Anti-IL-13RA2 monoclonal antibodies and uses thereof

Abstract

Antibodies or fragments thereof having binding specificity for the human interleukin-13 receptor subunit alpha 2 (IL-13RA2) protein are provided. These antibodies can bind to IL-13RA2 with high affinity, mediate antibody-dependent cellular cytotoxicity (ADCC), and effectively induce endocytosis. Methods and uses for treating cancer and inflammatory diseases are also provided. IL-13 signal transduction is regulated through a complex receptor system.

Description

[Background technology]

[0001] background Interleukin-13 receptor subunit alpha 2 (IL-13RA2 or IL-13Rα2), also known as cluster of differentiation 213A2 (CD213A2), is a membrane-bound protein that binds IL13. Unlike other receptor proteins, IL-13RA2 has a short cytoplasmic domain and does not appear to function as a signal mediator. However, this protein can regulate the effects of both IL-13 and IL-4, despite not directly binding to both.

[0002] IL-13 signaling is regulated through a complex receptor system. In non-hematopoietic cells, IL-13 associates with a heterodimeric receptor composed of IL-4Rα and IL-13Rα1. IL-13Rα1 binds IL-13 with low affinity. However, when complexed with IL-4Rα, it binds with much higher affinity and induces the effector functions of IL-13. IL-13RA2 is closely related to IL-13Rα1.

[0003] IL-13RA2 has been found to be overexpressed in various cancers, including pancreatic, ovarian, melanoma and malignant glioma. Cells with high IL-13RA2 expression can rapidly and efficiently deplete extracellular IL-13. Similarly, IL-13 responses are enhanced in mice lacking IL13RA2. Thus, IL-13RA2 can act as a decoy receptor for IL-13 and induce antagonist activity against IL-13.

[0004] IL-13RA2 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 example, in patients with nasal polyps (NP), IL-13RA2 may contribute to airway inflammation and aberrant remodeling, which are major pathological features. Summary of the Invention

[0006] overview Discovered herein are anti-IL-13RA2 antibodies that have high binding affinity to human IL-13RA2 protein and are efficient in mediating antibody-dependent cellular cytotoxicity (ADCC), inducing cytotoxicity and promoting cell renewal.Therefore, these antibodies can be suitably used to treat diseases such as cancer and inflammatory diseases, and act as target carriers for antibody-drug conjugates.

[0007] In one embodiment, an antibody or fragment thereof having specificity for human IL-13RA2 protein is provided. In some embodiments, the antibody or fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2 and CDRL3.

[0008] In some embodiments, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 25, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 26, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 27, the CDRL1 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 region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, and 54 to 57, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14, and 58 to 61. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 54, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 58.

[0010] In some embodiments, the CDRH1 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 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 comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, CDRH2 comprises the amino acid sequence of SEQ ID NO: 32.

[0011] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, and 62 to 65, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, and 66 to 69. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 62, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 66.

[0012] In some 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, 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 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.

[0013] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19, and 70 to 73, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 20, and 74 to 77. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 70, and the light chain variable region 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 CDRH3 comprises the 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 region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 21, and 78 to 81, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22, and 82 to 85. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 79, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 83.

[0016] Also provided in some embodiments are polynucleotides encoding the antibodies or fragments, antibody-drug conjugates based on the antibodies or fragments, and compositions comprising the antibodies or fragments thereof and a pharma- ceutically acceptable carrier.

[0017] Methods and uses for treating diseases and conditions are also provided. In one embodiment, a method of treating cancer or inflammation in a patient in need thereof is provided, comprising administering to the patient an antibody or fragment thereof of the present disclosure. [Brief description of the drawings]

[0018] [Figure 1A] 1A-B show the binding affinity of mouse antibodies to IL-13RA2 on A375 cells. [Figure 1B] 1A-B show the binding affinity of mouse antibodies to IL-13RA2 on A375 cells.

[0019] [Figure 2A] 2A-B show the binding affinity of rat antibodies to IL-13RA2 on A375 cells. [Figure 2B] 2A-B show the binding affinity of rat antibodies to IL-13RA2 on A375 cells.

[0020] [Diagram 3] FIG. 3 shows the binding affinity of certain chimeric antibodies to IL-13RA2 on A375 cells.

[0021] [Figure 4] FIG. 4 shows antibody-dependent cellular cytotoxicity (ADCC) of the chimeric antibody.

[0022] [Diagram 5] FIG. 5 shows the cell-killing activity of the chimeric antibody.

[0023] [Figure 6A] 6A-C show the internationalization of chimeric antibodies. [Figure 6B] 6A-C show the internationalization of chimeric antibodies. [Figure 6C] 6A-C show the internationalization of chimeric antibodies.

[0024] [Figure 7A] 7A-7J show the binding affinities of the humanized antibodies. [Figure 7B] 7A-7J show the binding affinities of the humanized antibodies. [Figure 7C] 7A-7J show the binding affinities of the humanized antibodies. [Figure 7D] 7A-7J show the binding affinities of the humanized antibodies. [Figure 7E] 7A-7J show the binding affinities of the humanized antibodies. [Figure 7F] 7A-7J show the binding affinities of the humanized antibodies. [Figure 7G] 7A-7J show the binding affinities of the humanized antibodies. [Figure 7H]7A-7J show the binding affinities of the humanized antibodies. [Figure 7I] 7A-7J show the binding affinities of the humanized antibodies. [Figure 7J] 7A-7J show the binding affinities of the humanized antibodies.

[0025] [Figure 8A] 8A-B show ADCC of humanized antibodies in IL-13RA2 overexpressing cell lines (A) and endogenous glioma cell lines (B). [Figure 8B] 8A-B show ADCC of humanized antibodies in IL-13RA2 overexpressing cell lines (A) and endogenous glioma cell lines (B).

[0026] [Figure 9A] 9A-C show the internationalization of selected humanized antibodies. [Figure 9B] 9A-C show the internationalization of selected humanized antibodies. [Figure 9C] 9A-C show the internationalization of selected humanized antibodies.

[0027] [Figure 10A] 10A-C show the in vitro cytotoxicity of antibody-drug conjugates using humanized antibodies. [Figure 10B] 10A-C show the in vitro cytotoxicity of antibody-drug conjugates using humanized antibodies. [Figure 10C] 10A-C show the in vitro cytotoxicity of antibody-drug conjugates using humanized antibodies.

[0028] [Figure 11A] 11A-B show the in vivo cytotoxicity of antibody-drug conjugates using humanized antibodies. [Figure 11B] 11A-B show the in vivo cytotoxicity of antibody-drug conjugates using humanized antibodies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description definition The term "a" or "an" entity refers to one or more of that entity. For example, "an antibody" should be understood to refer to one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0030] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as a plural number of "polypeptides" and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or may be produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. A polypeptide may be produced in any manner, including chemical synthesis.

[0031] "Homology", or "identity", or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence (which may be aligned for purposes of comparison). If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity (although preferably less than 25% identity) with one of the sequences of the present disclosure.

