are classified as either kappa or lambda (K, λ). Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains arc covalcnlly bondcd to each other, and the “tail” portions ofthe two heavy chains 30 are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cclls or genetically engincercd host cells. In the heavy chain. the amino acid scquences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

J0Ü40] Both the light and heavy chains are dividcd into régions of structural and functional homology. The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VK) and heavy (VH) chain portions détermine antigen récognition and spccificity. Conversely, the constant domains of the light chain (CK) and the heavy chain (CHl, CH2 or CH3) confer important biological properlies such as sécrétion, transplacental mobility, Fc receptor binding, complément binding, and the like. By convention the numbering of the constant région domains increases as they become more distal from the antigen-binding site or amino- terminus of the antibody. The N-terminal portion is a variable région and at the C-terminal portion is a constant région; thc CII3 and CK domains aclually comprise the carboxy-terminus of the heavy and light chain, rcspectively.

[0041] As indicated above, the variable région allows the antibody to selectively rccognizc and specifically bind épitopes on antigens. That is, the VK domain and VH domain, or subsel of the complementarity determining régions (CDRs), of an antibody combine to form the variable région that defines a three-dimensional antigen-binding site. T his quaternary antibody structure forms the antigen-binding site présent at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VK chains (i.e. CDR-Hl, CDR-H2, CDR-H3, CDR-LI, CDR-L2 and CDR-L3). In some instances, e.g., certain immunoglobulin molécules derived from camelid species or engineered based on camelid immunoglobulins, a complète immunoglobulin molécule may consisl of heavy chains only, with no light chains. See, e.g., Hamers-Castcrman ei al.. Nature 363:446-448 (l993).

[0042| In naturally occurring antibodies, the six “complementarity determining régions” or “CDRs” présent in each antigen-binding domain are short, non-contiguous sequences of amino acids thaï are specifically positioned to form the antigen-binding domain as the antibody assumes îts three dimensional configuration in an aqucous environment The remainder of lhe amino acids in the antigen-binding domains, referred to as “framework” régions, show less inter-molecular variability. The framework régions largely adopt a β-sheet conformation and thc CDRs form loops which connect, and in some cases form pari of, the β -sheet structure. Thus, framework régions act to form a scaffold lhat provides for posilioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigcnbinding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promûtes the noncovalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework régions, respectively, can be readily idcntified for any given heavy or light chain variable région by one of ordinary ski 11 in the art, since they hâve becn precisely defined (see “Sequences of Proteins of Immunological Intcrest. Rabat. E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Eesk, J. Mol Biol., 196:901-917 ( 1987)).

|0043] In the case where there are two or more définitions of a terni which is used and/or accepled within the art, the définition of the term as used herein is intended to include ail such meanings unless explicitly stated to the contrary. A spécifie example is the use of the term “complementarity determining région (“CDR”) to describe the non-contiguous antigen combining sites found within the variable région of both heavy and light chain polypeptides. This particular région has been described by Rabat et al., U.S. Depl. of Health and Human Services. “Sequences of Proteins of Immunological Intcrest (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporaled herein by référencé in their enlireties. The CDR définitions according to Rabat and Chothia include ovcrlapping or subsets of amino acid residues when comparcd against each other. Nevertheless. application of either définition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited référencés arc set forlh in the table below as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those ski lied in the art can routinely détermine which residues comprise a particular CDR given the variable région amino acid sequence of the antibody.

	Kahat	Chothia
CDR-H1	31-35	26-32
CDR-H2	50-65	52-58
CDR-H3	95-102	95-102
CDR-L1	24-34	26-32
CDR-L2	50-56	50-52
CDR-L3	89-97	91-96

10044| Rabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this

System of Kabat numbering” to any variable domain sequence, without rcliancc on any experimental data beyond the sequence itself. As used herein. Kabat numbering” refers to the numbering system set forth by Kabat el al., U.S. Dept. of Health and Human Services. “Sequence of Proteins of Immunological Interest” ( 1983 ).

[0045] In addition to table above, the Kabat number System describcs the CDR régions as follows: CDR-Hl begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine residue), includes approximately 5-7 amino acids, and ends ai the next tryptophan residue. CDR-H2 begins at the fifteenth residue after the end of CDR-Hl, includes approximately 16-l9 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the thirty third amino acid residue after the end of CDRH2; includes 3-25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid. CDR-Ll begins at approximately residue 24 (i.e., following a cysteine residue); includes approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the sixteenth residue after the end of CDR-Ll and includes approximately 7 residues. CDR-L3 begins al approximately the thirty third residue aller the end of CDR-L2 (i.e., following a cysteine residue); includes approximately 7-l l residues and ends at the sequence F or W-G-X-G, where X is any amino acid.

|0Q46J Antibodies disclosed herein may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goal, guinea pig, camel, llama, horsc, or chicken antibodies. In another embodiment. the variable région may be condricthoid in origin (cg., from sharks).

|0047| As used herein. the terni “heavy chain constant région” includes amino acid sequences derived from an immunoglobulin heavy' chain. A polypeptide comprising a heavy chain constant région comprises at least one of: a CHl domain, a hinge (e.g.. upper, middle, and/or lower hinge région) domain, a CH2 domain, a Cl I3 domain, or a variant or fragment thereof. For examplc, an antigen-binding polypeptide for use in the disclosurc may comprise a polypeptide chain comprising a CHl domain; a polypeptide chain comprising a CHl domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CHl domain and a CH3 domain; a polypeptide chain comprising a CHl domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CHl domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the disclosurc comprises a polypeptide

chain comprising a CH3 domain. Purifier, an anlibody l'or use in the disclosure may lack at least a portion of a CH2 domain (e.g, ail or part of a CH2 domain). As set forth above, it will be understood by one of ordinary skill in the art that the heavy chain constant région may bc modîfied such thaï they vary in amino acid sequence from the nalurally occumng immunoglobulin molécule.

|0048| The heavy chain constant région of an antibody discloscd herein may be derîved from different immunoglobulin molécules. For example, a heavy chain constant région of a polypeptide may comprise a CH1 domain derived from an IgG, molécule and a hinge région derived from an IgG₃ molécule. In another example, a heavy chain constant région can comprise a hinge région derived, in part, from an IgG| molécule and, in pari, from an IgGj molécule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG] molécule and, in part, from an lgÜ4 molécule.

(0049] As used herein, the term “light chain constant région” includes amino acid séquences derived from antibody light chain. Preferably, the light chain constant région comprises at least one of a constant kappa domain or constant lambda domain.

[00501 A “light chain-heavy chain pair” refers to the collection of a light chain and heavy chain that can form a dimer through a dîsulfïde bond between the CL domain of the light chain and the CFH domain ofthe heavy chain.

[0051 [ As previously indicated, the subunit structures and three-dimensional configuration of the constant régions of the varions immunoglobulin classes are well known. As used herein, the term “VII domain” includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CFH domain” includes the First (most amino terminal) constant région domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the Vil domain and is amino terminal to the hinge région of an immunoglobulin heavy chain molécule.

|0052| As used herein the term “CH2 domain” includes the portion of a heavy chain molécule thaï extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et cil., U.S. Dcpt. of Health and Hurnan Services, “Sequences of Proteins of Immunological Interest” (1983). The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched

AL carbohydrate chains are interposcd between the two CI 12 domains of an intact native IgG molécule. It is also well documentcd that the CH3 domain cxlends from the CI 12 domain to the C-terminal of the IgG molécule and comprises approximatcly 108 residucs.

[0053) As used herein, the term “hinge région” includcs the portion of a heavy chain molécule that joins the CI II domain to the CH2 domain. This hinge région comprises approximatcly 25 residues and is flexible, thus allowing the two N-terminal antigen-binding régions to move indepcndently. Hinge régions can bc subdivided inlo threc distinct domains: upper, middle, and lower hinge domains (Roux et al.. J. Immunol 161:4083 (1998)).

|0054| As used herein the term “disulfide bond” includcs the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molécules, the CHI and CK régions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering System (position 226 or 229, EU numbering System).

|0055] As used herein, the term “chimeric antibody” will be held to mean any antibody wherein the immunoreactive région or site is obtained or derived from a first species and the constant région (which may be intact, partial or modified in accordance with the instant disclosure) is obtained from a second species. In certain embodiments the target binding région or site will be from a non-human source (e.g. mouse or primate) and the constant région is human.

|0056| As used herein. “percent humanization” is calculated by determining the number of framework amino acid différences (i.e., non-CDR différence) between the humanized domain and the germline domain, subtracting that number from the total number of amino acids, and then dividing thaï by the total number of amino acids and multiplying by 100.

[0057] By “spccifically binds” or ‘'has specificity to, it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding emails somc complementarity between the antigen-binding domain and the epitope. According to this définition, an antibody is said to “spccifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it w'ould bind to a random, unrelated epitope. The terni “specificity” is used herein to quaiify the relative affinity bv which a certain antibody binds to a certain epitope. For example, antibody “A may be

Λ 3 deemed to havc a higher specificity for a given epitope than antibody “B,” or antibody “A” may bc said to bind to epitope “C” with a higher speci licity than it has for related epitope ‘D.’'

[0058] As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the objccl is to prevent or slow down (lesscn) an undesired physiological change or disorder, such as the progression of cancer. Bénéficiai or desired clinical results include, but are not limited to, alleviation of symptoms, diminishmcnt of extent of disease, stabilized (i.e., not worscning) State of disease, dclay or slowîng of disease progression, amelioration or palliation of the disease State, and remission (whether partial or total), whelher détectable or undetectablc. “Treatment’' can also mean prolonging survival as compared to expected survival if not recciving treatment. Those in need of treatment include those alrcady with the condition or disorder as well as those pronc to hâve the condition or disorder or those in which the condition or disorder is to bc prevented.

|0059| By “subject” or individual” or “animal” or “patient” or mammal,” is meant any subject. particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animais, farm animais, and zoo, sport, or pet animais such as dogs, cats, guinea pigs, rabbits, rats, micc, horscs, cattle, cows, and so on.

[0060] As used herein, phrases such as “to a patient in need of treatment” or “a subject in need of treatment” includes subjects, such as mammalian subjects, that would bcneftl from administration of an antibody or composition of the présent disclosure used. e.g.. for détection, for a diagnostic procedure and/or for treatment.

A nti-IL-l3RA 2 Antibodies

[0061] The présent disclosure provides anti-!L-13RA2 antibodies and fragments that hâve high affînity to the human IL-13RA2 protein, are potent in mediating ADCC and cylotoxicity, and can effectively induce endocytosis. In addition, both in vitro and in vivo testing has demonstrated the efficacies of these antibodies in killing 1L-13RA2-expressing cells, and treating cancer. These antibodies, therefore, are suitable agents for treating various diseases fealuring overexprcssed IL-13RA2, such as cancer and inflammatory diseases.

Lf

[0062] In accordance with one cmbodimcnt of the présent disclosure, thereforc, providcd are antibodies and antigen-binding fragments thereof that are able to bind to IL-13RA2. Examplc antibodies include those murine ones listed in Table 1 (e.g., 24G6D5, 34A5G9, 42E3C4. 51G2B3. 54A4F7, 57F8H3F1, 61H8C1, 62A8D8, 62F6A8, 72B4II4, 73A11E2 and 74B5E4), as well as humanized ones of Tables 8-11. Also includcd arc those that include the same CDRs as illustrated herein. In some embodiments. the disclosed antibodies and fragments include those that bind to the same epitope as those illustrated hcre, and those that compete with the instantly disclosed in binding to IL-13RA2.

|0063] In accordance with one embodimenl of the présent disclosure, providcd is an antibody or fragment thereof that includes the heavy chain and light chain variable domains with the CDR régions disclosed herein, as well as their biological équivalents.

|0Ü64] In one embodimenl, the CDRs are those of 61 H8C1 or its humanized counlerparts, as cxemplilïed in Tables 8B and 8D. In one embodimenl, the CDRH1 comprises lhe amino acid scquence of SEQ ID NO: 25 or a variant thereof having one. two, or three délétions, additions, substitutions or the combinations thereof, lhe CDRH2 comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDR1I3 comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having one, two, or three délétions, additions, substitutions or lhe combinations thereof, the CDRL1 comprises lhe amino acid sequence oi SEQ ID NO: 28 or a variant thereof having one, iwo, or three délétions, additions, substitutions or the combinations thereof, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 29 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 30 or a variant thereof having one, two, or three délétions, additions, substitutions or lhe combinations thereof.

[0065] In one embodimenl. the CDRII1 comprises the amino acid sequence of SEQ ID NO: 25, the CDRII2 comprises the amino acid sequence of SEQ ID NO: 26, the CDR113 comprises the amino acid sequence of SEQ ID NO: 27, lhe CDRL1 comprises lhe amino acid sequence of SEQ ID NO: 28, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 29, and the CDRE3 comprises the amino acid sequence of SEQ ID NO: 30.

|0066| Also provided, in somc embodiments. are those that includc the same CDRs as 6HI8C1 or its humanized counterparts. In some embodiments, the disclosed antibodies and fragments includc those that bind to the same epitope as 61H8C1 or its humanized counterparts, and those that compete with any of them in binding to IL-13RA2.

|0067] In some embodiments, the heavy chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO:l3 (mousc or chimcric) and 5457 (humanized), or a peptide having at least 90% sequence idcntity to an amino acid sequence selected from the group consisting of SEQ ID NO:l3 (mouse or chimcric) and 5457 (humanized).

|0068| In some embodiments, the light chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO:l4 (mousc or chimcric) and 5861 (humanized), or a peptide having at least 90% sequence identîty to an amino acid sequence selected from the group consisting of SEQ ID NO: 14 (mousc or chimcric) and 5861 (humanized).

|0069| In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:54 and the light chain variable région comprises the amino acid sequence of any one of SEQ ID NO: 58-61. In some embodiments, the heavy chain variable région comprises the amino acid sequence of any one of SEQ ID NO: 54-57 and the light chain variable région comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ 11) NO:54 and the light chain variable région comprises the amino acid sequence of SEQ ID NO:58.

|0070| In one embodiment, the CDRs are those of 57F8H3F1 or its humanized counterparts, as exemplified in Tables 9B and 9I). In one embodiment, the CDRl 11 comprises the amino acid sequence of SEQ ID NO: 31 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRH2 comprises the amino acid sequence ol SEQ ID NO: 32 or 49 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRH3 comprises the amino acid sequence ot SEQ ID NO: 33 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRI.I comprises the amino acid sequence of SEQ ID NO: 34 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 35 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 36 or a variant thereof having one, two, or three délétions, 5 additions, substitutions or the combinations thereof.

