Anti-EphA2 antibodies and their applications

Abstract

This invention relates to the field of antibodies, and particularly to anti-EphA2 antibodies and their applications. This invention provides anti-EPH receptor A2 (EphA2) antibodies and their uses. This application also provides chimeric and humanized antibody forms of anti-EphA2 antibodies. These antibodies have high affinity for the human EphA2 protein and are promising antibody molecules for the prevention and / or treatment of EphA2-related diseases, such as EphA2-related tumors, osteoporosis, Alzheimer's disease, etc.

Description

Technical Field

[0001] This invention relates to the field of antibodies, and particularly to anti-EphA2 antibodies and their applications. Background Technology

[0002] Tumorigenesis results from somatic cell gene mutations, with the tumor microenvironment, extracellular matrix, and immune system playing crucial roles in its development. Tumors can only progress or kill the host when cancerous cells evade immune surveillance and overcome immune control. Therefore, improving the human immune system and regulating the immune microenvironment are important means of treating or preventing tumor development. With a deeper understanding of tumor immune mechanisms, tumor immunotherapy has rapidly developed, becoming a treatment option for many patients with advanced cancer, significantly improving their survival and quality of life.

[0003] Pancreatic cancer is one of the most challenging malignant tumors to treat, characterized by its high invasiveness, early metastasis, and strong resistance to treatment. The average 5-year survival rate is only 4%. Currently, surgical resection is the only feasible treatment; however, 80%-85% of pancreatic cancer patients are diagnosed at an advanced stage, thus losing the opportunity for surgery. The poor prognosis of pancreatic cancer and other cancers has prompted researchers to search for new therapeutic targets.

[0004] EphA2 (Ephrin type-A receptor 2) is a tyrosine kinase receptor molecule identified in 1990, with EphrinA1 as its ligand. EphA2 protein is highly expressed in many tumors, such as skin cancer, gastrointestinal tumors, prostate cancer, ovarian cancer, and breast cancer, and is a molecular marker of poor prognosis in cancer patients. EphA2 exerts its effects in two modes: one is a ligand-independent oncogene mode, where EphA2 promotes cell proliferation and migration by activating downstream PI3K and Rho / Rac1 GTPase / MAPK signaling pathways; the other is an EphrinA1 ligand-dependent tumor suppressor mode, where EphrinA1-EphA2 binding can feedback-inhibit the MAPK pathway, suppressing tumor cell proliferation and migration. Professor Ye Hu et al. discovered that some pancreatic cancer patients develop resistance to the targeted drug gemcitabine. This resistance is transmitted between tumor cells through exosomes, and the internalization of exosomes is EphA2-dependent. EphA2 overexpression is also involved in mediating treatment resistance in breast cancer, cervical cancer, and melanoma. Recent studies have shown that inhibiting EphA2 can reduce the invasiveness of pancreatic cancer and increase the sensitivity of melanoma to vemurafenib and breast cancer to tamoxifen, suggesting that EphA2 may be a target for cancer immunotherapy.

[0005] In 2018, Japanese scientists developed an EphA2 agonist antibody. This antibody exhibits internalization properties, and in vitro experiments have demonstrated that EphA2 antibody conjugated with an immunotoxicant can kill melanoma cells. Currently, there are no therapeutic EphA2 antibodies on the market domestically or internationally; they are in the preclinical development stage. These data suggest that EphA2 may be a promising therapeutic target for pancreatic cancer and other EphA2-positive tumors.

[0006] Alzheimer's disease (AD) is a progressive neurodegenerative disease. Clinically, it is characterized by comprehensive dementia manifestations such as memory impairment, agnosia, and personality and behavioral changes. Current research on Alzheimer's disease mainly focuses on amyloid plaques and neurofibrillary tangles. Studies have found that Eph / Ephrin proteins are involved in the brain's learning and memory processes. Among them, EphB2 protein is involved in regulating the differentiation of presynaptic terminals. Soluble amyloid oligomers can alter the homeostasis of NMDA receptors, and EphB2 interacts with NMDA receptors, suggesting that Eph proteins may be involved in the progression of Alzheimer's disease. Other studies have found that the levels of miR-214 and EphrinA2 in the serum exosomes of osteoporosis patients are significantly upregulated. These exosomes interact with EphA2 receptors on osteoblast membranes through EphrinA2, thereby inhibiting osteoblast activity and causing osteoporosis. These data also suggest that EphA2 may be a target for the treatment of Alzheimer's disease and osteoporosis.