[0032] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence, meaning that, when aligned, that percentage of bases (or amino acids) are identical in comparing the two sequences.

[0033] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that has a degree of homology or sequence identity to the nucleotide sequence of the nucleic acid or its complement. A homolog of a double-stranded nucleic acid is a nucleic acid that is identical to or similar to its complement. It is intended to include nucleic acids having nucleotide sequences with a degree of homology (e.g., with the complement thereof). In one embodiment, a nucleic acid homolog is capable of hybridizing to a nucleic acid or its complement. Similarly, an "equivalent polypeptide" refers to a polypeptide having a degree of homology or sequence identity to the amino acid sequence of a reference polypeptide. In some embodiments, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, an equivalent polypeptide or polynucleotide has one, two, three, four, or five additions, deletions, substitutions, and combinations thereof, compared to a reference polypeptide or polynucleotide. In some embodiments, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridges) of the reference sequence.

[0034] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody, and any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide, including molecules that contain at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such include, but are not limited to, at least a portion of a heavy or light chain complementarity determining region (CDR), or ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or a binding protein.

[0035] The term "antibody fragment" or "antigen-binding fragment" as used herein refers to, for example, F(ab') 2 , F(ab) 2An antibody fragment is a portion of an antibody, such as Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0036] A "single chain variable fragment" or "scFv" is a fragment of the variable region of an immunoglobulin heavy chain (V H ) and the light chain variable region (V L In some embodiments, the regions are linked by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and serine or threonine for solubility, as well as V H N-terminus of V L The ScFv molecule may be linked to the C-terminus of either the ScFv or the C-terminus of the ScFv polypeptide, or vice versa. The protein retains the specificity of the original immunoglobulin, despite the removal of the constant region and the introduction of a linker. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0037] The term antibody encompasses a wide variety of biologically distinguishable polypeptides. 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 several subclasses within them (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of an antibody as IgG, IgM, IgA IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgG 5and the like are well characterized and known to provide functional specialization. Modified forms of each of these classes and isotypes are readily discernible to one of skill in the art in view of the present disclosure and are therefore within the scope of the present disclosure. All immunoglobulin classes are expressly within the scope of the present disclosure, and the following discussion is generally directed to immunoglobulin molecules of the IgG class. With respect to IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. These four chains are typically linked by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains beginning at the mouth of the "Y" and continuing through the variable region.

[0038] Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the present disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, such as Fab, Fab', and F(ab') 2 , Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments containing either the VK or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules of the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass of immunoglobulin molecule.

[0039] Light chains are classified as either kappa or lambda (K, λ). Each heavy chain class can be associated with either a kappa or lambda light chain. Generally, light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by either a hybridoma, a B cell, or a genetically engineered host cell. In the heavy chain, the amino acid sequences run from the N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

[0040] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, the variable domain of the light chain portion (VK) and the variable domain of the heavy chain portion (VH) both determine antigen recognition and specificity. Conversely, the constant domain of the light chain (CK) and the constant domain of the heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the numbering of the constant region domains increases as they move away from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CK domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0041] As shown above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope of an antigen. That is, the VK domain and the VH domain of an antibody, or a subset of complementarity determining regions (CDRs), combine to form the variable region that defines a three-dimensional antigen-binding site. The quaternary structure of the antibody forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of each of the VH and VK chains. In some cases, for example, certain immunoglobulin molecules derived from Camelidae species or engineered based on Camelidae immunoglobulins, the complete immunoglobulin molecule may be composed of only heavy chains without light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0042] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen-binding domain are short, non-contiguous amino acid sequences that occupy specific positions such that the antibody forms an antigen-binding domain when it adopts a three-dimensional arrangement in an aqueous environment. The remaining amino acids of the antigen-binding domain (called the "framework" regions) show less inter-molecular variability. The framework regions adopt a predominantly β-sheet structure, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. Thus, the framework regions serve to form a scaffold that provides for the positioning of the CDRs in the correct orientation by non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs determines a surface complementarity to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids which comprise the CDRs and framework regions, respectively, have been precisely defined (see Sequences of Proteins of Immunological Interest,'Kabat, E. et al., USDepartment of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987)), and can therefore be readily determined for any given heavy or light chain variable region by one of ordinary skill in the art.

[0043] In cases where there are two or more definitions of a term used and / or accepted in the art, the definition of that term as used herein is intended to include all of those meanings unless expressly indicated otherwise. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen binding sites found in the variable regions of both the heavy and light chain polypeptides. This particular region is described in Kabat et al., US Pept. of Health and Human Services, "Sequences of Proteins, of Immunological Interest'' (1983), and Chothia et al., J. MoI. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. The definitions of CDRs by Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, application of either definition to refer to the CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues that encompass the CDRs defined in each of the above cited references are set forth in the table below as a comparison. The exact number of residues that encompass a particular CDR will vary depending on the sequence and size of that CDR. One skilled in the art can routinely determine which residues encompass a particular CDR given the variable region amino acid sequence of an antibody. [Table 14]

[0044] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence without reliance on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0045] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at about amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), includes about 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the 15th residue after the end of CDR-H1, includes about 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at about the 33rd amino acid residue after the end of CDR-H2; includes 3-25 amino acids; and ends at the sequence WGXG (where X is any amino acid). CDR-L1 begins at about residue 24 (i.e., after the cysteine ​​residue); includes about 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at about the 16th residue after the end of CDR-L1 and includes about 7 residues. CDR-L3 begins at approximately the 33rd residue after the end of CDR-L2 (i.e., after the cysteine ​​residue); contains approximately 7 to 11 residues; and ends at the sequence F or WGXG (where X is any amino acid).

[0046] The antibodies disclosed herein can be of any animal origin, including birds and mammals.Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.In another embodiment, the variable region can be of condricthoid origin (e.g., shark origin).

[0047] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide that includes a heavy chain constant region includes at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the present disclosure may include a polypeptide chain that includes a CH1 domain; a polypeptide chain that includes a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain that includes a CH1 domain and a CH3 domain; a polypeptide chain that includes a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain that includes a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present disclosure includes a polypeptide chain that includes a CH3 domain. Furthermore, an antibody for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). As noted above, it will be understood by those skilled in the art that the heavy chain constant region may be modified so that it differs in amino acid sequence from that of a naturally occurring immunoglobulin molecule.

[0048] The heavy chain constant regions of the antibodies disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide may be derived from an IgG 1 The CH1 domain derived from the molecule and IgG 3 In another example, the heavy chain constant region may include a hinge region derived from an IgG molecule. 1 molecule, partially derived from IgG 3 In another example, the heavy chain portion may comprise, in part, a hinge region derived from an IgG molecule. 1 molecule, partially derived from IgG 4 The invention may include a chimeric hinge derived from a molecule.

[0049] As used herein, the term "light chain constant region" includes an amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region includes at least one of the constant kappa domain or the constant lambda domain.

[0050] A "light chain-heavy chain pair" refers to an assembly of a light chain and a heavy chain that can form a dimer through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0051] As indicated above, the subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino terminus of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.

