Anti-SLC34A2 monoclonal antibody and application thereof

Abstract

The present invention provides an antibody or fragment thereof having binding specificity for human solute carrier 34A2 (SLC34A2) protein. These antibodies are capable of binding to SLC34A2 with high affinity, and can mediate antibody dependent cytotoxicity (ADCC) and effectively induce endocytosis. Methods and uses for treating cancer are also provided.

Description

Background Art

[0001] Cell membrane transporters such as those belonging to the glucose transporter GLUT, ATP-binding cassette transporter ABC, and solute carrier transporter SLC families are frequently upregulated on cancer cells compared to adjacent normal cells. High levels of transporters have been found in a variety of solid tumors and are associated with poor survival. One of the potential molecular tumor markers may be the sodium-dependent phosphate transporter NaPi2b, encoded by the SLC34A2 (solute carrier 34A2) gene.

[0002] SLC34A2 encodes the type II Na / Pi co-transporter (NaPi2b), which is a multi-transmembrane sodium-dependent phosphate transporter responsible for transcellular inorganic phosphate absorption. NaPi2b is highly abundant in the brush border membrane of the small intestine, where it participates in the transcellular flux of inorganic phosphate across the apical membrane of epithelial cells. Altered expression of the sodium-dependent phosphate transporter NaPi2b has been reported in ovarian, lung, gastric, thyroid, and other cancers.

[0003] Currently, NaPi2b is the target of the therapeutic antibodies XMT-1536 and RG-7599, which are in clinical trials for the treatment of ovarian and lung cancer. There is a need to develop more effective and safer clinical candidates targeting this protein. Summary of the invention

[0004] It is found herein that anti-SLC34A2 antibodies have high binding affinity to human SLC34A2 protein and effectively mediate antibody-dependent cellular cytotoxicity (ADCC) and induce cytotoxicity. When compared to two benchmark antibodies XMT-1536 and RG-7599, the antibodies discovered herein exhibit higher general binding affinity, greatly improved cross-species reactivity, stronger ADCC and cytotoxicity. Therefore, these antibodies may be suitable for treating diseases such as cancer.

[0005] In one embodiment, an antibody or fragment thereof specific to human SLC34A2 protein is provided. In some embodiments, the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises heavy chain complementary determining regions CDRH1, CDRH2 and CDRH3, and the light chain variable region comprises light chain complementary determining regions CDRL1, CDRL2 and CDRL3.

[0006] In some embodiments, polynucleotides encoding the antibodies or fragments thereof, and compositions comprising the antibodies or fragments thereof and a pharmaceutically acceptable carrier are also provided.

[0007] Methods and uses for treating diseases and conditions are also provided.In one embodiment, a method of treating cancer in a patient in need thereof is provided, the method comprising administering to the patient an antibody or fragment thereof of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1A -B shows the binding affinity of the chimeric antibody to SLC34A2 endogenously expressed on OVCAR3 cells.

[0009] Figure 2A -B shows the binding affinity of the chimeric antibody to human SLC34A2 expressed on HEK293 cells.

[0010] Figure 3 The binding affinity of the chimeric antibody to human SLC34A2 on RMG-1 cells is shown.

[0011] Figure 4 The binding affinity of the chimeric antibody to rhesus SLC34A2 expressed on HEC293 cells is shown.

[0012] Figure 5 The binding affinity of the chimeric antibody to cynomolgus monkey SLC34A2 expressed on CHC-K1 cells is shown.

[0013] Fig. 6A -B shows the binding affinity of the chimeric antibody to rat (6A) and mouse (6B) SLC34A2 expressed on HEC293 cells.

[0014] Fig. 7A -B shows the ADCC activity of the chimeric antibody against HEC293 cells expressing human SLC34A2.

[0015] Fig. 8A -B shows the cytotoxic activity of the chimeric antibody against HEC293 cells expressing human SLC34A2.

[0016] Fig.9A -C shows the binding activity of PTM-derisked antibodies to HEC293 cells expressing human SLC34A2.

[0017] Fig.10 Shown are the binding activities of PTM de-risked antibodies to SLC34A2 endogenously expressed on OVCAR3 cells.

[0018] Fig.11 Shown are the binding activities of PTM de-risked antibodies to rhesus monkey SLC34A2 expressed on HEC293 cells.

[0019] Fig. 12A-D shows the binding activity of the humanized antibody to SLC34A2 endogenously expressed on OVCAR3 cells.

[0020] Fig.13A -D shows the binding activity of the humanized antibodies to human SLC34A2 on different types of cells.

[0021] Fig.14 The binding activity of the humanized antibodies to rhesus monkey SLC34A2 expressed on HEC293 cells is shown.

[0022] Fig.15 The binding activity of the humanized antibodies to rat SLC34A2 expressed on HEC293 cells is shown.

[0023] Fig.16 The binding activity of the humanized antibodies to mouse SLC34A2 expressed on HEC293 cells is shown.

[0024] Fig.17 The ADCC activity of the humanized antibodies on HEC293 cells expressing human SLC34A2 is shown.

[0025] Fig.18 The binding of selected humanized antibodies to SLC34A2 on different cell lines was compared with that of a benchmark antibody.

[0026] Fig.19 The cross-reactivity of selected humanized antibodies was compared with a benchmark antibody.

[0027] Fig. 20A -B compares the ADCC activities of the selected humanized antibodies and the benchmark antibodies against different cells. DETAILED DESCRIPTION

[0028] definition

[0029] It should be noted that the term "a" or "an" entity refers to one or more entities in that entity; for example, "antibody" should be understood to represent one or more antibodies. Therefore, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0030] As used herein, the term "polypeptide" is intended to include the singular "polypeptide" as well as the plural "polypeptide", and refers to a molecule composed of monomers (amino acids) linearly connected by amide bonds (also called peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, and does not refer to the specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to one or more chains of two or more amino acids are included in the definition of "polypeptide", and the term "polypeptide" can replace any of these terms or can be used interchangeably with any of these terms. The term "polypeptide" also refers to the product of post-expression modification of the polypeptide, and post-expression modification includes but is not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage or modification by non-naturally occurring amino acids. The polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It can be produced in any way, including by chemical synthesis.

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

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

[0033] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence with a certain degree of homology or sequence identity to the nucleotide sequence of the nucleic acid or its complement. The homologue of a double-stranded nucleic acid is intended to include a nucleic acid having a nucleotide sequence with a certain degree of homology to it or its complement. On the one hand, the homologue of a nucleic acid can hybridize with a nucleic acid or its complement. Similarly, "equivalent polypeptide" refers to a polypeptide with a certain degree of homology or sequence identity to the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In some aspects, compared with a reference polypeptide or polynucleotide, an equivalent polypeptide or polynucleotide has one, two, three, four or five additions, deletions, substitutions and combinations thereof. In some aspects, an equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of a 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. The antibody can be a complete antibody and any antigen-binding fragment or single chain thereof. Therefore, the term "antibody" includes any protein or peptide containing molecule that contains at least a portion of an immunoglobulin molecule that has biological activity that binds to an antigen. Such examples include, but are not limited to, a heavy or light chain complementary determining region (CDR) or a 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 at least a portion of a binding protein.

[0035] As used herein, the term "antibody fragment" or "antigen binding fragment" is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of the structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror isomers, 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] "Single-chain variable fragment" or "scFv" refers to the heavy chain (V H ) and light chain (V L ) fusion protein of the variable region of . In some aspects, these regions are connected with a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine to obtain flexibility, and rich in serine or threonine to obtain solubility, and V H The N-terminus of LThe C-terminus of the scFv is linked to the C-terminus of the scFv, and vice versa. Despite the removal of the constant region and the introduction of the linker, the protein retains the specificity of the original immunoglobulin. ScFv molecules are known in the art and are described, for example, in U.S. Pat. No. 5,892,019.

[0037] The term antibody includes polypeptides of various major classes that can be distinguished biochemically. It will be appreciated by those skilled in the art that heavy chains are classified as γ, μ, α, δ or ε, and some subclasses thereof (e.g., γ1-γ4). The nature of the chain determines the "class" of the antibody, which is IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgG5, etc. have been fully characterized and are known to confer functional specialization. According to the present disclosure, modified forms of each of these classes and isotypes are easily discernible to those skilled in the art and therefore fall within the scope of the present disclosure. All immunoglobulin classes clearly fall within the scope of the present disclosure, and the following discussion will generally be directed to the IgG class of immunoglobulin molecules. In terms of IgG, a standard immunoglobulin molecule comprises 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 daltons-70,000 daltons. The four chains are typically connected by disulfide bonds into a "Y" configuration, with the light chains starting at the mouth of the "Y" and bracketing the heavy chains throughout the variable region.