[0071] In one embodiment, the CDRHI comprises the amino acid sequence of SEQ ID NO: 31, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 32 or 49, the CDRI13 comprises the amino acid sequence of SEQ ID NO: 33, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 34, the CDRL2 comprises the amino acid sequence of SEQ ID NO: io 35, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 36. In one embodiment, the CDRHI comprises the amino acid sequence of SEQ ID NO: 31, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 32, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 33, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 34, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 35, and the CDRL3 15 comprises the amino acid sequence of SEQ ID NO: 36.

[0072| Also provided, in some embodiments, are those that include the saine CDRs as 57F8H3FI or its humanized counterparts. In some embodiments, the discloscd antibodics and fragments include those that bind to the same epitope as 57F8H3F1 or its humanized counterparts, and those that compete with any of them in binding to 1L-13RA2.

[0073] In some embodiments, the heavy chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO:11 (mouse or chimeric) and 6265 (humanized), or a peptide having at least 90% sequence identily to an amino acid sequence selected from the group consisting of SEQ ID NO: 11 (mouse or chimeric) and 6265 (humanized).

[0074] In some embodiments, the light chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12 (mouse or chimeric) and 6669 (humanized), or a peptide having al least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12 (mouse or chimeric) and 6669 (humanized).

[0075] In some embodiments. the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:62 and the light chain variable région comprises the amino acid sequence of any one of SEQ ID NO: 66-69. In some embodiments, the heavy chain variable région comprises the amino acid sequence of any one of SEQ ID NO: 62-65 and the light chain variable région comprises the amino acid sequence of SEQ ID NO: 66. In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:62 and the light chain variable région comprises the amino acid sequence of SEQ ID NO:66.

|0076| In one embodiment, the CDRs are those of 72B4H4 or ils humanized counterparts, as exemplified in Tables 10B and 10D. In one embodiment, the CDR1I1 comprises the amino acid sequence of SEQ ID NO: 37 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 38 or 50 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof. the CDRH3 comprises the amino acid sequence of SEQ ID NO: 39 or 51 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having one. two, or three délétions, additions, substitutions or the combinations thereof, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 42 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof.

|0077] In one embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 37, the CDRH2 comprises the amino acid sequence of SEQ ID NO; 38 or 50, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 39 or 51, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 40, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 41, and the CDRL3 comprises the amino acid sequence of SEQ ID NO; 42. In one embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 37. the CDRH2 comprises the amino acid sequence of SEQ ID NO: 38, the CDRH3 comprises the amino acid sequence of SEQ ID NO; 39, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 40, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 41, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 42.

[0078] Also provided, in some embodiments, arc those that include the samc CDRs as 72B4II4 or its humanized counterparts. In some embodiments, the disclosed antibodies and

6 fragments include those that bind to the sanie épitope as 72B4II4 or ils humanized counterparts, and those that compete with any of them in binding to IL-13RA2.

J0079] In some embodiments, the heavy chain variable région comprises an amino acid scquence selectcd from the group consisting of SEQ 11) NO: 19 (mouse or chimeric) and 7073 (humanized), or a peptide having at least 90% scquence identity to an amino acid sequence sclected from the group consisting of SEQ ID NO:l9 (mouse or chimeric) and 7073 (humanized).

|0Ü80] In some embodiments, the light chain variable région comprises an amino acid scquence selectcd from the group consisting of SEQ ID NO:20 (mouse or chimeric) and 7477 (humanized), or a peptide having at least 90% scquence identity to an amino acid sequence selectcd from the group consisting of SEQ ID NO: 20 (mouse or chimeric) and 7477 (humanized).

|008l] In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:70 and the light chain variable région comprises the amino acid sequence of any one of SEQ ID NO: 74-77. In some embodiments, the heavy chain variable région comprises the amino acid sequence of any one of SEQ ID NO: 70-73 and the light chain variable région comprises the amino acid scquence of SEQ ID NO: 74. In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:70 and the light chain variable région comprises the amino acid scquence of SEQ ID NO:74.

|0082] In one embodiment, the CDRs are those of 73A11E2 or ils humanized counterparts, as exemplified in Tables 11B and 111). In one embodiment, the CDRHI comprises the amino acid scquence of SEQ ID NO: 43 or a variant thereof having one, two, or three dcletions, additions, substitutions or the combinations thereof, the CDRII2 comprises the amino acid sequence of SEQ ID NO: 44 or 52 or a variant thereof having one, two, or three délétions, additions, substitutions or lhe combinations thereof, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 45 or a variant thereof having one. two, or three délétions, additions, substitutions or the combinations thereof, lhe CDRLl comprises the amino acid sequence of SEQ ID NO. 46 or a variant thereof having one, two. or three délétions, additions, substitutions or the combinations thereof. the CDRL2 comprises the amino acid sequence of SEQ ID NO: 47 or a variant thereof having one, two, or three

délétions, additions, substitutions or the combinations thereof, and the CDRL3 comprises the amino acid séquence of SEQ ID NO: 48 or 53 or a variant thereof having one, two, or three délétions, additions, substitutions or the combinations thereof.

|0083] In one embodiment, the CDRI II comprises the amino acid sequence of SEQ II) NO: s 43, the CDRII2 comprises the amino acid sequence of SEQ II) NO: 44 or 52. the CDRI I3 comprises the amino acid sequence of SEQ ID NO: 45, the CDRLl comprises the amino acid sequence of SEQ II) NO: 46, the CDRL2 comprises the amino acid sequence of SEQ II) NO: 47, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 48 or 53. In onc embodiment. the CDRI II comprises the amino acid sequence of SEQ II) NO: 43, the CDRII2 io comprises the amino acid sequence of SEQ ID NO: 44, the CDRI 13 comprises the amino acid sequence of SEQ II) NO: 45, the CDRLl comprises the amino acid sequence of SEQ ID NO:

, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 47, and the CDRL3 comprises the amino acid sequence of SEQ II) NO: 48.

[0084] Also provided, in some embodiments, are those that include the sanie CDRs as 15 73AI1E2 or its humanized countcrparts. In some embodiments, the disclosed antibodies and fragments include those that bind to the samc epitope as 73Λ1ΙΕ2 or ils humanized counterparts, and those that competc with any of them in binding to IL-13RA2.

|0085J In some embodiments. the heavy chain variable région comprises an amino acid sequence selected from the group consîsting of SEQ II) NO:21 (mouse or chimeric) and 7820 81 (humanized), or a peptide having at least 90% sequence identily to an amino acid sequence selected from the group consîsting of SEQ II) NO:21 (mouse or chimeric) and 7881 (humanized),

[0086] In some embodiments, the light chain variable région comprises an amino acid sequence selected from the group consîsting of SEQ 11) NO:22 (mouse or chimeric) and 8225 85 (humanized), or a peptide having at least 90% sequence identily to an amino acid sequence selected from the group consîsting of SEQ ID NC): 22 (mouse or chimeric) and 8285 (humanized).

|0087| In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ II) NO:79 and the light chain variable région comprises the amino acid sequence of any one of SEQ II) NC): 82-85. In some embodiments, the heavy chaîn variable région comprises the amino acid sequence of any onc of SEQ ID NO: 78-81 and the light

2. ο chain variable région comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:79 and the light chain variable région comprises the amino acid sequence of SEQ ID NO:83.

|0088] The antibodies that containcd these CDR régions, whether mouse, humanized or chimeric. had potent IL-13RA2 binding activities. As shown in Tables 8-l 1, certain residues within the CDR can be inodified to retain or improve the property or reduce their polcntial to hâve post-translational modifications (l’TMs). Such modilicd CDR can be referred to as affinity matured or de-rîsked CDRs (e.g., SEQ ID NO:49-53).

[0089] Modificd CDRs can include those having one, two or three amino acid addition, délétion and/or substitutions. In some embodiments, the substitutions can be conservative substitutions.

|0090] A conservative amino acid substitution” is one in which the amino acid residuc is replaced with an amino acid residue having a similar side chain. Familics of amino acid residues having similar side chains hâve been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, thréonine, tyrosine, cystéine), nonpolar side chains (e.g., alanine, valine. Icucine, isolcucinc. prolinc, phenylalanine, méthionine, tryptophan), beta-branched side chains (e.g., thréonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan. histidine). l'hus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the saine side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.

[0091] Non-lirniting examples of conservative amino acid substitutions arc providcd in the table below, where a similarity score of 0 or higher indicates conservative substitution between the two amino acids.

Table A. Amino Acid Similarily Matrix

	C	G	P	S	A	T	D	E	N	Q	H	K	R	V	M	1	L	F	Y	w
w	-8	-7	-6	2	-6	-5	-7	-7	-4	-5	-3	-3	2	6	-4	-5	-2	0	0	17
Y	0	-5	-5	-3	-3	-3	-4	-4	-2	-4	0	-4	-5	•2	-2	-1	-1	7	10
F	-4	-5	-5	-3	-4	-3	-6	-5	-4	-5	-2	-5	-4	1	0	1	2	9
L	-6	-4	-3	-3	-2	-2	-4	-3	-3	-2	-2	-3	-3	2	4	2	6
I	-2	-3	-2	-1	1	0	-2	-2	-2	-2	-2	-2	-2	4	2	5
M	-S	-3	-2	-2	-1	-1	-3	-2	0	-1	-2	0	0	2	6
V	-2	-1	-1	-1	0	0	-2	2	2	-2	-2	-2	-2	4
R	-4	-3	0	0	-2	-1	-1	-1	0	1	2	3	6
K	-5	2	-1	0	-1	0	0	0	1	1	0	5
H	-3	-2	0	-1	-1	-1	1	1	2	3	6
Q	-5	-1	0	-1	0	-1	2	2	1	4
N	-4	0	-1	1	0	0	2	1	2
E	-5	0	-1	0	0	0	3	4
D	-5	1	-1	0	0	0	4
T	-2	0	0	1	1	3
A	-2	1	1	1	2
S	0	1	1	1
P	-3	-1	6
G	-3	5
C	12

Table B. Conservative Amino Acid Substitutions

For Amino Acid	Substitution With
Alanine	D-Ala, Gly, Aib, β-Ala, L-Cys, D-Cys
Arginine	D-Arg, Lys, D Lys, Orn D-Orn
Asparagine	D-Asn, Asp, D-Asp, Glu, D-Glu Gin, D-GIn
Aspartic Acid	D-Asp, D-Asn, Asn, Glu, D-Glu, Gin, D-GIn
Cysteine	D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr, L-Ser, D-Ser
Glutamine	D-GIn, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp
Glutamic Acid	D-Glu, D-Asp, Asp, Asn, D-Asn, Gin, D-GIn
Glycine	Ala, D-Ala, Pro, D-Pro, Aib, β-Ala
Isoleucine	D-lle, Val, D-Val, Leu, D-Leu, Met, D-Met
Leucine	Val, D-Val, Met, D-Met, D-lle, D-Leu, lie
Lysine	D-Lys, Arg, D-Arg, Orn, D-Orn
Méthionine	D-Met, S-Me-Cys, Ile, D-lle, Leu, D-Leu, Val, D-Val
Phenylalanine	D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp
Proline	D-Pro
Serine	D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys
Threonine	D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Val, D-Val
Tyrosine	D-Tyr, Phe, D-Phe, His, D-His, Trp, D-Trp
Valine	D-Val, Leu, D-Leu, Ile, D-lle, Met, D-Met

* ' 21935 |0092] It will also be understood by one of ordinary skill in the art that antibodies as disclosed herein may be modified such that they vary in amino acid scqucnce from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid séquence derived from a designated prolein may be similar, e.g., hâve a certain percent identity to the starting sequence, e.g.. it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.

[0093] In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties not normally associalcd with an antibody. Exemplary modifications are describcd in more detail below. For example, an antibody of the disclosure may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g, PEG, a drug, a toxin. or a label).

|<)094] Antibodies, variants, or dérivatives thereof of the disclosure include dérivatives that are modified, i.e., by lhe covalent attachment of any type of molécule to lhe antibody such that covalent attachment does not prevent the antibody from binding to lhe epilope. For example, but not by way of limitation, the antibodies can be modified. e.g., by glycosylation, acétylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other prolein. etc. Any of numerous Chemical modifications may be carried out by known techniques, including, but not limited to spécifie Chemical cleavage, acétylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the antibodies may contain one or more non-classical amino acids.

]0095| In some embodiments, the antibodies may be conjugated to lherapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.

|0096] The antibodics may be conjugated or fused to a thcrapeutic agent, which may include délectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactivc thcrapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound cnhancing agent, a non-radioactive label, a combination thereof and other such agents known in lhe art.

