Antibodies targeting EPHA2 and their use in treatment of cancer

Abstract

The present invention relates to anti-EphA2 antibodies and the detection (or diagnosis) and treatment of cancer using the anti-EphA2 antibodies. The present invention creates anti-EphA2 antibodies, in particular single chain antibody fragments (scFvs) and humanised antibodies, which have the ability to bind to anti-EphA2 and inhibit angiogenesis, migration and cancer cell growth.

Description

Technical Field

[0001] The present invention relates to the fields of cancer detection (or diagnosis) and treatment. In particular, the present invention relates to antibodies targeting EphA2 and their use in cancer detection (or diagnosis) and treatment. Background Art

[0002] Cancer is the uncontrolled growth of abnormal cells anywhere in the body. These abnormal cells are called cancer cells, malignant cells or tumor cells. The interaction between the hepatocyte growth factor receptor (HGFR) and ephrin (Ephs / ephrin) controls a wide range of biological functions that have also been implicated in the pathogenesis of human cancer. EphA2 type (EphA2), a member of the tyrosine kinase family, interacts with ephrin (e.g., ephrin-A1) to trigger bidirectional signal transduction between cells. The interaction between EphA2 and ephrin-A1 results in the inhibition of Ras-MAPK activity, leading to the inhibition of tumor growth. In addition, studies have also confirmed that EphA2 overexpression can drive ligand-independent signal transduction and induce tumorigenesis. Therefore, it is believed that EphA2 can induce either negative or positive effects on tumor growth. During tumorigenesis, the regular interaction between EphA2 and ephrin-A1 is disrupted, leading to EphA2 overexpression and progression to cancer. The overexpression of EphA2 has been identified as a significant tumor target in the diagnosis and treatment of pancreatic cancer. Its higher gene expression is also associated with poor patient prognosis. In recent years, the anti-tumor activities of several tyrosine kinase inhibitors (TKIs) targeting EphA2 signaling have been evaluated. However, many of these TKIs have multiple targets, making their specificity for EphA2 a drawback in clinical development.

[0003] Therefore, there is also a need to develop antibodies with specific binding to EphA2. Summary of the Invention

[0004] The present invention provides a isolated anti-EphA2 antibody or antigen-binding portion thereof, which comprises at least one of the following: a light chain complementarity determining region 1 (L-CDR1) comprising the amino acid residues of SEQ ID NO:1 or a variant having an amino acid sequence with at least 80% identity to any of SEQ ID NO:1; a light chain complementarity determining region 2 (L-CDR2) comprising the amino acid residues of SEQ ID NO:2 or a variant having an amino acid sequence with at least 80% identity to any of SEQ ID NO:2; and a light chain complementarity determining region 3 (L-CDR3) comprising the amino acid residues of SEQ ID NO:3 or a variant having an amino acid sequence with at least 80% identity to any of SEQ ID NO:3; and at least one of the following: a heavy chain complementarity determining region 1 (H-CDR1) comprising the amino acid residues of SEQ ID NO:4 or a variant having an amino acid sequence with at least 80% identity to any of SEQ ID NO:4; a heavy chain complementarity determining region 2 (H-CDR2) comprising the amino acid residues of SEQ ID NO:5 or a variant having an amino acid sequence with at least 80% identity to any of SEQ ID NO:5; and a heavy chain complementarity determining region 3 (H-CDR3) comprising the amino acid residues of SEQ ID NO:6 or a variant having an amino acid sequence with at least 80% identity to any of SEQ ID NO:6; such that the isolated antibody or antigen-binding portion thereof binds to EphA2.

[0005] In some embodiments, the antibodies of the present invention include monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies. In some embodiments, the isolated anti-EphA2 antibody or antigen-binding portion thereof is a single-chain Fv (scFv), IgG, Fab, (Fab)2, or (scFv')2.

[0006] In some embodiments, the anti-EphA2 antibody or antigen-binding portion thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO:7 or 8 or a variant having at least 80% identity to SEQ ID NO:7 or 8, and a heavy chain comprising the amino acid sequence of SEQ ID NO:9 or 10 or a variant having at least 80% identity to SEQ ID NO:9 or 10. In some embodiments, the anti-EphA2 antibody or antigen-binding portion thereof comprises a light chain comprising the amino acid sequence shown in SEQ ID NO:7 or 8; and a heavy chain comprising the amino acid sequence of SEQ ID NO:9 or 10.

[0007] In some embodiments, the anti-EphA2 antibody or antigen-binding portion thereof comprises the amino acid sequence of SEQ ID NO: 11 or 12 or a variant having at least 80% identity to SEQ ID NO: 11 or 12. In some embodiments, the anti-EphA2 antibody or antigen-binding portion thereof comprises the amino acid sequence of SEQ ID NO: 11 or 12.

[0008] In other embodiments, the present invention provides an isolated antibody (scFv SD5) comprising a light chain having the amino acid sequence shown by the sequence of SEQ ID NO: 7 or a variant having at least 80% identity to SEQ ID NO: 7 and a heavy chain having the amino acid sequence shown by the sequence of SEQ ID NO: 9 or a variant having at least 80% identity to SEQ ID NO: 9. Preferably, the sequence identity as mentioned above is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0009] In another embodiment, the present invention provides a humanized antibody (humanized scFv hSD5) comprising a light chain having the amino acid sequence shown by SEQ ID NO: 8 or a variant having at least 80% identity to SEQ ID NO: 8 and a heavy chain having the amino acid sequence shown by SEQ ID NO: 10 or a variant having at least 80% identity to SEQ ID NO: 10.

[0010] In another embodiment, the present invention provides an isolated antibody (scFv SD5) comprising the amino acid sequence shown by SEQ ID NO: 11 or a variant having at least 80% identity to SEQ ID NO: 11. In another embodiment, the present invention includes a humanized antibody comprising the amino acid sequence shown by SEQ ID NO: 12 or a variant having at least 80% identity to SEQ ID NO: 12.

[0011] The present invention also provides an antibody-drug conjugate (ADC) comprising the anti-EphA2 antibody or antigen-binding portion thereof of the present invention and a drug-linker structure comprising an anti-tumor compound linked to the antibody via a linker.

[0012] In some embodiments, the anti-tumor compound is selected from auristatin (such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)), vincristine, vinblastine, methotrexate, platinum-based anti-tumor agents (cisplatin and its derivatives), doxorubicin, calicheamicin, dolastatin 10, maytansinoid, pyrrolobenzodiazepine dimer, camptothecin derivatives, duocarmycin, amanitin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, and Taxol and its derivatives. In some embodiments, the anti-tumor compound is MMAE.

[0013] The present invention provides a pharmaceutical composition comprising the anti-EphA2 antibody or an antigen-binding portion thereof or an ADC of the present invention and a pharmaceutically acceptable carrier or excipient.

[0014] In some embodiments, the pharmaceutical composition further comprises one or more additional anti-cancer agents or is used in combination therewith.

[0015] In some embodiments, the one or more additional anti-cancer agents are Gemcitabine.

[0016] The present invention also provides a method for treating or preventing EphA2-related cancer in an individual, which comprises administering to the individual the anti-EphA2 antibody or an antigen-binding portion thereof or an ADC of the present invention.

[0017] The present invention also provides a method for inhibiting the growth or cancer metastasis of EphA2-related cancer cells in an individual, which comprises administering to the individual the anti-EphA2 antibody or an antigen-binding portion thereof or an ADC of the present invention.

[0018] In some embodiments, the EphA2-related cancer is selected from cholangiocarcinoma, bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioma, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, stomach cancer, thymic cancer, and vulvar cancer. In some embodiments, the EphA2-related cancer is selected from bladder cancer, brain cancer, cholangiocarcinoma, colon cancer, gastric cancer, and pancreatic cancer.

[0019] In some embodiments, each of the compositions and treatment methods identified above may further include another anti-tumor drug and administration of one or more additional anti-tumor drugs.

[0020] The present invention further provides a method for detecting or diagnosing EphA2-related cancer or a high risk of future occurrence of cancer in an individual, or predicting metastasis or prognosis of cancer in an individual, or monitoring the progression of cancer in an individual diagnosed with EphA2-related cancer, which includes contacting a biological sample from the individual with the anti-EphA2 antibody of the present invention, quantifying the binding of the EphA2 antigen and the antibody in the sample, and comparing the binding with a reference value representing the binding between the anti-EphA2 antibody and the EphA2 antigen measured in a sample from a control individual not suffering from cancer.

[0021] The present invention further provides a kit for detecting or diagnosing EphA2-related cancer or a high risk of future occurrence of EphA2-related cancer in an individual, or predicting metastasis or prognosis of cancer, or monitoring cancer progression, which includes the anti-EphA2 antibody of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Exemplary embodiments are described in the accompanying drawings. The embodiments and drawings disclosed herein are to be considered illustrative rather than restrictive.

[0023] FIG. 1 shows the analysis of cancer types with high EphA2 gene expression using the GEPIA database.

[0024] FIGS. 2A to 2D show the analysis and evaluation of the correlation between EphA2 expression and pancreatic cancer.

[0025] FIGS. 3A to 3F show the characterization of anti-EphA2 scFv isolated using phage display technology.

[0026] FIGS. 4A to 4B show the cell growth inhibitory effects of these scFv tested at four concentrations on four strains of pancreatic cancer cells.

[0027] FIGS. 5A to 5D show the inhibitory effects of the isolated scFv on the proliferation and migration of PAAD cells.

[0028] Figures 6A to 6D show the binding specificity of the humanized antibody hSD5 to EphA2 and the induced tumor suppression signal transduction.

[0029] Figures 7A to 7D show the in vivo tumor growth inhibitory effect of the humanized IgG hSD5 in BxPc-3 xenograft mice.

[0030] Figures 8A to 8D show the in vivo tumor growth inhibitory effect of the humanized IgG hSD5 in Mia PaCa-2 xenograft mice.

[0031] Figures 9A to 9B show that the epitope definition of IgG hSD5 recognizes the active site of EphA2.

[0032] Figure 10 shows the growth inhibitory responses of different pancreatic cancer cell lines treated with serially diluted MMAE.

[0033] Figures 11A to 11D show the growth inhibitory responses of different pancreatic cancer cell lines by administration of serially diluted hSD5-ADC.

[0034] Figures 12A to 12B show the cell cycle changes of pancreatic cancer cells.

[0035] Figures 13A to 13B show the inhibitory effect on tumor growth in mice after administration of different concentrations of hSD5-ADC and control IgG-ADC tested using the BxPc-3 xenograft mouse model.

[0036] Figures 14A to 14C show the in vivo tumor growth inhibitory effect of hSD5-ADC in BxPc-3 xenograft mice.

[0037] Figures 15A to 15B show that anti-EphA2 hSD5 can recognize endogenous EphA2 molecules and inhibit cell growth on gastric cancer.

[0038] Figures 16A to 16B show the expression of EphA2 and the binding ability of anti-EphA2 hSD5 in GBM.

[0039] Figures 17A to 17C show that anti-EphA2 hSD5 can recognize endogenous EphA2 molecules and inhibit cell growth on cholangiocarcinoma and bladder cancer.

[0040] Figures 18A to 18C show that anti-EphA2 hSD5 can recognize endogenous EphA2 molecules and inhibit cell growth on colon cancer.

[0041] Figures 19A to 19C show that hSD5-ADC can inhibit tumor growth in an HCT116 xenograft mouse model. Detailed Description

[0042] The terms used herein are for describing particular embodiments of the present invention, but their use does not limit the present invention unless as set forth in the claims. It should be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory and do not limit the object claimed in the present application.

[0043] Terms such as "a / an" and "the" are not intended to refer only to a single entity, but include a general category for which a particular instance can be used to illustrate.

[0044] In the present application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other terms (such as "includes" and "included") is non-restrictive.

[0045] As used herein, the terms "tumor", "cancer", and "carcinoma" are used interchangeably and refer to all tumor cell growth and proliferation (whether malignant or benign), and all pre-cancerous and cancer cells and tissues.