[0038] The antibodies, antigen-binding polypeptides, variants or derivatives thereof disclosed herein include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized antibodies or chimeric antibodies, single-chain antibodies, epitope-binding fragments (e.g., Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv (scFv)), single-chain antibodies, disulfide-linked Fv (sdFv), fragments comprising VK or VH domains, fragments produced by Fab expression libraries, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies to LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules.

[0039] Light chains are classified as either kappa or lambda. Each heavy chain class can be combined with either a kappa or lambda light chain. Typically, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently bound to each other, and the "tail" portions of the two heavy chains are bound to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain.

[0040] Both the light chain and the heavy chain are divided into structural homology regions and functional homology regions. The terms "constant" and "variable" are used according to function. In this regard, it should be understood that the variable domains of the light chain (VK) and heavy chain (VH) portions determine antigen recognition and specificity. In contrast, the constant domains (CK) of the light chain and the constant domains (CH1, CH2 or CH3) of the heavy chain confer important biological properties, such as secretion, transplacental migration, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as they are farther and farther 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 contain the carboxyl termini of the heavy chain and light chain, respectively.

[0041] As shown above, variable region allows antibody selective recognition and specific binding to the epitope on the antigen. That is, the subset of the VK domain and VH domain or complementary determining region (CDR) of the antibody is combined to form a variable region that limits the three-dimensional antigen binding site. The four-level antibody structure forms an antigen binding site present at the end of each arm of Y. More specifically, the antigen binding site is limited by three CDRs (i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) on each chain in the VH chain and the VK chain. In some cases (for example, for some immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins), a complete immunoglobulin molecule can be composed only of 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 are specifically positioned to form an antigen binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen binding domain, known as the "framework" region, show less intermolecular variability. The framework region primarily adopts a β-sheet conformation, while the CDRs form loops that connect and in some cases form part of the β-sheet structure. Thus, the framework region serves to form a skeleton that positions the CDRs in the correct orientation through interchain, non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on an immunoreactive antigen. This complementary surface promotes non-covalent binding of an antibody to its cognate epitope. For any given heavy or light chain variable region, one of ordinary skill in the art can readily identify the amino acids comprising the CDR and framework regions, respectively, because they have been precisely defined (see "Sequences of Proteins of Immunological Interest", Kabat, E et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987)).

[0043] In the case of two or more definitions of terms used and / or accepted in the art, unless there is a clear contrary statement, the definition of the term as used herein is intended to include all such meanings. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-continuous antigen binding sites found in the variable region of both heavy chain polypeptides and light chain polypeptides. This specific region has been 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 entirety. The definition of CDR according to Kabat and Chothia includes overlapping or subsets of amino acid residues when compared to each other. However, the application of any definition of CDRs related to antibodies or their variants is intended to fall within the scope of the term as defined and used herein. The appropriate amino acid residues comprising the CDRs defined by each of the above-mentioned references are listed in the table below as a comparison. The exact number of residues comprising a particular CDR will vary depending on the sequence and size of the CDR. One skilled in the art can routinely determine which residues comprise a particular CDR based on the variable region amino acid sequence of an antibody.

[0044] Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3 95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

[0045] Kabat et al. also defined a numbering system applicable to the variable domain sequence of any antibody. One of ordinary skill in the art can clearly assign this "Kabat numbering" system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system proposed by Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0046] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 starts at about the 31st amino acid (i.e., about the 9th residue after the first cysteine ​​residue), includes about 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 starts at about 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 starts at about the 33rd amino acid residue after the end of CDR-H2; includes 3-25 amino acids; and ends with the sequence WGXG, where X is any amino acid. CDR-L1 starts at about the 24th residue (i.e., after the cysteine ​​residue); includes about 10-17 residues; and ends at the next tryptophan residue. CDR-L2 starts at about the 16th residue after the end of CDR-L1, and includes about 7 residues. CDR-L3 begins at about 33 residues after the end of CDR-L2 (ie, after the cysteine ​​residue); includes about 7-11 residues, and ends at the sequence F or WGXG, where X is any amino acid.

[0047] The antibodies disclosed herein may be from any animal origin, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies. In another embodiment, the variable region may be derived from a condricthoid (e.g., from a shark).

[0048] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. The polypeptide comprising the heavy chain constant region includes at least one of the following: 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, the antigen-binding polypeptide used in the present disclosure may include: a polypeptide chain containing a CH1 domain; a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain containing a CH1 domain and a CH3 domain; a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of the present disclosure includes a polypeptide chain containing a CH3 domain. In addition, the antibody used in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As described above, those of ordinary skill in the art will appreciate that the heavy chain constant regions can be modified such that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0049] The heavy chain constant region of the antibodies disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide may comprise a polypeptide derived from IgG l In another example, the heavy chain constant region may include a portion derived from IgG l In another example, the heavy chain portion may comprise a portion derived from an IgG l The molecule and the chimeric hinge are derived in part from the IgG4 molecule.

[0050] 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 comprises at least one of a constant kappa domain or a constant lambda domain.

[0051] A "light chain-heavy chain pair" refers to a collection of light chains and heavy chains that can form a dimer via a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0052] As indicated above, the subunit structures and three-dimensional configurations of the constant regions of the various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (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.

[0053] As used herein, the term "CH2 domain" includes the portion of the heavy chain molecule extending from about residue 244 to residue 360 ​​of, for example, an antibody using a conventional numbering scheme (residues 244 to 360, Kabat numbering system; residues 231-340, EU numbering system; see Kabat et al., USDept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)). The uniqueness of the CH2 domain is that it is not tightly paired with another domain. Instead, two N-connected branched sugar chains are inserted between the two CH2 domains of a complete native IgG molecule. It has also been well documented that the CH3 domain extends from the CH2 domain of an IgG molecule to the C-terminus and comprises approximately 108 residues.

[0054] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen binding regions to move independently. The hinge region can be subdivided into three different domains: the upper, middle and lower hinge domains (Roux et al., J. Immunol, 161: 4083 (1998)).

[0055] 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 region and the CK region are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).

[0056] As used herein, the term "chimeric antibody" will be taken to refer 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 complete, partial or modified, according to the present disclosure) is obtained from a second species. In certain embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.

[0057] As used herein, "percent humanization" is calculated by determining the number of framework amino acid differences (ie, non-CDR differences) between the humanized domain and the germline domain, subtracting that number from the total number of amino acids, then dividing by the total number of amino acids and multiplying by 100.

[0058] "Specific binding" or "specific for..." generally means that the antibody binds to the epitope via its antigen binding domain, and that the binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is considered to "specifically bind" to an epitope when it binds to the epitope via its antigen binding domain more easily than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which an antibody binds to an 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 considered to bind to epitope "C" with a higher specificity than it does to a related epitope "D".

[0059] As used herein, the term "treatment" refers to both therapeutic treatment and preventive or defensive measures, wherein the goal is to prevent or slow down (mitigate) undesirable physiological changes or disorders, such as cancer progression. Beneficial or desired clinical results include, but are not limited to, the alleviation of detectable or undetectable symptoms, the reduction of the extent of the disease, the stabilization of the disease state (i.e., no deterioration), the delay or slowing of disease progression, the improvement or alleviation of the disease state, and (partial or complete) relief. "Treatment" may also mean to extend survival compared to the expected survival in the absence of treatment. The situation requiring treatment includes the situation of already suffering from a condition or disorder, and the situation of being susceptible to a condition or disorder, or the situation of needing to prevent a condition or disorder.

[0060] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, livestock, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like.

[0061] As used herein, phrases such as "a patient in need of treatment" or "a subject in need of treatment" include subjects (such as mammalian subjects) who would benefit from administration of an antibody or composition of the present disclosure, for example, for detection, diagnostic procedures and / or therapy.

[0062] Anti-SLC34A2 Antibody

[0063] The present disclosure provides anti-SLC34A2 antibodies and fragments, which have high affinity for human SLC34A2 protein, effectively mediate ADCC and cytotoxicity, and can effectively induce endocytosis.

[0064] Furthermore, these antibodies have been extensively tested compared to two reference antibodies in clinical development, XMT-1536 and RG-7599. The new antibodies outperformed both reference antibodies in terms of general binding affinity, cross-species reactivity, ADCC and cytotoxicity. Therefore, these antibodies are suitable agents for treating various diseases characterized by overexpressed SLC34A2, such as cancer.

[0065] Therefore, according to one embodiment of the present disclosure, antibodies and antigen-binding fragments thereof that can bind to SLC34A2 are provided. Exemplary antibodies include those murine antibodies listed in Table 1 (e.g., 30-H9(B8)F9, 62-G10(E4)G8, 63-A11(D5)B4, 91-C9F1, 7-F4C4, 94-B10A8, 94-E9B12, 67-C9G11, 90-G10H7, 11-F6H8, 84-F2D10, 26-C5D2, 7-G1 0E11, 25-B11B9, 62-C10(B3)D2, 55-E5(B6)D12, 28-G3(C6)B9, 18-G1H4, 16-E5D10, 69-B11D8, 71-F7D4, 78-D1B10, 87-H12B7, 20-H2F1 and 95-H11A1D8), and the humanized antibodies in Tables 12-15. Also included are antibodies comprising the same CDRs as described herein. In some embodiments, the disclosed antibodies and fragments include antibodies and fragments that bind to the same epitope as the epitope shown herein, as well as antibodies and fragments that compete with the antibodies and fragments disclosed herein for binding to SLC34A2.

