Anti-EPHA2 antibody and related application thereof

Abstract

The invention discloses an anti-EPHA2 antibody and related application thereof, and belongs to the field of antibodies. The antibody provided by the invention can be specifically combined with an EPHA2 antigen and shows relatively high affinity. In-vivo and in-vitro experiments prove that the EPHA2-CD3 bispecific antibody constructed on the basis of the antibody has remarkable killing activity on various EPHA2 positive tumor cells, can be used for treating cancers and other diseases related to EPHA2 expression, and provides a key material for developing an immunotherapy strategy with efficient and lasting anti-tumor activity.

Description

Technical Field

[0001] This invention relates to the field of antibodies, and more specifically, to an antibody against EPHA2 and its related applications. Background Technology

[0002] EPHA2 is a key member of the Eph receptor tyrosine kinase family A subfamily. The encoding gene is located on human chromosome 1p36.13. The mature protein consists of an extracellular ligand-binding domain (LBD), a fibronectin type III repeat domain, a transmembrane domain, and an intracellular tyrosine kinase domain, and is an important membrane protein regulating intercellular signal transduction. Under physiological conditions, EPHA2 mediates cell migration, tissue boundary formation, and vascular homeostasis during embryonic development by binding to ephrin A1-A5 ligands. In adulthood, it exhibits low-level restricted expression in normal tissues (such as the proximal convoluted tubules of the kidney and mammary epithelium).

[0003] Clinical studies over the past decade have confirmed that EPHA2 exhibits "tumor-specific high expression" in various malignant tumors, and its expression level is directly related to the malignant phenotype of tumors and poor patient prognosis. Current EPHA2 targeting technologies face four major bottlenecks: "insufficient specificity, poor controllability of toxicity, limited efficacy, and narrow applicable population." The core issue lies in the lack of anti-EPHA2 antibodies with high affinity and high tumor specificity.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide an anti-EPHA2 antibody and its related applications.

[0006] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide an antibody against EPHA2 or an antigen-binding fragment thereof, comprising: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:6, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO:7; or, HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:14, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO:15.

[0007] Secondly, embodiments of the present invention provide an antibody conjugate comprising: the antibody or its antigen-binding fragment as described in the foregoing embodiments.

[0008] Thirdly, embodiments of the present invention provide a reagent or kit comprising: the anti-EPHA2 antibody or its antigen-binding fragment as described in the foregoing embodiments, or the antibody conjugate as described in the foregoing embodiments.

[0009] Fourthly, embodiments of the present invention provide an immunoconjugate or pharmaceutical composition comprising the anti-EPHA2 antibody or its antigen-binding fragment as described in the foregoing embodiments.

[0010] Fifthly, embodiments of the present invention provide the use of anti-EPHA2 antibodies or antigen-binding fragments thereof as described in the foregoing embodiments, antibody conjugates or immunoconjugates or pharmaceutical compositions as described in the foregoing embodiments in the preparation of products for the prevention or treatment of EPHA2-positive tumors, the preparation of products for the detection of EPHA2, or the detection of EPHA2 for non-disease treatment or diagnostic purposes.

[0011] In a sixth aspect, embodiments of the present invention provide an isolated nucleic acid or a vector containing said nucleic acid, which encodes the anti-EPHA2 antibody or its antigen-binding fragment described in the foregoing embodiments.

[0012] In a seventh aspect, embodiments of the present invention provide a recombinant cell containing the isolated nucleic acid described in the foregoing embodiments or a vector containing the nucleic acid.

[0013] Eighthly, embodiments of the present invention provide a method for preparing an antibody against EPHA2 or an antigen-binding fragment thereof as described in the foregoing embodiments, comprising: culturing the recombinant cells described in the foregoing embodiments.

[0014] The present invention has the following beneficial effects: The anti-EPHA2 antibody or its antigen-binding fragment provided by this invention has excellent antigen-binding activity, high specificity, and good affinity, with an affinity KD of 10. -9 The antibody against EPHA2 and the anti-CD3 antibody OKT3 were used to construct a bispecific antibody. Through in vivo and in vitro experiments, it was verified that the EPHA2-CD3 bispecific antibody constructed based on this antibody has significant killing activity against a variety of EPHA2-positive tumor cells. It can be used to treat cancers and other diseases related to EPHA2 expression, and provides key materials for developing immunotherapy strategies with efficient and durable anti-tumor activity.