[0097] The antibodies can be detcctably labelcd by coupling it to a chemiluminescent compound. The presence of lhe chemiluminescenl-lagged antigen-binding polypeptide is

then determined by detecting the presence of luminescence that arises during the course of a Chemical reaction. Examplcs of particularly useful chemi luminescent label ing compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium sait and oxalate ester.

[0098J The antibodies can also be detectably labeled using fluorescence emitting metals such as ^b2Eu, or olhers of the lanthanide sériés. These metals ean bc attachcd to the antibody using such métal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating varions moieties to an antibody are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hcllstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.J, Marcel Dekker, Inc., pp. 623- 53 (1987); Thorpc, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: Λ Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Détection And Therapy, Baldwin et al. (eds.), Academie Press pp. 303-16 (1985), and Thorpe et al., “The Préparation And Cytotoxic Properties Of Antibody-Toxin Conjugales”, hnmunol. Rev. (52:1 19-58 ( 1982)).

Bi-functional Molécules and Combination Thérapies |0099| IL·-13RA2 is overexpressed on tumor cells. As a tumor antigen largeting molécule, an antibody or anligen-binding fragment spécifie to IL-13RA2 can be cornbined with a second antigen-binding fragment spécifie to an immune cell, or an antigen-binding fragment spécifie to an immune checkpoint to generale a combination therapy or a bispccific antibody.

[0100} In some embodimenls, the immune cell is selected from the group consisting of a T cell. a B cell, a monocyte, a macrophage, a neutrophil, a dcndritic cell, a phagocyte, a natural killer cell, an eosinophil. a basophil, and a masl cell. Molécules on the immune cell which can be targeted include, for cxample, CCL1, CD3, CD16. CD19, CD28, and CD64. Other examples include PD-1,PD-L1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122. 41BB (also known as CD137), T1M3, OX-40 or OX40L, CD40 or CD40L, LIGHT, ;

ICOS/ICOSL, GITR/GITRL, HGU, CD27, VISTA, B7H3, B7U4, HEVM or BTLA (also known as CD272), killcr-cell immunoglobulin-like reccptors (KIRs), and CD47.

|0l0ll Different format of bispccific antibodies are also provided. In some embodiments. each of the antî-lL-l3RA2 fragment and the second fragment each is indepcndenlly selected 5 from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody. In some embodiments, the bispecific antibody further includes a Fc fragment.

|()l 02] Bifunctional molécules that include not just antibody or antigen binding fragment are also provided. As a tumor antigen targeting molécule, an antibody or antigen-binding fragment spécifie to IL-13RA2. such as those described hcre, can bc uombincd with an io immune cytokine or ligand optionally through a peptide linker. The linked immune cytokines or ligands include, but not limitcd to, IL-2, TL-3, IL-4, IL-5, IL-6, IL-7, IL-10. IL-12. IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL. Such bi-lunctional molécules can combine the immune checkpoint blocking effect with tumor site local immune modulation.

Antibody-Drug Conjugales |(>103| In some embodiments, the antibodies or fragments may be conjugatcd to thcrapeutic agents, prodrugs, peptides, prolcins, enzymes, viruscs, lipids, biological response modifiera, pharmaceutical agents, or PEG.

[0104] In one embodiment, the antibodies or fragments of the disclosurc arc covalently 20 attached to a drug moiety. The drug moiety may be, or be modified to include, a group reactive with a conjugation point on the antibody. For example, a drug moiety can be attached by alkylation (e.g., al lhe epsilon-amino group lysines or the N-tcrminus of antibodies), reductive amination of oxidized carbohydrate, transestérification between hydroxyl and carboxyl groups. amidation at amino groups or carboxyl groups, and 25 conjugation to thiols.

[0105] In some embodiments, lhe number of drug moictics, p, conjugatcd per antibody molécule ranges from an average of I to 8; 1 to 7, 1 to 6. I to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from an average of 2 to 8, 2 to 7, 2 to 6, 2 to 5. 2 to 4 or 2 to 3. In other embodiments, p is an average of I, 2, 3, 4. 5. 6, 7 or 8. In some embodiments, p 30 ranges from an average of about 1 to about 20, about 1 to about 10, about 2 to about 10, about lo about 9, about l to about 8, about l to about 7, about l to about 6, about l to about 5, about l lo about 4, about 1 to about 3, or about 1 to about 2. In sonie embodiments, p ranges from about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 Lo about 4 or about 2 to about 3.

[0106) For example, when Chemical activation of the protein resulls in formation of free thiol groups. the protein may be conjugated with a sulfhydryl réactive agent. In one aspect, the agent is one which is substantially spécifie for free thiol groups. Such agents include, for example, malcmide, haloacetamides (e.g., iodo, bromo or chloro), haloesters (e.g.. iodo, bromo or chloro), halomethyl ketones (e.g., iodo, bromo or chloro). benzylic halides (e.g., iodide, bromidc or chloride), vinyl sulfone and pyridyllhio.

|0107| The drug can be linked to the antibody or fragment by a linker. Suitablc linkers include. for example, cleavable and non-cleavable linkers. A cleavable linker is typically susceptible to cleavage under intraccllular conditions. Suitable cleavable linkers include, for cxample, a peptide linker cleavable by an intraccllular protease, such as lysosomal protease or an endosomal protease. In excmplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit), a phenylalanine-lysine (phe-lys) linker. or malcimidocapronic-valine-cilndine-p-aminobcnzyloxycarbonyl (mc-Val-Cit-PABA) linker. Another linker is Sulfosuccinimidyl-4-[ N-maleimidomelhyl]cyclohexane-1 -carboxylate (smcc). Sulfo-smce conjugation occurs via a maleimide group which reacls with sulfhydryls (thiols, —SU), while its Sulfo-NHS ester is réactivé loward primary amines (as found in Lysine and the protein or peptide N-tcrminus). Yet another linker is maleimidocaproyl (me). Other suitable linkers include linkers hydrolyzable at a spécifie pli or a pli range, such as a hydrazone linker. Additional suitable cleavable linkers include disulfide linkers. The linker may be covalently bound to the antibody to such an exlent that the antibody must be degraded intraccllularly in order for the drug to be rcleased e.g. the me linker and the like.

[0108] A linker can include a group for lînkage to the antibody. For example, linker can include an amino. hydroxyl, carboxyl or sulfhydryl réactivé groups (e.g., malcmide, haloacetamides (e.g., iodo, bromo or chloro), haloesters (e.g., iodo, bromo or chloro), halomethyl ketones (e.g.. iodo, bromo or chloro), benzylic halides (e.g., iodide, bromide or chloride), vinyl sulfone and pyridylthio).

|0l()9] In somc embodiments, the drug moiely is a cytotoxic or cytostatic agent, an immunosuppressive agent, a radioisotope, a toxin, or the like. The conjugale can be used for inhibiting the multiplication of a tumor cell or cancer cell. causing apoplosis in a tumor or cancer cell, or for treating cancer in a patient. The conjugale can be used accordingly in a 5 variety of settings for the treatmcnl of animal cancers. The conjugale can be used to deliver a drug to a tumor cell or cancer cell. Wilhout being bound by theory, in somc embodiments, the conjugale binds to or associâtes with a cancer cell expressing claudm 18.2, and the conjugale and/or drug can be taken up inside a tumor cell or cancer cell through receptor-mediated endocytosis.

w |0110| Once inside the cell, one or more spécifie peptide sequences within the conjugale (e.g., in a linker) arc hydrolytically cleaved by one or more lumor-cell or cancer-ccllassociated proteases, resulting in release of the drug. The relea.sed drug is then free to migrate within the cell and induce cytotoxic or cytostatic or other aclivilies. In somc embodiments, the drug is cleaved from the antibody outside the tumor cell or cancer cell, and the drug 15 subscquently pénétrâtes the cell, or acts al the cell surface.

|0111J Examples of drug moietics or payloads are selccted from the group consisting of DM1 (maytansine, N2'-deacetyl-N2'-(3-mercapto-l-oxopropyl)- or N2’-deacetyl-N2'-(3mercapto-l-oxopropyl)-maylansine), mc-MMAD (6-maleimidocaproyl· monomelhylauristatin-D or N-mcthyl-L-valyl-N-[( 1 S,2R)-2-methoxy-4-[(2S)-2-|( 1 R.2R)-1 20 methoxy-2-methyl-3-oxo-3-[| ( 1 S)-2-phenyl-1 -(2-lhiazolyl)cthyl]amino Ipropyl j-1 -pyr rolidinyl]-1 -[ ( 1 S)-l -methylpropyl|-4-oxobutyl]-N-methyl-(9CI)-L-vaiinamidc), mc-MMAF (maleimidocaproyl-monomethylaurislatin F or N-|6-(2,5-dihydro-2,5-dioxo-lIl·pyrrol-l-yl)l-oxohexyl]-N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3-mcthoxy-5-methyl-4(methylamino)heptanoyl-(aR. |JR,2S)-p-mcthoxy-a“niethyl-2-pyriOlidincpropanoyl-L25 phcnylalanine) and mc-Val-Cil-PABA-MMAE (ô-maleimidocaproyl-ValcCit-ipaminobenzyloxycarbonylj-monomcthylauristatin E or N-|[[4-[[N-|6-(2,5dihydro-2.5-dioxo1 H-pyrrol-l-yl)-l-oxohexyl]-T-valyl-N5-(aminocarbonyl)-Lomilhyijamino]phcnyI|methoxy]carbonyl|-N-meth yl-L-va!yl-N-|(lS,2R)-4-[(2S)-2[( I R,2R)-3-| |( I R,2S)-2-hydroxy-1 -mcthyl-2-phcnylclhyl |amino |-1 -mcthoxy-2-methyl-330 oxopropyl ]-1 -pyrrolidinyl ] -2-mcthoxy-l -[( 1 S)-l -methylpropyl]-4-oxobutyl ]-N-methyl-Lvalinamide). DM1 is a derivative of the tubulin inhibitor maytansine while MMAD, MMAE, and MMAF arc aurislatin dérivatives. In somc embodiments, the drug moiety is selccted from

ί.

the group consisting of mc-MMAF and mc-Val-Cit-ΡΛΒΑ-ΜΜΛΕ. In some embodiments, the drug moiety is a maytansinoid or an auristatin.

|0112j The antibodies or fragments may be conjugated or fused to a therapeutic agent, which may include détectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art.

|0113] The antibodies can be detectably labeled by coupling it to a chcmiluminescent compound. l'hc présence of the chemiluminescent-taggcd antigen-binding polypeptide is then determined by detccting the presence of luminescence that arises during the course of a Chemical reaction. Examples of particularly useful chcmiluminescent labeling compounds arc luminol. isoluminol, theromatic acridinium ester, imidazoie. acridinium sait and oxalate ester.

|0114] The antibodies can also be detectably labeled using fluorescence emiltîng mctals such as Eu, or others of the lanthanide sériés. These metals can be attachcd to the antibody using such métal chelating groups as diethylenetriamincpentacetic acid (DTPA) or ethylenediaminelctraacetic acid (EDTA). Techniques for conjugating various moieties to an antibody are well known, see, e.g., Amon et al., ‘'Monoclonal Antibodics For Immunotargeting Of Drugs In Cancer Thcrapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.. (eds.), Marcel Dckker, Inc., pp. 623- 53 (1987); Thorpc, “Antibody Carriers Of Cytotoxic Agents In Cancer Thcrapy: A Revicw”, in Monoclonal Antibodics '84: Biological And Clinical Applications, Pinchcra et al. (eds.), pp. 475-506 (1985); “Analysis. Results, And Future Prospective Of The Therapeutic Use Of Radiolabelcd Antibody In Cancer Thcrapy'’, in Monoclonal Antibodies For Cancer Détection And Thcrapy, Baldwin et al. (eds.), Academie Press pp. 303-16 (1985), and Thorpe et al., The Préparation And Cytotoxic Properties Of Antibody-Toxin Conjugales”, Immunol. Rev. (52:119-58 (1982)).

Polynueleolides Encoding the Antibodies and Methods of Preparing the Antibodies |0ll5| The présent disclosure also provides isolatcd polynueleolides or nucleic acid molécules encoding lhe antibodies, variants or dérivatives thereof of the disclosure. The polynueleolides of the présent disclosure may encode lhe entire heavy and lighl chain variable régions of the antigen-binding polypeptides, variants or dérivatives thereof on lhe same polynucleotide molécule or on separate polynucleotide molécules. Additionally, the polynueleolides of the présent disclosure may encode portions of the heavy and light chain variable régions of the antigen-binding polypeptides, variants or dérivatives thereof on the same polynucleotide molécule or on separate polynucleotide molécules.

|0ll6| Methods of making antibodies are well known in the art and described herein. In certain embodiments, both the variable and constant régions of the antigen-binding polypeptides of the présent disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies againsl a spécifie antigen can be prepared by administering the antigen to a transgcnic animal which has been modified to producc such antibodies in response to antigenic challenge, but whose endogenous loci hâve been disabled. Excmplary techniques thaï can be used to make such antibodies are described in U.S. palenls: 6, l 50,584; 6.458,592; 6,420,140 which arc incorporated by référencé in their enti relies.

Treatment Methods

[0117] As described herein, the antibodies. variants or dérivatives of the présent disclosure may be used in certain treatment and diagnostic methods.