[0066] According to one embodiment of the present disclosure, an antibody or a fragment thereof is provided, which comprises heavy chain and light chain variable domains having the CDR regions disclosed herein, and bioequivalents thereof.

[0067] In one embodiment, the CDRs are CDRs of 30-H9(B8)F9 or a humanized counterpart thereof (as exemplified in Table 12A). In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 51 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: 52 or 57 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: 53 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: 54 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: 55 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: 56 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof.

[0068] As shown in the experimental examples, the CDRH2 of 30-H9(B8)F9 (SEQ ID NO: 57) includes a NG dipeptide, which may undergo post-translational modification (PTM). Therefore, an N=>S mutation was performed to prevent such PTM, and the mutated CDRH2 was referred to as a PTM-de-risked CDR. However, experimental data have demonstrated that the PTM-de-risked version is as effective as the original version.

[0069] In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 51, CDRH2 comprises the amino acid sequence of SEQ ID NO: 52, CDRH3 comprises the amino acid sequence of SEQ ID NO: 53, CDRL1 comprises the amino acid sequence of SEQ ID NO: 54, CDRL2 comprises the amino acid sequence of SEQ ID NO: 55, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 56. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 51, CDRH2 comprises the amino acid sequence of SEQ ID NO: 57, CDRH3 comprises the amino acid sequence of SEQ ID NO: 53, CDRL1 comprises the amino acid sequence of SEQ ID NO: 54, CDRL2 comprises the amino acid sequence of SEQ ID NO: 55, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 56.

[0070] In some embodiments, antibodies or fragments comprising the same CDRs as 30-H9(B8)F9 or its humanized counterpart are also provided. In some embodiments, the disclosed antibodies and fragments include antibodies or fragments that bind to the same epitope as 30-H9(B8)F9 or its humanized counterpart, as well as antibodies or fragments that compete with either of them for binding to SLC34A2.

[0071] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO: 1 (mouse or chimeric) and 58-61 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 1 (mouse or chimeric) and 58-61 (humanized).

[0072] In some embodiments, the light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 2 (mouse or chimeric) and 62-65 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 2 (mouse or chimeric) and 62-65 (humanized).

[0073] In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, and the light chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 62-65. In some embodiments, the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 58-61, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62.

[0074] In one embodiment, the CDRs are CDRs of 62-G10(E4)G8 or humanized counterparts thereof (as exemplified in Table 13A). In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 66 or a variant thereof having one, two or three deletions, additions, substitutions, or combinations thereof, CDRH2 comprises the amino acid sequence of SEQ ID NO: 67 or 72 or a variant thereof having one, two or three deletions, additions, substitutions, or combinations thereof, CDRH3 comprises the amino acid sequence of SEQ ID NO: 68 or a variant thereof having one, two or three deletions, additions, substitutions, or combinations thereof, CDRL1 comprises the amino acid sequence of SEQ ID NO: 69 or a variant thereof having one, two or three deletions, additions, substitutions, or combinations thereof, CDRL2 comprises the amino acid sequence of SEQ ID NO: 70 or a variant thereof having one, two or three deletions, additions, substitutions, or combinations thereof, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 71 or a variant thereof having one, two or three deletions, additions, substitutions, or combinations thereof.

[0075] One of the CDRs, CDRH2, also includes a potential PTM site NG, so it was mutated to NA as a PTM-derisked version. Experimental data have shown that the PTM-derisked version is as effective as the original version.

[0076] In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 66, CDRH2 comprises the amino acid sequence of SEQ ID NO: 67, CDRH3 comprises the amino acid sequence of SEQ ID NO: 68, CDRL1 comprises the amino acid sequence of SEQ ID NO: 69, CDRL2 comprises the amino acid sequence of SEQ ID NO: 70, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 71. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 66, CDRH2 comprises the amino acid sequence of SEQ ID NO: 72, CDRH3 comprises the amino acid sequence of SEQ ID NO: 68, CDRL1 comprises the amino acid sequence of SEQ ID NO: 69, CDRL2 comprises the amino acid sequence of SEQ ID NO: 70, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 71.

[0077] In some embodiments, antibodies or fragments comprising the same CDRs as 62-G10(E4)G8 or its humanized counterpart are also provided. In some embodiments, the disclosed antibodies and fragments include antibodies or fragments that bind to the same epitope as 62-G10(E4)G8 or its humanized counterpart, as well as antibodies or fragments that compete with either of them for binding to SLC34A2.

[0078] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO: 3 (mouse or chimeric) and 73-76 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 3 (mouse or chimeric) and 73-76 (humanized).

[0079] In some embodiments, the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:4 (mouse or chimeric) and 77-80 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO:4 (mouse or chimeric) and 77-80 (humanized).

[0080] In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, and the light chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 77-80. In some embodiments, the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 73-76, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 79. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 79.

[0081] In one embodiment, the CDRs are CDRs of 63-A11(D5)B4 or a humanized counterpart thereof (as exemplified in Table 14A). In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 81 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: 82 or 87 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: 83 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: 84 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: 85 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: 86 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof.

[0082] One of the CDRs, CDRH2, also includes a potential PTM site NG, so it was mutated to SG as a PTM-derisked version. Experimental data have shown that the PTM-derisked version is as effective as the original version.

[0083] In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 81, CDRH2 comprises the amino acid sequence of SEQ ID NO: 82, CDRH3 comprises the amino acid sequence of SEQ ID NO: 83, CDRL1 comprises the amino acid sequence of SEQ ID NO: 84, CDRL2 comprises the amino acid sequence of SEQ ID NO: 85, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 86. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 81, CDRH2 comprises the amino acid sequence of SEQ ID NO: 87, CDRH3 comprises the amino acid sequence of SEQ ID NO: 83, CDRL1 comprises the amino acid sequence of SEQ ID NO: 84, CDRL2 comprises the amino acid sequence of SEQ ID NO: 85, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 86.

[0084] In some embodiments, antibodies or fragments comprising the same CDRs as 63-A11(D5)B4 or its humanized counterpart are also provided. In some embodiments, the disclosed antibodies and fragments include antibodies or fragments that bind to the same epitope as 63-A11(D5)B4 or its humanized counterpart, as well as antibodies or fragments that compete with either of them for binding to SLC34A2.

[0085] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO: 5 (mouse or chimeric) and 88-91 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 5 (mouse or chimeric) and 88-91 (humanized).

[0086] In some embodiments, the light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 6 (mouse or chimeric) and 92-95 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 6 (mouse or chimeric) and 92-95 (humanized).

[0087] In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 90, and the light chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 92-95. In some embodiments, the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 88-91, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 90, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 92.

[0088] In one embodiment, the CDRs are CDRs of 91-C9F1 or a humanized counterpart thereof (as exemplified in Table 15A). In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 96 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: 97 or 102 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: 98 or 103 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: 99 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: 100 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: 101 or a variant thereof having one, two or three deletions, additions, substitutions, or a combination thereof.

[0089] Two CDRs, CDRH2 and CDRH3, also include a potential PTM site NG, so they are mutated to ND and NA, respectively, as PTM-derisked versions. Experimental data have shown that the PTM-derisked versions are as effective as the original versions.

[0090] In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 96, CDRH2 comprises the amino acid sequence of SEQ ID NO: 97, CDRH3 comprises the amino acid sequence of SEQ ID NO: 98 or 103, CDRL1 comprises the amino acid sequence of SEQ ID NO: 99, CDRL2 comprises the amino acid sequence of SEQ ID NO: 100, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 101. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO: 96, CDRH2 comprises the amino acid sequence of SEQ ID NO: 97 or 102, CDRH3 comprises the amino acid sequence of SEQ ID NO: 98, CDRL1 comprises the amino acid sequence of SEQ ID NO: 99, CDRL2 comprises the amino acid sequence of SEQ ID NO: 100, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 101. In one embodiment, CDRH1 comprises the amino acid sequence of SEQ ID NO:96, CDRH2 comprises the amino acid sequence of SEQ ID NO:97, CDRH3 comprises the amino acid sequence of SEQ ID NO:98, CDRL1 comprises the amino acid sequence of SEQ ID NO:99, CDRL2 comprises the amino acid sequence of SEQ ID NO:100, and CDRL3 comprises the amino acid sequence of SEQ ID NO:101.

[0091] In some embodiments, antibodies or fragments comprising the same CDRs as 91-C9F1 or its humanized counterpart are also provided. In some embodiments, the disclosed antibodies and fragments include antibodies or fragments that bind to the same epitope as 91-C9F1 or its humanized counterpart, as well as antibodies or fragments that compete with either of them for binding to SLC34A2.