[0015] This invention conjugates an anti-EPHA2 antibody with val-cit-MMAE to construct an antibody-drug conjugate. The results show that EPHA2-MMAE has a significant killing effect on colorectal cancer cell lines that highly express EPHA2. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 In this embodiment of the invention, positive monoclonal phages that specifically bind to EphA2-His protein and HeLa-EPHA2 cells were screened using phage display technology. Figure 2 In this embodiment of the invention, SDS-PAGE analysis was performed to analyze the specific binding of purified anti-EPHA2 single-chain antibody to HeLa-EPHA2 cells and the immunofluorescence analysis. Figure 3 This is an analysis of the in vitro killing activity of EPHA2-CD3 against HeLa-EPHA2 cells in this embodiment of the invention; Figure 4 The present invention provides an example of SDS-PAGE analysis to determine the specific binding of purified recombinant chimeric anti-EPHA2 antibody to HeLa-EPHA2 cells using immunofluorescence analysis. Figure 5 This is a schematic diagram of the structure of the EPHA2-specific antibody-drug conjugate E75-MMAE in an embodiment of the present invention; Figure 6 This invention provides an in vitro cytotoxicity analysis of the EPHA2-specific antibody-drug conjugate E75-MMAE against colorectal cancer cell lines SK-CO-1, HT-29, GP2D, and SW480. Figure 7 This study analyzes the antitumor activity of the EPHA2-specific antibody-drug conjugate E75-MMAE against the HT29 subcutaneous tumor model in this embodiment of the invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0019] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. "Reaction" (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (JEColigan et al., eds., 1991), each of which is explicitly incorporated herein by reference.

[0020] This invention, through appropriate screening techniques and large-scale screening, ultimately obtained a specific anti-EPHA2 antibody. This anti-EPHA2 antibody and an anti-CD3 single-chain antibody were then used to construct a bispecific antibody, which still maintains good antigen-binding activity against EPHA2. In vivo and in vitro experiments confirmed that this bispecific antibody has strong killing activity against tumor cells with high EPHA2 expression, effectively improving treatment efficacy and providing a key material for the subsequent development of immunotherapies with highly efficient and durable anti-tumor activity.

[0021] Definition of noun The term "antibody" as used herein is used in the broadest sense and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antigen-binding fragments, provided they exhibit the desired biological activity, such as specific binding to the EPHA2 antigen or fragments thereof. An antigen-binding fragment, also known as a functional fragment of an antibody, typically has the same binding specificity as the antibody from which it originates. Antigen-binding fragments include any one selected from F(ab')2, Fab', Fab, Fv, and scFv of antibodies. Those skilled in the art will readily understand from the description of this invention that the aforementioned functional fragments of antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. The aforementioned antigen-binding fragments can also be obtained by recombinant genetic techniques, also known to those skilled in the art, or by synthesis using, for example, automated peptide synthesizers, such as those sold by Applied BioSystems.

[0022] In this article, the term "CDR" stands for "complementarity-determining region," which refers to the highly variable region of the heavy and light chains of an immunoglobulin, containing one or more or all of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to the antigen or epitope it recognizes.

[0023] The term "backbone region" in this article, synonymous with "framework region" or "FR region," refers to the region of the antibody's heavy chain variable region excluding the CDR region. The heavy chain backbone region can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3, and FR4), including the HFR1, HFR2, HFR3, and HFR4 backbone regions. The heavy chain variable region is obtained by arranging and connecting the following numbered CDRs with FRs (from the amino terminus to the carboxyl terminus): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0024] The “multivalent antibody” in this article is a polymer of a monovalent antibody that recognizes the same epitope and has a higher antigen affinity than the corresponding monovalent antibody.

[0025] The "multispecific antibody" in this article is a polymer of monovalent antibodies that bind to different targets or different binding regions on the same target, and has a stronger antigen recognition ability than the corresponding monovalent antibody.

[0026] The “chimeric antibody” mentioned in this article is usually an antibody formed by fusing the variable region of a non-human antibody with the constant region or backbone region of a human antibody, which can reduce the immune response induced by non-human antibodies.

[0027] The term "fusion antibody" in this article includes, but is not limited to, new fusion molecules formed by combining with other structures (such as BSA, IgG-Fc, etc.) through genetic engineering techniques, such as enzymes, antimicrobial peptides, or imaging substances that can prolong their half-life.

[0028] The term "treatment" in this invention includes preventing or alleviating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0029] For cancer, "treatment" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination thereof.

[0030] On one hand, embodiments of the present invention provide an antibody against EPHA2 or an antigen-binding fragment thereof, comprising: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:6, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO:7; or, The amino acid sequences are HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:14, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:15.

[0031] In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

[0032] In some embodiments, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO:1~4, STS, and SEQ ID NO:5, respectively; or, The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:9~12, RTS and SEQ ID NO:13, respectively.

[0033] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region.

[0034] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7; or, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:14; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:15.

[0035] In some embodiments, the antibody further includes a constant region.

[0036] In some embodiments, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0037] The light chain of a full-length antibody includes a variable region domain (VL) and a constant region domain (CL). VL is located at the amino terminus of the light chain, and the CL domain is located at the carboxyl terminus. The light chain includes a κ chain and a λ chain. The heavy chain of a full-length antibody includes a heavy variable region domain (VH) and a constant region domain (CH). VH is located at the amino terminus of the heavy chain, and the CH domain is located at the carboxyl terminus.