[0052] As used herein, the term "CH2 domain" includes the portion of a heavy chain molecule that extends from about residue 244 to residue 360 ​​of an antibody, using conventional numbering schemes (residues 244-360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., US Dept. of Health and Human 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 carbohydrate chains are inserted between the two CH2 domains in an intact native IgG molecule. It is also well established that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 residues.

[0053] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be divided into three separate domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

[0054] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 (positions 226 or 229, EU numbering system) using the Kabat numbering system.

[0055] As used herein, the term "chimeric antibody" refers to any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, a portion, or modified according to the present disclosure) is obtained from a second species. In certain embodiments, the target binding region or site is derived from a non-human source (e.g., mouse or primate) and the constant region is human.

[0056] As used herein, "percent humanization" is calculated by determining the number of framework amino acid differences (i.e., non-CDR differences) between the humanized domain and the germline domain, subtracting this number from the total number of amino acids, then dividing it by the total number of amino acids and multiplying by 100.

[0057] "Specifically binds" or "has specificity for" generally means that an antibody binds to an epitope through its antigen-binding domain, and that the binding requires some complementarity between the antigen-binding domain and the epitope. In accordance with this definition, an antibody is said to "specifically bind" to an epitope if it binds to that epitope more readily through its antigen-binding domain than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have a higher specificity for a given epitope than antibody "B", or antibody "A" may be said to bind epitope "C" with a higher specificity than it has for the related epitope "D".

[0058] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, where the objective is to prevent or slow down (alleviate) an undesired physiological change or disorder (e.g., the progression of cancer). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, a stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or palliative of the disease state, and remission (whether partial or complete remission). "Treatment" can also mean prolonging survival compared to the expected survival if not treated. Subjects in need of treatment include those already with a condition or disorder, as well as those prone to having the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0059] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals (e.g., dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.).

[0060] As used herein, phrases such as "to a patient in need of treatment" or "subject in need of treatment" include subjects (e.g., mammalian subjects) who would benefit from the administration of an antibody or composition of the disclosure, e.g., used for detection, for diagnostic procedures, and / or for treatment. Anti-IL-13RA2 antibody

[0061] The present disclosure provides anti-IL-13RA2 antibodies and fragments that have high affinity to human IL-13RA2 protein, are potent in mediating ADCC and cytotoxicity, and can effectively induce endocytosis.In addition, both in vitro and in vivo tests have demonstrated the effectiveness of these antibodies in killing IL-13RA2-expressing cells and treating cancer.Therefore, these antibodies are suitable agents for treating various diseases characterized by overexpression of IL-13RA2, such as cancer and inflammatory diseases.

[0062] Thus, according to one embodiment of the present disclosure, antibodies and antigen-binding fragments thereof capable of binding to IL-13RA2 are provided. Exemplary antibodies include the murine antibodies listed in Table 1 (e.g., 24G6D5, 34A5G9, 42E3C4, 51G2B3, 54A4F7, 57F8H3F1, 61H8C1, 62A8D8, 62F6A8, 72B4H4, 73A11E2, and 74B5E4), as well as the humanized antibodies in Tables 8-11. Also included are those that contain the same CDRs as those exemplified herein. In some embodiments, the disclosed antibodies and fragments include those that bind to the same epitopes as those exemplified herein, and those that compete with those disclosed immediately in binding to IL-13RA2.

[0063] According to one embodiment of the present disclosure, there is provided an antibody or fragment thereof, and biological equivalents thereof, comprising a heavy chain variable domain and a light chain variable domain having the CDR regions disclosed herein.

[0064] In one embodiment, the CDRs are those of 61H8C1 or their humanized counterparts, as illustrated in Tables 8B and 8D. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO:25 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRH2 comprises the amino acid sequence of SEQ ID NO:26 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRH3 comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRL1 comprises the amino acid sequence of SEQ ID NO:28 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, CDRL2 comprises the amino acid sequence of SEQ ID NO:29 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof, and CDRL3 comprises the amino acid sequence of SEQ ID NO:30 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof.

[0065] In one embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 25, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 26, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 27, the CDRL1 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.

[0066] In some embodiments, those that contain the same CDRs as 61H8C1 or its humanized counterpart are also provided. In some embodiments, the disclosed antibodies and fragments include those that bind to the same epitope as 61H8C1 or its humanized counterpart, and those that compete with either of them for binding to IL-13RA2.

[0067] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13 (murine or chimeric) and 54-57 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 13 (murine or chimeric) and 54-57 (humanized).

[0068] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 (murine or chimeric) and 58-61 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 14 (murine or chimeric) and 58-61 (humanized).

[0069] In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 54, and the light chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 58 to 61. In some embodiments, the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 54 to 57, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 54, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 58.

[0070] In one embodiment, the CDRs are those of 57F8H3F1 or their humanized counterparts, as illustrated in Tables 9B and 9D. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 31 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof, CDRH2 comprises the amino acid sequence of SEQ ID NO: 32 or 49 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof, CDRH3 comprises the amino acid sequence of SEQ ID NO: 33 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof, CDRL1 comprises the amino acid sequence of SEQ ID NO: 34 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof, CDRL2 comprises the amino acid sequence of SEQ ID NO: 35 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 36 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof.

[0071] In one embodiment, the CDRH1 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 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 comprises the amino acid sequence of SEQ ID NO: 36. In one embodiment, the CDRH1 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 comprises the amino acid sequence of SEQ ID NO: 36.

[0072] In some embodiments, those that contain the same CDRs as 57F8H3F1 or its humanized counterpart are also provided. In some embodiments, the disclosed antibodies and fragments include those that bind to the same epitope as 57F8H3F1 or its humanized counterpart, and those that compete with either of them for binding to IL-13RA2.

[0073] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:11 (murine or chimeric) and 62-65 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:11 (murine or chimeric) and 62-65 (humanized).

[0074] In one embodiment, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12 (murine or chimeric) and 66-69 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12 (murine or chimeric) and 66-69 (humanized).

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

[0076] In one embodiment, the CDRs are those of 72B4H4 or their humanized counterparts, as illustrated in Tables 10B and 10D. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 37 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof, CDRH2 comprises the amino acid sequence of SEQ ID NO: 38 or 50 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof, CDRH3 comprises the amino acid sequence of SEQ ID NO: 39 or 51 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof, CDRL1 comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof, CDRL2 comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 42 or a variant thereof having one, two or three deletions, additions, substitutions or a combination 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] In some embodiments, those that contain the same CDRs as 72B4H4 or its humanized counterpart are also provided. In some embodiments, the disclosed antibodies and fragments include those that bind to the same epitope as 72B4H4 or its humanized counterpart, and those that compete with either of them for binding to IL-13RA2.

[0079] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19 (murine or chimeric) and 70-73 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 19 (murine or chimeric) and 70-73 (humanized).

[0080] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 20 (murine or chimeric) and 74-77 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 20 (murine or chimeric) and 74-77 (humanized).

[0081] In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 70, and the light chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 74 to 77. In some embodiments, the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 70 to 73, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 74. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 70, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 74.