[0007] Therefore, providing anti-EphA2 antibodies and their applications are of significant practical importance. Summary of the Invention

[0008] In view of this, the present invention provides an anti-EphA2 antibody and its applications. This antibody, which specifically binds to EphA2, or its antigen-binding moiety, can induce EphA2-mediated anti-tumor immune responses and / or inhibit tumor growth and diseases such as Alzheimer's disease and osteoporosis.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides an antibody or antigen-binding moiety thereof that specifically binds to EphA2, comprising a heavy chain variable region;

[0011] The heavy chain variable region contains any one or more of the HCDR1, HCDR2, or HCDR3 sequences:

[0012] (I) The amino acid sequence of the HCDR1 has the amino acid sequence shown in SEQ ID No. 1, 7 or 13; or

[0013] The amino acid sequence of HCDR2 has the amino acid sequence shown in SEQ ID No. 2, 8 or 14; or

[0014] The amino acid sequence of the HCDR3 has the amino acid sequence shown in SEQ ID No. 3, 9 or 15;

[0015] or

[0016] (II) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as described in (I), and which has the same function as the amino acid sequence described in (I); or

[0017] (III) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (I) or (II).

[0018] In some specific embodiments of the present invention, the antibody or its antigen-binding portion includes a heavy chain variable region;

[0019] The heavy chain variable region contains any one or more of the HCDR1, HCDR2, or HCDR3 sequences:

[0020] (I-1) The amino acid sequence of HCDR1 has the amino acid sequence shown in SEQ ID No. 1; or

[0021] The amino acid sequence of HCDR2 has the amino acid sequence shown in SEQ ID No. 2; or

[0022] The amino acid sequence of HCDR3 is as shown in SEQ ID No. 3;

[0023] or

[0024] (II-1) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (I-1), and having the same function as the amino acid sequence described in (I-1); or

[0025] (III-1) An amino acid sequence having more than 90% identity with the amino acid sequence described in (I-1) or (II-1); or

[0026] (I-2) The amino acid sequence of the HCDR1 has the amino acid sequence shown in SEQ ID No. 7; or

[0027] The amino acid sequence of HCDR2 has the amino acid sequence shown in SEQ ID No. 8; or

[0028] The amino acid sequence of HCDR3 is as shown in SEQ ID No. 9;

[0029] or

[0030] (II-2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (I-2), and which has the same function as the amino acid sequence described in (I-2); or

[0031] (III-2) An amino acid sequence having more than 90% identity with the amino acid sequence described in (I-2) or (II-2); or

[0032] (I-3) The amino acid sequence of the HCDR1 is as shown in SEQ ID No. 13; or

[0033] The amino acid sequence of HCDR2 has the amino acid sequence shown in SEQ ID No. 14; or

[0034] The amino acid sequence of HCDR3 is as shown in SEQ ID No. 15;

[0035] or

[0036] (II-3) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (I-3), and having the same function as the amino acid sequence described in (I-3); or

[0037] (III-3) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (I-3) or (II-3).

[0038] In some specific embodiments of the present invention, the antibody or its antigen-binding portion further comprises a light chain variable region;

[0039] The light chain variable region comprises any one or more of the LCDR1, LCDR2, or LCDR3 sequences:

[0040] (I) The amino acid sequence of the LCDR1 has the amino acid sequence shown in SEQ ID No. 4, 10 or 16; or

[0041] The amino acid sequence of LCDR2 has the amino acid sequence shown in SEQ ID No. 5, 11 or 17; or

[0042] The amino acid sequence of the LCDR3 has the amino acid sequence shown in SEQ ID No. 6, 12 or 18;

[0043] or

[0044] (II) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as described in (I), and which has the same function as the amino acid sequence described in (I); or

[0045] (III) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (I) or (II).