[0038] In some embodiments, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0039] In some embodiments, the species source of the constant region is cattle, horses, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

[0040] In some embodiments, the constant region of the antibody includes: a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:18; and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:19.

[0041] In some embodiments, the antigen-binding fragment includes any one of Fab, Fab', F(ab')2, scFv, and Fv.

[0042] In some embodiments, when the antigen-binding fragment is scFv, the amino acid sequence of scFv is as shown in SEQ ID NO:8 or 16.

[0043] In some embodiments, the antibody is selected from any one of: monovalent antibody, multivalent antibody, multispecific antibody, chimeric antibody, and fusion antibody.

[0044] In some embodiments, the multispecific antibody includes any one of bispecific antibodies, trispecific antibodies, and tetraspecific antibodies.

[0045] Bispecific antibodies (BsAbs) are artificial antibodies that can simultaneously target two antigens or different epitopes of the same antigen. Bispecific antibodies can be IgG-like (full-length bispecific antibodies) or non-IgG-like bispecific antibodies that are non-full-length antibody constructs. Full-length bispecific antibodies typically retain the structure of a traditional monoclonal antibody (mAb) with two Fab arms and an Fc region, but the two Fab sites bind to different antigens. Non-full-length bispecific antibodies may lack the entire Fc region. These include chemically linked Fabs, Fab regions alone, and various types of bivalent and trivalent single-chain variable fragments (scFvs). Fusion proteins that mimic the variable domains of two antibodies also exist.

[0046] In some embodiments, bispecific antibodies include bispecific T-cell conjugating antibodies (BiTEs). The core function of bispecific T-cell conjugating antibodies (BiTEs) is to simultaneously target CD3 molecules on the surface of T cells and specific antigens on the surface of tumor cells through two different antigen-binding domains, thereby directly "recruiting" T cells to the vicinity of tumor cells, activating the killing function of T cells, and achieving specific clearance of tumor cells.

[0047] In some embodiments, the multispecific antibody includes: an anti-CD3 antibody and the anti-EPHA2 antibody or an antigen-binding fragment thereof.

[0048] In some embodiments, the anti-CD3 antibody comprises a single-chain antibody against CD3.

[0049] In some embodiments, the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO:17.

[0050] On the other hand, embodiments of the present invention provide an antibody comprising: the anti-EPHA2 antibody or its antigen-binding fragment as described in any of the foregoing embodiments.

[0051] In some embodiments, when the antibody is a bispecific antibody (bispecific T-cell conjugating antibody), the bispecific antibody may further include an anti-CD3 antibody.

[0052] Verification has shown that bispecific antibodies constructed from anti-EPHA2 antibodies or their antigen-binding fragments and anti-CD3 antibodies have good anti-tumor activity and can be used to prepare drugs for the prevention, diagnosis and treatment of at least one of the following diseases: breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, glioblastoma, head and neck cancer and osteosarcoma.

[0053] In some embodiments, the anti-EPHA2 antibody or its antigen-binding fragment and the anti-CD3 antibody are linked by a linker peptide.

[0054] In some embodiments, the anti-CD3 antibody comprises a single-chain antibody against CD3.

[0055] In some embodiments, the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO:17.

[0056] In some embodiments, the linker peptide is (G4S)n, where n is a non-zero natural number.

[0057] In some embodiments, n is 1 to 20.

[0058] In some embodiments, n is 3 or 4.

[0059] In some embodiments, the structure of the bispecific antibody (EPHA2-CD3) is represented as: VL1-(G4S)3-VH1-G4S-VH2-(G4S)3-VL2; wherein, VH1 is the heavy chain variable region of the anti-EPHA2 antibody, VL1 is the light chain variable region of the anti-EPHA2 antibody, VH2 is the heavy chain variable region of the anti-CD3 antibody, VL2 is the light chain variable region of the anti-CD3 antibody, (G4S)3 is a peptide linker, and VH2-(G4S)3-VL2 is a CD3 scFv.

[0060] On the other hand, embodiments of the present invention provide an antibody conjugate comprising: the anti-EPHA2 antibody or its antigen-binding fragment as described in any of the foregoing embodiments.

[0061] In some embodiments, the antibody conjugate further includes a label, purification tag, and / or solid-phase carrier conjugated to the anti-EPHA2 antibody or its antigen-binding fragment.

[0062] In some embodiments, the markers include fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0063] In some embodiments, fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5). Cy5.5, Cy3 and other similar dyes, Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar dyes) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar dyes).

[0064] In some embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0065] In some embodiments, radioactive isotopes include, but are not limited to, 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18F.

[0066] In some embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0067] In some embodiments, nanoparticle-based markers include, but are not limited to, nanoparticles and colloids; nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0068] In some embodiments, the solid support includes, but is not limited to, microspheres, plates, and membranes.