[0118] The présent disclosure is further directed to antibody-based thérapies which involvc administering the antibodies of the disclosure to a patient such as an animal, a mammal, and a human for treating one or more of the disorders or conditions described herein. Thcrapeutic compounds of lhe disclosure include, but are not limited to, antibodies of the disclosure (including variants and dérivatives thereof as described herein) and nucleic acids or polynueleolides encoding antibodies of the disclosure (including variants and dérivatives thereof as described herein).

|(ll 19] The antibodies of the disclosure can also be used to trcal or inhibit cancer. In some embodiments, the cancer cells in the patient express or overexpress IL-I3RA2. As providcd above, IL-13RA2 can be overexprcsscd in tumor cells, in panieular gaslric, pancreatic, esophagcal, ovarian, and lung lumors. Inhibition of IL-I3RA2 has been shown to be useful for treating the lumors.

|0l20] Accordingly, in some embodiments, provided arc mcthods for treating a cancer in a patient in need thereof. The method, in one embodiment, cntails administering to the patient an effective amounl ofan antibody ofthe présent disclosure. In some embodiments, at least one ofthe cancer cells (e.g., stromal cells) in the patient over-express IL-13RA2.

|0l21] Cellular thérapies, such as chimeric antigen receptor (CAR) T-cell thérapies, are also provided in the présent disclosure. A suitable cell can be used, that is put in contact with an anti-IL-!3RA2 antibody of the présent disclosure (or altematively engincered to express an anti-IL-l3RA2 antibody of the présent disclosure). In some embodiments. the antibody is presented in a chimeric antigen receptor (CAR). Upon such contact or engineering, the cell can lhen be introduccd to a cancer patient in need of a treatment. The cancer patient may hâve a cancer of any ofthe types as disclosed herein. The cell (e.g.. T cell) can be, for instance, a tumor-inlïltrating T lymphocyte, a CD4+ T cell, a CDS *· T cell. or the combination thereof, without limitation.

|0122] In some embodiments. the cell was isolated from the cancer patient him- or her-self. In some embodiments, the cell was provided by a donor or from a cell bank. When the cell is isolated from the cancer patient, undesired immune réactions can be minimized.

|0l23| Non-limiting examples of cancers include bladder cancer, breast cancer, colorectal cancer, endométrial cancer, esophagcal cancer, head and neck cancer, kidney cancer, leukemia. liver cancer, lung cancer, lymphoma, melanoma. pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is one or more of gaslric, pancreatic, esophagcal, ovarian, and lung cancers.

|0l24] Addilional discases or conditions associated with increased cell survival, that may be treated, prevenled, diagnoscd and/or prognosed with the antibodies or variants, or dérivatives thereof of the disclosure include, but are not limited to, progression, and/or métastasés of malignancies and related disorders such as leukemia (including aculc leukemias (e.g., acute lymphocytic leukemia. acute myclocytic leukemia (including myeloblastic, promyelocytic, myelomonocylic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myclocytic (granulocylic) leukemia and chronic lymphocytic leukemia)), polycythemia vera.

> 21935 lymphomas (e.g., Ilodgkin's discase and non-IIodgkin's discase), multiple mycloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including. but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma. liposarcoma, chondrosarcoma, ostéogénie sarcoma, chordoma, angiosarcoma. endolhcliosarcoma. lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma. rhabdomyo sarcoma. colon carcinoma. pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma. basai ccil carcinoma, adenocarcinoma. sweat gland carcinoma, sebaceous gland carcinoma. papillary carcinoma, papillary adenocarcinomas, cystadénocarcinome, medullary carcinoma, bronchogenic carcinoma, rénal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma. seminoma. embryonal carcinoma, Wilm's tumor. cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma. bladder carcinoma. épithélial carcinoma. glioma. astrocytoma, medulloblastoma, craniopharyngiome, ependymoma. pinealoma, hemangioblastoma, acouslic ncuroma, oligodendrogliome, menangioma. melandma, neuroblastome and retinoblastoma.

|0125] A spécifie dosage and trcalment regimen for any particular patient will dépend upon a variety of factors, including the particular antibodies, variant or dérivative thereof used. the patient's âge. body weight. general heaith. sex, and dict, and the time of administration, rate of excrétion, dmg combination, and the severily of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in lhe art. The amount will also dépend on the individual patient to be treated, the route of administration, lhe type of formulation, the characlcristics of lhe compound used, the severily of the discase, and the desired effect. The amount used can bc determined by pharmacological and pharmacokinelic principles well known in the art.

jOl26| In some embodiments, lhe antibodies of the présent disclosure can be used for treating inflammation or inflammatory discases. The inflammation, in some embodiments. may be caused by injury, infection, or autoimmune diseases. In some embodiments. the inflammation is a skin inflammation, such as nasal polyps (NP).

|0127] Methods of administration of the antibodies, variants or includc but arc not limited to intradetmal, intramuscular, intraperitoneal, intravenous, subeutaneous, intranasal. épidural, and oral routes. The antigen-binding polypeptides or compositions may bc administered by any convenicnt route, for examplc by infusion or bol us injection, by absorption through >

épithélial or mucocutaneous linings (e.g., oral mucosa. rectal and intestinal mucosa. etc.) and may bc administered together with other biologically active agents. Thus. pharmaceutical compositions containing the antigen-binding polypeptides of the disclosure may be administered orally, rectally, parenlerally, intracistemally, intravaginally. intrapcritoneally. topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.

|ÜI28| The terni “parentéral as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subeutaneous and intra-articular injection and infusion.

[0129J Administration can bc systemie or local. In addition, it may be désirable to inlroduce the antibodics of the disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular cathéter, for example, attached to a réservoir, such as an Ommaya réservoir. Pulmonary administration can also bc employcd, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

|0130| It may be désirable to adminîsler the antigen-binding polypeptides or compositions of the disclosure local ly to the area in need of treatment; this may bc achicvcd by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g.. in conjonction, with a wound dressing after surgery, by injection, by means of a cathéter, by means of a supposilory, or by means of an implant, said implant being of a porous, nonporous, or gelatinous material, including membranes, such as sialaslic membranes, or libers. Preferably, when administering a prolein, including an antibody, of the disclosure, care must be taken to use materials to which the protein does not absorb.

|(H311 The amount of the antibodics of the disclosure which will be effective in the treatment, inhibition and prévention of an inflammatory. immune or malignanl diseasc, disorder or condition can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employcd to help identify optimal dosage ranges. The précisé dose to be employcd in the formulation will also dépend on the roule of administration, and the seriousness of the discase, disorder or condition, and should bc decidcd according to the judgmenl of the practitioner and each patient's circumstanccs. Effective doses may be exlrapolated from dose-responsc curves derived from in vitro or animal mode! lest Systems.

|0132] As a general proposition, the dosage adminislered to a patient ofthe antigen-binding polypeptides of the présent disclosurc is typically 0.1 mg/kg to 100 mg/kg of the paticnt's body weight, between 0.1 mg/kg and 20 mg/kg of the patîenfs body weight, or l mg/kg to 10 mg/kg of the paticnt's body weight. Generally, human antibodies hâve a longer half-life within the human body than antibodies from other specics duc to the immune response to the forcign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible, Furlhcr, the dosage and frcqucncy of administration of antibodies of the disclosurc may bc redueed by enhancing uptake and tissuc pcnctralion (e.g.. into the brain) ofthe antibodies by modifications such as, for cxamplc, lipidation.

J0133] In an additional embodiment, the compositions ofthe disclosurc are administered in combination with cytokines. Cytokines that may be administered with the compositions of the disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-1Ü, IL-l 2, IL-l 3, IL-l 5, anti-CD40, CD40L, and TNF-a.

|0134| In additional embodiments, the compositions of the disclosure arc administered in combination with other thcrapeutic or prophylactic regimens, such as, for example, radiation therapy.

Diagnostic Met h ods

[0135] Over-expression of IL-13RA2 is observed in certain tumor samples, and patients having IL-l 3RA2-over-expressing cclls are likely responsive to treatments with the anti-IL13RA2 antibodies of the présent disclosure. Accordingly, the antibodies of the présent disclosure can also bc used for diagnostic and prognostic purposes.

|0136| A sample thaï preferably includes a cell can be obtained from a patient, which can bc a cancer patient or a patient desiring diagnosis. The cell be a cell of a tumor tissuc or a tumor block, a blood sample, a urine sample or any sample from the patient. Upon optional pretreatmenl of the sample, the sample can be incubated wilh an antibody of the présent disclosure under conditions allowing the antibody to interact with an IL-13RA2 prolcin potentially présent in the sample. Mcthods such as FUSA can be used, taking advantage of the anti-IL-13RA2 antibody, to detccl the presence ofthe IL-l3RA2 prolein in the sample.

|Ü137] Présence of the IL-13RA2 prolein in the sample (optîonally with the amount or concentration) can be used for diagnosis of cancer, as an indication that the patient is suitable for a treatment with the antibody, or as an indication that the patient has (or has not) responded to a cancer treatment. For a prognostic method, the détection can be donc at once, twîce or more, at certain stages, upon initiation of a cancer treatment to indicate the progress ofthe treatment.

Compositions

[0138] The présent disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody, and an acceptable carrier, ln some embodiments, the composition further includes a second anlicanccr agent (c.g., an immune checkpoint inhibitor).

[0139] In a spécifie embodiment. the term pharmaceutically acceptable” means approved by a rcgulatory agency of the Fédéral or a State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animais, and more particularly in humans. Further, a “pharmaceutically acceptable carrier” will generally be a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0140] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapcutic is administered. Such pharmaceutical carriers can be stérile liquids, such as water and oils, inciuding those of petroleum, animal, vegctable or synthetic origin. such as peanut oil, soybean oil, minerai oil. sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium sîearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, éthanol and the like. The composition, if desired, can also eontain minor amounts of welting or cmulsifying agents, or pli buffering agents such as acétates, citrates or phosphates. Antibactcrial agents such as benzyl alcohol or methyl parabens; antioxidanls such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacctic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned. These compositions can take the form of solutions, suspensions, émulsion, tablets, pi Ils, capsules, powders, sustained-release formulations and the like. The composition can be formulatcd as a suppository, with traditional binders and carriers such as triglycérides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnésium stéarate, sodium saccharine, cellulose, magnésium carbonate, etc. Examples of suitable pharmaceutical carriers arc dcscribcd in Reminglon's Pharmaceutical Sciences by E. W. Martin, incorporated hcrein by referencc. Such compositions will contain a thcrapeutically effective amounl of the antigen-binding polypeptide, preferably in purified form, together with a suitable amounl of carrier so as to provide lhe form for proper administration to lhe patient. The formulation should suit the mode of administration. The parental préparation can be cncloscd in ampoules, disposablc syringes or multiple dose vials made of glass or plastic.

[0141] In an embodiment. lhe composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration lo human beings. Typically, compositions for intravenous administration are solutions in stérile isotonie aqueous buffer. Where neccssary, lhe composition may also include a solubilizing agent and a local anesthetic such as lignocaine lo case pain al the siie of the injection. Generally. the ingrédients are supplied cither separatcly or mixed logether in unit dosage form. for example, as a dry lyophilized powder or water free concentrait- in a hermelically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be adminislcrcd by infusion, it can bc dispensed with an infusion boltle conlaining stérile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of stérile water for injection or saline can be provided so that the ingrédients may be mixed prior to administration.

|0142| 1 he compounds of the disclosure can be formulated as neulral or sali forms. Pharmaceutically acceptable salts include those formed with unions such as those derivcd from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derivcd from sodium, potassium, ammonium, calcium, ferrie hydroxides. isopropylamine, trielhylamine, 2-elhylamino éthanol, histidinc, procaine, etc.

EXAMPLES

Example 1: Génération of murine and rat monoclonal antibodies against human II 13RA2

[0143] The human IL-13KA2 prolcin was used to immunize different strains of mice and rats.

and hybridomas were gencrated accordingly. IL-I3R.A2 positive binders were selected and subcloned. Subscquently, in vitro binding and functional screening were carried out and lead antibodies with highest binding affinity and strongest functional potency were identified.

[0144] The VH/VL sequences of 12 lead murine antibodies arc provided in Table I below.