[0046] As used herein, the term "biological sample" refers to a sample obtained from a patient. For example, biological samples can be obtained from blood, tissue (such as tumor), serum, feces, urine, sputum, cerebrospinal fluid, nipple aspirate, and supernatant from cell lysates.

[0047] As used herein, the term "diagnosis" means identifying the presence or nature of a pathological condition and includes identifying an individual at risk of developing cancer. Diagnostic methods vary in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive ("true positives"). Diseased individuals not detected by the assay are "false negatives". Individuals who are not diseased and test negative in the assay are called "true negatives". The "specificity" of a diagnostic assay is the proportion determined to be correctly identified as negative (e.g., the percentage of individuals who are not diseased and are correctly identified as not having the condition).

[0048] As used herein, the terms "detection", "detecting", and the like can be used in the case of detecting a biomarker or detecting cancer (e.g., when a positive assay result is obtained). In the latter case, "detection" and "diagnosis" are considered synonyms.

[0049] The "test amount" of a marker refers to the amount of the marker present in the sample being tested.

[0050] The "control amount" of the label can be any amount or range of amounts to be compared with the measured amount of the label.

[0051] The term "at risk" is intended to mean at increased risk compared to a normal individual or compared to a control group. Thus, an individual "at risk of developing cancer" is at increased risk compared to the normal population, and an individual "at risk of cancer recurrence" can be considered at increased risk of recurrence compared to the risk of recurrence among all treated cancer patients.

[0052] As used herein, the terms "increased risk" or "elevated risk" mean any statistically significant increase in, for example, the probability that the body will develop cancer or its recurrence.

[0053] As used herein, the term "prognosis" refers to the prediction of the likelihood of cancer-attributable death or progression (including recurrence, metastatic spread, and drug resistance of neoplastic diseases such as ovarian cancer). The term "poor prognosis" means that, despite the use of standards of care for treating cancer (e.g., surgery, radiation, chemotherapy), the survival period and the prospects for recovery of the disease are unlikely. A poor prognosis is the category of patients whose survival period is less than the median survival period.

[0054] As used herein, the term "metastasis" is defined as the spread of cancer from one part of the body to another. A tumor formed by the spread cells is called a "metastatic tumor" or a "metastasis".

[0055] As used herein, the term "metastasis risk" is a prognostic indication that cancer in a particular patient, especially a human patient, will progress to a metastatic state based on statistical predictors. Actual progression to a metastatic state is not required, and treatment modalities are expected to be employed to attempt to delay or prevent the realization of such risk.

[0056] As used herein, the expression "reference value" refers to a laboratory value used as a reference for a numerical / data obtained from a sample taken from an individual.

[0057] As used herein, "determination of a level", "determining a level", or "measuring a level" generally refers to calculating the amount or concentration of a particular substance, or quantifying the intensity of a signal from a probe that represents the amount or concentration of a particular substance.

[0058] As used herein, the term "antibody" is used in the broadest sense and specifically encompasses, for example, a single monoclonal antibody (including agonist, antagonist, and neutralizing antibodies), an antibody composition having multi-epitope specificity, a polyclonal antibody, a single-chain anti-antibody, and fragments of an antibody (see below), so long as it specifically binds to the native polypeptide and / or exhibits the biological or immunological activity of the present invention. According to one embodiment, the antibody binds to the oligomeric form of the target protein, such as the trimeric form. The phrase "functional fragment or analog" of an antibody is a compound having the same qualitative biological activity as the antibody to which it refers. For example, a functional fragment or analog of an antibody of the present invention may be one that specifically binds to EGFR. In one embodiment, the antibody may prevent or substantially reduce the ability of EGFR to induce cell proliferation.

[0059] As used herein, the term "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from components of its natural environment. The components of its natural environment are materials that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody will be purified (1) to greater than 95% by weight antibody, as determined by the Lowry method, and optimally greater than 99% by weight, (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequenator, or (3) to homogeneity, by SDS-PAGE, under reducing or non-reducing conditions, using Coomassie blue or preferably silver staining. Isolated antibodies include antibodies in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibodies will be prepared by at least one purification step.

[0060] As used herein, "percent amino acid sequence identity (%)" and "homology" with respect to a peptide, polypeptide, or antibody sequence refer to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. The alignment for the purpose of determining the percent amino acid sequence identity can be achieved in various ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the alignment, including any algorithms known in the art for achieving the maximum alignment over the full length of the sequences being compared.

[0061] As used herein, the term "Fab" refers to an antigen-binding fragment of an Ig (however produced), including the variable domain and the first constant domain.

[0062] As used herein, the term "Fv" is the smallest antibody fragment that contains a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy-chain variable region and one light-chain variable region that are tightly, non-covalently associated. From the folding of these two domains emerge six hypervariable loops (3 loops from each of the H and L chains) that contribute amino acid residues for antigen binding and confer the antibody-binding specificity of the antibody. However, even a single variable domain (or half of the Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind an antigen, although with a lower affinity than the entire binding site.

[0063] As used herein, the term "single-chain Fv" (also abbreviated as "sFv" or "scFv") is an antibody fragment that contains the VH and VL antibody domains linked in a single polypeptide chain. Preferably, the sFv polypeptide further contains a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0064] As used herein, the term "complementary determining region" (CDR) refers to the non-contiguous antigen-combining sites found within the variable regions of heavy-chain and light-chain polypeptides. The CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where these definitions include overlapping or subsets of amino acid residues when compared to each other.

[0065] As used herein, the term "humanized antibody" refers to a recombinant protein in which the complementarity determining regions (CDRs) of an antibody from one species; e.g., a murine or chicken antibody, are transferred from the heavy and light variable chains of the antibody of that species to the human heavy and light variable domains (framework regions). The constant domains of the antibody molecule are derived from those of human antibodies. In some cases, certain residues in the framework regions of the humanized antibody (particularly those that contact or are proximate to the CDR sequences) may be modified, e.g., replaced with the corresponding residues from the original murine, rodent, sub-human primate or other antibody. The humanized antibody can be achieved by various methods, including (a) grafting only the non-human CDRs onto human framework and constant regions, with or without retention of critical framework residues, or (b) transplanting these entire non-human variable domains, but "masking" them with human-like portions by replacing surface residues. Such methods useful for practicing the present invention include those disclosed in Padlan, Mol. Immunol., 31(3):169-217 (1994).

[0066] As used herein, the term "chimeric antibody" refers to a recombinant protein containing the variable domains of both the heavy and light antibody chains (including the complementarity determining regions (CDRs) derived from an antibody of one species (preferably a murine antibody or a chicken antibody, more preferably a murine antibody)), while the constant domains of these antibody molecules are derived from those of human antibodies.

[0067] As used herein, the term "treatment / treating" of a disease is a method for obtaining a beneficial or desired result (including a clinical result). For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of one or more symptoms caused by the disease, elimination of the extent of the disease, stabilization of the disease (e.g., preventing or delaying the worsening of the disease), prevention or delay of the spread of the disease (e.g., metastasis), prevention or delay of the recurrence of the disease, delay or slowing of the progression of the disease, improvement of the disease state, provision of remission (partial or complete) of the disease, reduction of the dose of one or more other drugs required to treat the disease, delay of the progression of the disease, increase or improvement of the quality of life, increase in weight gain and / or prolongation of survival. "Treatment" also encompasses a reduction in the pathological consequences (such as, e.g., tumor volume) of cancer. The methods provided herein cover any one or more of these embodiments of treatment.

[0068] As used herein, the term "administer / administration" refers to the act of injecting or otherwise physically delivering a substance (such as a formulation of the present invention) to a patient when it is present in vitro, such as by mucosal, intradermal, intravenous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When treating a disease or its symptoms, the substance is typically administered after the onset of the disease or its symptoms. When preventing a disease or its symptoms, the substance is typically administered before the onset of the disease or its symptoms.

[0069] As used interchangeably herein, the terms "individual", "subject", "host" and "patient" refer to a mammal, including (but not limited to) murine (rats, mice), non-human primates, humans, dogs, cats, ungulates (such as horses, cows, sheep, pigs, goats), etc.

[0070] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of an anti-EphA2 antibody in an individual that is sufficient to effect such treatment of a disease upon administration to a mammal or other individual.

[0071] The development of next-generation antibody drugs is a remarkable trend in current cancer treatment. The present invention aims to develop anti-EphA2 antibodies and construct antibody-drug conjugates (ADCs) against EphA2-related cancers; particularly pancreatic cancer. Targeting tumor-specific antigen (EphA2) with an antibody to inhibit cancer cell growth and induce endocytosis, and conjugating the antibody with an anti-tumor compound (such as the small molecule monomethyl auristatin E (MMAE)) will result in more effective tumor cytotoxicity.

[0072] The present invention establishes anti-EphA2 antibodies, specifically single-chain antibody fragments (scFv) and humanized antibodies, which have the ability to bind to EphA2 and inhibit angiogenesis and cancer cell growth.

[0073] In another embodiment, the present invention provides a isolated anti-EphA2 antibody or antigen-binding portion thereof, comprising at least one of the following: a light chain complementarity determining region 1 (L-CDR1) comprising the amino acid residues of SEQ ID NO:1 or a variant having an amino acid sequence with at least 80% identity to any of SEQ ID NO:1; a light chain complementarity determining region 2 (L-CDR2) comprising the amino acid residues of SEQ ID NO:2 or a variant having an amino acid sequence with at least 80% identity to any of SEQ ID NO:2; and a light chain complementarity determining region 3 (L-CDR3) comprising the amino acid residues of SEQ ID NO:3 or a variant having an amino acid sequence with at least 80% identity to any of SEQ ID NO:3; and at least one of the following: a heavy chain complementarity determining region 1 (H-CDR1) comprising the amino acid residues of SEQ ID NO:4 or a variant having an amino acid sequence with at least 80% identity to any of SEQ ID NO:4; a heavy chain complementarity determining region 2 (H-CDR2) comprising the amino acid residues of SEQ ID NO:5 or a variant having an amino acid sequence with at least 80% identity to any of SEQ ID NO:5; and a heavy chain complementarity determining region 3 (H-CDR3) comprising the amino acid residues of SEQ ID NO:6 or a variant having an amino acid sequence with at least 80% identity to any of SEQ ID NO:6; such that the isolated antibody or antigen-binding portion thereof binds to EphA2. Preferably, the sequence identity as mentioned above is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0074] The amino acid sequences of the complementarity determining regions in the light and heavy chains are listed below.

[0075] CDR of the light chain L-CDR1 L-CDR2 L-CDR3 SGSYG (SEQ ID NO:1) DND (SEQ ID NO:2) GSADSSTYVGM (SEQ ID NO:3)

[0076] CDR of the heavy chain

[0077] In some embodiments, the isolated anti-EphA2 antibody is a monoclonal antibody, chimeric antibody, humanized antibody or human antibody. In some embodiments, the isolated anti-EphA2 antibody is a single-chain antibody (such as Fv (scFv), IgG, Fab, (Fab)2 or (scFv')2).

[0078] According to the present invention, examples of the amino acids of the light and heavy chains of the antibodies of the present invention are listed below.

[0079] In some embodiments, the present invention provides a light chain comprising an amino acid sequence comprising SEQ ID NO:7 or 8.

[0080] In some embodiments, the present invention provides a heavy chain comprising an amino acid sequence comprising SEQ ID NO:9 or 10.

[0081] In other embodiments, the present invention provides a separated antibody (scFv SD5) which comprises a light chain having an amino acid sequence shown by the sequence comprising SEQ ID NO:7 or a variant having at least 80% identity with SEQ ID NO:7, and a heavy chain having an amino acid sequence shown by the sequence comprising SEQ ID NO:9 or a variant having at least 80% identity with SEQ ID NO:9. Preferably, the sequence identity mentioned above is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0082] In another embodiment, the present invention provides a humanized antibody (humanized scFv hSD5) which comprises a light chain having an amino acid sequence shown by SEQ ID NO:8 or a variant having at least 80% identity with SEQ ID NO:8 and a heavy chain having an amino acid sequence shown by SEQ ID NO:10 or a variant having at least 80% identity with SEQ ID NO:10.