[0092] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:7 (mouse or chimeric) and 104-107 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO:7 (mouse or chimeric) and 104-107 (humanized).

[0093] In some embodiments, the light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 8 (mouse or chimeric) and 108-111 (humanized), or a peptide having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO: 8 (mouse or chimeric) and 108-111 (humanized).

[0094] In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 105, and the light chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 108-111. In some embodiments, the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 104-107, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 110. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 105, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 110.

[0095] It will be appreciated that the CDRs may be modified to include CDRs with one, two or three amino acid additions, deletions and / or substitutions. In some embodiments, the substitutions may be conservative substitutions.

[0096] "Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with 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), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, a non-essential amino acid residue in an immunoglobulin polypeptide is preferably replaced by another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0097] Non-limiting examples of conservative amino acid substitutions are provided in the following table, where a similarity score of 0 or above indicates the presence of a conservative substitution between two amino acids.

[0098] Table A. Amino acid similarity matrix

[0099] C G P S A T D E N Q H K R V M I L F Y W W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 K -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 P -3 -1 6 G -3 5 C 12

[0100] Table B. Conservative amino acid substitutions

[0101]

[0102]

[0103] It will also be understood by those of ordinary skill in the art that the antibodies disclosed herein can be modified so that they differ in amino acid sequence from the naturally occurring binding polypeptides from which they are derived. For example, the polypeptide or amino acid sequence derived from a given protein can be similar, for example, having a certain percentage of identity with the starting sequence, for example, it can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the starting sequence.

[0104] In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, an 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).

[0105] Antibodies, variants or derivatives thereof disclosed herein include modified derivatives, i.e., derivatives in which any type of molecule is covalently attached to the antibody such that the covalent attachment does not prevent the antibody from binding to the epitope. For example, but not limited to, the antibody may be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by 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. In addition, the antibody may contain one or more non-classical amino acids.

[0106] Antibodies can be detectably labeled by coupling with a chemiluminescent compound. The presence of the chemiluminescent labeled antigen-binding polypeptide is then determined by detecting the presence of luminescence that occurs during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

[0107] Fluorescent emitting metals such as 152Eu or other lanthanides allow the antibody to be detectably labeled. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic 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. 243-256 (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. 623-653 (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 The Therapeutic Use Of "Radiolabeled Antibodies In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press, pp. 303-316 (1985).

[0108] Bifunctional molecules and combination therapies

[0109] SLC34A2 is overexpressed on tumor cells. As tumor antigen targeting molecules, antibodies or antigen-binding fragments specific for SLC34A2 can be combined with a second antigen-binding fragment specific for immune cells or an antigen-binding fragment specific for immune checkpoints to generate combination therapy or bispecific antibodies.

[0110] In some embodiments, immune cells are selected from T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils and mast cells. Molecules that can be targeted on immune cells include, for example, CCL1, CD3, CD16, CD19, CD28 and CD64. Other examples include PD-1, PD-L1, CTLA-4, LAG-3 (also referred to as CD223), CD28, CD122, 4-1BB (also referred to as CD137), TIM3, OX-40 or OX40L, CD40 or CD40L, LIGHT, ICOS / ICOSL, GITR / GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also referred to as CD272), killer cell immunoglobulin-like receptor (KIR) and CD47.

[0111] Different forms of bispecific antibodies are also provided. In some embodiments, each anti-SLC34A2 fragment and the second fragment are each 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.

[0112] Also provided are bifunctional molecules that include not only antibodies or antigen binding fragments. As tumor antigen targeting molecules, antibodies or antigen binding fragments specific for SLC34A2, such as antibodies or antigen binding fragments described herein, can be optionally combined with immune cytokines or ligands through peptide linkers. The connected immune cytokines 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 regulation at the tumor site.

[0113] Polynucleotide encoding antibody and method for preparing antibody

[0114] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding antibodies, variants or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure can encode the entire heavy chain and light chain variable regions of antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on different polynucleotide molecules. In addition, the polynucleotides of the present disclosure can encode portions of the heavy chain and light chain variable regions of antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on different polynucleotide molecules.

[0115] Methods for preparing antibodies are well known in the art and are described herein. In certain embodiments, the variable and constant regions of the antigen-binding polypeptides disclosed herein are all fully human. Fully human antibodies can be prepared using techniques described in the art and as described herein. For example, fully human antibodies against specific antigens can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to an antigen attack, but whose endogenous loci have been invalidated. Exemplary techniques for preparing such antibodies are described in U.S. Patents 6,150,584, 6,458,592, and 6,420,140, ​​which are incorporated by reference in their entirety.

[0116] Treatment

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

[0118] The present disclosure also relates to antibody-based therapies, which involve administering the antibodies of the present disclosure to patients such as animals, mammals and humans for the treatment of one or more disorders or conditions described herein. The therapeutic compounds of the present disclosure include, but are not limited to, antibodies of the present disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the present disclosure (including variants and derivatives thereof as described herein).

[0119] The antibodies disclosed herein can also be used to treat or inhibit cancer. In some embodiments, cancer cells in a patient express or overexpress SLC34A2. As described above, SLC34A2 can be overexpressed in tumor cells, particularly in gastric, pancreatic, esophageal, ovarian and lung tumors. It has been shown that inhibition of SLC34A2 can be used to treat tumors.

[0120] Thus, in some embodiments, a method for treating cancer in a patient in need thereof is provided. In one embodiment, the method requires administering to the patient an effective amount of an antibody of the present disclosure. In some embodiments, at least one of the cancer cells (e.g., stromal cells) in the patient overexpresses SLC34A2.

[0121] The present disclosure also provides cell therapy, such as chimeric antigen receptor (CAR) T cell therapy. Suitable cells can be used to contact them with the anti-SLC34A2 antibodies disclosed herein (or alternatively, engineered to express the anti-SLC34A2 antibodies disclosed herein). In some embodiments, the antibody is present in a chimeric antigen receptor (CAR). After such contact or engineering, the cells can be introduced into a cancer patient in need of treatment. Cancer patients may suffer from any type of cancer disclosed herein. Cells (e.g., T cells) can be, for example, tumor infiltrating T lymphocytes, CD4+T cells, CD8+T cells, or a combination thereof, but are not limited thereto.

[0122] In some embodiments, cells are isolated from cancer patients themselves. In some embodiments, cells are provided by donors or come from cell banks. When cells are isolated from cancer patients, undesirable immune responses can 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] Other diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed and / or prognosed with the antibodies of the present disclosure, or variants or derivatives thereof, include, but are not limited to, progression and / or metastasis of malignancies and related conditions such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia)) and chronic leukemias (e.g., chronic myeloid (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 such as fibrosarcomas, myxosarcoma, liposarcoma, tumors, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangioma, lymphangioendotheliosarcoma, synovioma, 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, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumors, 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 dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, variant or derivative thereof, the patient's age, weight, general health, sex and diet, time of administration, excretion rate, drug combination and severity of the specific disease being treated. The judgment of these factors by the medical caregiver is within the ordinary skill of the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles known in the art.

[0126] The method of administration of antibodies and variants includes but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. Antigen-binding polypeptides or compositions can be applied by any convenient route, such as by infusion or push injection, absorbed by epithelial or mucocutaneous lining (e.g., oral mucosa, rectum and intestinal mucosa, etc.), and can be applied together with other bioactive agents. Therefore, pharmaceutical compositions containing antigen-binding polypeptides of the present disclosure can be applied by oral, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, local (such as by powder, ointment, drops or transdermal patch), oral administration or as oral spray or nasal spray.

[0127] The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0128] Administration may be systemic or local. In addition, it is desirable to introduce the antibodies of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injections; intraventricular injections may be performed, for example, by an intraventricular catheter connected to a reservoir (such as an Ommaya reservoir). Pulmonary administration may also be employed, for example, by use of an inhaler or nebulizer and formulation with an aerosolizing agent.

[0129] It may be desirable to administer the antigen-binding polypeptides or compositions of the present disclosure locally to the area in need of treatment; this may be accomplished, for example but not limited to, by local infusion during surgery, topical application (e.g., in conjunction with a wound dressing after surgery), by injection, by catheter, by suppository, or by implant, which is a porous, non-porous, or gel-like material, including membranes (such as sialastic membranes) or fibers. Preferably, when administering proteins of the present disclosure (including antibodies), care must be taken to use materials that do not absorb the protein.

[0130] The amount of the antibodies disclosed herein that will effectively treat, inhibit and prevent an immune or malignant disease, disorder or condition can be determined by standard clinical techniques. In addition, in vitro assays can optionally be used to help identify the optimal dosage range. The precise dose used in the formulation will also depend on the route of administration and the severity of the disease, disorder or condition, and should be determined according to the physician's judgment and the circumstances of each patient. The effective dose can be extrapolated from a dose-response curve derived from an in vitro or animal model test system.