Table 1. VH/VL sequence of the lead murine antibodies

N:imc	Sequence (CDRs are undcrlined)	SF.Q 11) MO:
24G6DS VH	QVQLQQSGPELVKPGTSVKMSCKASGYTFTSNYIHWVKQRPGQGLEWIGW IYPGDGSIKYNEKFKGKTTLTADKSSSTVYMLLSSLTSEDSAIYFCAVLR RLDYWGQGTTLTVSS	1
24G6D5 VL	DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLlh'Y ASQSISGIPSRFSGSGSGT DFT LSINSVETED PGMYFCQQSNSWPLTFGA GTKLELK	2
34A5G9 VH	EVHLQQSGPELVKPGASVKÎSCKTSGYTFTEYTMHWVKQSHGKSLEWIGR INPNNGIAEYKQTFKDKAT LAV DKS S S TAYME FRSLTS E D3AVY YCARGE GYGYWFAYWGQGTLVTVSA	3
34ASG9 VL	D1QMTQSPASLSASVGETVTITCRTSDNIYSYLAWYQQRQGKSPQLLVSN ARTLAEGVPSRFSGSGSGTQFSLK1NSLQPEDFGIYYCQHHSGTPFTFGS GTNLEIE	4
42E3C4 VH	EVKLVESGGGLVRPGGSLKISCAASGFTFSTYGMSWVRQTPEKRLEWVAT ISGGGSYTYYPDSVKGR FTIS R DNAKN N L Y LQMS S LRS E DTALY YCA RRD LRSAMDYW GQGT S VT VS S	5
42E3C4 VL	DIVMTQSQKFMST S VGDRVSVTCKASQNVDINVAWYQQK PGQS PKALIY S ASFRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPLTFGG GTKLEIK	6
51G2B3 VH	QVQLQQSGPELVKPGASVKÎSCKASGYAFSDSWMNWVKQRPGQGLEWTGR IYPGDGDTNYNGKFKGKATLTADKS S N TA YMQLS S LT S V DSAVYFCARSW NGSPWFPYWGQGTLVTVSA	7
51G2B3 VL	DVLMTQT PLS LPVSLG DQASISCRSSQTIAHRNGNTYLEWYLQKPGQSPK LLLYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVT WTFGGGTKLEIK	8
54A4F7 VH	EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWVAT ISGGGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLRS EDTALYYCARRD LRSAMDHWGQGTSVTVSS	9
54A4F7 VL	DIVMTQSQKFMSTSVGDRVSVTCRA8QNVDINVAWYQQKPGQSPK3LIYS ASYRYSGVP DRFTGSGSGT DFTLTISNVQS EDLAE Y FCQQYNSYPLTFGG GTKLEIR	10
57F8H3F1 VH	QVQLQQPGAELVRPGASVKLSCKASGYTFTNYWMNWIKQRPEQGLEWIGR IDPYETETHYNQKFRDRAILTVDKSS 3TAFMQLS G LTS E DSAVYYCARGY GHYYGVDYWGQGTSVTVSS	11
57F8H3F1 VL	DIVLTQS PAS LAV S LGQRATIS CRASENVGNFGIS FVN'/J FQQKPGQ P P Kl,	12

	LIYAASNLGSGVPARFSGSGSGTD FS LNIH PMEEDDAAMYFCQQSKGVPW TFGGGTKLEIK
61H8C1 VH	QVQLQQSGAELARPGASVMLSCKASGYTFTSYWTQWIKQRPGQGLEGIGA IYPGDDDTSYNQKFKGKATLTADKSSSTAYMQTNSLTSEDSAVYYCARGA TLLRPFPYWGQGTLVTVSA	13
61H8C1 VL	DIVMTQSH KFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYS ASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYSCQQHYSIPPTFGG GTKLEIK	14
62A8D8 VH	EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWVAT ISGGGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARRD LRSAMDYWGQGTSVTVSS	15
62A8D8 VL	DIVMTQSQKFMSTSVGDRVSVTCKASQNVDINVAWYQQKPGQSPKALIYS ASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEFFCQQYNNYPLTFGG GTKLEIK	16
62F6A8 VH	QVQIQQSGAELARPGASVMLSCKASGYTFTDYWMQWIKQRPGQGLEGIGA IYPGDGDTAYNQKFRGKATLTADKS S STAYMQLN S LAS E DSAVY FCARGA TLLRPFPFWGQGTLVTVSA	17
62F6A8 VL	D1VMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLLYS ASYRYAGV PDRFTGSGSÜT D FT FTISSVQAEDLAVYSCQQHYSIPPTFGG GTKLEIK	18
72B4H4 VH	QVQLQQPGAERVRPGASVKLSCKASGYTFTNYWMNWVKQRPEQGLEWIGR IDPYAGE THYNQKFKDKAILTVDKSSSTAYMQLRSLTSEDSAVYYCARGY GDSYGMDHWGQGTSVTVS S	19
72B4H4 VL	DIVLTQS PASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQP PKI LIYAASRQGSGD PAR FS GS G S GT D FSLNIH PLE EDDTAMYFCQQSKEVPW TFGGGTKLEIK	20
73A11E2 VH	EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWVAT ISGGGSYTYYPD SVKGRFT T SRDNARNNLYLQMNS LRSEDTALYYCARRD LRSAMDYWGQGTSVTVS S	21
73A11E2 VL	DIVMTQSQKFMSTSVGDRVSVTCKASQNVDINVAWYQQKPGQSPKALIYS ASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEFFCQQYNSYPLTFGG GTKLEIK	22
74BSE4 VH	EVKLVESGGGLVKPGGSLRLSCAASGFTFSTYGMSWVRQTPEKRLEWVAT ISGGGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARRD LRSAMDYWGQGTSVTVSS	23
74B5E4 VL	DIVMTQSQK FMSTSVG DRVSVTCKASQNVDINVAWYQQKPGQS PKALIYS ASYRYSGVPDRFTGSGSGTD FTLTISNVQSEDLAEYFUQQYNTYPLTFGG GTKLEIK	24

Example 2. Binding of Murine Antibodies to IL-13RA2 Protein |0145] This example examined the mouse antibodies, 28 of which. for their ability to bind the human IL-I3RA2 protein expressed on A375 cells.

[0146] A375 cells that endogenously expressed IL-13RA2 were harvestcd from flasks. 100 μΐ of ]χ10^ή cells/ml of cells were ineubated with primary antibodies in 4-fold serial dilutions starting from 100 nM to 0.006 nM for 30 minutes on ice. After being washcd with 200 μΙ of FACS buffer iwice, cells were ineubated with secondary antibody for 30 minutes on icc. Cells were washcd with 200 μΙ of FACS buffer twicc and analyzed by FACS.

[0147] The results of the study (FIG. IA-B and Table 2) show that lhe purificd murine antibodies could bind to endogenously expressed IL-1 3RA2 on A375 cells at high F.C50.

Table 2. Binding Affinity of Purified Mouse Antibodies

Antibody	EC50(nM)	Top (MFI)
62A8D8	0.90	333889
62F6A8	0.68	217495
70E6D10	1.18	254063
51A2G9	0.92	244257
46C3E3	1.75	249095
51G2B3	0.87	242845
45B5G6	0.72	188842
48F3H9	0.41	187915
66A2D7	1.89	246499
71G3C1	2.16	222342
73A11E2	0.71	223279
42E3C4	0.52	235111
72B4H4	0.76	261794
34A5G9	0.67	248373
10C6H11	1.24	216888
65F11D11	3.18	302092
61H8C1	0.41	202985
54A4F7	0.84	267980
53A10G6	1.25	224833
53G4D1	1.23	239435
67B1E10	1.32	225194
57A8F3D4	0.80	201907
47A8H10H5	0.60	201116
63E10E12B12	1.93	212433
57F8H3F1	0.70	246001
63E10C8D7	1.00	233188
24G6D5	0.68	225833
74B5E4	0.60	267529

Examplc 3. Binding of Rat Antibodies to IL-13RA2 Protcin |0148] This example examined the rat antibodies, 24 of which, for their ability to bind the human IL-13RA2 protein expressed on A375 cells.

|0149| Λ375 cells that endogenously expressed IL-13RA2 were harvested from flasks. 100 μΙ of 1 * 10⁶ cells/ml of cells were incubated with primary antibodies in 4-fold serial dilutions starting from 100 nM to 0.006 nM for 30 minutes on icc. After being washed with 200 μΐ of FACS buffer twice, cells were incubated with secondary antibody for 30 minutes on icc. Cells were washed with 200 μΐ of FACS buffer twice and analyzed by FACS.

[0150] The results of the study (FIG. 2A-B and Table 3) show that the purified ra! antibodies eould bind to endogenously expressed 1L-13RA2 on A375 cells al high EC50.

Table 3. Binding Affinity of Purified Rat Antibodies

Antibody	ECS0 (nM)	Top (MFI)
94F6G2	1.60	177701
86A2G11	2.14	164044
156G9E4	2.07	174584
119E6G12	1.38	174436
88D8G8	1.70	166609
98A12F3	0.96	165026
98B1C5	2.58	170793
139D9D6	1.56	174468
117H1F11	6.61	178554
104G6A2	2.10	158706
156C3G7	1.15	167075
1OOF2C11	0.85	163131
1Û0B4E2	1.14	171789
150H10E5	NA	19998886
154F3B3	2.71	174154
132C5A1	1.89	165490
1O1E2E3	1.35	179217
101F10B2	2.64	167453
123A10B11A12	1.34	171246
94F3B6B8	1.32	162963
99F7A10F4	4.49	167342
94F3B6C8	1.38	157537
94F3B8D5	1.36	162611
152H5E9H11	1.82	165451

Example 4. Binding of Chimeric Antibodies to IL-13RA2 Protcin

[0151] This example prepared and examincd chimeric antibodies (with human Fc) prepared from the murine ones, for their ability to bind the human 1L-13RA2 protein expressed on A375 cells.

[0152] The murine VII and VK genes were produced synthelically and thon respcctively io cloned into vectors containing the human gamma 1 and human kappa constant domains. The purified chimeric antibodies were produced from transfecled CHOs cells.

|0153| A375 cells that endogenously expressed IL-13RA2, were harvested from llasks. 100 μΙ of 1 xlO⁶ cells/ml of cells were incubated with primary chimeric antibodies in 3-fold serial dilutions starting from 300 nM to 0.002 nM for 30 minutes on ice. Aller being washed with 15 200 μΐ of FACS buffet twicc, cells were incubated with secondary antibody for 30 minutes on ice. Cells were washed with 200 μΐ of FACS buffer twicc and analyzcd by FACS

|0154| As shown in FIG. 3 and Table 4, the chimeric antibodies bound to human IL-13RA2 at different EC50.

Table 4. Binding Affinity of Chimeric Rat Antibodies

Antibody	EC50(nM)	Top(MFI)
XÎ24G6D5 hlgGl	0.88	15173
XÎ34A5G9 hlgGl	0.26	16673
XÎ42E3C41 hlgGl	0.71	17081
xi51G2B3 hlgGl	0.64	15528
x:62A8D8 hlgGl	0.82	17291
XÎ62F6A8 hlgGl	0.48	15815
XÎ42E3C4-2 hlgGl	NA	~351045
XÎ61H8C1 hlgGl	0.47	15582
X154A4F7 hlgGl	0.63	18048
XÎ57F8H3F1 hlgGl	0.99	17646
XÎ72B4H4 hlgGl	0.85	16871
XÎ73A11E2 hlgGl	0.65	17964
XÎ74B5E4 hlgGl	0.70	18142

Example 5. Antibody-Dcpcndcnt Cellular Cytotoxicity (ADCC) of Chimeric Antibodies |(H55| This cxamplc measured the antibody-dependent cellular cytotoxicity (ADCC) of thc chimeric antibodies.

|0156] l'he ADCC Reporter Bioassay uses an alternative rcadout at an earlier point in ADCC MOA pathway activation: the activation of gene transcription through the NFAT (nuclear factor of activatcd T-cells) pathway in the effector ccll. In addition, the ADCC Reporter Bioassay uses engineered Jurkat cells slably expressing thc FcyRIIla receptor. VI58 (high affinity) variant, and an NFAT response element driving expression of firefly luciferase as effector cells. Antibody biological activity in ADCC MOA is quantified through the luciferase produced as a resuit of NFAT pathway activation; luciferase activity in the effector cell is quantified with luminescence rcadout. Signal is high, and assay background is low.

|(H57] Serial dilutions of IL-13RA2 chimeric monoclonal antibody were incubated for 6 hours of induction at 37°C with engineered Jurkat effector cells (ADCC Bioassay Effector Cells). with ADCC Bioassay Target Cells (A375 ccll line). Luciferase activity was quantified using Bio-GloTM Reagent.

|0158] The results are presented in FIG. 4 and Table 5. As shown. ail these chimeric antibodies exhibited very strong ADCC activities.

μ ο

Table 5. Binding Affînity of Chimeric Antibodies

Antibody	ECS0{nM)	Top (MFI)
xi24G6D5 hlgGl	0.18	1543889
XÎ34A5G9 hlgGl	1.83	329309
XI42E3C41 hlgGl	0.38	445302
XÎ51G2B3 hlgGl	0.75	1056643
XÎ54A4F7 hlgGl	0.51	532808
XÎ57F8H3F1 hlgGl	0.48	695892
XÎ61H8C1 hlgGl	0.26	716855
XÎ62A8D8 hlgGl	0.32	943767
XÎ62F6A8 hlgGl	-0.4233	384827
XÎ72B4H4 hlgGl	0.43	412773
XÎ73A11E2 hlgGl	1.02	464841
XÎ74B5E4 hlgGl	- 0.3929	534397

Example 6. Cytotoxie Activities of the Chimeric Antibodies |(H59j This example measured the cclI killing activity of the chimeric antibodies.

|(H60| Λ375 cells that cndogenously expresscd IE-I3RA2 were seeded to a 96-well plate al

3000 cells per welL After overnight incubation, each diluted sample which lias been conjugated with MMAE was added. After 5 days incubation, cell viability was measured by CellTiter-GIo reagent. The luciferase activity was detected by Envison.

[0161I As shown in FIG. 5 and fable 6. these chimeric antibodies ail hâve very strong io cytotoxie activities.

Table 6. Cytotoxie Activity of Chimeric Antibodies

Antibody	ICS0(nM)	Top (MFI)
XÎ24G6D5 hlgGl	1.00	109775
XÎ34A5G9 hlgGl	0.19	98834
XÎ42E3C4-1 hlgGl	0.06	88587
XÎ51G2B3 hlgGl	0.27	96866
XÎ54A4F7 hlgGl	0.11	130274
XÎS7F8H3F1 hlgGl	0.07	152191
XÎ61H8C1 hlgGl	0.33	152919
xi62A8D8 hlgGl	1.01	134240
XÎ62F6A8 hlgGl	0.36	128503
XÎ72B4H4 hlgGl	0.08	146238
XÎ73A11E2 hlgGl	0.03	175106
XÎ74B5E4 hlgGl	1.62	128406

Examplc 6. Endocytosis of the Chimeric Antibodies |Ü162| This examplc texted the chimeric antibodies for their ability to inducc internalization upon binding to surface IE-13RA2.

|(H63| The method uses a new hydrophitic and bright pli sensor dyc (pHAb dyc). which is not fluorescent al neutral pH but becomcs highly fluorescent at acidic pif with inlernaiization. ft can be used to detect the process of cndocytosis. CHO-K.1/HIL-13RA2, 1 IEK-293/H_II>13RA2 cells which overexpress human IL-13RA2, and A375 endogenously expressed human IL-13RA2 as the target cells, the détection antibody labcled with pHAb dye was added to evaluate the cndocytosis of of IL-13RA2 chimeric monoclonal antibody in vitro.