[0083] In another embodiment, the present invention provides a separated antibody (scFv SD5) which comprises the following sequence: The linker in the scFv can be any linker known in the art. In some embodiments, the linker has a sequence of less than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In some embodiments, the linker comprises the amino acids of a glycine-serine (GS) linker, RGRGRGRGRSRGGGS or GQSSRSS.

[0084] In another embodiment, the present invention provides a separated antibody (scFv SD5) which comprises the amino acid sequence shown by SEQ ID NO:11 or a variant having at least 80% identity with SEQ ID NO:11. In another embodiment, the present invention includes a humanized antibody which comprises the amino acid sequence shown by SEQ ID NO:12 or a variant having at least 80% identity with SEQ ID NO:12. scFv SD5 (SEQ ID NO:11) ( GQSSRSS is the linker sequence ) Humanized scFv hSD5 (SEQ ID NO:12)

[0085] Techniques well known in the art for preparing monoclonal antibodies substantially against any target antigen are used. See, for example, Kohler and Milstein, Nature 256:495 (1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, Volume 1, pages 2.5.1 to 2.6.7 (John Wiley & Sons 1991). Briefly, monoclonal antibodies can be obtained by injecting a composition containing the antigen into a mouse or chicken, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting a positive clone that produces an antibody against the antigen, culturing the clone that produces an antibody against the antigen, and isolating the antibody from these hybridoma cultures.

[0086] Various techniques, such as generating chimeric or humanized antibodies, may involve procedures for antibody cloning and construction. The antigen-binding variable light and variable heavy chain sequences of the antibody of interest can be obtained by various molecular cloning procedures, such as RT-PCR, 5'-RACE, and cDNA library screening. The variable heavy or light chain sequence genes of an antibody from cells expressing a murine antibody can be cloned and sequenced by PCR amplification. To confirm their authenticity, these cloned VL and VH genes can be expressed as chimeric antibodies in cell culture, as described by Orlandi et al. (Proc. Natl. Acad. Sci., USA, 86:3833 (1989)). Based on the variable heavy or light chain gene sequences, humanized antibodies can be designed and constructed, as described by Leung et al. (Mol. Immunol., 32:1413 (1995)).

[0087] Chimeric antibodies are recombinant proteins in which the variable regions of human antibodies have been replaced by, for example, the variable regions of murine antibodies, including the complementarity-determining regions (CDRs) of murine antibodies. Chimeric antibodies exhibit reduced immunogenicity and increased stability when administered to an individual. Methods for constructing chimeric antibodies are well known in the art (e.g., Leung et al., 1994, Hybridoma 13:469).

[0088] Chimeric monoclonal antibodies can be humanized by transferring the chicken CDRs from the heavy and light variable chains of chicken immunoglobulins to the corresponding variable domains of human antibodies. The chicken framework regions (FRs) in the chimeric monoclonal antibodies are also replaced with human FR sequences. To maintain the stability and antigen specificity of the humanized monoclonal, one or more human FR residues can be replaced with the corresponding murine residues. The humanized monoclonal antibodies can be used for therapeutic treatment of individuals. Techniques for generating humanized monoclonal antibodies are well known in the art (see, for example, Jones et al., 1986, Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen et al., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad. Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest et al., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993, 150:2844).

[0089] The anti-EphA2 antibodies and antibody fragments can be generated in vitro from immunoglobulin variable domain (V) gene repertoires of unimmunized donors using phage display technology. According to this technique, antibody V domain genes are cloned in-frame into the major or minor coat protein gene of a filamentous phage such as M13 or fd, and functional antibody fragments are displayed on the surface of the phage particles. Since the filamentous particles contain a single-stranded DNA copy of the phage genome, genes encoding antibodies that display those properties can also be selected based on the functional properties of the antibodies. Thus, phage mimics some of the properties of B cells. Phage display can be carried out in various forms, as reviewed, for example, in Johnson, Kevin S. and Chiswell, David J., Curr. Opin Struct. Biol. 3:564-571 (1993). V-gene fragments from several sources can be used for phage display. Clackson et al., Nature 352:624-628 (1991) isolated various anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. In other embodiments, ribosome display technology can be used to generate anti-EphA2 antibodies and in vitro antibody fragments.

[0090] A variety of techniques have been developed for generating antibody fragments. Traditionally, these fragments have been derived via proteolytic digestion of intact antibodies. However, these fragments can now be produced directly in recombinant host cells, for example, using the nucleic acids encoding anti-EphA2 antibodies of the present invention. Fab, Fv, and scFv antibody fragments can all be expressed and secreted from Escherichia coli, thus allowing the direct production of large quantities of these fragments. Anti-EphA2 antibody fragments can also be isolated from antibody phage libraries as discussed above. Alternatively, Fab'-SH fragments can be directly recovered from Escherichia coli and chemically conjugated to form F(ab')2 fragments. According to another method, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. The production of Fab and F(ab')2 antibody fragments with increased in vivo half-life is described in US 5,869,046. In other embodiments, the selected antibody is a single-chain Fv fragment (scFv).

[0091] The nucleic acids encoding the polypeptides described herein can be modified without eliminating their biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those skilled in the art and include, for example, termination codons, addition of a methionine at the amino terminus to provide initiation, sites, additional amino acids placed at either terminus to create well-known restriction sites, or additional amino acids that facilitate purification steps (such as poly His). In addition to recombinant methods, the antibodies of the present invention can also be constructed in whole or in part using standard peptide synthesis well known in the art.

[0092] As a modification to the purification protocol for diabodies, these heavy and light chain regions are separately solubilized and reduced and then combined in a refolding solution. Exemplary yields are obtained when the two proteins are mixed in a molar ratio such that no more than a 5-fold molar excess of one protein relative to the other is present. After completion of redox-shuffling, excess oxidized glutathione or other oxidized low molecular weight compounds can be added to the refolding solution.

[0093] In addition to recombinant methods, the antibodies and their variants disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid-phase synthesis of polypeptides can be achieved by attaching the C-terminal amino acid of the sequence to an insoluble support and then sequentially adding the remaining amino acids in the sequence. Techniques for solid-phase synthesis are described by Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Volume 2: Special Methods in Peptide Synthesis, Part A. Pages 3 to 284; Merrifield et al., J. Am. Chem. Soc. 85: 2149-2156, 1963, and Stewart et al., Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chem. Co., Rockford, Ill., 1984. Proteins of greater length can be synthesized by condensation of the amino and carboxyl termini of shorter fragments.

[0094] In preliminary data, the present invention has isolated a specific antibody SD5 targeting the structural active site of EphA2 through phage display technology. The results have confirmed that the antibody SD5 can significantly inhibit the growth and migration of cancer cells, leading to the molecular degradation of EphA2 and inducing the endocytosis of targeted cells. Therefore, the antibody has the potential and value for development into an ADC. In preliminary experiments, the humanized antibody incorporating MMAE, designated hSD5-ADC, showed excellent tumor-killing effects in vitro and in vivo, inducing apoptosis of cancer cells. These experimental results demonstrate and support the value of continued development of hSD5-ADC. In addition, the present invention has verified the therapeutic effect of the ADC through whole-cell and animal experiments. It is believed that the ADC drug can provide a more effective and comprehensive therapeutic effect against EphA2-related cancers (especially pancreatic cancer and even other EphA2-related cancers).

[0095] The present invention also provides an ADC comprising an anti-EphA2 antibody or an antigen-binding portion thereof of the present invention and a drug-linker structure comprising an anti-tumor compound linked to the antibody via a linker.

[0096] The anti-EphA2 antibody or an antigen-binding portion thereof of the present invention can be bound to an anti-tumor compound via a linker structure portion to prepare an anti-EphA2 antibody-drug conjugate. The anti-tumor compound is not particularly limited as long as it has a substituent or partial structure that can be linked to the linker structure.

[0097] Examples of such anti-tumor compounds include, but are not limited to, auristatins such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), vincristine, vinblastine, methotrexate, platinum-based anti-tumor agents (cisplatin and its derivatives), doxorubicin, calicheamicin, dolastatin 10, maytansinoids, pyrrolobenzodiazepine dimers, camptothecin derivatives, duocarmycin, ptericin, daunomycin, mitomycin C, bleomycin, cytarabine, and paclitaxel and its derivatives.

[0098] In the ADCs of the present application, there is no particular limitation on the linker structure that binds the anti-EphA2 antibody to the drug, as long as the ADC can be used. The linker structure can be appropriately selected and used according to the purpose of use.

[0099] Also provided are pharmaceutical compositions comprising the anti-EphA2 antibody or ADC of the present invention.

[0100] Certain embodiments relate to pharmaceutical compositions comprising the anti-EphA2 antibody or ADC of the present invention and a pharmaceutically acceptable carrier or excipient. The term "pharmaceutically acceptable carrier" is intended to include, but is not limited to, any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid known to those skilled in the art. Diluents such as polyols, polyethylene glycols and dextrans can be used to increase the biological half-life of the conjugate.

[0101] The pharmaceutical compositions of the present invention can be formulated according to known methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A.), and can also contain pharmaceutically acceptable carriers and additives. Examples include, but are not limited to, surfactants, excipients, colorants, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flow promoters and flavoring agents, and other commonly used carriers can be appropriately used. Specific examples of carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannose, starch, cross-linked carboxymethyl cellulose calcium, cross-linked carboxymethyl cellulose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc.

[0102] One embodiment relates to a method for treating or preventing EphA2-related cancer in an individual, which comprises administering to the individual an anti-EphA2 antibody or ADC of the present invention. Alternatively, one embodiment relates to the use of an anti-EphA2 antibody or ADC of the present invention in the manufacture of a medicament for treating or preventing an angiogenesis disorder in an individual.

[0103] Another embodiment relates to a method for inhibiting the growth of EphA2-related cancer cells or the metastasis of EphA2-related cancer in an individual, which comprises administering to the individual an anti-EphA2 antibody or ADC of the present invention. Alternatively, another embodiment relates to the use of an anti-EphA2 antibody or ADC of the present invention in the manufacture of a medicament for inhibiting the growth of EphA2-related cancer cells or the metastasis of EphA2-related cancer in an individual.

[0104] In some embodiments, the EphA2-related cancer is selected from cholangiocarcinoma, bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioma, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, stomach cancer, thymic cancer, and vulvar cancer.

[0105] The method also comprises administering the anti-EphA2 antibody or ADC of the present invention simultaneously with or after other standard therapies, wherein the standard therapies are selected from the group consisting of radiotherapy, surgery, and chemotherapy.

[0106] The anti-EphA2 antibody, ADC, or a pharmaceutical composition thereof can be administered intravenously, intraperitoneally, intraarterially, intrathecally, intravesically, or intratumorally. Those of ordinary skill in the art will appreciate that the effective amount of the anti-EphA2 antibody can be determined empirically. It should be understood that when administered to a human patient, the total daily dosage of the anti-EphA2 antibody or composition will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dosage for any particular patient will depend on various factors: the type and extent of the cellular response to be achieved; the activity of the particular anti-EphA2 antibody, ADC, or composition employed; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and excretion rate of the anti-EphA2 antibody; the duration of the treatment; drugs used in combination with or in conjunction with the anti-EphA2 antibody, ADC, or composition; and similar factors well known in the medical arts.