[0046] In some specific embodiments of the present invention, the antibody or its antigen-binding portion further comprises a light chain variable region;

[0047] The light chain variable region comprises any one or more of the LCDR1, LCDR2, or LCDR3 sequences:

[0048] (I-1) The amino acid sequence of the LCDR1 has the amino acid sequence shown in SEQ ID No. 4; or

[0049] The amino acid sequence of LCDR2 has the amino acid sequence shown in SEQ ID No. 5; or

[0050] The amino acid sequence of the LCDR3 is as shown in SEQ ID No. 6;

[0051] or

[0052] (II-1) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (I-1), and having the same function as the amino acid sequence described in (I-1); or

[0053] (III-1) An amino acid sequence having more than 90% identity with the amino acid sequence described in (I-1) or (II-1); or

[0054] (I-2) The amino acid sequence of the LCDR1 has the amino acid sequence shown in SEQ ID No. 10; or

[0055] The amino acid sequence of LCDR2 has the amino acid sequence shown in SEQ ID No. 11; or

[0056] The amino acid sequence of the LCDR3 is as shown in SEQ ID No. 12;

[0057] or

[0058] (II-2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (I-2), and which has the same function as the amino acid sequence described in (I-2); or

[0059] (III-2) An amino acid sequence having more than 90% identity with the amino acid sequence described in (I-2) or (II-2); or

[0060] (I-3) The amino acid sequence of the LCDR1 has the amino acid sequence shown in SEQ ID No. 16; or

[0061] The amino acid sequence of LCDR2 has the amino acid sequence shown in SEQ ID No. 17; or

[0062] The amino acid sequence of the LCDR3 is as shown in SEQ ID No. 18;

[0063] or

[0064] (II-3) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (I-3), and having the same function as the amino acid sequence described in (I-3); or

[0065] (III-3) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (I-3) or (II-3).

[0066] In some specific embodiments of the present invention, the antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region;

[0067] The heavy chain variable region contains any one or more of the HCDR1, HCDR2, or HCDR3 sequences:

[0068] (I) The amino acid sequence of HCDR1 has the amino acid sequence shown in SEQ ID No. 1; or

[0069] The amino acid sequence of HCDR2 has the amino acid sequence shown in SEQ ID No. 2; or

[0070] The amino acid sequence of HCDR3 is as shown in SEQ ID No. 3;

[0071] or

[0072] (II) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as described in (I), and which has the same function as the amino acid sequence described in (I); or

[0073] (III) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (I) or (II);

[0074] The light chain variable region comprises any one or more of the LCDR1, LCDR2, or LCDR3 sequences:

[0075] (IV) The amino acid sequence of the LCDR1 has the amino acid sequence shown in SEQ ID No. 4; or

[0076] The amino acid sequence of LCDR2 has the amino acid sequence shown in SEQ ID No. 5; or

[0077] The amino acid sequence of the LCDR3 is as shown in SEQ ID No. 6;

[0078] or

[0079] (V) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (IV), and having the same function as the amino acid sequence described in (IV); or

[0080] (VI) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (IV) or (V).

[0081] In some specific embodiments of the present invention, the antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region;

[0082] The heavy chain variable region contains any one or more of the HCDR1, HCDR2, or HCDR3 sequences:

[0083] (I) The amino acid sequence of the HCDR1 is as shown in SEQ ID No. 7; or

[0084] The amino acid sequence of HCDR2 has the amino acid sequence shown in SEQ ID No. 8; or

[0085] The amino acid sequence of HCDR3 is as shown in SEQ ID No. 9;

[0086] or

[0087] (II) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as described in (I), and which has the same function as the amino acid sequence described in (I); or

[0088] (III) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (I) or (II);

[0089] The light chain variable region comprises any one or more of the LCDR1, LCDR2, or LCDR3 sequences:

[0090] (IV) The amino acid sequence of the LCDR1 is as shown in SEQ ID No. 10; or

[0091] The amino acid sequence of LCDR2 has the amino acid sequence shown in SEQ ID No. 11; or

[0092] The amino acid sequence of the LCDR3 is as shown in SEQ ID No. 12;

[0093] or

[0094] (V) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (IV), and having the same function as the amino acid sequence described in (IV); or

[0095] (VI) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (IV) or (V).