[0082] In one embodiment, the CDRs are those of 73A11E2 or their humanized counterparts, as illustrated in Tables 11B and 11D. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof, CDRH2 comprises the amino acid sequence of SEQ ID NO: 44 or 52 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof, CDRH3 comprises the amino acid sequence of SEQ ID NO: 45 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof, CDRL1 comprises the amino acid sequence of SEQ ID NO: 46 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof, CDRL2 comprises the amino acid sequence of SEQ ID NO: 47 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 48 or 53 or a variant thereof having one, two or three deletions, additions, substitutions or a combination thereof.

[0083] In one embodiment, 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 CDRH3 comprises the 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 one embodiment, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 43, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 44, the CDRH3 comprises the 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.

[0084] In some embodiments, those that contain the same CDRs as 73A11E2 or its humanized counterpart are also provided. In some embodiments, the disclosed antibodies and fragments include those that bind to the same epitope as 73A11E2 or its humanized counterpart, and those that compete with either of them for binding to IL-13RA2.

[0085] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 21 (mouse or chimeric) and 78-81 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 21 (mouse or chimeric) and 78-81 (humanized).

[0086] In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22 (mouse or chimeric) and 82-85 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 22 (mouse or chimeric) and 82-85 (humanized).

[0087] In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 79, and the light chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 82 to 85. In some embodiments, the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 78 to 81, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 79, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 83.

[0088] Antibodies containing these CDR regions had strong IL-13RA2 binding activity, whether murine, humanized, or chimeric. As shown in Tables 8-11, specific residues within the CDRs may be modified to retain or improve properties or to reduce the likelihood of having post-translational modifications (PTMs). Such altered CDRs may be referred to as affinity matured CDRs or risk-reduced CDRs (e.g., SEQ ID NOs: 49-53).

[0089] Modified CDRs may include those with one, two, or three amino acid additions, deletions, and / or substitutions. In some embodiments, the substitutions may be conservative substitutions.

[0090] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have 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, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, the string of amino acids may be replaced with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0091] Non-limiting examples of conservative amino acid substitutions are shown in the table below, where a similarity score of 0 or higher indicates a conservative substitution between the two amino acids. [Table A] [Table B-1] [Table B-2]

[0092] Those skilled in the art will also understand that the antibodies disclosed herein may be modified to differ in amino acid sequence from the naturally occurring binding polypeptide from which they are derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., 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 that are not normally associated with antibodies. Exemplary modifications are described in more detail below. For example, the antibody of the present 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).

[0094] The antibodies, variants, or derivatives of the present disclosure include derivatives that are modified, i.e., modified by the covalent attachment of any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from binding to its epitope. For example, but not by way of limitation, the antibody may be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a number of chemical modifications may be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Additionally, the antibody may contain one or more non-classical amino acids.

[0095] In some embodiments, the antibody may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.

[0096] Antibodies may be conjugated or fused to therapeutic agents (which may include a detectable label, such as a radioactive label), immunomodulatory agents, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents (which may be drugs or toxins), ultrasound enhancing agents, non-radioactive labels, combinations thereof, and other such agents known in the art.

[0097] The antibody can be detectably labeled by coupling to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0098] The antibody may also be 152Fluorescence-emitting metals such as Eu, or other metals of the lanthanum series may be used to detectably label the antibody. These metals may be attached to the antibody using metal chelating groups such as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to antibodies 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); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:A Review'', in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);''Analysis, Results, And Future Prospective See, "Of The Therapeutic Use Of Radiolabeled Antibodies In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. (52:119-58 (1982)). Bifunctional molecules and combination therapies

[0099] IL-13RA2 is overexpressed on tumor cells. As a tumor antigen targeting molecule, the antibody or antigen-binding fragment specific for IL-13RA2 can be combined with a second antigen-binding fragment specific for immune cells or an antigen-binding fragment specific for immune checkpoints to create a combination therapy or bispecific antibody.

[0100] In some embodiments, the immune cells are selected from the group consisting of T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils, and mast cells. Molecules on immune cells that can be targeted include, for example, CCL1, CD3, CD16, CD19, CD28, and CD64. Other examples include PD-1, PD-L1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122, 4-1BB (also known as CD137), TIM3, OX-40 or OX40L, CD40 or CD40L, LIGHT, ICOS / ICOSL, GITR / GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272), killer cell immunoglobulin-like receptors (KIRs), and CD47.

[0101] Different formats of bispecific antibodies are also provided. In some embodiments, each of the anti-IL-13RA2 fragment and the second fragment is independently selected from a Fab fragment, a single chain variable fragment (scFv), or a single domain antibody. In some embodiments, the bispecific antibody further comprises an Fc fragment.

[0102] Bifunctional molecules that do not simply include antibodies or antigen-binding fragments are also provided. As tumor antigen targeting molecules, antibodies or antigen-binding fragments specific for IL-13RA2 (such as those described herein) can be linked to immunocytokines or ligands, optionally by peptide linkers. The linked immunocytokines or ligands include, but are not limited to, IL-2, IL-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 bifunctional molecules can combine immune checkpoint blockade effects with local immune modulation at the tumor site. Antibody-drug conjugates

[0103] In some embodiments, the antibody or fragment may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.

[0104] In one embodiment, the antibody or fragment of the present disclosure is covalently attached to a drug moiety. The drug moiety may be a group reactive with a conjugation point on the antibody, or may be modified to contain a group reactive with a conjugation point on the antibody. For example, drug moieties may be attached by alkylation (e.g., at the ε-amino group of lysine or the N-terminus of the antibody), reductive amination of oxidized carbohydrates, transesterification between hydroxyl and carboxyl groups, amidation at amino or carboxyl groups, and conjugation to thiols.

[0105] In some embodiments, the number p of conjugated drug moieties per antibody molecule is in the range of 1 to 8 on average; 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is in the range of 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 on average. In other examples, p is in the range of 1, 2, 3, 4, 5, 6, 7, or 8 on average. In some embodiments, p is in the range of about 1 to about 20, about 1 to about 10, about 2 to about 10, about 2 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2 on average. In some embodiments, p is in the range of about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, or about 2 to about 3.

[0106] For example, when a free thiol group is formed by chemical activation of a protein, the protein can be conjugated with a sulfhydryl-reactive reagent. In one embodiment, the reagent is substantially specific for free thiol groups. Such reagents include, for example, malemides, haloacetamides (e.g., iodine, bromine, or chloride), haloesters (e.g., iodo, bromo, or chloro), halomethylketones (e.g., iodo, bromo, or chloro), benzylhalides (e.g., iodide, bromide, or chloride), vinylsulfones, and pyridylthios.