[0107] Each of the compositions and treatment methods identified above may additionally include another anti-tumor drug and administration of one or more other anti-tumor drugs. Anti-tumor drugs suitable for use with the present invention include (but are not limited to) agents that induce apoptosis, agents that inhibit adenosine deaminase function, agents that inhibit pyrimidine biosynthesis, agents that inhibit purine ring biosynthesis, agents that inhibit nucleotide interconversion, agents that inhibit ribonucleotide reductase, agents that inhibit thymidine monophosphate (TMP) synthesis, agents that inhibit dihydrofolate reduction, agents that inhibit DNA synthesis, agents that form adducts with DNA, agents that damage DNA, agents that inhibit DNA repair, agents that intercalate into DNA, agents that deaminate asparagine, agents that inhibit RNA synthesis, agents that inhibit protein synthesis or stability, agents that inhibit microtubule synthesis or function, and the like. Examples of additional anti-tumor drugs include (but are not limited to) 1) alkaloids, including microtubule inhibitors (such as vincristine, vinblastine, vindesine, etc.), microtubule stabilizers (such as paclitaxel (TAXOL), docetaxel, etc.), and chromatin function inhibitors, including topoisomerase inhibitors, such as epipodophyllotoxin (such as etoposide (VP-16), teniposide (VM-26), etc.), and agents that target topoisomerase I (such as camptothecin, irinotecan (CPT-11), etc.); 2) covalent DNA-binding agents (alkylating agents), including nitrogen mustards (such as mechlorethamine, chlorambucil, cyclophosphamide, ifosphamide, busulfan (MYLERAN), etc.), nitrosoureas (such as carmustine, lomustine, semustine, etc.), and other alkylating agents (such as temozolomide, dacarbazine, hydroxymethylmelamine, thiotepa, mitomycin, etc.);3) Monovalent DNA-binding agents (antitumor antibiotics), including nucleic acid inhibitors (such as actinomycin (actinomycin D), etc.), anthracyclines (such as daunomycin (daunomycin), doxorubicin, idarubicin (idarubicin)), etc.), anthraquinones (such as anthracycline analogs, such as mitoxantrone, etc.), bleomycin (BLENOXANE), etc., and plicamycin (mithramycin), etc.; 4) Antimetabolites, including antifolates (such as methotrexate, FOLEX, MEXATE, etc.), purine antimetabolites (such as 6-mercaptopurine (6-MP, PURINETHOL), 6-thioguanine (6-TG), azathioprine, acyclovir, ganciclovir, chlorodeoxyadenosine, 2-chlorodeoxyadenosine (CdA), and 2'-deoxycoformycin (pentostatin), etc.), pyrimidine antagonists (such as fluoropyrimidines (such as 5-fluorouracil (ADRUCIL), 5-fluorodeoxyuridine (FdUrd)), etc.), and cytosine arabinoside (such as CYTOSAR (ara-C), fludarabine, etc.); 5) Enzymes, including L-asparaginase and hydroxyurea, etc.; 6) Hormones, including glucocorticoids, antiestrogens (such as tamoxifen, etc.), non-steroidal antiandrogens (such as flutamide, etc.), and aromatase inhibitors (such as anastrozole (ARIMIDEX), etc.); 7) Platinum compounds (such as cisplatin and carboplatin, etc.); 8) Monoclonal antibodies conjugated with anticancer drugs, toxins, and / or radionuclides, etc.; 9) Biological response modifiers (such as interferons (such as IFN-α, etc.) and interleukins (such as IL-2, etc.), etc.); 10) Adoptive immunotherapy; 11) Hematopoietic growth factors; 12) Agents that induce tumor cell differentiation (such as all-trans retinoic acid, etc.); 13) Gene therapy techniques; 14) Antisense therapy techniques; 15) Tumor vaccines; 16) Therapies targeting tumor metastasis (such as batimastat, etc.); 17) Angiogenesis inhibitors; 18) Proteasome inhibitors (such as VELCADE); 19) Inhibitors of acetylation and / or methylation (such as HDAC inhibitors); 20) Modulators of NFκB; 21) Inhibitors of cell cycle regulation (such as CDK inhibitors); and 22) Modulators of p53 protein function.

[0108] The present invention also shows an association between EphA2 levels and cancer severity; thus, a high level of expression of EphA2 or a fragment thereof in a biological sample, as compared to the reference expression level of EphA2 or a fragment thereof in a control sample, indicates a predicted metastasis or poor prognosis. The present invention unexpectedly finds that the anti-EphA2 antibody of the present invention can be used as an indicator for diagnosing or predicting the prognosis, metastasis, or increased risk of future cancer occurrence in an individual. Accordingly, the present invention provides a method for detecting or diagnosing a high risk of cancer or future occurrence of cancer, or predicting metastasis or prognosis of cancer in an individual, which comprises contacting a biological sample from the individual with the anti-EphA2 antibody of the present invention, quantifying the binding of the EphA2 antigen to the antibody in the sample, and comparing the binding with a reference value representing the binding between the anti-EphA2 antibody and the EphA2 antigen measured in a sample from a control individual not suffering from cancer.

[0109] In one embodiment, the biological sample can be cells, tissues, organs, organ samples, tissue biopsies, blood, plasma, serum, ascetic fluid, lymphocytes, urine, bone marrow fluid, lymph fluid, saliva, lachrymal fluid, mucosal fluid, amniotic fluid, or a combination thereof.

[0110] Detectable labels suitable for binding to antibodies and other binding reagents include radioisotopes, fluorescent labels, enzyme-substrate labels, chromogenic labels, chemiluminescent labels, and colloidal gold particles.

[0111] Examples of measurement methods include (but are not limited to) fluorescence immunoassay (FIA) methods, enzyme immunoassay (EIA) methods, radioimmunoassay (RIA) methods, Western blotting methods, dot blot, immunohistochemical assays, fluorescence-activated cell sorting (FACS), in vivo imaging, and radioimaging assays.

[0112] The present invention further provides a method for monitoring the progression of cancer in an individual already diagnosed with cancer. In some embodiments, the monitoring can be used to evaluate whether a particular treatment is successful.

[0113] In some embodiments, monitoring cancer progression includes determining a first level of EphA2 or a fragment thereof in a first biological sample obtained from an individual diagnosed with cancer by an anti-EphA2 antibody of the present invention; and determining a second level of EphA2 or a fragment thereof in a second biological sample obtained from the individual by the anti-EphA2 antibody of the present invention after a predetermined time period; comparing the first and second levels of EphA2 or a fragment thereof; wherein a higher level of EphA2 or a fragment thereof in the second sample compared to the first sample indicates disease progression and deterioration. Similarly, a decrease in the level of EphA2 or a fragment thereof in the second sample compared to the first sample indicates improvement.

[0114] The diagnostic method of the present invention can be combined with known methods for diagnosing cancer.

[0115] Another embodiment of the present invention encompasses a kit for detecting or diagnosing cancer or a high risk of future occurrence of cancer, or predicting metastasis or prognosis of cancer, or monitoring cancer progression in an individual. It is usually in a package containing all the elements (optionally including instructions for use). The package can be segmented so that the components are not mixed until needed. The individual components can be separately packaged within the kit. The kit can contain reagents necessary for detecting the expression level of a marker gene. A kit is any article (such as a package or container) that contains at least one reagent, such as an antibody reagent, for specifically detecting and / or measuring the expression level of a marker gene in a sample.

[0116] The present invention encompasses various kits with different components. Generally, the kit will include components for quantifying EphA2 or more biomarkers in an individual. In another embodiment, the kit will include components for collecting biological samples, components for quantifying EphA2 or more biomarkers in the biological samples, and instructions for use of the kit contents. In certain embodiments, the kit includes components for quantifying the amount of a biomarker. In other embodiments, the components for quantifying the amount of a biomarker include reagents necessary for detecting the amount of the biomarker.

[0117] Numerous embodiments of the present invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the present invention. Therefore, the following examples are intended to illustrate but not limit the scope of the present invention as described in the claims. Examples

[0118] Materials and Methods

[0119] Cell Culture and Animal Immunization

[0120] Human PAAD cell lines AsPc-1, BxPc-3, Panc-1, and Mia PaCa-2 were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). All cell lines were cultured according to the ATCC standard protocol and cultured at 37°C in a humidified 5% CO2 atmosphere. Female white leghorn (Gallus domesticus) and non-obese diabetic mice with severe combined immunodeficiency (NOD / SCID) were purchased from the Laboratory Animal Center (Taiwan, China), and were housed in the animal facility of Taipei Medical University.

[0121] Bioinformatics analysis

[0122] To investigate the association between EphA2 gene expression and the development of PAAD, we used the Gene Expression Profiling Interactive Analysis (GEPIA; http: / / gepia.cancer-pku.cn) database to analyze the differences in EphA2 expression in clinical cancer samples and normal samples and to analyze the correlation between EphA2 expression and PAAD survival rate (Nucleic Acids, Res 45(W1):W98-W102 (2017)).

[0123] Antibody library construction and biopanning

[0124] Based on the crystal structure of the Aprin A1–EphA2 complex (Protein Data Bank [PDB] ID: 3CZU), we designed a peptide immunogen for the EphA2 target molecule; the peptide immunogen (EphA2pep) contains the epitope 1GWDLMQNIMNDMPIYMYSV and the epitope 2VSSDFEARHV, which are linked together by using the linker GGGGGGS. EphA2pep contains six consecutive repeats. After gene synthesis (GENEWIZ), it was constructed on the pET21a vector (Novagen) and transformed into the Escherichia coli BL-21(DE3) strain to express it as a recombinant protein. After purification by using Ni2+-charged agarose gel (GE Healthcare Life Sciences), the recombinant EphA2pep protein was used for animal immunization. Female White Leghorn chickens were immunized by intramuscular injection of 50 μg of the recombinant EphA2pep protein mixed with adjuvant each time. During immunization, we used Freund’s complete adjuvant (Sigma-Aldrich) for the first time and Freund’s incomplete adjuvant (Sigma Aldrich) at all other times. The immunization schedule included four immunizations at 7-day intervals. Chicken spleens were harvested 7 days after the final immunization to construct a scFv antibody library. The library was constructed according to a published protocol with minor modifications (J Immunol Methods., 242(1-2):159-181(2000)).

[0125] For panning, the recombinant EphA2 protein was pre-coated onto the wells of a microtiter plate at 4 °C overnight. The next day, the EphA2 protein was removed, and the wells were blocked with 3% BSA for 1 h at room temperature. Then, the recombinant library phage solution (1011 phage particles) was added to the wells and incubated for 2 h at room temperature. Unbound phages in the supernatant were removed, and the wells were washed 10 times by pipetting phosphate-buffered saline with 0.05% Tween 20 (PBST). Subsequently, the bound phages were eluted with 0.1 M HCl–glycine (pH 2.2) / 0.1% BSA elution buffer and neutralized with 2 M Tris basic buffer. The eluted phages were immediately used to infect Escherichia coli ER2738 strain for recombinant phage amplification. The amplified phages were precipitated and recovered by a previous method (Proc Natl Acad Sci., 88(18):7978-7982 (1991)) and used in the next round of panning. The panning procedure was repeated four times to effectively enrich anti-EphA2 binding phages. After panning, total library DNA was purified and transformed into Escherichia coli strain TOP 10F’ (Invitrogen, a non-suppressor strain) for scFv expression. The expressed scFv was further purified using Ni2+-charged agarose gel according to the manufacturer's instructions (GE Healthcare LifeScience).

[0126] Sequence analysis

[0127] To sequence the scFv clones of interest, we used the ompseq primer (5’-AAGACAGCTATCGCGATTGCAGTG-3’) complementary to the outer membrane protein A (ompA) signal sequence upstream of the light chain variable region. Next, the website International ImMunoGeneTics information system / V-QUEry and Standardization (http: / / imgt.org) was used to compile and analyze the sequence data on the basis of germline genes.

[0128] Enzyme-linked immunosorbent assay

[0129] The wells of a microtiter plate were coated with the recombinant EphA2 protein (0.5 μg / well) overnight at 4 °C. These wells were blocked with 5% skim milk and then scFv or phage was added to these wells for 1 hour at room temperature. After washing these wells with PBST, the bound scFv or phage was then detected and developed using horseradish peroxidase (HRP)-conjugated goat anti-chicken light chain antibody (Bethyl Laboratories) or HRP-conjugated anti-M13 antibody (GE Healthcare Life Science). Finally, the substrate 3,5,5-tetramethylbenzidine dihydrochloride (TMB) was added for signal visualization. The reaction was stopped by adding 1 N HCl, and the absorbance was measured by determining the optical density (OD) at 450 nm.