[0096] In some specific embodiments of the present invention, the antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region;

[0097] The heavy chain variable region contains any one or more of the HCDR1, HCDR2, or HCDR3 sequences:

[0098] (I) The amino acid sequence of HCDR1 has the amino acid sequence shown in SEQ ID No. 13; or

[0099] The amino acid sequence of HCDR2 has the amino acid sequence shown in SEQ ID No. 14; or

[0100] The amino acid sequence of HCDR3 is as shown in SEQ ID No. 15;

[0101] or

[0102] (II) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids as described in (I), and which has the same function as the amino acid sequence described in (I); or

[0103] (III) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (I) or (II);

[0104] The light chain variable region comprises any one or more of the LCDR1, LCDR2, or LCDR3 sequences:

[0105] (IV) The amino acid sequence of the LCDR1 has the amino acid sequence shown in SEQ ID No. 16; or

[0106] The amino acid sequence of LCDR2 has the amino acid sequence shown in SEQ ID No. 17; or

[0107] The amino acid sequence of the LCDR3 is as shown in SEQ ID No. 18;

[0108] or

[0109] (V) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence described in (IV), and having the same function as the amino acid sequence described in (IV); or

[0110] (VI) An amino acid sequence that has more than 90% identity with the amino acid sequence described in (IV) or (V).

[0111] In some specific embodiments of the present invention, the antigen-binding portion is selected from: Fab fragment, Fab' fragment, F(ab')2 fragment, scFv fragment, Fd fragment or single-domain antibody.

[0112] In some specific embodiments of the present invention, the antibody is a murine antibody, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 19, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 22; or

[0113] The antibody is a chimeric antibody, optionally comprising a heavy chain with an amino acid sequence as shown in SEQ ID No. 20, and / or a light chain with an amino acid sequence as shown in SEQ ID No. 23; or

[0114] The antibody is a humanized antibody, and optionally, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 21, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 24.

[0115] In some specific embodiments of the present invention, the EphA2 is a primate EphA2; preferably, the primate EphA2 is selected from human EphA2 or monkey EphA2.

[0116] In some specific embodiments of the present invention, the substitution, deletion or addition of one or more amino acids is two, three, four or five.

[0117] In some specific embodiments of the present invention, the antibody or its antigen-binding portion further includes a constant region, wherein the constant region of the heavy chain of the antibody or its antigen-binding portion is any one of human IgG1, IgG2, IgG3 or IgG4; and the constant region of the light chain of the antibody or its antigen-binding portion is κ-type or λ-type.

[0118] The present invention also provides the use of the antibody or its antigen-binding portion in the preparation of medicaments for the prevention and / or treatment of EphA2-related diseases.

[0119] In some specific embodiments of the present invention, the EphA2-related disease is one or more of tumors, osteoporosis, or Alzheimer's disease; preferably, the tumor is selected from one or more of the following: gastric cancer, pancreatic cancer, intestinal cancer, esophageal cancer, liver cancer, ovarian cancer, lung cancer, and bladder cancer, or metastases of the above tumors.

[0120] The antibodies or antigen-binding moieties that specifically bind to EphA2 provided by this invention can induce EphA2-mediated anti-tumor immune responses and / or inhibit tumor growth and diseases such as Alzheimer's disease and osteoporosis. Attached Figure Description

[0121] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0122] Figure 1 FACS screening for anti-human EphA2 hybridoma antibodies;

[0123] Figure 2 FACS screening shows that candidate clonal antibodies compete with ligands for binding;

[0124] Figure 3 FACS identification of anti-EphA2 chimeric antibody clones; (A) binding of 13E4 and 1D6 antibodies to human colon cancer cells HCT116; (B) binding of 6D5 antibody to human colon cancer cells HCT116;

[0125] Figure 4(A) shows the binding identification results of anti-EphA2 humanized 13E4 antibody with human hepatocellular carcinoma cells HepG2; Figure 4(B) shows the binding identification results of anti-EphA2 humanized 13E4 antibody with human lung cancer cells HCC827; Figure 4(C) shows the binding identification results of anti-EphA2 humanized 13E4 antibody with human breast cancer cells MDA-MB-231.