[0069] In some embodiments, the solid support includes any one or more of magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0070] On the other hand, embodiments of the present invention provide a reagent or kit comprising: an anti-EPHA2 antibody or its antigen-binding fragment as described in any of the foregoing embodiments, or an antibody conjugate as described in any of the foregoing embodiments.

[0071] On the other hand, embodiments of the present invention provide an immunoconjugate or pharmaceutical composition comprising the anti-EPHA2 antibody or its antigen-binding fragment as described in any of the foregoing embodiments.

[0072] In some embodiments, the immune conjugate further includes a therapeutic agent.

[0073] In some embodiments, the therapeutic agent comprises at least one of: a mitotic inhibitor, a chemotherapeutic agent, a radionuclide, a photosensitizer, a photothermal agent, an immune checkpoint inhibitor, a toxin, a factor, a kinase inhibitor, an antibody that inhibits a second signaling molecule, a PD-L1 inhibitor, and a PD-1 / PD-L1 monoclonal antibody.

[0074] In some embodiments, the mitotic inhibitor includes at least one of dolalastatin, aristatin, maytansine, and phytoalkaloids.

[0075] In some embodiments, the arithmetic is monomethyl arithmetic F (MMAF) or monomethyl arithmetic E (same as "monomethyl arithmetic E", MMAE).

[0076] In some embodiments, the anti-EPHA2 antibody or its antigen-binding fragment is conjugated to the arithmetic (load) via a linker to form an antibody-drug conjugate (ADC).

[0077] In some embodiments, the connector includes a detachable connector.

[0078] In some embodiments, the linker is selected from any one or a combination of valine-citrulline (Val-Cit), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-alanine (Val-Ala), valine-lysine (Val-Lys), valine-arginine (Val-Arg), phenylalanine-citrulline (Phe-Cit), phenylalanine-lysine (Phe-Lys), and phenylalanine-arginine (Phe-Arg).

[0079] In some embodiments, the chemotherapeutic drugs described above are selected from any one or more of taxanes, vinca alkaloids, anthracyclines, epipodophyllotoxins, tyrosine kinase inhibitors, fulapindo, irinotecan and its metabolite SN-38, topotecan, teniposide, etoposide, imatinib, gefitinib, danuseltide, doxorubicin, daunorubicin, mitoxantrone, methotrexate, camptothecin, and saquinavir.

[0080] In some embodiments, the photosensitizer is selected from: (1) 5-aminolevulinic acid (ALA) or its derivatives; (2) photosensitizing compounds containing a tetrapyrrole ring; (3) traditional Chinese medicine photosensitizers; or (4) combinations of ALA or its derivatives with compounds in (2) or (3), respectively.

[0081] In some embodiments, the photothermal agent is selected from IR-780, IR-783, IR-805, IR-808, IR-825, IR-1045, IR-1048, IR-1061 and IR-26.

[0082] In some embodiments, the inhibitory second signaling molecule may be PD-1; CTLA-4; or PD-1 and CTLA-4.

[0083] In some embodiments, the PD-1 / PD-L1 monoclonal antibody drug is selected from at least one of the following groups: nivolumab, pembrolizumab, pitilizumab, lambrolizumab, BMS-936559, atezolizumab, AMP-224, AMP224, AUNP12, BGB108, MCLA134, MEDI0680, PDROO1, REGN2810, SHR1210, STIAl110, STIAl1110, TSR042, BMS-936558, BGB-A317, BCD-100, and JS001.

[0084] In some embodiments, the therapeutic agent further includes a cytotoxic agent.

[0085] In some embodiments, the pharmaceutical composition includes at least one of a pharmaceutical excipient, a carrier, and a diluent. The carrier is a pharmaceutically acceptable carrier, including but not limited to fillers, lubricants, disintegrants, binders, flow aids, etc.

[0086] In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to, one or a combination of polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose and its derivatives, hydroxypropylcellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, dicalcium phosphate, calcium stearate, sodium stearoyl fumarate, silicon dioxide, titanium dioxide, talc, and indigo.

[0087] On the other hand, embodiments of the present invention provide the use of anti-EPHA2 antibodies or antigen-binding fragments thereof as described in any of the foregoing embodiments, antibody conjugates or immunoconjugates or pharmaceutical compositions as described in any of the foregoing embodiments in the preparation of products for the prevention or treatment of EPHA2-positive tumors, the preparation of products for the detection of EPHA2, or the detection of EPHA2 for non-disease treatment or diagnostic purposes.

[0088] In some embodiments, the EPHA2-positive tumor includes at least one of breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, glioblastoma, head and neck cancer, and osteosarcoma.

[0089] In some embodiments, the product includes at least one of immune cells, reagents, kits, pharmaceuticals, and pharmaceutical compositions.