[0130] Western blot and immunoprecipitation assays

[0131] After electrophoresis using sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) or native polyacrylamide gel (native-PAGE), the recombinant EphA2 protein was transferred to a nitrocellulose membrane (GE Healthcare Life Sciences), and the membranes were incubated with the purified scFv antibody to determine the binding reactivity. These membranes were blocked with 5% skim milk and then incubated with scFv for 1 hour at room temperature. After washing with PBST, the membranes were detected and developed using HRP-conjugated goat anti-chicken light chain antibody. Finally, 3,3'-diaminobenzidine substrate was added for color development until the desired color intensity was achieved.

[0132] For immunoprecipitation assays, 300 μg of each PAAD cell lysate was incubated overnight at 4 °C with 50 μg of anti-EphA2 scFv (scFv fused with His-tag). The next day, 30 μL of Ni2+-charged agarose gel was added to the mixture and incubated at 4 °C for 1 hour. After washing three times with NiNTA wash buffer (50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole, pH 8.0), the scFv-bound agarose gel beads were resuspended in 50 μL of PBS buffer. Subsequently, the agarose gel solution was denatured at 95 °C for 10 minutes and analyzed by SDS-PAGE. After transferring the proteins to a polyvinylidene difluoride membrane, the membrane was probed overnight at 4 °C with anti-EphA2 antibody (R&D Systems) and anti-His (Proteintech Group) antibody. The next day, after washing with PBST, the membrane was incubated with HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories). Finally, chemiluminescent substrate was added for luminal signal detection.

[0133] Flow cytometry analysis

[0134] Four PAAD cells, AsPc-1, BxPc-3, Panc-1, and Mia PaCa-2, with endogenous EphA2 molecule expression were analyzed by flow cytometry to determine the binding reactivity of the indicated scFv. Freshly prepared cancer cells were harvested and washed twice with PBS. Then, individual scFv was added and incubated at room temperature for 1 hour. The bound scFv was visualized using goat anti-chicken light chain antibody and donkey anti-goat antibody conjugated with fluorescein isothiocyanate (FITC; Jackson ImmunoResearch Laboratories). In the assay, irrelevant scFv was used as a negative control, and commercially available goat anti-EphA2 antibody was used as a positive control (R&D Systems). Results were analyzed using a FACS can flow cytometer (BD Biosciences, Systems and Reagents).

[0135] To detect the binding specificity of the scFv, 293T cells were transfected with EphA1-A8 plasmids individually to overexpress different EphA molecules. The cell lines were freshly prepared and washed with FACS buffer (2% FBS in PBS). These cells were seeded at 1×105 cells / well into 96-well U-bottom plates and incubated with the scFv at 4 °C for 1 hour. After washing the plates with FACS buffer, the anti-hemagglutinin (HA) antibody was added for scFv binding detection (scFv was fused with the HA tag) and then developed using a FITC-conjugated secondary antibody. Finally, cell binding signals were analyzed using a FACS can flow cytometer.

[0136] Cell proliferation assay

[0137] PAAD cell proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation assay kit (Promega). These cells were seeded at a density of 5000 cells / well in 96-well culture plates for attachment. Then, various concentrations of the scFv were added to the cell cultures and incubated for 5 days. Finally, MTS and phenazine methosulfate solutions were added and incubated for 90 minutes for development. After adding the SDS reagent to stop the reaction, the absorbance of each well was measured by determining the OD at 490 nm.

[0138] Cell migration and scratch wound healing assay

[0139] PAAD cells were seeded at 5×104 cells / well in 24-well Transwell cell migration plates (Corning) and incubated with the scFv for 2 days. In this assay, recombinant ephrin-A1 (Sino Biological) was added as a positive control. After scFv treatment, the cells were fixed with ice-cold 100% methanol for 10 minutes and stained with 0.01% crystal violet at room temperature for 1 hour. After washing the plates with ddH2O, the upper-layer cells were removed using a cotton swab. Cell staining was imaged using a microscope and analyzed using ImageJ. For the scratch wound healing assay, PAAD cells were seeded in 6-well culture plates. The seeded cells in the wells were scratched with a pipette tip to simulate a wound. After treatment with the scFv and incubation for 36 or 72 hours, the cells were imaged using a microscope. The wound area was analyzed using ImageJ.

[0140] For scratch wound healing assay, PAAD cells were seeded in 6-well culture plates. These seeded cells in the wells were scratched with a pipette tip to simulate a wound. After treatment with scFv and incubation for 36 or 72 hours, the cells were imaged using a microscope. The wound area was analyzed using ImageJ.

[0141] Molecular signaling

[0142] To determine molecular signaling in BxPc-3 and Mia PaCa-2 cells, these cells were cultured in 6-well culture plates and treated with scFv antibodies at the indicated concentrations for 24 hours. The cells were harvested and lysed using lysis buffer [50 mM Tris–HCl (pH 7.5), 50 mM NaCl, 5 mM ethylenediaminetetraacetic acid, and 1% Triton X-100], and then a protease inhibitor mixture (Roche Applied Science) was added. The protein concentration of the cell lysates was determined by Coomassie Plus (Bradford) protein assay (Thermo Fisher Scientific). Samples were run on reducing SDS-PAGE for Western blot analysis and detected using antibodies against p-EphA2, EphA2, p-AKT, AKT, p-ERK, ERK, p-FAK, FAK, pSTAT3, STAT3 (Cell Signaling Technology), and β-actin (GeneTex).

[0143] Antibody internalization assay

[0144] BxPc-3 cells were seeded on coverslips placed in the wells of 6-well culture plates. These BxPc-3 seeded slides were incubated with epratuzumab A1-Fc (Sino Biological) or IgG hSD5 at 37 °C or 4 °C for 1 hour. After washing with PBS, the cells were fixed with 100% ice-cold methanol for 10 minutes. Next, the cells were stained with FITC-conjugated anti-human Fc antibody and subsequently mounted using ProLong Diamond Antifade Mountant containing 4′,6-diamidino-2-phenylindole for nuclear counterstaining (Invitrogen). The cells were imaged using a confocal microscope (Leica Microsystems).

[0145] Tumor xenograft model

[0146] Freshly prepared BxPc-3 and Mia PaCa-2 cancer cells were harvested during the logarithmic growth phase and resuspended in PBS for tumor implantation. Subsequently, each NOD / SCID mouse was inoculated with cancer cells (5×10 6 for BxPc-3 and 1×10 7 for Mia PaCa-2) for tumor formation. Tumor size was measured twice a week, and the volume was calculated as follows: V = 0.5lw2, where l = length and w = width. When the tumor size was approximately 100 mm3, the animals were divided into groups receiving the following: (a) vehicle alone, by intravenous injection (i.v.), once a week (qwk); (b) control human IgG1, 20 mg / kg, by i.v. qwk; (c) and (d) hSD5 IgG1, 2 or 20 mg / kg, by i.v. qwk; (e) and (f) gemcitabine, 20 or 100 mg / kg, by i.v., twice a week (biw); or (g) a combination of hSD5 IgG1, 2 mg / kg, by i.v. qwk and gemcitabine, 20 mg / kg, by i.v. Biw. At the end of the experiment, these anti-tumor effects were quantified by expressing tumor growth inhibition (TGI; %) by dividing the tumor volume in the treatment group by the tumor volume in the control group and multiplying by 100. The mice were also frequently examined for obvious signs of adverse drug-related side effects.

[0147] Immunohistochemical staining

[0148] Pancreatic tissue microarray slides (USBiomax, PA483e) were used to detect and analyze EphA2 expression in clinical samples. In the xenograft animal model, excised BxPc-3 and Mia PaCa-2 tumors were fixed in formalin, embedded in paraffin, and sectioned for immunohistochemical staining (IHC). Commercial antibodies (Cell signaling, Dako; Agilent Technologies; and Abcam) were used to stain the EphA2 molecule, the cell proliferation marker Ki-67, and the apoptosis marker cleaved caspase 3. The stained effects were observed using a Zeiss Axioskop-2 microscope (Carl Zeiss).

[0149] Interaction residue definition

[0150] We used peptide enzyme-linked immunosorbent assay (ELISA) to identify the engineered epitopes on EphA2; these BSA-conjugated peptides EphA2pep_P1 (BSA-CGGGWDLM QNIMNDMPIYMYSV) and EphA2pep_P2 (BSA-CGGGGGGSVSSDFEARHV) represent two fragments of the linear epitopes engineered on EphA2, respectively. EphA2pep_P1P2 (BSA-CGGGWDLMQNIMNDMPIYMYSVGGGGGGSVSSDFEARHV) represents the two linear epitope sequences linked by a linker. These peptides were synthesized and conjugated to BSA (Kelowna International Scientific). The BSA-conjugated peptides were individually coated onto 96-well microplates overnight at 4°C. After blocking the wells with 3% BSA, IgG hSD5 was added and incubated. Next, an HRP-conjugated anti-HA tag antibody (Cell Signaling Technology) was used to detect the bound scFv. Finally, TMB substrate was added for development, and the reaction was stopped by adding 1N HCl. Absorbance was measured by determining the OD at 450 nm. A dot blot assay was used to detect the binding of IgG hSD5 to the synthesized peptides; 1 mL of individual peptides (1 mg / mL; in triplicate) was dropped onto the NC membrane and allowed to fully absorb at RT. Blocking with 3% BSA was carried out for 1 hour at RT. Then, IgG hSD5 (10 μg / mL) was added, and the reaction was allowed to proceed for 1 hour at RT. After washing the membrane, the HRP-conjugated anti-HA tag antibody was added, and the reaction was allowed to proceed for 1 hour at RT. Finally, DAB was used to initiate a color reaction.

[0151] Statistical analysis

[0152] Data are represented as mean ± standard error of the mean and analyzed using GraphPad Prism (GraphPad Software). One-way analysis of variance was used for statistical comparison between groups, followed by a post hoc Tukey’s honest significant difference test. A P value of less than 0.05 was considered significant.

[0153] Example 1. The GEPIA database was used to analyze cancer types with high EphA2 gene expression. Cancer samples are red dots, and normal samples are green dots

[0154] The GEPIA database was used to analyze cancer types with high expression of the EphA2 gene, including cervical squamous cell carcinoma, colon adenocarcinoma, glioblastoma, ovarian serous cystadenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, and thymoma. These red dots represent cancer samples, and these green dots represent normal samples. (Figure 1)

[0155] It has been found that the cancer types with high expression of the EpHA2 gene are cervical squamous cell carcinoma, colon adenocarcinoma, glioblastoma, ovarian serous cystadenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, and thymoma. The organs involved in the digestive tract are the stomach, pancreas, and colorectum, indicating the correlation between the expression of EphA2 and the digestive organs.

[0156] Example 2. Analysis and evaluation of the correlation between EphA2 expression and pancreatic cancer (PAAD)

[0157] The GEPIA database was used to analyze the difference in EphA2 gene expression in pancreatic cancer samples (red bars) and normal samples (gray bars). (Figure 2A). The red bars represent cancer samples (n = 179), and the gray bars represent normal samples (n = 171). A significant increase in the gene expression of EphA2 was found in PAAD samples.

[0158] Figure 2B illustrates the correlation between EphA2 gene expression and survival in pancreatic cancer patients. Through the analysis of survival correlation, it was found that patients with high EphA2 gene expression (red line) had a lower survival rate than those with low EphA2 gene expression (blue line). (Figure 2B)

[0159] To analyze the expression of the EphA2 molecule, four PAAD cell lines, AsPc-1, BxPc-3, Panc-1, and Mia PaCa-2, and a normal pancreatic endothelial cell line, hTERT-HPNE, were used by Western blotting to compare the expression of EphA2 in cancer cells. (Figure 2C) All four PAAD cell lines showed higher EphA2 protein expression than that of the control hTERT-HPNE cell line.