[0131] As a general suggestion, the dosage of the antigen-binding polypeptide of the present disclosure administered to a patient is generally 0.1 mg / kg to 100 mg / kg patient weight, 0.1 mg / kg to 20 mg / kg patient weight, or 1 mg / kg to 10 mg / kg patient weight. Typically, due to the immune response to exogenous polypeptides, the half-life of human antibodies in the human body is longer than antibodies from other species. Therefore, lower doses of human antibodies and less frequent administration are generally possible. In addition, the dosage and frequency of administration of the antibodies of the present disclosure can be reduced by enhancing the uptake and tissue penetration (e.g., into the brain) of the antibodies by modification (e.g., lipidation).

[0132] In other embodiments, the compositions of the present disclosure are administered in combination with cytokines. Cytokines that can be administered with the compositions of the present disclosure 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, anti-CD40, CD40L, and TNF-α.

[0133] In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or preventive regimens (eg, radiation therapy).

[0134] Diagnostic Methods

[0135] Overexpression of SLC34A2 is observed in certain tumor samples, and patients with cells that overexpress SLC34A2 may respond to treatment with the anti-SLC34A2 antibodies of the present disclosure. Thus, the antibodies of the present disclosure may also be used for diagnostic and prognostic purposes.

[0136] Preferably, the sample including the cells can be obtained from a patient, which can be a cancer patient or a patient in need of diagnosis. The cells are cells of tumor tissue or tumor mass, blood sample, urine sample or any sample from the patient. After optional pretreatment of the sample, the sample can be incubated with the antibody of the present disclosure under conditions that allow the antibody to interact with the SLC34A2 protein that may be present in the sample. The presence of the SLC34A2 protein in the sample can be detected using methods such as ELISA using anti-SLC34A2 antibodies.

[0137] The presence of SLC34A2 protein in a sample (optionally in a specific amount or concentration) can be used for diagnosis of cancer, as an indication that the patient is suitable for treatment with an antibody, or as an indication that the patient has (or has not) responded to cancer treatment. For prognostic methods, one, two or more tests can be performed at certain stages after the start of cancer treatment to indicate the progress of treatment.

[0138] Composition

[0139] 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 also includes a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0140] In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, more particularly in humans. In addition, a "pharmaceutically acceptable carrier" is generally any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0141] The term "carrier" refers to a diluent, adjuvant, excipient or solvent used together with the therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oil, including oils from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition can also include a small amount of wetting agent or emulsifier, or pH buffers such as acetate, citrate or phosphate. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting tension such as sodium chloride or glucose may also be considered. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition may be made into suppositories with conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will include a therapeutically effective amount of an antigen-binding polypeptide, preferably an antigen-binding polypeptide in purified form, and a suitable amount of a carrier in order to provide a form suitable for administration to a patient. The formulation should be suitable for the mode of administration. Parenteral formulations may be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0142] In one embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to a human being according to conventional steps. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic (such as lidocaine) to alleviate pain at the injection site. Typically, these components are provided separately or mixed together in a unit dosage form, for example, in the form of a lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or a pouch) indicating the amount of the active agent. In the case where the composition is administered by infusion, it can be distributed with an infusion bottle equipped with sterile pharmaceutical grade water or saline. In the case where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that each component is mixed before administration.

[0143] The compounds of the present disclosure may be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations, such as salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0144] Example

[0145] Example 1: Generation of mouse monoclonal antibodies against human SLC34A2

[0146] Different strains of mice were immunized with human SLC34A2 protein and hybridomas were generated accordingly. SLC34A2 positive binders were selected and subcloned. Subsequently, in vitro binding and functional screening were performed to identify the lead antibodies with the highest binding affinity and strongest functional potency.

[0147] The VH / VL sequences of the 25 lead mouse antibodies are provided in Table 1 below.

[0148] Table 1. VH / VL sequences of lead mouse antibodies

[0149]

[0150]

[0151]

[0152]

[0153] Example 2. FACS binding of chimeric antibodies to human SLC34A2

[0154] In this example, flow cytometry was used to evaluate the cell-based binding of a chimeric antibody (hlgG1) prepared from a murine antibody on cells expressing human SLC34A2.

[0155] Briefly, OVCAR3 cells or HEK293 cells expressing human SLC34A2 were incubated with chimeric antibodies (starting from 200 nM, 4-fold dilution, 8 points). After incubation at 4°C for 60 minutes, the cells were washed twice with FACS buffer and then stained with fluorescently conjugated secondary antibodies for 30 minutes at 4°C. Afterwards, the cells were washed twice and analyzed by flow cytometry.

[0156] Research results Figure 1A -B and Figure 2A-B and Tables 2-3) show that compared with the reference antibody XMT-1536, all chimeric antibodies can bind to human SLC34A2 with high affinity, except for chimeric 62-C10(B3)D2 and 95-H11A1D8 which showed a slight decrease at the highest concentration.

[0157] Table 2. Binding affinity of chimeric antibodies to OVCAR3 cells

[0158]

[0159]

[0160] Table 3. Binding affinity of chimeric antibodies to HEK293 cells expressing human SLC34A2

[0161]

[0162]

[0163] Based on the above cell-based binding results, some chimeric antibodies were selected for further cell-based binding to cells expressing human SLC34A2 using flow cytometry. RMG-1 cells were incubated with the selected chimeric antibodies (starting from 200nM, 4-fold dilution, 8 points). After incubation at 4°C for 60 minutes, the cells were washed twice with FACS buffer and then stained with a fluorescently coupled secondary antibody at 4°C for 30 minutes. Then, the cells were washed twice and analyzed by flow cytometry.

[0164] Research results Figure 3 and Table 4) show that the chimeric antibody can bind to human SLC34A2 with high affinity compared to the reference antibody XMT-1536.

[0165] Table 4. Binding affinity of selected chimeric antibodies to RMG-1 cells

[0166] Antibody <![CDATA[EC 50 (nM)]]> Top(MFI) xi7F4C4 hIgG1 5.03 8461 xi94B10A8 hIgG1 2.58 9373 xi94E9B12hIgG1 9.05 10003 xi90G10H7 hIgG1 2.77 9922 xi84F2D10hIgG1 4.33 8596 xi26C5D2 hIgG1 2.52 7062 xi55E5B6D12hIgG1 3.48 9502 xi28G3C6B9 hIgG1 4.73 9424 xi30H9B8F9 hIgG1 0.99 9470 xi62G10E4G8 hIgG1 3.65 9371 xi63A11D5B4 hIgG1 5.32 9768 xi18G1H4 hIgG1 7.91 9800 xi16E5D10hIgG1 3.76 9495 xi69B11D8 hIgG1 1.70 9201 xi71F7D4 hIgG1 8.36 10204 xi78D1B10hIgG1 2.10 9860 xi91C9F1 hIgG1 1.30 9233 xi87H12B7 hIgG1 9.11 9657 hIgG1 isotype NA NA XMT-1536 4.87 9114

[0167] Example 3. Cross-reactive binding of chimeric antibodies to rhesus monkey and cynomolgus monkey SLC34A2 proteins

[0168] In this example, flow cytometry was used to assess cell-based binding of chimeric antibodies on cells expressing rhesus and cynomolgus monkey SLC34A2.

[0169] Briefly, HEK293 engineered cells expressing rhesus SLC34A2 (HEK293 / Rhesus_SLC34A2) were incubated with chimeric antibodies (starting from 200 nM, 4-fold dilution, 8 points). After incubation at 4°C for 60 minutes, the cells were washed twice with FACS buffer and then stained with fluorescently conjugated secondary antibodies for 30 minutes at 4°C. Afterwards, the cells were washed twice and analyzed by flow cytometry.

[0170] Research results Figure 4 and Table 5) showed that most chimeric antibodies could bind to rhesus monkey SLC34A2 with high affinity.

[0171] Table 5. Cross-reactivity with rhesus monkey binding

[0172] Antibody <![CDATA[EC 50 (nM)]]> Top(MFI) xi7F4C4 hIgG1 3.09 13074 xi94B10A8 hIgG1 2.24 13671 xi94E9B12hIgG1 2.38 18712 xi90G10H7 hIgG1 2.19 18502 xi84F2D10hIgG1 46.04 8844 xi26C5D2 hIgG1 NA NA xi55E5B6D12hIgG1 2.46 15942 xi28G3C6B9 hIgG1 3.19 13396 xi30H9B8F9 hIgG1 1.26 17011 xi62G10E4G8 hIgG1 2.53 11975 xi63A11D5B4 hIgG1 2.56 15148 xi18G1H4 hIgG1 2.90 14349 xi16E5D10hIgG1 2.72 14803 xi69B11D8 hIgG1 1.68 11619 xi71F7D4 hIgG1 NA ～5396799 xi78D1B10hIgG1 1.58 15959 xi91C9F1 hIgG1 0.95 12220 xi87H12B7 hIgG1 2.85 18333 hIgG1 isotype NA NA XMT-1536 2.54 11913

[0173] Likewise, the binding of the chimeric antibody to cynomolgus monkey SLC34A2 was measured, and the results are shown in Figure 5 and Table 6. The results showed that most chimeric antibodies could bind to cynomolgus monkey SLC34A2 with high affinity.