[0090] On the other hand, embodiments of the present invention provide an isolated nucleic acid or a vector containing said nucleic acid, which encodes the anti-EPHA2 antibody or its antigen-binding fragment as described in any of the foregoing embodiments.

[0091] Considering the degeneracy of codons, the gene sequence encoding the above antibodies can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody; alternatively, the gene can be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0092] In some embodiments, the vector containing the nucleic acid includes a recombinant vector. The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, which can refer to any recombinant polynucleotide construct that can directly introduce the target DNA fragment into the host cell for target gene expression via transformation, transfection, or transduction.

[0093] In some embodiments, the recombinant vector is a plasmid or a virus; the virus is an adenovirus, adeno-associated virus, retrovirus, lentivirus, or oncolytic virus.

[0094] On the other hand, embodiments of the present invention provide a recombinant cell containing the isolated nucleic acid described in any of the foregoing embodiments or a vector containing the nucleic acid.

[0095] In some embodiments, the recombinant cells include prokaryotic or eukaryotic cells. The prokaryotic cells include, but are not limited to, bacterial cells, such as *Escherichia coli*; the eukaryotic cells include, but are not limited to, yeast cells, insect cells, animal cells, or plant cells; the yeast cells may be, but are not limited to, *Pichia pastoris* or *Saccharomyces cerevisiae* cells. Optionally, the recombinant cells may be 293 cells, 293T cells, 293FT cells, CHO cells, or Per6 cells. The 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins and are well known to those skilled in the art.

[0096] Furthermore, embodiments of the present invention provide a method for preparing an anti-EPHA2 antibody or its antigen-binding fragment as described in any of the foregoing embodiments, which includes: culturing the recombinant cells described in any of the foregoing embodiments.

[0097] The present invention does not specifically limit the culture conditions of the host cells; culture conditions that enable the host cells to express and produce the antibody can be obtained based on conventional technical knowledge.

[0098] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0099] All animal experiments are conducted in accordance with animal ethics guidelines and are carried out only after being approved by the relevant institution's animal experiment ethics committee.

[0100] Example 1 1. Preparation of recombinant EPHA2 protein The nucleic acid sequence encoding human EPHA2 (NM_004431.5) was synthesized by Beijing Yiqiao Shenzhou Technology Co., Ltd. It was amplified by PCR and subcloned into the pcDNA3.1 expression vector (Invitrogen). Then, the extracellular domains of EPHA2 were subcloned into pcDNA3.1 expression vectors carrying either an Fc or His tag at the C-terminus. The Fc tags included human Fc (hFc) and mouse Fc (mFc). After transient transfection with 293FT, the protein was cultured in FreeStyle™ serum-free medium (Life Technologies) in shake flasks for 5–7 days. The supernatant was collected, centrifuged, and ultrafiltered. The recombinant EPHA2 protein carrying either the Fc or His tag was then purified by Protein A / G or nickel column affinity chromatography and molecular sieve chromatography. 2. Preparation of stable cell lines expressing human EPHA2 antigen The full-length sequence encoding human EPHA2 was constructed into a lentiviral vector carrying plvx. HeLa cells were cultured in DMEM containing 10% fetal bovine serum. Cells were transfected using Lipo3000 (Life Technologies) transfection reagent, and after 48 hours, they were sorted by flow cytometry and cultured into 96-well plates for screening and identification of stable single-clone cell lines. HeLa cells stably expressing EPHA2 (HeLa-EPHA2) were preserved.

[0101] Example 2 1. Preparation of anti-EPHA2 monoclonal antibody: (1) Animal immunization Five- to six-week-old female Balb / c mice were used as immunization animals, with an immunization dose of 100 μg per mouse. For the initial immunization, 100 μl of Freund's complete adjuvant (Sigma) was mixed with an equal volume of recombinant EPHA2 protein, thoroughly emulsified, and then injected subcutaneously at multiple sites. Every two weeks, an equal volume of Freund's incomplete adjuvant (Sigma) was mixed with the recombinant protein, thoroughly emulsified, and then injected subcutaneously at multiple sites. Four booster immunizations were administered. Ten days after the final booster immunization, blood was collected by tail cutting to detect antibody titers. Three days before lymph node collection, mice were given a 100 μg intraperitoneal pulse of recombinant protein.

[0102] (2) Phage library construction and antigen panning Total RNA was extracted from mouse lymph nodes using the TRIZOL method. cDNA was then obtained by reverse transcription using a reverse transcription kit (catalog 18080051, SuperScript III First-Strand Synthesis System, Invitrogen) with the RNA as a template. The VH and VL sequences were then amplified by PCR using cDNA as a template. The scFv sequence was amplified using out-F and out-R primers. The approximately 750 bp band was separated and recovered by agarose gel electrophoresis.

[0103] The PCR product and pMECS vector were digested and recovered using SfiI enzyme, and then ligated using T4 DNA ligase.