[0160] IHC staining using tissue arrays was performed to analyze the expression of EphA2 molecules in clinical pancreatic cancer tissues and normal pancreatic tissues (Figure 2D). It was found that EphA2 levels were higher in the cell membranes of clinical PAAD tissue specimens than in normal pancreatic tissues. In the specimens stained in Figure 2D, tissue microarray slides containing 38 pancreatic cancer tissues and 8 normal pancreatic tissues were used for IHC analysis. Among the 38 cases, 37 were adenocarcinomas with high, medium, and low levels of differentiation in terms of histopathology. Only one case was classified as squamous cell carcinoma. Careful microscopic studies by pathologists showed that EphA2 molecules in 34 of the 37 adenocarcinomas were strongly stained in a diffuse cytoplasmic staining pattern, while the squamous cell carcinoma was completely negative; local staining was observed in the acinus region but not in the ductal compartments of 5 out of 8 normal pancreatic tissues. In two of the three adenocarcinomas with low levels of differentiation, a diffuse, cytoplasmic, and local and typical membrane staining pattern was observed, indicating that EphA2 is a membrane biomarker that is often recognized in cancer cells with extremely high histopathological grades.

[0161] These results indicate that the EphA2 biomarker may be cancer-specific and applicable to IHC staining for the diagnosis and prognostic purposes of pancreatic adenocarcinoma; it exhibits a characteristic cytoplasmic staining pattern in most cancer cases. For poorly differentiated adenocarcinoma of the pancreas, EphA2 tends to translocate to the cell surface or cell membrane of high-grade cancer cells.

[0162] Example 3. Characterization of anti-EphA2 scFv isolated using phage display technology.

[0163] To enable these isolated antibodies to recognize the activation site on EphA2, discontinuous fragments were designed as immunogens based on the published EphA2 complex structure. Based on the X-ray crystal structure with PDB id: 3CZU, the design of discontinuous antigenic peptides in the active site of EphA2 was carried out. Aprillin-A1 is in gray, EphA2 is in blue-green, and the red part is two antigenic peptides (epitope 1 and epitope 2) located on EphA2. EphA2pep was designed by linking the two antigenic peptides. (Figure 3A) As shown in Figure 3A, the structure of the complex protein (PDB ID: 3CZU) formed by EphA2 (blue-green) and Aprillin-A1 (gray) was analyzed. The binding site of Aprillin-A1 was amplified to within the range and determine the amino acids in the EphA2 molecule that can lead to interactions. Based on this information, a peptide sequence with discontinuous fragments (red: epitope 1 and epitope 2) is established, and a linker sequence (GGGGGGGS) is used to connect EphA2pep: GWDLMQNMNDMPIYMYSVGGGGGGSVSSDFEARHV. After expressing the 6-repeat peptide sequence as a recombinant protein, chickens are then immunized. Subsequently, a highly complex library of scFv antibodies and isolated specific antibodies is constructed using phage display technology. The number of bound phages is found to increase by approximately 60-fold between the first and the last rounds (data not shown), indicating that specific binding strains are enriched by panning.

[0164] Use phage ELISA to test the amplified phage antibody library after each round of panning, where "naïve" represents the original antibody library and "M13" represents wild-type phage. (Figure 3B) As shown in Figure 3B, in phage ELISA, the amplified phage library obtained after the first round of panning already contains enriched specific strains, which exhibit a significant binding reaction to EphA2 compared to the initial antibody library (naïve) and wild-type M13 phage.

[0165] Four representative scFvs are serially diluted to test the percentage of binding to the EphA2 molecule. (Figure 3C) After gene sequencing, we serially diluted four isolated representative scFvs to test the binding reaction with EphA2. The EC50 values of scFv SA1 and SD5 are similar at 1.7 and 1.8 nM, and the EC50 values of scFv SG3 and SH2 are 33.6 and 94 nM, respectively; the difference in binding ability is due to the interaction of the CDR sequences of different antibodies on the EphA2 molecule.

[0166] Use the scFv SA1 and SD5 to identify EphA2 under reducing (SDS-PAGE) and non-reducing (native-PAGE) conditions. Ctrl is the result detected using a commercial Ab. (Figure 3D) The scFv SA1 and SD5 with the best EC50 values are selected for subsequent tests. By using NativePAGE (red arrow), it is determined that scFv SA1 and SD5 can recognize EphA2 in its native form but not in its denatured form, indicating that the epitopes recognized by the scFv are conformational epitopes.

[0167] The endogenous EphA2 molecules on four pancreatic cancer cell lines were identified by flow cytometry analysis using the scFv SA1 and SD5. PC used a commercial anti-EphA2 antibody, and NC was a control group without added antibody. (Figure 3E) These results indicate that these scFvs exhibit significant binding responses to the EphA2 molecules on cancer cells.

[0168] To further explore the ability to bind to endogenous EphA2 molecules, the native EphA2 molecules in the lysates of four pancreatic cancer cell lines were pulled down by immunoprecipitation assay using the scFv SA1 and SD5; NC was an irrelevant scFv without reaction. (Figure 3F) Compared with the irrelevant scFv in the control group (which does not bind to EphA2), both scFv SA1 and SD5 captured the free EphA2 molecules from the lysates of the four strains of PAAD cells, and the binding effect of scFv SD5 was better than that of SA1.

[0169] Example 4. Inhibitory effects of isolated scFv on the proliferation and migration of PAAD cells.

[0170] As shown in Figures 4A to 4B, a cell viability analysis assay (MTS assay) was performed to observe the growth inhibitory effects of scFv SA1 and SD5 on four PAAD cell lines by adding different concentrations of the antibody to cell cultures. At the specified concentrations, scFv SA1 and SD5 interacted with cancer cells for 5 days, and the effects on the growth of cancer cells were observed. By the 5th day of the antibody reaction, it was found that scFv SA1 exhibited an inhibitory effect of approximately 18% to 24% on the cancer cell lines AsPc-1 and BxPc-3. However, at a concentration of 20 mM, scFv SD5 inhibited the growth of three cell lines, AsPc-1, Panc-1, and Mia PaCa-2, by approximately 80%. In addition, it inhibited the growth of the cell line BxPc-3 by 58.5%. A dose-dependent response was observed at different antibody concentrations; the results indicate that the binding of scFv SD5 to EphA2 on the surface of PAAD cells can inhibit the growth of cancer cells.

[0171] Since it has been confirmed that the molecular regulation of EphA2 promotes the migration of cancer cells, both transwell migration assay and wound healing assay were used to determine whether scFv SA1 and SD5 inhibit the migration of PAAD cells. The results of the transwell migration analysis are presented in Figure 5A. The experimental responses of these cancer cells, BxPc-3 and Mia PaCa-2, showed that after treatment with scFv SA1 and SD5 at a dose of 20 mM for 2 days, the number of migrating cancer cells was effectively reduced. The same conditions of epirubicin-A1 treatment were given as the control group in the experiment. (Figure 5A)

[0172] The quantitative percentages illustrate the migration inhibitory effects of the scFv on four pancreatic cancer cells in the transwell migration assay. The experimental results of scFv SD5 are better than those of scFv SA1. The scFv SD5 can inhibit the migration of cancer cell lines Panc-1 and BxPc-3 by 65% and 91%, respectively. (Figure 5B)

[0173] The results of the wound healing assay are presented in Figure 5C. Based on the experimental responses of cancer cells BxPc-3 and Mia PaCa-2, the treatments of scFv SA1 and SD5 inhibited the migration of cancer cells after 36 and 72 hours of treatment with scFv. The same concentration of Aprinone-A1 treatment was given as a control group in the experiment. (Figure 5C)

[0174] The quantitative percentages illustrate the migration inhibitory effects of the scFv on four pancreatic cancer cells in the wound healing assay. The experimental results of scFv SD5 are superior to those of scFv SA1; scFv SD5 exhibits higher reactivity against cancer cell lines Mia PaCa-2 and BxPc-3, and the inhibitory effects are 65% and 67%, respectively. Therefore, we selected scFv SD5 for subsequent experiments. (Figure 5D)

[0175] Example 5. Binding specificity of humanized antibody hSD5 to EphA2 and induced tumor suppression signal transduction.

[0176] Figure 6A confirms the binding reactivity of humanized scFv hSD5 to cells overexpressing different Eph family proteins (EphA1-A8). To improve the clinical applicability of the antibody, humanization of chicken-derived scFv SD5 was performed. Since these Ephs family molecules (EphA1–A8) have multiple roles in human cell physiology, it was necessary to determine the specificity of the isolated antibody for the EphA2 molecule. By using cells overexpressing EphA1–A8 molecules, it was confirmed that humanized scFv hSD5 specifically binds to the EphA2 molecule and does not exhibit cross-binding reactivity to other family proteins. (Figure 6A)

[0177] Figure 6B demonstrates the endocytosis of humanized IgG hSD5 upon treatment on pancreatic cancer cell BxPC3. The intact IgG hSD5 was expressed for endocytosis-based experiments. After treating PAAD cell BxPc-3 with IgG hSD5, it was cultured at 4 °C and 37 °C for 1 hour for observation. Ephrin-A1, the ligand of EphA2, was used as a positive control for comparison. The control Ab was a commercial anti-EphA2 IgG antibody that could induce endocytosis. These red arrows show that Ephrin-A1 and IgG hSD5 were endocytosed from the cell membrane into the cytoplasm after targeting the EphA2 molecule.

[0178] Culturing these cells at 4 °C will cause these cells to enter a resting state. Unlike in the control IgG group, the reaction of the antibody used to recognize EphA2 occurs on the cell membrane at 4 °C and 37 °C. When the IgG antibody hSD5 is administered at 37 °C, these antibodies enter into these cells through the cell membrane and enter into the cytoplasm (indicated by red arrows) through endocytosis. These experimental results are similar to the results of the experiment administering Ephrin A1 (EphA2 ligand). (Figure 6B) Using biolayer interferometry analysis, we analyzed the kon and koff parameters of IgG hSD5 targeting the EphA2 protein; the calculated affinity (KD) of IgG hSD5 was 2.06 nM.

[0179] As shown in Figure 6C, changes in molecular signaling in cancer cells were observed after administering scFv hSD5. These cancer cells BxPc-3 and Mia PaCa-2 degraded the EphA2 molecule 6 hours after administering scFv and showed a dose-dependent response. The lysosome-associated proteins LAMP1 and LAMP2 also showed upregulated levels in the two cancer cell lines, meaning that scFv hSD5 can enter the cells through endocytosis after acting on EphA2 and the lysosomes are involved in protein degradation. (Figure 6C)

[0180] After treating cancer cells BxPc-3 and Mia PaCa-2 with different concentrations of humanized scFv hSD5 for 24 hours, the molecular signaling of the cells was analyzed. As shown in Figure 6D, EphA2 was almost completely degraded, and the amount of pEphA2 also decreased. Regarding the signals pERK and pAKT, which are related to cancer cell proliferation and metastasis, also showed similar decreases. The two signals of pSTAT3 and pFAK (signals related to cancer cell survival and adhesion) in the two cancer cell lines were observed to decrease in a dose-dependent manner. Treating with scFv hSD5 led to similar changes in the molecular signaling of the two cancer cell lines. (Figure 6D)

[0181] The above examples show that the binding of the antibody hSD5 to the EphA2 molecule on the surface of cancer cells causes the degradation of the EphA2 molecule and induces endocytosis of the cells, which enables the antibody molecule to enter the cytoplasm (Figure 6D); this is similar to the process in which the antibody binds at the EphA2 active site and produces a similar response targeting epirubicin A1. The observations indicate that hSD5 can be developed into an antibody-drug conjugate. In preliminary experiments, it was found that labeling hSD5 with the small molecule MMAE was effective in inducing apoptosis in cancer cells.

[0182] Example 6. In vivo tumor growth inhibitory effect of humanized IgG hSD5 in BxPc-3 xenograft mice.