[0164] Serial dilutions of IL-13RA2 chimeric monoclonal antibodies were incubatcd for 24 hours at 37°C. Luciferase activity was detected.

|0165| The results are shown in FIG. 6A-C and Table 7, which demonstrate thaï these chimeric antibodies hâve very strong endocytosis activitics.

Table 7. Inlernaiization of Chimeric Antibodies

Antibody	293 ECSO (nM)	293 Top(MFI)	CHO-K1 ECso(nM)	CHO-K1 Top (MFI)	A375 EC50 (nM)	A375 Top(MFl)
XÎ24G6D5 hlgGl	2.76	23713	0.86	12681	0.61	4114
XÎ34A5G9 hlgGl	1.78	25655	1.38	13863	3.63	8610
XÎ42E3C4-1 hlgGl	1.56	25623	1.35	18711	0.49	4695
XÎ51G2B3 hlgGl	3.84	28949	1.84	17110	1.32	6384
XÎ54A4F7 hlgGl	1.65	22791	1.42	17996	0.66	5505
XÎ57F8H3F1 hlgGl	2.42	26010	2.37	19197	0.74	2725
XÎ61H8C1 hlgGl	1.49	24686	1.02	16949	~ 0.4412	3824
XÎ62A8D8 hlgGl	1.88	23262	1.58	17204	1.00	5553
XÎ62F6A8 hlgGl	1.73	22304	1.17	15857	~ 0.4473	4531
XÎ72B4H4 hlgGl__ XÎ73A11E2 hlgGl	2-18____ 1.98	19_54Ê,_ 23481	2.92 1.52	J.8705_____ 17880	1.06___ 0.77	5262___ 4311
XÎ74B5E4 hlgGl	1.31	21621	0.91	17727	~ 0.4370	5173

Example 7. Humanization of Mouse Antibodies

[0166] Four of the murine antibodies, 61H8C1, 57F8H3F1, 72B4H4 and 73Λ11Ε2, were selected for humanization.

[0167] The amino acid sequences of the VH and VL of each murine antibody were compared against the available database of human Ig gene sequences to fînd the overall best-matching human germline Ig gene sequences. The CDRs of the murine antibodics were lhen grafted into the matched human sequences. The cDNA was synthesized and used to produce the humanized antibodies. Certain back mutations from the murine antibodies were lhen introduced back to the humanized antibodies. Certain amino acids were mutated lo reducc lhe chance of post-lranslational modification.

[0168] The amino acid sequences ol'the humanized antibodies arc provided in Tables 8-11 below.

Humanized sequences

Λ. 61H8C1

Table 8A. Huntanization of 6IH8C1 - VH

Na me	Sequence	SEQ ID NO;
61H8C1 VH	QVQLQQSGAELARPGASVMLSCKASGYTFTSYWTQWIKQRPGQGL EGIGAIYPGDDDTSYNQKFKGKATLTADKSSSTAYMQLNSLTSED SAVYYCARGATLLRPFPYWGQGTLVTVSA	13
VH_I (CDR giaiting)	QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWTQWVRQAPGQGL EWMGAIYPGDDDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSED TAVYYCARGATLLRPFPYWGQGTLVTVSS	54
VH 2	QVQLVQSGAEVAKPGASVKVSCKASGYTFTSYWTQWIRQAPGQGL EGMGAIYPGDDDTSYNQKFKGRVTMTADTSTSTVYMELSSLRSED TAVYYCARGATLLRPFPYWGQGTLVTVSS	55
VH 3	QVQLVQSGAEVAKPGASVKVSCKASGYTFTSYWTQWIRQAPGQGL EGIGAIYPGDDDTSYNQKFKGRATLTADKSTSTVYMQL,SSLRSED TAVYYCARGATLLRPFPYWGQGTLVTVSS	56
VHJ	QVQLVQSGAEVAKPGASVKLSCKASGYTFTSYWTQWIKQAPGQGL EGIGAIY PG DDDT SYNQKFKGKATLTADKST S TAYMQLS S L RS ED TAVYYCARGATLLRPFPYWGQGTLVTVSS	57

Table 8B. CDR Sequences

CDR	Séquence	SEQ ID NO:
CDR-HI	SYWTQ	25
CDR-H2	AIYPGDDDTSYNQKFKG	26
CDR-H3	GATLLRPFPY	27

Table 8C. Humanization of 61H8CI — VL

Name	Sequence	SEQ ID NO:
61H8CI VL.	DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPK LLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYSCQQ HYSIPPTFGGGTKLEIK	14
VLJ (CDR grafting)	DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPK LLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ HYSIPPTFGQGTKLEIK	58
VL_2	D1QMTQSPSSLSTSVGDRVTITCKASQDVSTAVAWYQQKPGKSPK LLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYSCQQ HYSIPPTFGQGTKLEIK	59
VLJ	DIQMTQSPSSLSTSVGDRVTITCKASQDVSTAVAWYQQKPGKSPK LLIYSASYRYTGVPDRFSGSGSGTDFTFTISSLQPEDFATYSCQQ HYSIPPTFGQGTKLEIK	60
VLJ	DIVMTQSPSSLSTSVGDRVTITCKASQDVSTAVAWYQQKPGKSPK LLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQPEDFATYSCQQ HYSIPPTFGQGTKLEIK	61

Table 8D. CDR Sequenees

CDR	Sequence	SEQ ID NO:
CDR-L1	KASQDVSTAVA	28
CDR-L2	SASYRYT	29
CDR-L3	QQHYSIPPT	30

Table 8E. Humanized antibodies

VH VL	61H8C1 VH_1	61H8C1 VH_2	61H8C1 VH_3	61H8C1 VH_4
61H8C1 VL_1	H1L1	H2L1	H3L1	H4L1
61H8C1 VL_2	H1L2	H2L2	H3L2	H4L2
61H8C1 VL_3	H1L3	H2L3	H3L3	H4L3
61H8C1 VL_4	H1L4	H2L4	H3L4	H4L4

B. 57F8H3F1

Table 9A. Humanization of 57F8H3F1 — VH

Nanie	Sequence	SEQ 11) NO:
57F8H3F1 VH	QVQLQQ PGAELVRPGAS VKLS C KASG YT F’TNYWMNW IKQRPEQGL EWIGRIDPYETETHYNQKFRDRAILTVDKSSSTAFMQLSGLTSED SAVYYCARGYGHYYGVDYWGQGTSVTVSS	11
VH i iCDR grafting)	QVQLVQSGAEVKKPGASVKVSCKASGYTF’TNYWMNWVRQAPGQGL EWMGRIDAYETETHYNQKFRDRVTMTRDTSTSTVYMELSSLRSED TAVYYCARGYGHYYGVDYWGQGTLVTVSS	62
VII 2	QVQLVQSGAEVVKPGASVKVSCKASGYTFTNYWMNWVRÛAPGQGI. EWIG1UDAYETETHYNQKFRDRVTMTVDTSTSTVYMELSSLRSED TAVYYCARGYGHYYGVDYWGQGTLVTVSS	63
VH 3	QVQLVQSGAEVVKPGASVKLSCKASGYTFTNYWMNWVKQAPGQGL EWIGRIDAYETETHYNQKFRDRATLTVDKSTSTVYMELSSLRSED TAVYYCARGYGHYYGVDYWGQGTLVTVSS	64
VH _4	QVQLVQSGAEVVKPGASVKLSCKASGYTFTNYWMNWVKQRPGQGL EWIGRIDAYETETHYNQKFRDRAILTVDKSTSTAYMELRSLRSED TAVYYCARGYGHYYGVDYWGQGTLVTVSS	65 . J

Table 9B. CDR Sequenees

CDR	Sequence	SEQ ID ΝΌ: 31
CDR-Hl	NYWMN
CDR-H2	RIDPYETETHYNQKFRD	49
RIDAYETETHYNQKFRD	32
CDR-H3	GYGHYYGVDY	.33

Table 9C. Humanization of 57F8H3FI - VL

Namc Sequence £sEQIDNO: ~| /4

57F8H3F! VL	DIVLTQS PASLAVSLGQ RATISCRASENVGNFGISFVNWFQQKPG QPPKLLIYAASNLGSGVPARFSGSGSGTDFSLNIHPMEEDDAAMY FCQQSKGVPWTFGGGTKLEIK	12
VL_ 1 (CDR grafting)	DIQMTOSPSSLSASVGDRVTITCRASENVGNFGISFVNWYQQKPG KAPKLLIYAASNLGSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSKGVPWTFGQGTKVEIK	66
VL_2	DIQMTQSPSSLSASVGDRVTITCRASENVGNFGISFVNWFQQKPG KAPKLLIYAASNLGSGDPSRFSGSGSGTDFTLTISSLQPEDFATY FCQQSKGVPWT FGQGT KVE1K	67
VLJ	DIQLTQSPSSLSASVGDRVTITCRASENVGNFGISFVNWFQQKPG KAPKLLIYAASNLGSGDPARFSGSGSGTDFTLTISSLQPEDFATY FCQQSKGVPWTFGGGTKVEIK	68
VL_4	DIVLTQS PSS LSASVGDRATITCRASENVGNFGISFVNWFQQKPG KPPKLLIYAASNLGSGDPARFSGSGSGTDFTLTISSLQPEDFATY FCQQS KGV PWT FGGGT KLEIK	69

Table 9D. CDR Seq uen ces

CDR	Sequence	SEQ ID NO:
CDR-LI	RASENVGNFGISFVN	34
COR-L2	AASNLGS___________	35
CDR-L3	QQSKGVPWT	36

Table 9E. Humanized antibodies

VH VL	57F8H3F1 VH_1	57F8H3F1 VH_2	57F8H3F1VH_3	57F8H3F1 VH_4
57F8H3F1 VL_1	H1L1	H2L1	H3L1	H4L1
57F8H3F1 VL_2	H1L2	H2L2	H3L2	H4L2
57F8H3F1 VL_3	H1L3	H2L3	H3L3	H4L3
57F8H3F1 VL_4	H1L4	H2L4	H3L4	H4L4

C. 72B4H4

Table 10A. Humanisation of 72B4H4 - VH

Na nie	Sequence	SEQ II) NO:
72114114 VH	QVQLQQPGAERVRPGASVKLSCKASGYTFTNÏWMNWVKQRPEQGL EWIGRIDPYAGETHYNQKFKDKAILTVDKSSSTAYMQLR5LTSED SAVYYCARGYGDSYGMDHWGQGTSVTVSS	19
Vil 1 (CDR grafting)	QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMNWVRQAPGQGL EWMGRIDAYAGETHYNQKFKDRVTMTRDTSTSTVYMELSSLRSED TAVYYCARGYGDAYGMDHWGQGTLVTVSS	70
VH 2	QVQLVQSGAEVVKPGASVKVSCKASGYTFTNYWMNWVRQAPGQGL EWIGRIDAYAGETHYNQKFKDRVTMTVDTSTSTVYMELSSLRSED TAVYYCARGYGDAYGMDHWGQGTLVTVSS	71
VH 3	QVQLVQSGAEVVKPGASVKLSCKASGYTFTNYWMNWVKQAPGQGL EWIGRIDAYAGETHYNQKFKDRATLTVDKS'TSTVYMELSSLRSED TAVYYCARGYGDAYGMDHWGQGTLVTVSS	72
VH 4	QVQLVQSGAEVVKPGASVKLSCKASGYTFTNYWMNWVKQRPGQGL	73

Μ· 5

E WIGRIDAYAGET H YNQKFK DRAILTVDKS TS TAYME LRSLRS E D TAVYYCARGYGDAYGMDHWGQGTLVTVSS

Table 10 B. CDR Sequences

CDR	Sequence	SEQ (D NO:
COR-H 1	NYWMN	37
CDR-H2	RIDPYAGETHYNQKFKD	50
RIDAYAGETHYNQKFKD	38
CDR-H3	GYGDSYGMDH	51
GYGDAYGMDH	39

Table 10C. Humanization of72B4H4 - VL

Name	Sequence	SEQ 11) NO:
72B4H4 VL	DIVLTQSPAS LAVS LGQRATIS CRASESVDNYGISEMNWFQQKPG QPPKLLIYAASRQGSGDPARFSGSGSGTDFSLNIHPLEEDDTAMY FCQQSKEVPWTFGGGTKLEIK	20
VLJ (CDR graiting)	DIQMTQS PSS LSASVGDRVTITC RAS ESVDNYGIS FM NWYQQKPG KAPKLLIYAASRQGSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSKEVPWTFGQGTKVEIK	74
VL. 2	DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPG KAPKLLIYAASRQGSGDPSRFSGSGSGTDFTLTISSLQPEDFATY FCQQSKEVPWTFGQGTKVEIK	75
VI _3	DIQLTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPG KAPKLLÏYAASRQGSGDPARFSGSGSGTDFTLTISSLQPEDFATY FC QQS KEV PWT FGGGT KVEIK	76
VLJ	DIVLTQSPSSLSASVGDRATITCRASESVDNYGISFMNWFQQKPG KPPKLLIYAASRQGSGDPARFSGSGSGTDFTLTISSLQPEDFATY FCQQSKEVPWTFGGGTKLEIK	77