[0174] Table 6. Cross-reactivity with cynomolgus monkeys

[0175]

[0176]

[0177] Example 4. Cross-reactivity with rat SLC34A2 protein

[0178] In this example, flow cytometry was used to assess cell-based binding of chimeric antibodies on cells expressing rat SLC34A2.

[0179] Briefly, HEK293 engineered cells expressing rat and mouse SLC34A2 (HEK293 / Rat_SLC34A2, HEK293 / Mouse_SLC34A2) were incubated with chimeric antibodies (10ug / ml). After incubation at 4°C for 60 minutes, the cells were washed twice with FACS buffer and then stained with fluorescently conjugated secondary antibodies for 30 minutes at 4°C. Afterwards, the cells were washed twice and analyzed by flow cytometry.

[0180] Research results Fig. 6A -B) shows that most chimeric antibodies can bind rodent SLC34A2 with high affinity.

[0181] Example 5. ADCC against cells expressing human SLC34A2

[0182] This example evaluates the ability of chimeric antibodies to induce antibody-dependent cellular cytotoxicity activity.

[0183] The ADCC reporter gene bioassay used here uses another reading at an earlier time point of ADCC MOA pathway activation: activation of gene transcription by NFAT (nuclear factor of activated T cells) pathway in effector cells. In addition, the ADCC reporter gene bioassay uses engineered Jurkat cells that stably express FcγRIIIa receptors, V158 (high affinity) variants, and NFAT response elements that drive firefly luciferase expression as effector cells. The antibody biological activity in ADCC MOA is quantified by luciferase produced by NFAT pathway activation; Luciferase activity in effector cells is quantified with luminescent readings. The signal is high, and the background is low.

[0184] Serial dilutions of the chimeric antibody were incubated with engineered Jurkat effector cells (ADCC bioassay effector cells) and ADCC bioassay target cells (expressing SLC34A2) for 6 hours at 37°C. TM Luciferase Assay Buffer Reagent quantifies luciferase activity. Results are shown in Fig. 7A -B and Table 7. As shown, all the chimeric antibodies tested showed significant ADCC inducing activity.

[0185] Table 7. ADCC of chimeric antibodies

[0186]

[0187]

[0188] Example 6. Cytotoxic effect on cells expressing human SLC34A2

[0189] This example examines the cytotoxic effect of the chimeric antibody.

[0190] HEK293 engineered cells expressing human SLC34A2 (HEK293 / H_SLC34A2) were seeded into 96-well plates. Cells were treated with chimeric antibody complexes at corresponding concentrations for 5 days. Cell viability was determined by CellTiter-Glo reagent. Luciferase activity was detected by Envison.

[0191] result( Fig. 8A -B and Table 8) show that these chimeric antibodies have highly efficient killing activity.

[0192] Table 8. Cytotoxic activity

[0193] Antibody <![CDATA[IC 50 (nM)]]> inhibition% xi7F4C4 hIgG1 0.67 86.5 xi94B10A8 hIgG1 0.70 97.5 xi94E9B12hIgG1 0.85 83.6 xi90G10H7 hIgG1 0.28 103.1 xi84F2D10hIgG1 0.61 101.9 xi26C5D2 hIgG1 1.06 82.9 xi55E5B6D12hIgG1 0.69 96.7 xi28G3C6B9 hIgG1 0.36 86.4 xi30H9B8F9 hIgG1 0.84 91.7 xi62G10E4G8 hIgG1 0.42 97.2 xi63A11D5B4 hIgG1 0.66 99.0 xi18G1H4 hIgG1 0.36 82.7 xi16E5D10hIgG1 0.78 93.1 xi69B11D8 hIgG1 0.67 86.9 xi71F7D4 hIgG1 0.83 99.4 xi78D1B10hIgG1 0.50 94.0 xi91C9F1 hIgG1 0.80 97.4 xi87H12B7 hIgG1 0.35 82.6 hIgG1 isotype NA 92.3 XMT-1536 0.72 100.4

[0194] Example 7. CDR mutations to prevent post-translational modifications

[0195] Six murine antibodies, 90G10H7, 55E5B6D12, 30H9B8F9, 62G10E4G8, 63A11D5B4 and 91C9F1, were selected for humanization and amino acid mutation to prevent potential post-translational modifications (PTMs), which are referred to herein as PTM-derisked sequences.

[0196] First, the effect of the PTM-risk-free VH chain on target binding was tested. Flow cytometry was used to evaluate the cell-based binding of PTM antibodies on cells expressing human SLC34A2. Briefly, HEK293 / H_SLC34A2 cells were incubated with PTM antibodies (starting at 200nM, 4-fold dilution, 8 points). After incubation at 4°C for 60 minutes, the cells were washed twice with FACS buffer and then stained with a fluorescently coupled secondary antibody at 4°C for 30 minutes. Afterwards, the cells were washed twice and analyzed by flow cytometry.

[0197] Research results Fig.9A -C and Table 9) show that the humanized / PTM-derisked antibodies can bind to human SLC34A2 with high affinity.

[0198] Table 9. Binding to HEK293 expressing SLC34A2

[0199] Antibody <![CDATA[EC 50 (nM)]]> Top(MFI) hIgG1 isotype NA NA XMT-1536 2.50 27716 xi30H9B8F9 hIgG1 1.14 32228 30H9B8F9 VH_P1 / VL hIgG1 0.89 31744 30H9B8F9 VH_P2 / VL hIgG1 2.04 31036 30H9B8F9 VH_P3 / VL hIgG1 0.86 31483 xi62G10E4G8 hIgG1 1.95 30330 62G10E4G8 VH_P1 / VL hIgG1 7.31 33356 62G10E4G8 VH_P2 / VL hIgG1 2.63 29055 62G10E4G8 VH_P3 / VL hIgG1 2.73 30766 xi63A11D5B4 hIgG1 2.13 30449 63A11D5B4 VH_P1 / VL hIgG1 1.48 30393 63A11D5B4 VH_P2 / VL hIgG1 3.74 30993 63A11D5B4 VH_P3 / VL hIgG1 1.26 31638 xi90G10H7 hIgG1 1.14 32228 90G10H7.p1 hIgG1 0.89 31744 90G10H7.p2 hIgG1 2.04 31036 90G10H7.p3 hIgG1 0.86 31483 90G10H7.p4 hIgG1 1.95 30330 90G10H7.p5 hIgG1 7.31 33356 90G10H7.p6 hIgG1 2.63 29055 90G10H7.p7 hIgG1 2.73 30766 90G10H7.p8 hIgG1 2.13 30449 xi55E5B6D12hIgG1 1.48 30393 55E5B6D12.p1 hIgG1 3.74 30993 55E5B6D12.p2 hIgG1 1.26 31638 xi91C9F1 hIgG1 1.57 28615 91C9F1.p1 hIgG1 0.68 28205 91C9F1.p2 hIgG1 2.07 26916

[0200] Similarly, these humanized / PTM-derisked antibodies were tested for binding to OVCAR3 cells that endogenously express the protein. Fig.10 and Table 10. The results again confirmed the binding affinity of these antibodies.

[0201] Table 10. Binding to OVCAR3 cells

[0202] Antibody <![CDATA[EC 50 (nM)]]> Top(MFI) hIgG1 isotype NA NA XMT-1536 3.8 58447 xi30H9B8F9 hIgG1 1.1 69144 30H9B8F9 VH_P1 / VL hIgG1 1.5 70424 xi62G10E4G8 hIgG1 5.8 71096 62G10E4G8 VH_P3 / VL hIgG1 9.1 72506 xi63A11D5B4 hIgG1 9.2 78584 63A11D5B4 VH_P1 / VL hIgG1 7.5 75229 xi90G10H7 hIgG1 3.9 72926 90G10H7.p1 hIgG1 2.5 73591 90G10H7.p4 hIgG1 2.2 71848 xi55E5B6D12hIgG1 5.1 71368 55E5B6D12.p2 hIgG1 28 67983 xi91C9F1 hIgG1 1.6 63258 91C9F1.p1 hIgG1 1 63499

[0203] Binding of these antibodies to rhesus monkey SLC34A2 was also tested using the procedure described above. Fig.11 and Table 11, confirming the high affinity of these PTM de-risked sequences.