[0183] Administration of IgG hSD5 (20 mg / kg, iv, qwk, indicated as blue filled triangles) and gemcitabine (100 mg / kg, iv, biw, indicated as orange filled squares) was used to test the inhibitory effect on BxPc-3 tumor growth, n = 6, TGI represents tumor growth inhibition. (Figure 7A) Administration of IgG hSD5 (2 mg / kg, iv, qwk, indicated by solid black triangles), gemcitabine (20 mg / kg, iv, biw, indicated by black open squares), and an experimental group treated with a simultaneous combination of IgG and gemcitabine (indicated by solid red diamonds) was used to test the inhibitory effect on BxPc-3 tumor growth. (Figure 7B) During the treatment, the weight changes of each mouse group were tracked. (Figure 7C) The expression levels of EphA2, the cell proliferation marker Ki67, and the apoptosis-related marker cleaved caspase 3 in the excised tumor groups were analyzed by IHC staining. (Figure 7D)

[0184] Xenograft mice were used to evaluate the growth inhibitory effect of IgG hSD5 in vivo. In BxPc-3 xenograft mice, compared with the control IgG treatment (which showed no inhibitory effect), the treatment based on IgG hSD5 significantly inhibited tumor growth in vivo. The TGI of the IgG hSD5 treatment at 20 mg / kg, iv, qwk was 53.1%, and that of gemcitabine at 100 mg / kg, iv, qwk was 59.8% (Figure 7A). In the same experiment, the TGI of gemcitabine (20 mg / kg, iv, biw) was 34.6%. However, when the low-dose IgG hSD5 and gemcitabine were combined, a synergistic effect was observed, where the TGI was 57.4% (Figure 7B), and no change in the body weight of the mice was observed (Figure 7C). In addition, IHC staining was performed on the tissue sections of the removed tumors to observe the expression levels of EphA2 in the BxPc-3 tumors and the cell proliferation marker Ki-67 in the tissues. Compared with the results of the control IgG group, the treatment significantly reduced the expression levels of EphA2 and Ki-67 in the tumors (Figure 7D)

[0185] Example 7. In vivo tumor growth inhibitory effect of humanized IgG hSD5 in Mia PaCa-2 xenograft mice.

[0186] Mia PaCa-2 xenograft mice were also used to determine the inhibitory effect of IgG hSD5 on tumor growth in vivo. The administrations of IgG hSD5 (20 mg / kg, iv, qwk, indicated as blue-filled inverted triangles) and gemcitabine (100 mg / kg, iv, biw, indicated as orange-filled squares) were used to test the inhibitory effect on BxPc-3 tumor growth, n = 6, TGI represents tumor growth inhibition. (Figure 8A) The administrations of IgG hSD5 (2 mg / kg, iv, qwk, indicated by solid black triangles), gemcitabine (20 mg / kg, iv, biw, indicated by black open squares), and the experimental group treated with the simultaneous combination of IgG and gemcitabine (indicated by solid red diamonds) were used to treat the growth inhibitory effect of BxPc-3 on tumors. (Figure 8B) During the treatment, the weight changes of each mouse group were tracked. (Figure 8C) The expression levels of EphA2, the cell proliferation marker Ki67, and the apoptosis-related marker cleaved caspase-3 in the excised tumor groups were analyzed by IHC staining. (Figure 8D)

[0187] After administration of 20 mg / kg IgG hSD5, the TGI was 63.2%, while after administration of 100 mg / kg IgG gemcitabine, the TGI was 73.7% (Figure 8A). When gemcitabine (20 mg / kg) was administered, the TGI was 38.7% (Figure 8B). However, similar to the therapeutic effect observed in BxPc-3 tumor-bearing mice, a significant synergistic effect was observed when a combination therapy consisting of low-dose IgG hSD5 and gemcitabine was administered, and the TGI was 76.8%. No side effects or changes in body weight were observed in the mice (Figure 8C). The results of IHC staining of Mia PaCa-2 tumor tissue sections showed that the expression levels of EphA2 and Ki-67 in the tissue were significantly reduced. These results confirmed that the IgG hSD5 targeting EphA2 effectively inhibited tumor growth in vivo (Figure 8D).

[0188] As can be seen from Examples 6 and 7, the administration of the combination of hSD5 (2 mg / kg, iv, qwk) and gemcitabine (20 mg / kg, iv, biw) produced a strong synergistic effect on the cancer cells BxPc-3 and Mia PaCa-2. These results indicate that the use of low doses in combination therapy can lead to tumor growth inhibition, and it also indicates the potential of the antibody hSD5 for therapeutic applications. Gemcitabine is a first-line therapeutic drug for pancreatic cancer; it can inhibit DNA synthesis after entering cells, resulting in cytotoxicity (J Clin Oncol., 15(6):2403-2413 (1997); Mol Pharm., 10(2):430-444 (2013)). However, when combined with the inhibitory effect induced by hSD5 targeting EphA2 on the surface of cancer cells, a more comprehensive therapeutic effect can be achieved in PAAD.

[0189] Example 8. Epitope definition of IgG hSD5 identifies the active site of EphA2.

[0190] To determine whether the antibody hSD5 can recognize two antigen fragments simultaneously, peptide synthesis of the two antigen fragments was performed to test the binding reaction of the antibody IgG hSD5. ELISA was used to test the binding reactivity of the antibody hSD5 to the synthetic peptides. The two short peptides located at the designed activation site of EphA2 were EphA2pep_P1 and EphA2pep_P2, respectively. The long peptide formed by linking the two short peptides was EphA2pep_P1P2. NC1pep and NC2pep were two irrelevant peptides as negative controls. EphA2 ECD was the recombinant EphA2 extracellular domain protein.

[0191] The IgG hSD5 exhibits a binding reaction to the long peptide (EphA2pep_P1P2) linking the two antigen fragments, and IgG hSD5 individually recognizes the two synthetic short peptides (EphA2pep_P1 and EphA2pep_P2). In addition, IgG hSD5 does not exhibit a cross-binding reaction to the two irrelevant peptides. These experimental results indicate the positions where the antibody hSD5 binds to the designed antigen fragments, and the antibody hSD5 interacts with both antigen fragments; the interaction with EphA2pep_P1 is stronger than the interaction with EphA2pep_P2, indicating that the conformational antigen determinants on the antibody structure can induce an immune response. (Figure 9A)

[0192] Figure 9B is a dot blot assay used to test the binding reaction of the antibody hSD5 to synthetic peptides. By using the dot blot assay, experimental findings similar to those of peptide ELISA were obtained; IgG hSD5 can recognize the antigen fragments EphA2pep_P1 and EphA2pep_P2, and its binding reaction to the peptide EphA2pep_P1 is stronger than the binding reaction to EphA2pep_P2. (Figure 9B)

[0193] In the therapeutic strategy targeting EphA2, using soluble EphrinA1 or fusing recombinant EphrinA1 to human IgG Fc for dimerization can effectively promote the phosphorylation and degradation of EphA2 and ultimately inhibit the growth of tumor cells (Biochem Biophys Res Commun. 320(4):1096 - 1102(2004)). However, EphrinA1 interacts with multiple Eph family molecules, and these factors can produce adverse side effects, thus limiting its efficacy. Through the structural design of the immunogen, we prepared a specific antibody targeting the binding site of EphrinA1 at the activation site on EphA2; these test results indicate that the antibody can bind to the conformational antigen determinant formed by two discontinuous surfaces (Figure 9A and Figure 9B). Therefore, the antibody hSD5 can induce a positive tumor growth inhibitory effect similar to that of EphA2 targeted by EphrinA1. As confirmed in Figure 6A of Example 5 above, the hSD5 specifically binds to EphA2 and does not cross-react with other Eph family proteins. The results reflect the advantages of the antibody: preventing adverse side effects and promoting the inhibitory reaction of neutralizing the EphA2 molecule.

[0194] Example 9. Growth inhibitory responses of different pancreatic cancer cell lines treated with serially diluted MMAE.

[0195] As shown in Figure 10, the hTERT - HPNE is a normal pancreatic endothelial cell line.

[0196] Monomethyl auristatin E or MMAE is 100 to 1000 times more potent than doxorubicin (adriamycin / Rubex) and cannot be used as a drug by itself. However, as part of an antibody-drug conjugate or ADC, MMAE is linked to a monoclonal antibody (mAb) that recognizes the expression of a specific marker in cancer cells and directs MMAE to specifically target cancer cells. (Int J Mol Sci. 21(9):3286(2020)) In the example, the efficacy of MMAE in pancreatic cancer cell lines AsPc-1, BxPc-3, Mia PaCa-2. In contrast, it only shows a mild effect of growth inhibition in the normal pancreatic cell line hTERT HPNE. (Figure 10)

[0197] Example 10. Administration of serially diluted hSD5-ADC and growth inhibition responses in different pancreatic cancer cell lines.

[0198] As can be seen from Figures 11A to 11D, clearly, administration of hSDS alone does not provide an ideal inhibitory effect on various pancreatic cell lines. (hTERT HPNE, AsPC-1, BxPc-3 and Mia PaCa-2), while administration of hSD5-ADC provides a significant effect on the growth inhibition responses of these pancreatic cell lines. Since hSD5-ADC has a limited effect on normal pancreatic cell lines, it can be concluded that hSD5-ADC has shown high selectivity for pancreatic cancer cell lines (AsPC-1, BxPc-3 and Mia PaCa-2).

[0199] Example 11. Cell cycle changes in pancreatic cancer cells

[0200] Figures 12A to 12B illustrate the percentage of cell populations in different stages of the cell cycle as stacked histograms. hSD5 is the same antibody without MMAE as a comparison group. Larger apoptotic cell populations (indicated by the Sub-G1 bar) in the BxPc-3 (Figure 12A) and MiaPaCa-2 (Figure 12B) cell lines after treatment with different doses of hSD5-ADC indicate that hSD5-ADC is effective in inducing cancer cell death. A dose-dependence of the hSD5-ADC response was also observed.

[0201] Example 12. Using the BxPc-3 tumor xenograft mouse model to test the inhibitory effect on tumor growth in mice after administration of different concentrations of hSD5-ADC and the control group IgG-ADC.

[0202] Tumor size was measured during antibody therapy. Antibodies were administered by i.v. injection into the tail vein once a week, n = 5. %TGI is the percentage of tumor growth inhibition. (Figure 13A) Changes in the body weight of mice in each group were recorded during antibody treatment. (Figure 13B)

[0203] As can be seen from Figure 13A, the inhibitory effect on tumor growth after administration of IgG-ADC was limited, while the inhibitory effect on tumor growth after administration of hSD5-ADC was more significant. (The TGI of hSD5-ADC treatment at 1 mg / kg, iv, qwk was 56%). A dose-dependent trend was also observed.

[0204] As can be seen from Figure 13B, obviously, no side effects or weight changes were observed in the mice.

[0205] Example 13. In vivo tumor growth inhibitory effect of hSD5-ADC on BxPc-3 xenograft mice.

[0206] The hSD5-ADC was administered to the mice (2 mg / kg, iv, qwk) to test the tumor growth inhibitory effect. IgG-ADC was used as the experimental control group, and under the same administration conditions, n = 4. (Figure 14A) During the treatment, the weight changes of each mouse group were tracked. (Figure 14B) After the experiment was completed, the tumors in the mice were removed for recording. (Figure 14C)

[0207] As can be seen from Figure 14A and Figure 14C, the administration of IgG-ADC had only a mild inhibitory effect on tumor growth, while the administration of hSD5-ADC had a significant inhibitory effect on tumor growth. As can be seen from Figure 14B, obviously, no side effects or weight changes were observed in the mice.

[0208] Example 14. Anti-EphA2 hSD5 can recognize endogenous EphA2 molecules and inhibit cell growth on gastric cancer.