Table 10D. CDR Sequences

CDR	j Sequence	SEQ ID NO:
CDR-L1	1 RASESVDNYGISFMN	40
CDR-L2	| AASRQGS	41
CDR-L3	[ QQSKEVPWT	42

Table 10E. Humanized antibodies

VH VL	72B4H4 VH_1	72B4H4 VH_2	72B4H4 VH_3	72B4H4 VH_4
72B4H4 VL_1	H1L1	H2L1	H3L1	H4L1
72B4H4 VL_2	H1L2	H2L2	H3L2	H4L2
72B4H4 VL_3	H1L3	H2L3	H3L3	H4L3
72B4H4 VL_4	H1L4	H2L4	H3L4	H4L4

D.73AHE2

Table J ΙΑ. Humanization of 73Al 1E2 - VH

Namc	Sequence	SEQ il) NO:
7.3AI1E2 VH	EVKLVESGGGL·VKPGGSL·KLSCAASGFTFSSYGMSWVRQTPEKRL EWVATISGGGSYTYYPDSVKGRFTIS RDNARNNLY LQMNS LRS E D T ALYY CARRDLRSAMDYWGQG T S V TVS S	21
VHJ (CDR grafting)	QVQLVQSGAEVKKPGASVKVSCKASGFTFSSYGMSWVRQAPGQGL EWMGTISGGGSYTYYPESVKGRVTMTTDTSTSTAYMELRSLRSDD TAVYYCARRDLRSAMDYWGQGTLVTVSS	78
VHJ	QVQLVQSGAEVVKPGASVKVSCKASGFTFSSYGMSWVRQAPGQGL EWVGTISGGGSYTYYPESVKGRFTITRDTSTSTAYMELRSLRSDD TAVYYCARRDLRSAMDYWGQGTLVTVSS	79
VHJ	QVQLVQSGAEVVKPGASVKLSCKASGFTFSSYGMSWVRQAPGQGL EWVATISGGGSYTYYPESVKGRFTITRDNSTSTLYMEMRSLRSDD TAVYYCARRDLRSAMDYWGQGTLVTVSS	80
VHJ	QVKLVES GAEVVKPGASVKLSCKASG FT FS S Y GMSWVRQAPGQRL EWVATISGGGSYTYYPESVKGRFTITRDNSTSTLYLEMRSLRSDD TAVYYCARRDLRSAMDYWGQGTLVTVSS	81

Table II B. CDR Séquences

CDR	Sequence	SEQ ID NO:
CDR-HI	SYGMS	43
CDR-H2	TlSGGGSYTYYPDSVKG	52
TlSGGGSYTYYPESVKG	44
CDR-H3	RDLRSAMDY	45

Table I1C. Humanization of73Al!E2 - VL

N a nie	Sequence	SEQ ID NO:
73AI ] E2 VL	DIVMTQSQKFMSTSVGDRVSVTCKASQNVDINVAWYQQKPGQSPK ALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEFFCQQ YNSYPLTFGGGTRLEIK	22
VL J (CDR grafting)	DWMTQSPLSLPVTLGQPASISCKASQNVDINVAWFQQRPGQSPR RLIYSASYRYSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQ YNAYPLTFGQGTKLEIK	92
VLJ	DIVMTQSPLSLPVTLGQPASISCKASQNVDINVAWYQQRPGQSPR ALIYSASYRYSGVPDRFSGSGSGTDFTLKISRVEAEDVGVFYCQQ YNAYPLT FGQGTKLEIK	93
VL J	DIVMTQSPLSLPTTLGQPASISCKASQNVDINVAWYQQRPGQSPR ALIYSASYRYSGVPDRFSGSGSGTDFTLTISRVEAEDVGVFYCQQ YNAYPLTFGGGTKLEIK	84
VLJ	DIVMTQSPLSLPTTLGQPVSVSCKASQNVDINVAWYQQRPGQSPR ALIYSASYRYSGVPDRFSGSGSGTDFTLTISRVEAEDVAVFFCQQ YNAYPLTFGGGTKLEIK	85

Table 11D. CDR Sequences

CDR	Sequence	SEQ ID NO:
CDR-L1	KASQNVDINVA	46

CDR-L2	SASYRYS	47
CDR-L3	QQYNSYPLT	53
QQYNAYPLT	48

Table 11E. Humanized antibodies

VH VL	73A11E2 VH_1	73A11E2VH_2	73A11E2VH_3	73A11E2 VH_4
73A11E2 VL_1	H1L1	H2L1	H3L1	H4L1
73A11E2 VL_2	H1L2	H2L2	H3L2	H4L2
73A11E2 VL_3	H1L3	H2L3	H3L3	H4L3
73A11E2 VL_4	H1L4	H2L4	H3L4	H4L4

Examplc 8. Binding Affinity of Humanized Antibodies |0169] This example tested the binding affinity of the humanized antibodies.

|0170] A375 eells that endogcnously expressed IL-13RA2, were harvestcd from fiasks. 100 μΙ of 1 x 10⁶ cclls/ml of eells were incubated with primary humanized antibodics in 4-fold serial dilutions starting from 200 nM to 0.003 nM for 30 minutes on icc. After being washcd with 200 μΐ of TACS buffer twice, eells were incubated with secondary antibody for 30 io minutes on ice. Cells were washcd with 200 μΐ of FACS buffer twice and analyzed by FACS.

[01711 The results are presenled in FIG. 7A- and Table 12. As shown. the humanized antibodies can bind to human IL-13RA2 at high EC50.

Table 12A. Binding Affinity of Humanized Versions of61H8Cl

Antibody	ECS0 (nM)	Top (MFI)
hlgGl Isotype	N.A.	N.A.
XÎ61H8C1 hlgGl	0.16	22167
61H8C1 H1L1 hlgGl	0.14	20253
61H8C1 H1L2 hlgGl	0.15	19637
61H8C1 H1L3 hlgGl	0.20	20025
61H8C1 H1L4 hlgGl	0.17	19776
61H8C1 H2L1 hlgGl	0.16	22421
61H8C1 H2L2 hlgGl	0.15	22358
61H8C1 H2L3 hlgGl	0.19	22140
61H8C1 H2L4 hlgGl	0.18	21925

Table 12B. Binding Affinity of Humanized Versions of 61H8C1 (cont’d)

Antibody	ECS0(nM)	Top(MFl)
hlgGl Isotype	N.A.	N.A.
XÎ61H8C1 hlgGl	0.16	21465
61H8C1 H3L1 hlgGl	0.15	20615
61H8C1 H3L2 hlgGl	0.17	22336

61H8C1H3L3 hlgGl	0.15	20148
61H8C1 H3L4 hlgGl	0.20	21962
blH8Cl H4L1 hlgGl	0.17	22219
61H8C1 H4L2 hlgGl	0.14	21148
61H8C1 H4L3 hlgGl	0.18	20846
61H8C1 H4L4 hlgGl	0.19	21550

Table 12C. Binding Affinity of Hunianized Versions of 57F8H3F1

Antibody	ECS0(nM)	Top(MFl)
XI57F8H3F1 hlgGl	0.33	7344
57F8H3F1 H1L1 hlgGl	0.63	7015
57F8H3F1 H1L2 hlgGl	0.78	7509
57F8H3F1 H1L3 hlgGl	0.81	7536
57F8H3F1 H1L4 hlgGl	0.94	7815
57F8H3F1 H2L1 hlgGl	0.38	6910
57F8H3F1 H2L2 hlgGl	0.89	7090
57F8H3F1H2L3 hlgGl	0.92	7013
57F8H3F1H2L4 hlgGl	1.01	7144

Table 12D. Binding Affinity of Hunianized Versions of 57F8H3FI (cont'd)

Antibody	ECS0(nM)	Top(MFI)
XÎ57F8H3F1 hlgGl	0.41	7340
57F8H3F1 H3L1 hlgGl	0.71	8157
57F8H3F1 H3L2 hlgGl	1.25	7424
57F8H3F1 H3L3 hlgGl	1.62	7614
57F8H3F1 H3L4 hlgGl	1.30	7609
57F8H3F1 H4L1 hlgGl	0.42	7353
57F8H3F1 H4L2 hlgGl	0.75	7335
57F8H3F1 H4L3 hlgGl	0.80	7184
57F8H3F1 H4L4 hlgGl	0.91	7267

Table I2E. Binding Affinity of Hunianized Versions of 72B4H4

Antibody	EC50(nM)	Top (MF!)
XÎ72B4H4 hlgGl	0.36	8877
72B4H4H1L1 hlgGl	0.54	8438
72B4H4H1L2 hlgGl	0.81	8536
72B4H4H1L3 hlgGl	1.00	8902
72B4H4 H1L4 hlgGl	0.98	9045
72B4H4 H2L1 hlgGl	2.75	8357
72B4H4 H2L2 hlgGl	1.48	8699
72B4H4 H2L3 hlgGl	0.83	8420
72B4H4 H2L4 hlgGl	1.18	8634

Table 12F. Binding Affinity of Hunianized Versions of 72B4H4 (cont’d)

Antibody	ECS0 (nM)	Top (MF1)
XÎ72B4H4 hlgGl	0.5813	8499
72B4H4H3L1 hlgGl	1.137	8829
72B4H4H3L2 hlgGl	1.436	8551
72B4H4 H3L3 hlgGl	1.662	8777
72B4H4 H3L4 hlgGl	1.363	8769

72B4H4 H4L1 hlgGl	0.5522	8266
72B4H4 H4L2 hlgGl	0.9756	8613
72B4H4H4L3 hlgGl	0.8109	8467
72B4H4 H4L4 hlgGl	1.183	8181

Table I2G. Binding Affinity of Humanized Versions of 73A11E2

Antibody	ECS0 (nM)	Top (MFI)
XÎ73A11E2 hlgGl	0.26	8139
73A11E2 H1L1 hlgGl	82.75	389.7
73A11E2 H1L2 hlgGl	~ 23774557481	~412109
73A11E2 H1L3 hlgGl	~ 250.6	~4149
73A11E2 H1L4 hlgGl	~ 2.678e+017	~ 294650
73A11E2 H2L1 hlgGl	0.6	5960
73A11E2 H2L2 hlgGl	0.67	7664
73A11E2 H2L3 hlgGl	0.69	7269
73A11E2 H2L4 hlgGl	1.61	7019

Table 12H. Binding Affinity of Humanized Versions of73AllE2 (cont’d)

Antibody	ECm (nM)	Top(MFI|
XÎ73A11E2 hlgGl	0.3586	7701
73A11E2 H3L1 hlgGl	1.072	5980
73A11E2 H3L2 hlgGl	2.224	7368
73A11E2 H3L3 hlgGl	1.494	7423
73A11E2 H3L4 hlgGl	1.157	7285
73A11E2 H4L1 hlgGl	0.9434	5835
73A11E2 H4L2 hlgGl	1.122	7347
73A11E2 H4L3 hlgGl	0.9092	6985
73A11E2 H4L4 hlgGl	1.117	6839

Table 121. Binding Affinity of Humanized Versions of 73A11E2 (cont’d)

Antibody	ECm (nM)	Top(MFl)
XÎ73A11E2 hlgGl	0.45	17083
73A11E2 H1L1 hlgGl	- 708625	~ 49273
73A11E2 H1L2 hlgGl	178.4	1029
73A11E2 H1L3 hlgGl	-521.7	~ 1490
73A11E2 H1L4 hlgGl	-388.5	996.9
73A11E2 H2L1 hlgGl	0.56	13314
73A11E2 H2L2 hlgGl	0.67	15705
73A11E2 H2L3 hlgGl	0.67	15470
73A11E2 H2L4 hlgGl	0.66	15306

Table 12J. Binding Affinity of Humanized Versions of 73A11E2 (cont’d)

Antibody	ECs0(nM)	Top (MFI)
XÎ73A11E2 hlgGl	0.45	17083
73A11E2 H3L1 hlgGl	1.37	15156
73A11E2 H3L2 hlgGl	1.53	15878
73A11E2 H3L3 hlgGl	1.17	15883
73A11E2 H3L4 hlgGl	1.29	16583
73A11E2 H4L1 hlgGl	1.20	14883

73A11E2 H4L2 hlgGl	1.31	16290
73A11E2 H4L3 hlgGl	1.45	16196
73A11E2 H4L4 hlgGl	1.51	16454

Example 9. ADCC of the Humanized Antibodies |01721 l’his example measured lhe anlibody-dependent cellular cytoloxicity (ADCC) of the humanized antibodies.

[0173] The ADCC Reporter Bioassay uses an alternative readout al an earlier point in ADCC MOA pathway activation: lhe activation of gene transcription through the NI AT (nuclear factor of activalcd T-cclls) pathway in the effector celi. In addition, the ADCC Reporter Bioassay uses engineered Jurkat cclls stably expressing the EcyRlIla receptor, VI58 (high affmity) variant, and an NFAT responsc élément driving expression of fireily luciferase as effector ceils. Antibody biological activity in ADCC MOA is quanlilied through the luciferase produccd as a rcsult of NFAT pathway activation; luciferase activity in the effector celi is quantificd with luminescence readout. Signal is high, and assay background is low.

[0174] Serial dilutions of IL-13RA2 humanized monoclonal antibody werc incubated for 6 hours of induction at 37°C with engineered Jurkat effector ceils (ADCC Bioassay Effector Colis), with ADCC Bioassay Target Ceils (A375 celi line). Luciferase activity was quantified using Bio-GloTM Reagent.

|0l75] The rcsulls show that these humanized antibodies hâve very strong ADCC activitics.

|0176] lhe results arc presented in FIG. SA (1L-13RA2 overexpression celi fines) and SB (Endogenous Gliomas celi lines). As shown, ail thèse humanized antibodies exhibited very' strong ADCC activities.