[0107] The drug may be linked to the antibody or fragment by a linker. Suitable linkers include, for example, cleavable linkers and non-cleavable linkers. Cleavable linkers are typically susceptible to cleavage in intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that can be cleaved by intracellular proteases, such as lysosomal or endosomal proteases. In an exemplary embodiment, the linker may be a dipeptide linker, such as a valine-citrulline (val-cit), phenylalanine-lysine (phe-lys) linker, or a maleimidocapronic-valine-citrulline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PABA) linker. Another linker is sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (smcc). Sulfo-smcc conjugation occurs through a maleimide group that reacts with sulfhydryls (thiols, -SH), while the Sulfo-NHS ester is reactive to primary amines (such as those found in lysines and the N-terminus of proteins or peptides). Yet another linker is maleimidocaproyl (mc). Other suitable linkers include linkers that can be hydrolyzed at a particular pH or pH range, such as hydrazone linkers. Further suitable cleavable linkers include disulfide linkers. Linkers, such as the mc linkers, can be covalently attached to antibodies to the extent that the antibody must be degraded within the cell in order for the drug to be released.

[0108] The linker may contain a group for linkage to an antibody. For example, the linker may contain an amino, hydroxyl, carboxyl, or sulfhydryl reactive group (e.g., malemide, haloacetamide (e.g., iodo, bromo, or chloro), haloester (e.g., iodo, bromo, or chloro), halomethylketone (e.g., iodo, bromo, or chloro), benzylhalide (e.g., iodide, bromide, or chloride), vinylsulfone, and pyridylthio).

[0109] In some embodiments, the drug moiety is a cytotoxic or cytostatic agent, an immunosuppressant, a radioisotope, a toxin, or the like. The conjugate can be used to inhibit tumor or cancer cell multiplication, to induce apoptosis in tumor or cancer cells, or to treat cancer in a patient. The conjugate can be used appropriately in a variety of situations for the treatment of cancer in animals. The conjugate can be used to deliver drugs to tumor or cancer cells. Without being bound by theory, in some embodiments, the conjugate binds to or associates with cancer cells that express claudin 18.2, and the conjugate and / or drug can be internalized by the tumor or cancer cells by receptor-mediated endocytosis.

[0110] Once inside the cell, one or more specific peptide sequences in the conjugate (e.g., in the linker) are hydrolytically cleaved by one or more tumor cell or cancer cell-associated proteases, resulting in the release of the drug. The released drug is then free to migrate within the cell and induce cytotoxic or cytostatic, or other activity. In some embodiments, the drug is cleaved from the antibody outside the tumor or cancer cell, after which the drug enters the cell or acts at the cell surface.

[0111] Examples of drug moieties or payloads are DM1 (maytansine, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)- or N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine), mc-MMAD (6-maleimidocaproyl-monomethylauristatin D or N-methyl-L-valyl-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[(1S )-2-phenyl-1-(2-thiazolyl)ethyl]amino]propyl]-1-pyrrolidinyl]-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-(9Cl)-L-valinamide), mc-MMAF (maleimidocaproyl-monomethylauristatin F or N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3-methoxy-5-methyl mc-Val-Cit-PABA-MMAE (6-Maleimidocaproyl-ValcCit-(p-aminobenzyloxycarbonyl)-monomethylauristatin E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L-valyl-N5-(αR,βR,2S)-β-methoxy-α-methyl-2-pyrrolidinepropanoyl-L-phenylalanine) DM1 is a derivative of the tubulin inhibitor maytansine, while MMAD, MMAE, and MMAF are auristatin derivatives.In some embodiments, the drug moiety is selected from the group consisting of mc-MMAF and mc-Val-Cit-PABA-MMAE, hi some embodiments, the drug moiety is a maytansinoid or an auristatin.

[0112] The antibodies or fragments may be conjugated or fused to therapeutic agents (which may include a detectable label, such as a radioactive label), immunomodulatory agents, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents (which may be drugs or toxins), ultrasound enhancing agents, non-radioactive labels, combinations thereof, and other such agents known in the art.

[0113] The antibody can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0114] The antibody may also be 152Fluorescence-emitting metals, such as Eu, or other metals of the lanthanide series, may be used to detectably label the antibody, which may be attached to the antibody using metal chelating groups such as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to antibodies 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); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of Therapeutic ... See, Therapeutic Use Of Radiolabeled Antibodies In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. (52:119-58 (1982)). Polynucleotides encoding antibodies and methods for preparing antibodies

[0115] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies, variants or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, variant or derivative thereof, on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, variant or derivative thereof, on the same polynucleotide molecule or on separate polynucleotide molecules.

[0116] Methods for producing antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptide of the present disclosure are fully human. Fully human antibodies can be produced using techniques described in the art and as described herein. For example, fully human antibodies against a particular antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen exposure, but whose endogenous locus has been disabled. Exemplary techniques that can be used to produce such antibodies are described in U.S. Patent Nos. 6,150,584; 6,458,592; and 6,420,140, ​​which are incorporated by reference in their entirety. Treatment

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

[0118] The present disclosure is also directed to antibody-based therapies, including administering the antibodies of the present disclosure to patients, such as animals, mammals, and humans, to treat one or more of the disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, the antibodies of the present disclosure (including their variants and derivatives as described herein), and nucleic acids or polynucleotides encoding the antibodies of the present disclosure (including their variants and derivatives as described herein).

[0119] The antibody of the present disclosure can also be used to treat or inhibit cancer. In some embodiments, the cancer cells of the patient express or overexpress IL-13RA2. As mentioned above, IL-13RA2 can be overexpressed in tumor cells, particularly in gastric tumors, pancreatic tumors, esophageal tumors, ovarian tumors, and lung tumors. Inhibition of IL-13RA2 has been shown to be useful in treating said tumors.

[0120] Thus, in some embodiments, a method for treating cancer in a patient in need thereof is provided, which in one embodiment involves administering to the patient an effective amount of an antibody of the present disclosure. In some embodiments, at least one cancer cell (e.g., a stromal cell) in the patient overexpresses IL-13RA2.

[0121] Cell therapy (e.g., chimeric antigen receptor (CAR) T cell therapy) is also provided in the present disclosure. Suitable cells contacted with (or alternatively engineered to express) the anti-IL-13RA2 antibodies of the present disclosure may be used. In some embodiments, the antibodies are presented in a chimeric antigen receptor (CAR). Once such contacted or engineered, the cells may then be introduced into a cancer patient in need of treatment. The cancer patient may have any type of cancer disclosed herein. The cells (e.g., T cells) may be, but are not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof.

[0122] In some embodiments, the cells are isolated from the cancer patient themselves. In some embodiments, the cells are provided by a donor or from a cell bank. If the cells are isolated from the cancer patient, unwanted immune responses may be minimized.

[0123] Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal 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 gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer.

[0124] Additional disorders or conditions associated with increased cell survival that may be treated, prevented, diagnosed, and / or prognosed by the disclosed antibodies or variants or derivatives thereof include, but are not limited to, leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)), and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors (including, but not limited to, sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and sarcoma). , chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor and / or metastasis of malignant tumors and related disorders, including, but not limited to, tumors, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0125] The specific administration and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, variant or derivative thereof used, the patient's age, weight, general health, sex, and diet, as well as the time of administration, excretion rate, drug combination, and the severity of the particular disease being treated. The judgment of such factors by the medical caregiver is within the skill of the art. The amount will also depend on the individual patient being treated, the route of administration, the type of formulation, the nature of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0126] In some embodiments, the antibodies of the present disclosure can be used to treat inflammation or inflammatory diseases. In some embodiments, the inflammation can be caused by injury, infection, or autoimmune disease. In some embodiments, the inflammation is skin inflammation, such as nasal polyps (NP).