[0209] Flow cytometry was used to analyze the reaction of hSD5 IgG in recognizing endogenous EphA2 molecules on three strains of gastric cancer cell lines. PC represents the determination of EphA2 expression on cancer cells using a commercial Ab; NC represents the control group of an irrelevant isotype IgG1 antibody. The hSD5 was used to analyze the recognition of endogenous EphA2 molecules on three strains of gastric cancer cells by flow cytometry. The results indicated that the hSD5 exhibited a significant binding reaction to EphA2 molecules on gastric cancer cells. (Figure 15A)

[0210] The growth inhibitory effects of hSD5-ADC on three strains of gastric cancer cells were tested at different concentrations. The cancer cells were interacted with at the designated concentrations for 3 days, and the effects on the growth of the cancer cells were observed by MTS assay. As can be seen from Figure 15B, obviously, administration of hSDS alone could not provide an ideal inhibitory effect on the three different gastric cancer cells (SNU-16, N87, and MKN-45), while administration of hSD5-ADC provided a significant effect on the growth inhibitory response of these gastric cancer cell lines. The results showed that hSD5-ADC had demonstrated high selectivity against gastric cancer cell lines. In addition, dose-dependent inhibition of hSD5 and hSD5 ADC was observed, and the growth inhibitory effect of hSD5-ADC on the three strains of gastric cancer cells was more significant than that of hSD5. (Figure 15B)

[0211] Example 15. Expression of EphA2 and Binding Ability of Anti-EphA2 hSD5 in GBM

[0212] To analyze the expression of the EphA2 molecule, four brain tumor cell lines GBM8901, LN229, T98G, and U87MG and one normal cell line SVGp12 were used to compare the expression of EphA2 in cancer cells. Compared with the normal SVGp12 cell line, all four brain tumor cell lines showed moderate or higher EphA2 protein (Figure 16A)

[0213] Flow cytometry was used to analyze the reaction of hSD5 IgG in recognizing endogenous EphA2 molecules on four strains of brain tumor cell lines. PC represents the use of a commercial Ab to determine the expression of EphA2 on cancer cells; NC represents the control group of an irrelevant isotype IgG1 antibody. (Figure 16B)

[0214] The endogenous EphA2 molecules on four brain tumor cell lines were analyzed by flow cytometry using the hSD5. PC used a commercial anti-EphA2 antibody, and NC was the control group of an irrelevant isotype IgG1. The results indicated that the hSD5 showed a significant binding reaction to the EphA2 molecules on brain cancer cells.

[0215] Example 16. Anti-EphA2 hSD5 Can Recognize Endogenous EphA2 Molecules and Inhibit Cell Growth Based on Cholangiocarcinoma and Bladder Cancer.

[0216] Western blot was used to analyze the expression of the EphA2 molecule in four cholangiocarcinoma cell lines HuCCT1, ssp-25, RBE, TFK, and one bladder cancer cell PC-3. (Figure 17A) Compared with the control cell line, four cholangiocarcinoma and one bladder cancer cell line showed moderate to high expression of the EphA2 protein. The results of the expression of the EphA2 molecule were correlated with the binding reaction to the EphA2 molecule in the four cholangiocarcinoma cell lines.

[0217] Flow cytometry was used to analyze the reaction of hSD5 IgG with endogenous EphA2 molecules on four strains of cholangiocarcinoma cell lines and bladder cancer cell lines at different concentrations. PC indicates the use of a commercial Ab to determine the expression of EphA2 on cancer cells. (Figure 17B) The results indicate that these hSD5s exhibit a significant binding reaction to EphA2 molecules on cancer cells.

[0218] The growth inhibitory effect of hSD5 scFv on four strains of cholangiocarcinoma cells was tested under different conditions. At the specified concentration, hSD5 scFv interacted with cancer cells for 5 days, and the effect on the growth of cancer cells was observed by MTS assay. (Figure 17C)

[0219] By the 5th day of the antibody reaction, at a concentration of 20 mM, hSD5 scFv showed a growth inhibitory effect of approximately 40% to 70% on the four cell lines HuCCT1, ssp-25, RBE, and TFK, based on the cancer cell line %. A dose-dependent response was observed at different antibody concentrations; the results indicate that the binding of hSD5 scFv to EphA2 on the surface of cholangiocarcinoma cells can inhibit the growth of cancer cells.

[0220] Example 17. Anti-EphA2 hSD5 can recognize endogenous EphA2 molecules and inhibit cell growth on colon cancer.

[0221] Western blot was used to analyze the expression of EphA2 molecules in three colon cancer cells HCT116, SW480, and SW460 and one normal colon endothelial cell FHC. (Figure 18A) Compared with the control cell line, all three colon cancer cell lines showed higher expression of EphA2 protein.

[0222] Flow cytometry was used to analyze the reaction of hSD5 IgG with endogenous EphA2 molecules on three strains of gastric cancer cell lines. PC indicates the use of a commercial Ab to determine the expression of EphA2 on cancer cells; NC indicates a control group of irrelevant isotype IgG1 antibody. (Figure 18B) The results indicate that these hSD5s exhibit a significant binding reaction to EphA2 molecules on cancer cells.

[0223] The growth inhibitory effect of hSD5-ADC on three strains of colon cancer cells was tested at different concentrations. It interacted with cancer cells for 3 days at the specified concentration, and the effect on the growth of cancer cells was observed by MTS analysis. (Figure 18C)

[0224] As can be seen from Figure 18C, obviously, administration of hSDS alone cannot provide an ideal inhibitory effect on three different colon cancer cells (HCT116, SW480, and SW460), while administration of hSD5-ADC provides a significant effect on the growth inhibitory response of these colon cancer cell lines. The results indicate that hSD5-ADC has shown high selectivity against colon cancer cell lines. In addition, dose-dependent inhibition of hSD5 and hSD5 ADC was observed, and the growth inhibitory effect of hSD5-ADC on the three strains of colon cancer cells was more significant than that of hSD5.

[0225] Example 18. hSD5-ADC can inhibit tumor growth in an HCT116 xenograft mouse model.

[0226] The growth inhibitory effects of hSD5-ADC (2 mg / kg, iv, qwk, solid red square markers), control IgG-ADC (2 mg / kg, iv, qwk, solid blue triangle markers), and PBS group (solid black circular markers) on the HCT116 tumor volume were observed. (Figure 19A) The tumor weights of HCT116 xenograft tumors after treatment with hSD5-ADC and control IgG-ADC (*p < 0.05). (Figure 19B) After administration of the antibody, the weight changes of HCT116 xenograft mice were recorded. (Figure 19C). As can be seen from Figures 19A to 19B, the inhibitory effect on tumor growth after administration of IgG-ADC was limited, while the inhibitory effect on tumor growth after administration of hSD5-ADC was more significant. (The TGI of hSD5-ADC treatment at 2 mg / kg, iv, qwk was 60.7%). As can be seen from Figure 19C, obviously, no side effects or weight changes were observed in the mice.

Claims

1. An isolated anti-EphA2 antibody or antigen-binding portion thereof, comprising: A light chain complementarity-determining region 1 (L-CDR1) comprising the amino acid residues of SEQ ID NO:1 or a variant having an amino acid sequence with at least 95% identity to any of SEQ ID NO:1; a light chain CDR2 (L-CDR2) comprising the amino acid residues of SEQ ID NO:2 or a variant having an amino acid sequence with at least 95% identity to any of SEQ ID NO:2; and a light chain CDR3 (L-CDR3) comprising the amino acid residues of SEQ ID NO:3 or a variant having an amino acid sequence with at least 95% identity to any of SEQ ID NO:3; and A heavy chain complementarity-determining region 1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO:4 or a variant having an amino acid sequence with at least 95% identity to any of SEQ ID NO:4; a heavy chain CDR2 (H-CDR2) comprising the amino acid residues of SEQ ID NO:5 or a variant having an amino acid sequence with at least 95% identity to any of SEQ ID NO:5; and a heavy chain CDR3 (H-CDR3) comprising the amino acid residues of SEQ ID NO:6 or a variant having an amino acid sequence with at least 95% identity to any of SEQ ID NO:6; such that the isolated antibody or antigen-binding portion thereof binds to EphA2.

2. The anti-EphA2 antibody or antigen-binding portion thereof according to claim 1, which is a monoclonal antibody, chimeric antibody, humanized antibody or human antibody.

3. The anti-EphA2 antibody or antigen-binding portion thereof according to claim 1, which is a single-chain Fv (scFv), IgG, Fab, (Fab)2 or (scFv')2.

4. The anti-EphA2 antibody or antigen-binding portion thereof according to claim 1, comprising: A light chain comprising the amino acid sequence of SEQ ID NO:7 or 8 or a variant having at least 95% identity to SEQ ID NO:7 or 8; and A heavy chain comprising the amino acid sequence of SEQ ID NO:9 or 10 or a variant having at least 95% identity to SEQ ID NO:9 or 10.

5. The anti-EphA2 antibody or antigen-binding portion thereof according to claim 4, comprising: A light chain comprising the amino acid sequence of SEQ ID NO:7 or 8; and A heavy chain comprising the amino acid sequence of SEQ ID NO:9 or 10.

6. The anti-EphA2 antibody or antigen-binding portion thereof according to claim 1, which comprises the amino acid sequence of SEQ ID NO:11 or 12 or a variant having at least 95% identity to SEQ ID NO:11 or 12.

7. The anti-EphA2 antibody or antigen-binding portion thereof according to claim 6, which comprises the amino acid sequence of SEQ ID NO:11 or 12.

8. The anti-EphA2 antibody or antigen-binding portion thereof according to any one of claims 1 to 7, wherein the antibody is a humanized antibody.

9. An antibody-drug conjugate (ADC) comprising the anti-EphA2 antibody or antigen-binding portion thereof according to any one of claims 1 to 8 and a drug-linker structure comprising an anti-tumor compound linked to the antibody via a linker.

10. The antibody-drug conjugate according to claim 9, wherein the anti-tumor compound is selected from auristatins (such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)), vincristine, vinblastine, methotrexate, platinum-based anti-tumor agents (cisplatin and its derivatives), doxorubicin, calicheamicin, dolastatin 10, maytansinoids, pyrrolobenzodiazepine dimers, camptothecin derivatives, duocarmycins, amanitin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, and paclitaxel (Taxol), and derivatives thereof.

11. The antibody-drug conjugate according to claim 9, wherein the anti-tumor compound is MMAE.

12. A pharmaceutical composition comprising the anti-EphA2 antibody according to any one of claims 1 to 8 or the ADC according to claim 9 or 10 and a pharmaceutically acceptable carrier or excipient.

13. The pharmaceutical composition according to claim 12, further comprising one or more additional anti-cancer agents or used in combination therewith.

14. The pharmaceutical composition according to claim 13, wherein the one or more additional anti-cancer agents are Gemcitabine.

15. A method for treating or preventing EphA2-related cancer in an individual, comprising administering to the individual a therapeutically effective amount of the anti-EphA2 antibody according to any one of claims 1 to 8 or the ADC according to claim 9 or 10.

16. A method for inhibiting the growth or metastasis of EphA2-related cancer cells in an individual, comprising administering to the individual a therapeutically effective amount of the anti-EphA2 antibody according to any one of claims 1 to 8 or the ADC according to claim 9 or 10.

17. The method according to claim 15 or 16, wherein the EphA2-related cancer is selected from cholangiocarcinoma, bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioma, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, stomach cancer, thymic cancer, and vulvar cancer.

18. The method according to claim 15 or 16, wherein the EphA2-related cancer is selected from bladder cancer, brain cancer, cholangiocarcinoma, colon cancer, gastric cancer, and pancreatic cancer.

19. The method according to claim 15 or 16, further comprising an additional anti-cancer agent.

20. The method according to claim 19, wherein the additional anti-cancer agent is gemcitabine.

21. A kit for detecting or diagnosing EphA2-related cancer or a high risk of future occurrence of EphA2-related cancer in an individual, or predicting cancer metastasis or prognosis, or monitoring cancer progression, comprising an EphA2 antibody or an antigen-binding portion thereof according to any one of claims 1 to 8.

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