[0204] Table 11. Binding to rhesus monkey SLC34A2

[0205] Antibody <![CDATA[EC 50 (nM)]]> Top(MFI) hIgG1 isotype NA NA XMT-1536 5.8 27020 xi30H9B8F9 hIgG1 1.9 47919 30H9B8F9 VH_P1 / VL hIgG1 2 47355 xi62G10E4G8 hIgG1 3.7 39085 62G10E4G8 VH_P3 / VL hIgG1 4.3 36475 xi63A11D5B4 hIgG1 4.6 45513 63A11D5B4 VH_P1 / VL hIgG1 3.9 39411 xi90G10H7 hIgG1 3.5 47439 90G10H7.p1 hIgG1 2.4 48938 90G10H7.p4 hIgG1 2.3 48591 xi55E5B6D12hIgG1 4.9 46426 55E5B6D12.p2 hIgG1 ～21808 ～121986 xi91C9F1 hIgG1 3.9 15455 91C9F1.p1 hIgG1 2.8 26430

[0206] Example 8. Humanized Antibodies

[0207] Based on the above results, four of the above antibodies, 30H9B8F9, 62G10E4G8, 63A11D5B4 and 91C9F1, continued to be developed through humanization.

[0208] The amino acid sequence of VH and VL of each mouse antibody is compared with the available human Ig gene sequence database to find the best matching human germline Ig gene sequence overall. The CDR of the mouse antibody is then transplanted into the matching human sequence. Synthesize cDNA and use it to produce humanized antibodies. Then some back mutations from the mouse antibody are introduced back to the humanized antibody.

[0209] The amino acid sequences of the humanized antibodies are provided in Tables 12-15 below.

[0210] Humanization and PTM de-risking sequences

[0211] A.30-H9(B8)F9

[0212] Table 12A. Humanized sequences

[0213]

[0214] Table 12B. CDR sequences

[0215]

[0216] Table 12C. Humanized Antibodies

[0217] 30-H9(B8)F9.p1 VH_1 VH_2 VH_3 VH_4 VL_1 Z1 Z5 Z9 Z13 VL_2 Z2 Z6 Z10 Z14 VL_3 Z3 Z7 Z11 Z15 VL_4 Z4 Z8 Z12 Z16

[0218] B.62-G10(E4)G8

[0219] Table 13A. Humanized sequences

[0220]

[0221]

[0222] Table 13B. CDR sequences

[0223]

[0224] Table 13C. Humanized Antibodies

[0225] 62-G10(E4)G8.p3 VH_1 VH_2 VH_3 VH_4 VL_1 Z1 Z5 Z9 Z13 VL_2 Z2 Z6 Z10 Z14 VL_3 Z3 Z7 Z11 Z15 VL_4 Z4 Z8 Z12 Z16

[0226] C.63-A11(D5)B4

[0227] Table 14A. Humanized sequences

[0228]

[0229]

[0230] Table 14B. CDR sequences

[0231]

[0232] Table 14C. Humanized Antibodies

[0233] 63-A11(D5)B4.p1 VH_1 VH_2 VH_3 VH_4 VL_1 Z1 Z5 Z9 Z13 VL_2 Z2 Z6 Z10 Z14 VL_3 Z3 Z7 Z11 Z15 VL_4 Z4 Z8 Z12 Z16

[0234] D.91-C9F1

[0235] Table 15A. Humanized sequences

[0236]

[0237]

[0238] Table 15B. CDR sequences

[0239]

[0240] Table 15C. Humanized Antibodies

[0241] 91-C9F1.p1 VH_1 VH_2 VH_3 VH_4 VL_1 Z1 Z5 Z9 Z13 VL_2 Z2 Z6 Z10 Z14 VL_3 Z3 Z7 Z11 Z15 VL_4 Z4 Z8 Z12 Z16

[0242] Binding of these humanized antibodies to OVCAR3 cells was evaluated as described above, and the results are shown in Figure 12A -D and Tables 16A-D.

[0243] Table 16A. Binding of humanized antibodies to OVCAR3 - 30H9B8F9

[0244]

[0245]

[0246] Table 16B. Binding of humanized antibodies to OVCAR3 - 62G10E4G8

[0247] Antibody <![CDATA[EC 50 (nM)]]> Top(MFI) hIgG1 isotype NA NA XMT-1536 6.82 28971 xi62G10E4G8 hIgG1 3.73 27650 62G10E4G8.p3 hIgG1 6.98 30347 62G10E4G8.p3.z1 hIgG1 943.80 87738 62G10E4G8.p3.z2 hIgG1 32.88 29698 62G10E4G8.p3.z3 hIgG1 17.33 31289 62G10E4G8.p3.z4 hIgG1 16.27 29908 62G10E4G8.p3.z5 hIgG1 ～1361 ～230025 62G10E4G8.p3.z6 hIgG1 8.06 28157 62G10E4G8.p3.z7 hIgG1 6.08 26810 62G10E4G8.p3.z8 hIgG1 8.61 29963 62G10E4G8.p3.z9 hIgG1 24.07 31332 62G10E4G8.p3.z10hIgG1 4.87 27396 62G10E4G8.p3.z11hIgG1 4.32 28200 62G10E4G8.p3.z12hIgG1 2.96 25440 62G10E4G8.p3.z14hIgG1 4.58 27907 62G10E4G8.p3.z15hIgG1 3.76 27300 62G10E4G8.p3.z16hIgG1 10.95 31916

[0248] Table 16C. Binding of humanized antibodies to OVCAR3 - 63A11D5B4

[0249]

[0250]

[0251] Table 16D. Binding of humanized antibodies to OVCAR3 - 91C9F1

[0252] Antibody <![CDATA[EC 50 (nM)]]> Top(MFI) hIgG1 isotype NA NA XMT-1536 2.68 32389 xi91C9F1 hIgG1 1.77 32062 91C9F1.p1 hIgG1 2.29 32342 91C9F1.p1.z1 hIgG1 3.78 34220 91C9F1.p1.z2 hIgG1 3.32 34537 91C9F1.p1.z3 hIgG1 4.04 35456 91C9F1.p1.z4 hIgG1 1.12 34629 91C9F1.p1.z5 hIgG1 2.95 36861 91C9F1.p1.z6 hIgG1 1.72 36117 91C9F1.p1.z7 hIgG1 1.67 37440 91C9F1.p1.z8 hIgG1 2.13 35657 91C9F1.p1.z9 hIgG1 2.72 37027 91C9F1.p1.z10hIgG1 2.32 38122 91C9F1.p1.z11hIgG1 1.41 30997 91C9F1.p1.z12hIgG1 1.77 34468 91C9F1.p1.z14hIgG1 3.87 36194

[0253] In another test, flow cytometry was used to evaluate cell-based binding of hu antibodies on cells expressing human SLC34A2. Briefly, HEK293 / H_SLC34A2, OVCAR3, and RMG-1 cells were incubated with hu antibodies. After incubation at 4°C for 60 minutes, the cells were washed twice with FACS buffer and then stained with a fluorescently conjugated secondary antibody at 4°C for 30 minutes. Afterwards, the cells were washed twice and analyzed by flow cytometry.

[0254] Research results Figure 13A -D and Tables 17A-D) show that the hu antibody can bind to human SLC34A2 with high affinity.

[0255] Table 17A. Binding to Cell Lines - 30H9B8F9

[0256]

[0257] Table 17B. Binding to cell lines - 62G10E4G8

[0258]

[0259] Table 17C. Binding to Cell Lines - 63A11D5B4

[0260]

[0261] Table 17D. Binding to Cell Lines - 91C9F1

[0262]

[0263] In further analysis, the binding affinity of these antibodies to rhesus monkey / rat / mouse SLC34A2 was evaluated using the above method. Figures 14 - 16 As shown in Table 18, these humanized antibodies retained high affinity.

[0264] Table 18. Binding to rhesus monkey SLC34A2

[0265] Antibody <![CDATA[EC 50 (nM)]]> Top(MFI) hIgG1 isotype NA NA XMT-1536 2.6 11488 xi30H9B8F9 hIgG1 0.9 17418 30H9B8F9.p1 hIgG1 0.8 17001 30H9B8F9.p1.z5 hIgG1 0.7 15993 30H9B8F9.p1.z6 hIgG1 1.1 15133 30H9B8F9.p1.z9 hIgG1 0.9 16265 30H9B8F9.p1.z13hIgG1 0.8 15597 xi62G10E4G8 hIgG1 1.8 13309 62G10E4G8.p3 hIgG1 5 14367 62G10E4G8.p3.z8 hIgG1 2.5 12206 62G10E4G8.p3.z11hIgG1 3.4 14730 62G10E4G8.p3.z15hIgG1 1.4 14363 xi63A11D5B4 hIgG1 2.3 15157 63A11D5B4.p1 hIgG1 0.7 13775 63A11D5B4.p1.z5 hIgG1 3.9 11134 63A11D5B4.p1.z9 hIgG1 1.6 11777 63A11D5B4.p1.z15hIgG1 4.7 10342 xi91C9F1 hIgG1 1.2 11351 91C9F1.p1 hIgG1 0.8 7973 91C9F1.p1.z4 hIgG1 3.2 6719 91C9F1.p1.z6 hIgG1 8.8 8156 91C9F1.p1.z7 hIgG1 4.2 9495 91C9F1.p1.z10hIgG1 4.2 9233

[0266] Example 8. ADCC of humanized antibodies

[0267] This example measures the antibody-dependent cellular cytotoxicity (ADCC) of humanized antibodies.