Example 10. F.ndoeytosis of the Humanized Antibodies

[0177] This example texled lhe chimeric antibodies for their ability to induce internalization upon binding to surface 1L-I3RA2.

|OI78| The method uses a new hydrophilic and bright pli sensor dye (pHAb dye). which is not lluorcscent at ncutral piI but becomes highly fluorescent at acidic pH with internalization. It can be used to detecl the proccss of endocylosis. CHO-K1/HJL-13RA2. I lEK-293/H_IL13RA2 ceils which overexpress human IL-13RA2, and A375 cndogenously expressed human

IL-13RA2 as the target cells, the détection antibody iabelcd with pHAb dye was added to evaluatc the cndocytosis of of IL-13RA2 chimeric monoclonal antibody in vitro.

[0179] Serial dilutions of selected humanized antibodies were incubated for 24 hours at 37°C. Luciferase activity was detectcd.

[0180] The results arc shown in FIG. 9A (IL-13RA2 overexpression cell Unes), and 9B-C (endogenous cell lines), which demonstrate that these humanized antibodies hâve vcry strong cndocytosis activities.

Examplc 11. In vitro Cytotoxicity of Antibody-Drug Conjugales

H)181| This exampic tested the cyloloxicity of humanized antibodies conjugatcd to toxic drugs (ADC) on different Gliomas cells.

[0182] A375 and U251 cells that cndogenously expressed IL-13RA2, IIEK-293 cells that stably expressed IL-13RA2 were seeded to a 96-well plate at 3000 cells per well. After overnight incubation, each diluted sample which has been conjugatcd with MMAE was added. After 5 days incubation, cell viability was measured by CeU liter-Glo reagent. The luciferase activity was detectcd by Envison.

[0183| As shown in FIG. 10A-C, these ADC hâve very strong cytotoxic activities.

Exampk· 12. In vivo Cytotoxicity of Antibody-Drug Conjugales |0184| This cxample tested the in vivo cytotoxicity of humanized antibodies conjugated to MMAE (ADC).

[0185) In this study, 6-8 weeks fcmale NCG mice (Jiangsu Jicui Yaokang Biotcchnology Co.. Ltd) were used. Each mouse was inoculated subcutaneously at the right axillary (latéral) with A375 tumor cell (5*106) in 0.1 ml of PBS with matrigel (V:V=I:I ) for tumor development. The animais were grouped when tumor volume reach around 70mm³, which is the 5th day after cell inoculation, then treatment started for the effïcacy study.

|0186| Testing articles at dose of 3 mg/kg was administrated via intravenous (i.v.) at day 0, day 7, and day 14. The experiment was terminated at day 23. Tumor sizes were measured three limes weckly in tw'o dimensions using a caliper and the volume was expressed in mm³ using the formula: V = 0.5 a x b² where a and b were the long and short diameters of the tumor, respectively. The tumor sizes were then used for the calculations of T/C (%) values. T/C (%) was calculated using the formula: T/C % = (Ti-T0)/ (Vi-VO) *100 . Ti was the average tumor volume of a treatment group on a given day. TO was the average tumor 5 volume of the treatment group on the first day of treatment, Vi was the average tumor volume of the vehicle control group on the samc day with Ti, and V0 was the average tumor volume ofthe vehicle group on the first day of treatment. TGI was calculated for each group using the formula: TGI (%) = [100-T/C]. Data points represent group (n=5) mcan, error bars represent standard error ofthe mean (SEM). p value was calculated based on tumor size by tio test, compared with PBS control.

[0187] FIG. 11A shows body weight changes of the tumor-bearing mice in different groups. Data points represent group mean body weights. Error bars represent standard error of the mcan (SEM). FIG. 11B and Table 13 show tumor growth curves of A375 tumor-bearing mice post administration of testing ADCs. As shown in thèse figures, the ADC exhibited 15 strong in vivo cytotoxic activities.

Table 13. Tumor growth measuremeut post treatments

T/C (%)	Days after treatment	0	2	5	7	9	13	16	20	23
G2	-	71.58	23.20	-10.55	-10.69	-8.24	-6.18	-2.29	1.37
G3	-	98.78	21.32	7.86	-3.80	-1.39	-0.72	3.21	12.67
G4	-	60.43	33.37	5.46	-3.84	-3.40	-0.58	2.86	10.72
G5	-	84.85	22.37	-0.35	-4.15	-4.71	-2.48	3.39	10.43

TGI (%)	Days after treatment	0	2	5	7	9	13	16	20	23
G2	-	28.42	76.80	110.55	110.69	108.24	106.18	102.29	98.63
G3	-	1.22	78.68	92.14	103.80	101.39	100.72	96.79	87.33
G4	-	39.57	66.63	94.54	103.84	103.40	100.58	97.14	89.28
G5	-	15.15	77.63	100.35	104.15	104.71	102.48	96.61	89.57

P value	Days after treatment	0	2	5	7	9	13	16	20	23
G2	0.9515	0.2847	0.0038	o.oooi	0.0000	0.0001	0.0000	0.0002	0.0001
G3	0.9733	0.9455	0.0009	0.0003	0.0000	0.0002	0.0000	0.0002	0.0002
G4	0.9704	0.1019	0.0033	0.0002	0.0000	o.oooi	0.0000	0.0002	0.0002
G5	0.9061	0.5923	0.0014	0.0002	0.0000	0.0001	0.0000	0.0002	0.0002

* * *

[0188| The présent disclosure is not to be limîted in scope by the spécifie embodiments described which are intended as single illustrations of îndividual aspects ofthe disclosure, and any compositions or methods which are functionally équivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the présent disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the présent 5 disclosure cover the modifications and variations of this disclosure provided they corne w'ithin the scope of the appendcd daims and their équivalents.

|0l89| Ail publications and patent applications mentioned in this spécification arc herein incorporated by reference to the saine extent as if each individual publication or patent application was specîfically and individually indicated to bc incorporated by reference.

Claims (28)

1. An antibody or fragment thereof having bînding speeificity to a human interleukin 13 receptor subunit alpha 2 (IL-13RA2) protein, wherein thc antibody or fragment thereof comprises a heavy chain variable région comprising heavy chain complcmcntarity determining régions CDRI 11, CDRH2, and CDRH3 and a light chain variable région light chain comprising complcmcntarity determining régions CDRLI, CDRL2, and CDRL3, and wherein:

(a) the CDRIII comprises the amino acid sequence of SEQ ID NO: 25, the CDRI 12 comprises the amino acid sequence of SEQ ID NO: 26, the CDRI 13 comprises the amino acid sequence of SEQ ID NO: 27, the CDRLI comprises the amino acid sequence of SEQ ID NO: 28, thc CDRL2 comprises the amino acid sequence of SEQ ID NO: 29, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 30, (b) thc CDRI 11 comprises thc amino acid sequence of SEQ ID NO: 31, the CDRI 12 comprises thc amino acid sequence of SEQ ID NO: 32 or 49, the CDRI 13 comprises the amino acid sequence of SEQ ID NO: 33, thc CDRI J comprises the amino acid sequence of SEQ ID NO: 34, the CDRL2 comprises the amino acid séquence of SEQ ID NO: 35, and thc CDRL3 comprises the amino acid sequence of SEQ ID NO: 36, (c) the CDRI II comprises the amino acid sequence of SEQ ID NO: 37, the CDRI 12 comprises the amino acid sequence of SEQ ID NO: 38 or 50, the CDRI 13 comprises the amino acid sequence of SEQ ID NO: 39 or 51, the CDRLI comprises the amino acid sequence of SEQ ID NO: 40, thc CDRL2 comprises the amino acid sequence of SEQ ID NO: 41, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 42, or (d) the CDRI 11 comprises the amino acid sequence of SEQ ID NO: 43, the CDRI 12 comprises the amino acid sequence of SEQ ID NO: 44 or 52, the CDRI 13 comprises the amino acid sequence of SEQ ID NO: 45, the CDRLI comprises the amino acid sequence of SEQ ID NO; 46, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 47, and thc CDRL3 comprises the amino acid sequence of SEQ ID NO: 48 or 53.

2. The antibody or fragment thereof of claim 1, wherein the CDRI11 comprises the amino acid sequence of SEQ II) NO: 25, the CDRH2 comprises the amino acid sequence of SEQ II) NO: 26, the CDRH3 comprises the amino acid sequence of SEQ II) NO: 27, the CDRLl comprises the amino acid sequence of SEQ II) NO: 28. the CDRL2 comprises the amino acid sequence of SEQ II) NO: 29, and the CDRL3 comprises the amino acid sequence ofSEQ II) NO: 30.

3. The antibody or fragment thereof of claim 2, wherein the heavy chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13 and 54-57, and the light chain variable région comprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 14 and 58-61.

4. The antibody or fragment thereof of claim 2, wherein the heavy chain variable région comprises the amino acid sequence ofSEQ ID NO:54, and the light chain variable région comprises the amino acid sequence ofSEQ ID NO:58.

5. 1 he antibody or fragment thereof of claim 1, wherein the CDRI11 comprises the amino acid sequence ofSEQ ID NO: 31, the CDRII2 comprises the amino acid sequence of SEQ ID NO: 32 or 49, the CDRI13 comprises the amino acid sequence of SEQ ID NO: 33, the CDRLl comprises the amino acid sequence ofSEQ ID NO: 34, the CDRL2 comprises the amino acid sequence ofSEQ ID NO: 35, and the CDRL3 comprises the amino acid sequence ofSEQ ID NO: 36.

6. The antibody or fragment thereof of claim 5, wherein lhe CDRI 12 comprises lhe amino acid sequence ofSEQ ID NO: 32.

7. The antibody or fragment thereof of claim 5, wherein the heavy chain variable région comprises an amino acid sequence selected from the group consisting ofSEQ II) NO: 11 and 62-65, and the light chain variable région comprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 12 and 66-69.

8. The antibody or fragment thereof of claim 5, wherein the heavy chain variable région comprises the amino acid sequence ofSEQ II) NO:62, and the light chain variable région comprises the amino acid sequence ofSEQ ID NO:66.

9. The antibody or fragment thereof of claim l, wherein the CDRHl comprises the amino acid sequence of SEQ ID NO: 37, the CDRH2 comprises the amino acid séquence of SEQ ID NO: 38 or 50, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 39 or 51, the CDRLl comprises the amino acid sequence of SEQ ID NO: 40, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 41, and the CDRL3 comprises the amino acid sequence of SEQ ID NO: 42.

10. The antibody or fragment thereof of claim 9, wherein the CDRH2 comprises the amino acid sequence of SEQ ID NO: 38, and the CDRII3 comprises the amino acid sequence ofSEQ ID NO: 39.

11. The antibody or fragment thereof of claim 9, wherein the heavy chain variable région comprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 19 and 70-73, and the light chain variable région comprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 20 and 74-77.

12. The antibody or fragment thereof of claim 9, wherein the heavy chain variable région comprises the amino acid sequence ofSEQ ID NO:70, and the light chain variable région comprises the amino acid sequence ofSEQ ID NO:74.

13. The antibody or fragment thereof of claim 1, wherein the CDRHl comprises the amino acid sequence ofSEQ ID NO: 43, the CDRI 12 comprises the amino acid sequence of SEQ ID NO: 44 or 52, the CDRI 13 comprises the amino acid sequence ofSEQ ID NO: 45, the CDRLl comprises the amino acid sequence ofSEQ ID NO: 46, the CDRL2 comprises the amino acid sequence ofSEQ ID NO: 47, and the CDRL3 comprises the amino acid sequence ofSEQ ID NO: 48 or 53.

14. The antibody or fragment thereof of claim 13, wherein the CDRI 12 comprises the amino acid sequence ofSEQ ID NO: 44, and the CDRL3 comprises the amino acid sequence ofSEQ IDNO: 48.

15. The antibody or fragment thereof of claim 13, wherein the heavy chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 21 and 78-81, and the light chain variable région comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22 and 82-85.

16. The antibody or fragment thereof of claim 13, wherein the heavy chain variable région comprises the amino acid sequence of SEQ ID NO:79, and the light chain variable région comprises the amino acid sequence of SEQ ID NO:83.

17. The antibody or fragment thereof of any one of claims 1-16, which is humanized.

18. An antibody or fragment thereof, which has binding specificity to a human interleukin 13 receptor subunit alpha 2 (IL-13RA2) protein and binds to the same epilopc on the IL13RA2 protein as the antibody or fragment thereof of claim 1, or competes with the antibody or fragment thereof of claim 1 in binding to the IL-13RA2 protein.

19. The antibody or fragment of any one of claims 1-18, which is capable of mediating antibody-dependent cellular cytotoxicity (ADCC).

20. The antibody or fragment thereof of any one of claims 1-19, which further has a binding specificity to a second target protein.

21. An antibody-drug conjugale, comprising an antibody or fragment thereof of any one of claims 1 -20 conjugatcd to an antineoplastic agent.

22. A composition comprising the antibody or fragment thereof of any one of claims 1 -20 and a pharmaceutically acceptable carrier.

23. One or more polynucleotide encoding the antibody or fragment thereof of any one of claims 1-20.

24. A cell that comprises the one or more polynucleotide of claim 23.

25. The antibody or fragment thereof of any one of claims l -20 for use in the treatment of cancer or inflammation in a patient in need thereof.

26. Use of the antibody or fragment thereof of any one of claims l -20 for the préparation of a médicament for treating cancer or inflammation.

27. The antibody or fragment thereof of claim 25 or the use of claim 26, wherein the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endométrial cancer, leukemia, lymphoma, pancreatic cancer, small ccll lung cancer, non-small cell lung cancer, breast cancer, uréthral cancer, head and neck cancer, gastrointestinal cancer, slomach cancer, oesophageal cancer, ovarian cancer, rénal cancer, melanoma, prostate cancer and thyroid cancer.

28. The antibody or fragment thereof of claim 25 or the use of claim 26, wherein the inflammation is caused by an infection or an autoimmune disease.