[0104] The ligation product was added to E. coli TG1 competent cells and electroporated to allow it to enter TG1 cells. Immediately after electroporation, SOC medium was added and the cells were incubated at 37°C and 200 rpm for 1 h. The cells were then plated on LB / AMP-GLU plates and incubated at 37°C for 6-8 h. The bacterial colony was collected and 1 / 3 volume of 50% glycerol was added to obtain the prepared phage library.

[0105] (3) Determination of phage library diversity and library capacity The electroconversion product was diluted 10-fold and then plated on LB / AMP-GLU plates. After incubation at 37°C for 12 h, the number of transformants was calculated, and the final library capacity was 8.9 × 10⁻⁶. Phage library.

[0106] Using the prepared phage library as the antibody source, three rounds of screening were conducted using phage display technology. From the plates selected in the third round of screening, 96 clones were randomly chosen for expansion culture. Single-chain antibodies that specifically bind to EPHA2 protein and HeLa-EPHA2 cells were identified using monoclonal ELISA and flow cytometry.

[0107] The results showed that 90 out of 96 clones were positive. Sequencing analysis of the positive clones yielded two specific single-chain antibodies against EPHA2. Two optimal single clones (E28 and E75) were ultimately identified for subsequent affinity and functional assays. Figure 1 As shown.

[0108] The sequences of two specific single-chain antibodies against EPHA2 are shown in Table 2. Antibodies E28 and E75scFv specifically bind to HeLa-EPHA2 cells as shown in Table 2. Figure 2 As shown.

[0109] Example 3 In vitro killing assay of EPHA2-CD3 bispecific antibody.

[0110] E28 scFv and E75 scFv were linked to the CD3 molecule Orthoclone OKT3, which targets the surface of human T cells, via a (GGGGS)3 linker peptide. The OKT3 sequence was then linked to the expression vector pcDNA3.1-MCS-His via homologous recombination to construct recombinant E28 / CD3BiTE and E75 / CD3 BiTE expression vectors. The amino acid sequence of OKT3 was used (as shown in SEQ ID NO:17 below). The EPHA2-CD3 BiTE bispecific antibody was purified by nickel affinity chromatography and molecular sieve chromatography.

[0111] By transiently transfecting 293FT, using Serum-free culture medium (Life Technologies) was cultured in shake flasks for 5-7 days. The supernatant was collected, centrifuged and ultrafiltered, and then purified by Protein A / G affinity chromatography and molecular sieve column chromatography to obtain the corresponding types of anti-EPHA2 recombinant chimeric antibodies E28 mAb and E75 mAb.

[0112] Preparation of effector and target cells: Peripheral blood was collected from healthy donors, PBMCs were isolated, and T cells were isolated using the Miltenyi T Cell Isolation Kit. The isolated T cells were cultured in X-VIVO (LONZA) medium containing 5% AB serum. TC-coated 6-well plates were pre-incubated at 37°C for 2 hours with 1 ml of coating solution containing 50 ng / ml anti-human CD3 antibody (PeproTech) and 50 ng / ml CD28 antibody (PeproTech). The coating solution was removed before use. Cells were seeded at 1 ml / well into antibody-coated 6-well plates and cultured for 48 hours. IL-2 (100 U / mL) and IL-15 (10 ng / mL) were then added as activating factors for continued culture. Target cells were HT29 colorectal cancer cells with high EPHA2 expression, cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum (Gibco).

[0113] Cell co-culture experiment: HeLa-EPHA2 tumor cells in logarithmic growth phase were added to 96-well plates and cultured overnight. Then, 0 ng / ml (Control) or 10 ng / ml of bispecific antibody was added. Simultaneously, T cells were added at an effector-to-target ratio of 2:1 (T cells to tumor cells). Bright-field images were taken after 24 hours. Figure 3 As shown.

[0114] The results showed that, compared with the control group without the addition of bispecific antibodies, the EPHA2-CD3 bispecific antibodies (E28, E75) had a significant killing effect on HeLa-EPHA2 tumor cells.

[0115] Example 4 Expression of recombinant chimeric antibody against EPHA2 and its binding to HeLa-EPHA2.

[0116] The VH and VL gene fragments of monoclonal E28 and E75 were concatenated into recombinant vectors containing the human hIgG1 heavy chain constant region and the human IgGκ light chain constant region, respectively, to obtain recombinant chimeric antibody heavy chain expression vectors and light chain expression vectors. These vectors were then transiently transfected at 293FT and used... Serum-free culture medium (Life Technologies) was cultured in shake flasks for 5-7 days. The supernatant was collected, centrifuged and ultrafiltered, and then purified by Protein A / G affinity chromatography and molecular sieve column chromatography to obtain the corresponding types of anti-EPHA2 recombinant chimeric antibodies E28 mAb and E75 mAb.

[0117] The human IgG1 heavy chain constant region sequence used in the EPHA2 recombinant chimeric antibody is shown in SEQ ID NO:18.