[0127] Methods of administration of antibodies, variants include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding polypeptides or compositions may be administered by any conventional route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Thus, pharmaceutical compositions comprising antigen-binding polypeptides of the present disclosure may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (by powders, ointments, drops, or transdermal patches), bucally, or as an oral or nasal spray.

[0128] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0129] Administration can be systemic or local. In addition, it may be desirable to introduce the antibodies of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be facilitated by an intraventricular catheter (e.g., attached to a reservoir, e.g., an Ommaya reservoir). Pulmonary administration can also be employed (e.g., by using an inhaler or nebulizer, and formulation with an aerosolizing agent).

[0130] It may be desirable to administer an antigen-binding polypeptide or composition of the disclosure locally to the area requiring treatment; this may be achieved, for example (and not by way of limitation), by localized infusion during surgery, by topical application after surgery (e.g., in combination with) a wound dressing, by injection, by catheter, by suppository, or by implant (said implants being porous, non-porous, or gelatinous materials, including membranes such as sialastic membranes, or fibers). Preferably, when administering proteins (including antibodies) of the disclosure, care must be taken to use materials to which the proteins do not absorb.

[0131] The amount of the antibody of the present disclosure that is effective in treating, inhibiting, and preventing inflammatory, immune, or malignant diseases, disorders, or conditions can be determined by standard clinical techniques. In addition, in vitro assays can be employed as necessary to help determine optimal dosage ranges. The exact dose to be employed in the formulation also depends on the route of administration and the severity of the disease, disorder, or condition, and should be determined according to the judgment of the physician and each patient's circumstances. Effective doses can be estimated from dose-response curves derived from in vitro or animal model test systems.

[0132] As a general proposition, the dosage of an antigen-binding polypeptide of the present disclosure administered to a patient is typically 0.1 mg / kg to 100 mg / kg (patient's body weight), 0.1 mg / kg to 20 mg / kg (patient's body weight), or 1 mg / kg to 10 mg / kg (patient's body weight). In general, human antibodies have a longer half-life in the human body than antibodies derived from other species due to the immune response to the foreign polypeptide. This often allows for lower dosages of human antibodies and less frequent administration. Additionally, the dosage and frequency of administration of an antibody of the present disclosure can be reduced by enhancing uptake (e.g., into the brain) and tissue passage of the antibody through modifications (e.g., lipidation, etc.).

[0133] In additional embodiments, the compositions of the present disclosure are administered in combination with cytokines that may be administered with the compositions of the present disclosure, including, but not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0134] In additional embodiments, compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens, such as radiation therapy. Diagnostic methods

[0135] Overexpression of IL-13RA2 has been observed in certain tumor samples, and patients with IL-13RA2-overexpressing cells may be responsive to treatment with the anti-IL-13RA2 antibodies of the present disclosure. Thus, the antibodies of the present disclosure may also be used for diagnostic and prognostic purposes.

[0136] A sample, preferably including cells, may be obtained from a patient, which may be a cancer patient or a patient seeking diagnosis. The cell may be a cell from a tumor tissue or tumor mass, a blood sample, a urine sample, or any sample from a patient. After sample pretreatment as required, the sample may be incubated with an antibody of the present disclosure under conditions that allow interaction of the antibody with IL-13RA2 protein potentially present in the sample. To detect the presence of IL-13RA2 protein in a sample, a method such as ELISA may be used utilizing an anti-IL-13RA2 antibody.

[0137] The presence of IL-13RA2 protein in the sample (optionally together with the amount or concentration) can be used for cancer diagnosis, as an indication that the patient is suitable for treatment with the antibody, or as an indication that the patient has responded (or has not responded) to cancer treatment. For prognostic methods, detection can be performed once, twice, or more times at a certain stage once cancer treatment has been started, to indicate the progress of the treatment. composition

[0138] The present disclosure also provides pharmaceutical compositions. Such compositions include an effective amount of an antibody and an acceptable carrier. In some embodiments, the composition further includes a second anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0139] In certain embodiments, the term "pharmaceutical acceptable" means approved by a regulatory agency of the federal or state government, or listed in the United States Pharmacopeia or a generally recognized pharmacopoeia for use in animals, and more specifically, in humans. Moreover, a "pharmaceutical acceptable carrier" is generally a non-toxic solid, semi-solid, 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 a pharmaceutical is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral 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 stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if necessary, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates. Antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for regulating tonicity such as sodium chloride or dextrose are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions may be formulated as suppositories, using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, incorporated herein by reference. Such compositions contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation should be suitable for the mode of administration.The parental preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0141] In some embodiments, the composition is formulated according to the usual procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. In general, the ingredients may be supplied separately or mixed together in unit dosage form (e.g., as a dry lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachette, indicating the amount of active agent). If the composition is to be administered by injection, it may be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed prior to administration.

[0142] The compounds of the present disclosure can be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. EXAMPLES

[0143] Working Example Example 1: Generation of mouse and rat monoclonal antibodies against human IL-13RA2 Human IL-13RA2 protein was used to immunize different strains of mice and rats, thereby generating hybridomas. IL-13RA2 positive binders were selected and subcloned. In vitro binding and functional screening were then performed to identify lead antibodies with the highest binding affinity and strongest functional capacity.

[0144] The VH / VL sequences of the 12 lead murine antibodies are provided in Table 1 below. [Table 1-1] [Table 1-2] Example 2. Binding of mouse antibodies to IL-13RA2 protein

[0145] In this example, 28 of the murine antibodies were tested for their ability to bind to human IL-13RA2 protein expressed on A375 cells.

[0146] A375 cells that endogenously express IL-13RA2 were harvested from a flask. 100 μl of 1 × 10 6 Cells at 10 ...

[0147] The results of the study (FIGS. 1A-B and Table 2) show that the purified murine antibody can bind to endogenously expressed IL-13RA2 on A375 cells with a high EC50. [Table 2] Example 3. Binding of rat antibodies to IL-13RA2 protein

[0148] In this example, 24 of the rat antibodies were tested for their ability to bind to human IL-13RA2 protein expressed on A375 cells.

[0149] A375 cells that endogenously express IL-13RA2 were harvested from a flask. 100 μl of 1 × 10 6 Cells at 10 ...

[0150] The results of the study (FIGS. 2A-B and Table 3) show that the purified rat antibody can bind to endogenously expressed IL-13RA2 on A375 cells with a high EC50. [Table 3] Example 4. Binding of chimeric antibodies to IL-13RA2 protein

[0151] In this example, chimeric antibodies (with human Fc) prepared from murine antibodies were prepared and tested for the ability to bind to human IL-13RA2 protein expressed on A375 cells.