[0268] ADCC reporter gene bioassay uses another reading at an earlier time point of ADCC MOA pathway activation: activation of gene transcription by NFAT (nuclear factor of activated T cells) pathway in effector cells. In addition, ADCC reporter gene bioassay uses engineered Jurkat cells that stably express FcγRIIIa receptors, V158 (high affinity) variants, and NFAT response elements that drive firefly luciferase expression as effector cells. The antibody biological activity in ADCC MOA is quantified by luciferase produced by NFAT pathway activation; Luciferase activity in effector cells is quantified with luminescent readings. The signal is high, and the background is low.

[0269] Serial dilutions of hu antibodies were incubated with engineered Jurkat effector cells (ADCC bioassay effector cells) and ADCC bioassay target cells (expressing SLC34A2) at 37°C for 6 hours. TM Luciferase Assay Buffer Reagent quantifies luciferase activity.

[0270] Results Picture 17 and Table 19, confirming the strong ADCC inducing activity of these humanized antibodies.

[0271] Table 19. ADCC activity

[0272] antibody <![CDATA[EC 50 (nM)]]> Top(MFI) hIgG1 isotype ～33.32 14765 XMT-1536 0.76 126843 RG-7599 ～1.310 15689 xi30H9B8F9 hIgG1 1.26 108750 30H9B8F9.p1 hIgG1 2.52 126415 30H9B8F9.p1.z5 hIgG1 0.72 135100 30H9B8F9.p1.z6 hIgG1 1.25 134931 30H9B8F9.p1.z9 hIgG1 1.53 144513 30H9B8F9.p1.z13hIgG1 0.96 142916 xi62G10E4G8 hIgG1 4.56 100880 62G10E4G8.p3 hIgG1 4.23 122270 62G10E4G8.p3.z11hIgG1 3.78 116421 62G10E4G8.p3.z15hIgG1 5.39 122634 63A11D5B4.p1 hIgG1 2.83 113949 63A11D5B4.p1.z5 hIgG1 0.95 116223 63A11D5B4.p1.z9 hIgG1 1.69 116236 63A11D5B4.p1.z15hIgG1 1.47 121606 xi91C9F1 hIgG1 1.96 132927 91C9F1.p1 hIgG1 3.31 143117 91C9F1.p1.z4 hIgG1 3.14 137231 91C9F1.p1.z6 hIgG1 1.77 125245 91C9F1.p1.z7 hIgG1 2.12 123589 91C9F1.p1.z10hIgG1 2.37 138028

[0273] Example 9. Testing of selected humanized antibodies

[0274] Based on previous results, four humanized antibodies were selected for further development, including 30-H9(B8)F9.p1.z5, 62-G10(E4)G8.p3.z15, 63-A11(D5)B4.p1.z9, and 91-C9F1.p1.z7.

[0275] The candidate antibodies were further tested in comparison to benchmark antibodies XMT-1536 and RG-7599. Picture 18 Comparison of binding to human SLC34A2 on different cell types is shown. Picture 19 The cross-reactivity of the antibodies with SLC34A2 proteins from different species was compared. Picture 20A -B compared their ADCC inducing activities.

[0276] The above results are summarized in Table 20, which shows that the antibodies developed by the present invention are significantly superior to the benchmarks (XMT-1536 and RG-7599) in many aspects.

[0277] Table 20. Summary of comparison

[0278]

[0279]

[0280] ***

[0281] The present disclosure is not limited by the scope of the specific embodiments, which are intended to be a single illustration of the various aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It is apparent to those skilled in the art that various modifications and variations may be made to the methods and compositions of the present disclosure without departing from the essence or scope of the present disclosure. Therefore, the present disclosure is intended to cover modifications and variations of the present disclosure, provided that these modifications and variations are within the scope of the appended claims and their equivalents.

[0282] 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. An antibody or fragment thereof having binding specificity to human solute carrier 34A2 (SLC34A2) protein, wherein the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises heavy chain complementary determining regions CDRH1, CDRH2 and CDRH3, and the light chain variable region comprises light chain complementary determining regions CDRL1, CDRL2 and CDRL3, and wherein: (a) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 51, The CDRH2 comprises the amino acid sequence of SEQ ID NO: 52 or 57, The CDRH3 comprises the amino acid sequence of SEQ ID NO:53, The CDRL1 comprises the amino acid sequence of SEQ ID NO: 54, The CDRL2 comprises the amino acid sequence of SEQ ID NO: 55, and The CDRL3 comprises the amino acid sequence of SEQ ID NO:56, (b) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 66, The CDRH2 comprises the amino acid sequence of SEQ ID NO: 67 or 72, The CDRH3 comprises the amino acid sequence of SEQ ID NO: 68, The CDRL1 comprises the amino acid sequence of SEQ ID NO: 69, The CDRL2 comprises the amino acid sequence of SEQ ID NO: 70, and The CDRL3 comprises the amino acid sequence of SEQ ID NO: 71, (c) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 81, The CDRH2 comprises the amino acid sequence of SEQ ID NO: 82 or 87, The CDRH3 comprises the amino acid sequence of SEQ ID NO: 83, The CDRL1 comprises the amino acid sequence of SEQ ID NO: 84, The CDRL2 comprises the amino acid sequence of SEQ ID NO: 85, and The CDRL3 comprises the amino acid sequence of SEQ ID NO: 86, or (d) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 96, The CDRH2 comprises the amino acid sequence of SEQ ID NO: 97 or 102, The CDRH3 comprises the amino acid sequence of SEQ ID NO: 98 or 103, The CDRL1 comprises the amino acid sequence of SEQ ID NO: 99, The CDRL2 comprises the amino acid sequence of SEQ ID NO: 100, and The CDRL3 comprises the amino acid sequence of SEQ ID NO:

101.

2. The antibody or fragment thereof according to claim 1, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 51, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 52, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 53, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 54, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 55, and the CDRL3 comprises the amino acid sequence of SEQ ID NO:

56.

3. The antibody or fragment thereof according to claim 2, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 58-61, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 62-65.

4. The antibody or fragment thereof according to claim 2, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

62.

5. The antibody or fragment thereof according to claim 1, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 66, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 67 or 72, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 68, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 69, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 70, and the CDRL3 comprises the amino acid sequence of SEQ ID NO:

71.

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

67.

7. The antibody or fragment thereof according to claim 5, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 73-76, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 77-80.

8. The antibody or fragment thereof according to claim 5, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 76, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

79.

9. The antibody or fragment thereof according to claim 1, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 81, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 82 or 87, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 83, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 84, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 85, and the CDRL3 comprises the amino acid sequence of SEQ ID NO:

86.

10. The antibody or fragment thereof according to claim 9, wherein the CDRH2 comprises the amino acid sequence of SEQ ID NO:

82.

11. The antibody or fragment thereof according to claim 9, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 88-91, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 92-95.

12. The antibody or fragment thereof according to claim 9, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 90, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

92.

13. The antibody or fragment thereof according to claim 1, wherein the CDRH1 comprises the amino acid sequence of SEQ ID NO: 96, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 97 or 102, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 98 or 103, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 99, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 100, and the CDRL3 comprises the amino acid sequence of SEQ ID NO:

101.

14. The antibody or fragment thereof according to claim 13, wherein the CDRH2 comprises the amino acid sequence of SEQ ID NO: 97, and the CDRH3 comprises the amino acid sequence of SEQ ID NO:

98.

15. The antibody or fragment thereof according to claim 13, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 104-107, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 108-111.

16. The antibody or fragment thereof according to claim 13, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 105, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

110.

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

18. An antibody or a fragment thereof, which has binding specificity to human solute carrier 34A2 (SLC34A2) protein and binds to the same epitope on the SLC34A2 protein as the antibody or fragment thereof according to claim 1, or competes with the antibody or fragment thereof according to claim 1 for binding to the SLC34A2 protein.

19. A composition comprising the antibody or fragment thereof according to any one of claims 1 to 18, and a pharmaceutically acceptable carrier.

20. One or more polynucleotides encoding the antibody or fragment thereof according to any one of claims 1-18.

21. A cell comprising one or more polynucleotides according to claim 20.

22. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient the antibody or fragment thereof according to any one of claims 1-18.

23. Use of the antibody or fragment thereof according to any one of claims 1 to 18 for preparing a medicament for treating cancer.

24. The method of claim 22 or the use of claim 23, wherein the cancer is selected from 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, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer and thyroid cancer.

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