[0118] The human IgGκ light chain constant region sequence is shown in SEQ ID NO:19.

[0119] HeLa-EPHA2 cells were seeded in 24-well cell culture dishes. The next day, recombinant chimeric antibodies E28 mAb and E75 mAb were used as primary antibodies, and AF594-labeled Goat Anti-human IgG (Beyotime Biotechnology Co., Ltd.) was used as secondary antibodies. The cells were observed and photographed using fluorescence confocal microscopy.

[0120] like Figure 4 As shown, the chimeric antibodies E28 mAb and E75 mAb can specifically bind to HeLa-EPHA2 cells.

[0121] Example 5 In vitro binding affinity and kinetic experiments.

[0122] This embodiment uses the surface plasmon resonance (SPR) method and analyzes the data using a GE Biacore 8K instrument. Using a kit provided by Biacore, the EPHA2-His recombinant protein was covalently linked to the CM5 (GE) chip using the standard amino-coupled method. The test antibody was then serially diluted at different concentrations in the same buffer solution and injected. After injection, the antibody was regenerated using the regeneration reagent provided in the kit. Data analysis and acquisition were performed using the Biacore 8K's accompanying analysis software. The results are shown in Table 1.

[0123] Table 1. In vitro binding affinity and kinetic analysis of antibody and antigen

[0124] Example 6 Preparation of EPHA2-specific antibody-drug conjugates.

[0125] This invention selects a chimeric antibody E75 mAb with high affinity and reacts it with TCEP at 25°C for 2 hours to reduce the disulfide bonds between antibody chains. After reduction, the antibody is fed into the reaction at a molar ratio of 1:6 to val-cit-MMAE drug and coupled at 25°C for 3 hours. After coupling, the obtained ADC sample is centrifuged at 4000 rpm for 5 minutes, and the supernatant is collected. The sample is then replaced with ADC storage buffer using a 30 KD ultrafiltration tube. Ultrafiltration is required at least 3 times to remove uncoupled small molecules. After detecting the concentration of the antibody-conjugated drug, it is filtered through a 0.22 μm filter membrane in a clean bench. The antibody-conjugated drug is aliquoted in the dark and stored at -80°C.

[0126] Example 7 In vitro antitumor activity of EPHA2-specific antibody-drug conjugate.

[0127] HT29, GP2D, SW40, and SK-CO-1 were added at 100 μL per well, 5.0 × 10⁻⁶. 3 Cells were evenly seeded at a density in 96-well cell culture plates and cultured overnight. The next day, E75-MMAE (Example 6) was added to the cells at the following concentration gradients: 0, 0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 μM. Each concentration was repeated in triplicate. After 72 h of drug uptake, 10 μL of 5 mg / mL MTT solution was added to each well in the dark, and the cells were incubated at 37°C for another 4 h. After the culture was completed, the drug-containing medium was discarded, and 150 μL of LDMSO was added to each well. After shaking the plate for 5 min, the absorbance of each well was measured at 570 nm. The ADC sample E75-MMAE was obtained by conjugating VCMMAE with the recombinant chimeric antibody EPHA2 (E75 mAb). Cell activity was compared with the control IgG-MMAE, revealing that IgG-MMAE had virtually no cell-killing ability, while the ADC sample E75-MMAE exhibited excellent tumor cell-killing ability. This indicates that conjugation of the antibody with VCMMAE significantly enhances the anti-tumor effect.

[0128] Example 8 Anti-tumor experiments using xenograft mouse models.

[0129] This embodiment uses a xenograft mouse model to evaluate the in vivo antitumor activity of the EPHA2-targeting antibody-drug conjugate E75-MMAE (Example 6). An immunodeficient mouse model was used for evaluation.

[0130] NCG severe immunodeficiency mouse model: NCG severe immunodeficiency mice were purchased from the Institute of Model Animals, Nanjing University. 2×10 6 HT29 cells in the logarithmic growth phase were subcutaneously in the right posterior back of NCG mice. After approximately 6 days, the tumor grew to 200 mm. 3 Subsequently, mice with uniformly sized tumors were randomly divided into groups of 5 mice each. A control group received the same volume of physiological saline as the administered medication. Administration was via tail vein injection, with the following dosages based on mouse weight: EPHA2 monoclonal antibody 5 mg / kg, E75-MMAE 5 mg / kg, and E75-MMAE 10 mg / kg, administered every 3 days for a total of 4 doses. Mice were weighed and tumor size measured every 3 days. The average tumor volume was calculated using the formula V = 1 / 2 (L × W²), where L represents the length of the tumor and W represents its width. When the tumor volume reached 2000 mm², the tumor was considered the target. 3 If obvious ulceration occurs on the tumor surface, the mouse is euthanized, and the animal experiment ends. All data are mean ± SD. P-values ​​were obtained using an unpaired two-tailed Student's t-test. Experimental results are as follows: Figure 7 As shown.