[0152] The mouse VH and VK genes were synthetically produced and then cloned into a vector containing the human gamma 1 and human kappa constant domains, respectively. Purified chimeric antibody was produced from transfected CHO cells.

[0153] A375 cells that endogenously express IL-13RA2 were harvested from a flask. 100 μl of 1 × 10 6Cells at 1000 cells / ml were incubated with primary chimeric antibodies in 3-fold serial dilutions from 300 nM to 0.002 nM for 30 min on ice. After washing twice with 200 μl of FACS buffer, cells were incubated with secondary antibodies for 30 min on ice. Cells were washed twice with 200 μl of FACS buffer and analyzed by FACS.

[0154] As shown in FIG. 3 and Table 4, the chimeric antibodies bound to human IL-13RA2 with different EC50s. [Table 4] Example 5. Antibody-dependent cellular cytotoxicity (ADCC) of chimeric antibodies

[0155] In this example, the antibody-dependent cellular cytotoxicity (ADCC) of the chimeric antibody was measured.

[0156] The ADCC reporter bioassay uses an alternative readout at an earlier time point in the ADCC MOA pathway activation: activation of gene transcription via the NFAT (nuclear factor of activated T cells) pathway in effector cells. In addition, the ADCC reporter bioassay uses engineered Jurkat cells as effector cells that stably express the FcγRIIIa receptor, the V158 (high affinity) mutant, and an NFAT response element that drives expression of firefly luciferase. Antibody bioactivity in the ADCC MOA is quantified by luciferase produced as a result of NFAT pathway activation; luciferase activity in the effector cells is quantified with a luminescent readout. The signal is high and the background of the assay is low.

[0157] Serial dilutions of IL-13RA2 chimeric monoclonal antibodies were incubated with engineered Jurkat effector cells (ADCC Bioassay Effector Cells) and ADCC Bioassay Target Cells (A375 cell line) for 6 hours at 37° C. Luciferase activity was quantified using Bio-Glo™ Reagent.

[0158] The results are presented in Figure 4 and Table 5. As shown, all of these chimeric antibodies exhibited very strong ADCC activity. [Table 5] Example 6. Cytotoxic activity of chimeric antibodies

[0159] In this example, the cell-killing activity of the chimeric antibody was measured.

[0160] A375 cells that endogenously expressed IL-13RA2 were seeded at 3000 cells / well in a 96-well plate. After overnight incubation, each diluted sample conjugated with MMAE was added. After 5 days of incubation, cell viability was measured by CellTiter-Glo reagent. Luciferase activity was detected by Envison.

[0161] As shown in FIG. 5 and Table 6, all of these chimeric antibodies have very strong cytotoxic activity. [Table 6] Example 6. Endocytosis of chimeric antibodies

[0162] In this example, chimeric antibodies are described for their ability to induce internalization upon binding to surface IL-13RA2.

[0163] This method uses a novel hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH upon internalization. It can be used to detect the process of endocytosis. Using CHO-K1 / H_IL-13RA2, HEK-293 / H_IL-13RA2 cells overexpressing human IL-13RA2, and A375 cells endogenously expressing human IL-13RA2 as target cells, detection antibody labeled with pHAb dye was added to evaluate the endocytosis of IL-13RA2 chimeric monoclonal antibody in vitro.

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

[0165] The results are shown in Figures 6A-C and Table 7 and demonstrate that these chimeric antibodies have very strong endocytic activity. [Table 7] Example 7. Humanization of mouse antibodies

[0166] Four of the murine antibodies, 61H8C1, 57F8H3F1, 72B4H4 and 73A11E2, were selected for humanization.

[0167] The amino acid sequences of VH and VL of each mouse antibody were compared with the available database of human Ig gene sequences to find the best overall match of the human germline Ig gene sequence. The CDRs of the mouse antibodies were then grafted onto the matching human sequences. cDNA was synthesized and used to generate humanized antibodies. Specific back mutations from the mouse antibodies were then introduced back into the humanized antibodies. Specific amino acids were mutated to reduce the probability of post-translational modifications.

[0168] The amino acid sequences of the humanized antibodies are provided in Tables 8-11 below. Humanized sequences A.61H8C1

Table 8A

Table 8B

Table 8C

Table 8D

Table 8E

Table 9A

Table 9B

Table 9C

Table 9D

Table 9E

Table 10B

Table 10C

Table 10D

[0169] In this example, the binding affinity of the humanized antibody was examined.

[0170] A375 cells that endogenously express IL-13RA2 were harvested from a flask. 100 μl of 1 × 10 6 Cells at 1000 cells / ml were incubated with primary humanized antibodies in 4-fold serial dilutions from 200 nM to 0.003 nM for 30 min on ice. After washing twice with 200 μl of FACS buffer, cells were incubated with secondary antibodies for 30 min on ice. Cells were washed twice with 200 μl of FACS buffer and analyzed by FACS.

[0171] The results are presented in Figure 7A and Table 12. As shown, the humanized antibody can bind to human IL-13RA2 with a high EC50. [Table 12A] [Table 12B] [Table 12C] [Table 12D] [Table 12E] [Table 12F] [Table 12G] [Table 12H] [Table 12I] [Table 12J] Example 9. ADCC of humanized antibodies

[0172] In this example, the antibody-dependent cellular cytotoxicity (ADCC) of a humanized antibody was measured.

[0173] The ADCC reporter bioassay uses an alternative readout at an earlier time point in the ADCC MOA pathway activation: activation of gene transcription via the NFAT (nuclear factor of activated T cells) pathway in effector cells. In addition, the ADCC reporter bioassay uses engineered Jurkat cells as effector cells that stably express the FcγRIIIa receptor, the V158 (high affinity) mutant, and an NFAT response element that drives expression of firefly luciferase. Antibody bioactivity in the ADCC MOA is quantified by luciferase produced as a result of NFAT pathway activation; luciferase activity in the effector cells is quantified with a luminescent readout. The signal is high and the background of the assay is low.

[0174] Serial dilutions of IL-13RA2 humanized monoclonal antibody were incubated with engineered Jurkat effector cells (ADCC Bioassay Effector Cells) and ADCC Bioassay Target Cells (A375 cell line) for 6 hours at 37° C. Luciferase activity was quantified using Bio-Glo™ Reagent.

[0175] The results show that these humanized antibodies have very strong ADCC activity.

[0176] The results are shown in Figure 8A (IL-13RA2 overexpressing cell lines) and Figure 8B (endogenous glioma cell lines). As shown, all of these humanized antibodies exhibited very strong ADCC activity. Example 10. Endocytosis of humanized antibodies

[0177] In this example, chimeric antibodies are described for their ability to induce internalization upon binding to surface IL-13RA2.

[0178] This method uses a novel hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH upon internalization. It can be used to detect the process of endocytosis. Using CHO-K1 / H_IL-13RA2, HEK-293 / H_IL-13RA2 cells overexpressing human IL-13RA2, and A375 cells endogenously expressing human IL-13RA2 as target cells, detection antibody labeled with pHAb dye was added to evaluate the endocytosis 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 detected.