[0131] Depend on Figure 7 The results showed that, compared with the control group without administration, the E75MMAE group had a significant inhibitory effect on the growth of HT29 tumor cells, and the tumor inhibition effect was more obvious with the increase of the administration concentration. Compared with the control group, there was no significant difference in the change of body weight of mice after administration.

[0132] The sequence information involved in this application is shown in the table below.

[0133] Table 2. Sequence Information

[0134] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody against EPHA2 or an antigen-binding fragment thereof, characterized in that, It includes: The amino acid sequences are HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:6, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO:7; or, The amino acid sequences are HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:14, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:

15.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one of the following systems: Kabat, Chothia, IMGT, AbM, or Contact. Optionally, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO:1~4, STS, and SEQ ID NO:5, respectively; or, The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:9~12, RTS and SEQ ID NO:13, respectively.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7; or, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:14; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

15. Optionally, the antibody further includes a constant region; Optionally, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans; Optionally, the heavy chain constant region is selected from the heavy chain constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; and / or; the light chain constant region is selected from the κ-type or λ-type light chain constant region; Optionally, the constant region of the antibody includes: a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:18; and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:19; Optionally, the antigen-binding fragment includes any one of: Fab, Fab', F(ab')2, scFv, and Fv; Optionally, when the antigen-binding fragment is scFv, the amino acid sequence of scFv is as shown in SEQ ID NO:8 or 16; Optionally, the antibody is selected from any one of: monovalent antibody, multivalent antibody, multispecific antibody, chimeric antibody, and fusion antibody; Optionally, the multispecific antibody includes any one of bispecific antibodies, trispecific antibodies, and tetraspecific antibodies; Optionally, the multispecific antibody includes: an anti-CD3 antibody and the anti-EPHA2 antibody or an antigen-binding fragment thereof; Optionally, the anti-CD3 antibody includes a single-chain antibody against CD3; Optionally, the amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO:

17.

4. An antibody conjugate, characterized in that, It includes: the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3; Optionally, the antibody conjugate may further include a label, purification tag, and / or solid-phase carrier conjugated to the anti-EPHA2 antibody or its antigen-binding fragment.

5. A reagent or kit, characterized in that, It includes: The antibody against EPHA2 as described in any one of claims 1 to 3, or the antigen-binding fragment thereof, or the antibody conjugate as described in claim 4.

6. An immunoconjugate or pharmaceutical composition, characterized in that, It includes the anti-EPHA2 antibody or its antigen-binding fragment as described in any one of claims 1 to 3; Optionally, the immune conjugate further includes a therapeutic agent; Optionally, the therapeutic agent includes at least one of the following: mitosis inhibitors, chemotherapeutic drugs, radionuclides, photosensitizers, photothermal agents, immune checkpoint inhibitors, toxins, factors, kinase inhibitors, antibodies against inhibitory second signaling molecules, PD-L1 inhibitors, and PD-1 / PD-L1 monoclonal antibody drugs; Optionally, the mitotic inhibitor includes at least one of dolalastatin, aristatin, maytansine, and phytoalkaloids; Optionally, the alistatin is monomethylalistatin F or monomethylalistatin E; Optionally, the anti-EPHA2 antibody or its antigen-binding fragment is conjugated to the alistatin via a linker; Optionally, the connector includes a detachable connector; Optionally, the linker is selected from any one or more combinations of: valine-citrulline (Val-Cit), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-alanine (Val-Ala), valine-lysine (Val-Lys), valine-arginine (Val-Arg), phenylalanine-citrulline (Phe-Cit), phenylalanine-lysine (Phe-Lys), and phenylalanine-arginine (Phe-Arg); Optionally, the pharmaceutical composition may further include at least one of a pharmaceutical excipient, a carrier, and a diluent.

7. The use of the anti-EPHA2 antibody or its antigen-binding fragment as described in any one of claims 1 to 3, the antibody conjugate as described in claim 4, or the immunoconjugate or pharmaceutical composition as described in claim 6 in the preparation of products for the prevention or treatment of EPHA2-positive tumors, the preparation of products for the detection of EPHA2, or the detection of EPHA2 for non-disease treatment or diagnostic purposes; Optionally, the EPHA2-positive tumors include at least one of breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, glioblastoma, head and neck cancer, and osteosarcoma; Optionally, the product includes: At least one of immune cells, reagents, kits, drugs, and drug compositions.

8. An isolated nucleic acid or a vector containing said nucleic acid, characterized in that, It encodes the anti-EPHA2 antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

9. A recombinant cell, characterized in that, It contains the isolated nucleic acid as described in claim 8 or a vector containing said nucleic acid.

10. The method for preparing the anti-EPHA2 antibody or its antigen-binding fragment as described in any one of claims 1 to 3, characterized in that, It includes: Cultivate the recombinant cells as described in claim 9.

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