[0180] The results are shown in FIG. 9A (IL-13RA2 overexpressing cell line) and FIG. 9B-C (endogenous cell line) and demonstrate that these humanized antibodies have very strong endocytic activity. Example 11. In vitro cytotoxicity of antibody-drug conjugates

[0181] In this example, the cytotoxicity of humanized antibodies conjugated to toxic drugs (ADCs) against different glioma cells was tested.

[0182] A375 and U251 cells that endogenously express IL-13RA2, and HEK-293 cells that stably express IL-13RA2 were seeded at 3000 cells / well in a 96-well plate. After overnight incubation, each diluted sample conjugated with MMAE was added. After 5 days of incubation, cell viability was measured by CellTiter-Glo reagent. Luciferase activity was detected by Envison.

[0183] As shown in Figures 10A to 10C, these ADCs have very strong cytotoxic activity. Example 12. In vivo cytotoxicity of antibody-drug conjugates

[0184] In this example, the in vivo cytotoxicity of a humanized antibody conjugated to MMAE (ADC) was examined.

[0185] In this study, 6-8 week-old female NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were used. Each mouse was subcutaneously inoculated in the right axilla (outer side) with 0.1 ml of PBS containing Matrigel (V:V=1:1) and A375 tumor cells (5 × 106) for tumor development. The tumor volume was approximately 70 mm 3 When the total cell mass index (T1) was reached, 5 days after cell inoculation, the animals were grouped and then treatment was initiated for efficacy testing.

[0186] Test article at a dose of 3 mg / kg was administered intravenously (iv) on days 0, 7 and 14. The experiment was terminated on day 23. Tumor size was measured three times weekly in two dimensions using calipers and volume was calculated using the formula V=0.5a×b 2 where a and b are the long and short diameters of the tumor, respectively, to calculate the tumor volume in cubic millimeters (mm 3 This tumor size was then used to calculate the T / C(%) value. T / C(%) was calculated using the formula T / C%=(T i -T 0 ) / (V i -V 0 )×100(T i is the mean tumor volume of the treatment group on a given day, and T 0 is the mean tumor volume of the treatment group on the first day of treatment, and V is the mean tumor volume of the treatment group on the first day of treatment. i is the mean tumor volume of the vehicle control group on the same day as 0 TGI was calculated using the formula TGI(%)=[100-T / C] for each group. Data points represent group (n=5) means and error bars represent standard error of the mean (SEM). p-values ​​were calculated based on tumor size by t-test compared to PBS control.

[0187] Figure 11A shows the body weight change of tumor-bearing mice in different groups. Data points represent group mean body weight. Error bars represent standard error of the mean (SEM). Figure 11B and Table 13 show the tumor growth curves of A375 tumor-bearing mice after administration of test ADCs. As shown in these figures, the ADCs showed strong in vivo cytotoxicity activity. [Table 13] * * *

[0188] The present disclosure is not limited in scope by the specific embodiments described (which are intended to be an illustration of one of the individual aspects of the present disclosure), and any compositions and methods that are functionally equivalent are within the scope of the present disclosure. It is clear to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit and scope of the present disclosure. Thus, the present disclosure is intended to cover modifications and variations of the present disclosure, provided that such modifications and variations are within the scope of the appended claims or their equivalents.

[0189] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. An antibody or fragment thereof having binding specificity for human interleukin-13 receptor subunit alpha 2 (IL-13RA2) protein, comprising a heavy chain variable region comprising heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2 and CDRL3, (a) the CDRH1 comprises the amino acid sequence of SEQ ID NO:25; said CDRH2 comprising the amino acid sequence of SEQ ID NO:26; the CDRH3 comprises the amino acid sequence of SEQ ID NO:27; said CDRL1 comprising the amino acid sequence of SEQ ID NO:28; said CDRL2 comprises the amino acid sequence of SEQ ID NO:29; and said CDRL3 comprising the amino acid sequence of SEQ ID NO: 30; (b) the CDRH1 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 CDRH3 comprises the amino acid sequence of SEQ ID NO: 33; said CDRL1 comprising the amino acid sequence of SEQ ID NO:34; said CDRL2 comprises the amino acid sequence of SEQ ID NO:35; and said CDRL3 comprising the amino acid sequence of SEQ ID NO: 36; (c) 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; said CDRL1 comprising the amino acid sequence of SEQ ID NO:40; said 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 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 CDRH3 comprises the amino acid sequence of SEQ ID NO:45; said CDRL1 comprising the amino acid sequence of SEQ ID NO:46; said CDRL2 comprises the amino acid sequence of SEQ ID NO:47; and An antibody or fragment thereof, wherein the CDRL3 comprises the amino acid sequence of SEQ ID NO: 48 or 53.

2. The antibody or fragment thereof of claim 1, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 25, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 26, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 27, the CDRL1 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.

3. 3. The antibody or fragment thereof of claim 2, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 54-57, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 58-61.

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

58.

5. The antibody or fragment thereof of claim 1, wherein the CDRH1 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 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 comprises the amino acid sequence of SEQ ID NO:

36.

6. The antibody or fragment thereof of claim 5, wherein the CDRH2 comprises the amino acid sequence of SEQ ID NO:

32.

7. 6. The antibody or fragment thereof of claim 5, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 and 62-65, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 66-69.

8. The antibody or fragment thereof of claim 5, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:62 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

66.

9. The antibody or fragment thereof of claim 1, wherein 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.

10. The antibody or fragment thereof of claim 9, wherein 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.

11. 10. The antibody or fragment thereof of claim 9, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 and 70-73, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 74-77.

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

74.

13. The antibody or fragment thereof of claim 1, wherein 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 CDRH3 comprises the 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.

14. The antibody or fragment thereof of claim 13, wherein 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.

15. The antibody or fragment thereof of claim 13, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 and 78-81, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 and 82-85.

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

83.

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

18. An antibody or fragment thereof having binding specificity for human interleukin-13 receptor subunit alpha 2 (IL-13RA2) protein and binding to the same epitope on the IL-13RA2 protein as the antibody or fragment thereof described in claim 1, or competing with the antibody or fragment thereof described in claim 1 in binding to the IL-13RA2 protein.

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

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

21. An antibody-drug conjugate comprising the antibody or fragment thereof according to any one of claims 1 to 20 conjugated to an anti-neoplastic agent.

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

23. 21. One or more polynucleotides encoding the antibody or fragment thereof according to any one of claims 1 to 20.

24. 24. A cell comprising one or more polynucleotides of claim 23.

25. 21. A method of treating cancer or inflammation in a patient in need thereof, comprising administering to the patient an antibody or fragment thereof according to any one of claims 1 to 20.

26. Use of an antibody or a fragment thereof according to any one of claims 1 to 20 for the preparation of a medicament for treating cancer or inflammation.

27. 27. The method according to claim 25 or the use according to claim 26, wherein the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer and thyroid cancer.

28. 27. The method according to claim 25 or the use according to claim 26, wherein the inflammation is caused by an infection or an autoimmune disease.

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