[0126] Figure 5(A) shows the binding activity of 11D6 with human / mouse EphA2 protein; Figure 5(B) shows the binding activity of 6D5 with human / mouse EphA2 protein.

[0127] Figure 6 The effect of anti-EphA2 antibody 6D5 on osteoclast-related gene activity was investigated. Detailed Implementation

[0128] This invention discloses an anti-EphA2 antibody and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0129] definition

[0130] As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains: two heavy chains (H) and two light chains (L) linked by disulfide bonds, and its multimers (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated VH) and a heavy chain constant region (abbreviated CH). The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated VL) and a light chain constant region (abbreviated CL). The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with conserved regions called framework regions (FRs). In some embodiments, from the N-terminus to the C-terminus, both the light chain and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0131] As used herein, the term "antigen-binding moiety" of an antibody refers to a portion or segment of the complete antibody molecule responsible for binding an antigen. The antigen-binding moiety may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. The antigen-binding moiety of an antibody can be prepared from the complete antibody molecule using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering. Non-limiting examples of antigen-binding moieties include: Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-chain Fv (scFv) molecules, single-domain antibodies, dAb fragments, and the smallest recognition unit (e.g., isolated CDR) consisting of amino acid residues mimicking the hypervariable region of an antibody. The term "antigen-binding moiety" also includes other engineered molecules such as biantibodies, triantibodies, tetraantibodies, and microantibodies. For example, the Fd fragment mentioned in this article refers to an antibody fragment composed of VH and CH1 domains; the Fv fragment is composed of VL and VH domains in the single arm of the antibody; and the dAb fragment (Ward et al., Nature 1989; 341: 544-546) is composed of VH domains.

[0132] It is well known to those skilled in the art that complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on antibody affinity and specificity. There are two common ways to define the CDR sequence for VH or VL: the Kabat definition and the Chothia definition, for example, see Kabat et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, MD. (1991); Al-Lazikani et al., J Mol Biol 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). For a given antibody's variable region sequence, the CDR sequence in the VH and VL sequences can be determined according to either the Kabat or Chothia definition. In the embodiments of this application, the Kabat definition of the CDR sequence is used. In this paper, the CDR1, CDR2 and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2 and HCDR3, respectively; the CDR1, CDR2 and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2 and LCDR3, respectively.

[0133] For a given antibody's variable region sequence, the CDR region sequence can be analyzed in various ways, such as using the online software Abysis (http: / / www.abysis.org / ).

[0134] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope, or the ability of an antibody to bind to a specific antigen with an affinity at least twice that of a nonspecific antigen. However, it should be understood that antibodies can specifically bind to two or more antigens associated with their sequence. For example, the antibody of this application can specifically bind to human EphA2.

[0135] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for the possibility of naturally occurring mutations in a small number of individuals. Monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remaining portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies, provided they exhibit the desired biological activity (see, U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

[0136] As used herein, the term "mouse antibody" refers to any antibody in which all domain sequences are mouse sequences. Such antibodies can be produced via hybridomas.

[0137] As used herein, the term "chimeric antibody" refers to an antibody comprising segments derived from two or more different antibodies. In some embodiments, one or more CDRs are derived from mouse anti-EphA2 antibodies. In other embodiments, all CDRs are derived from mouse anti-EphA2 antibodies. In some embodiments, CDRs derived from more than one mouse anti-EphA2 antibody are combined in the chimeric antibody. For example, the chimeric antibody may comprise CDR1 from the light chain of a first mouse anti-EphA2 antibody, CDR2 from the light chain of a second mouse anti-EphA2 antibody, and CDR3 from the light chain of a third mouse anti-EphA2 antibody, and CDRs from the heavy chain may be derived from one or more other anti-EphA2 antibodies. Furthermore, the framework regions may be derived from the same anti-EphA2 antibody or from one or more different individuals.

[0138] As used herein, the term "humanized antibody" refers to a CDR-transplanted antibody, specifically an antibody generated by transplanting a mouse CDR region sequence into the variable region framework of a human antibody. The aim is to overcome the strong immune side effects induced in humans by chimeric antibodies that carry a large number of protein components from other species, such as mice.

[0139] As used in this article, the term "nucleic acid molecule" can refer to DNA molecules and RNA molecules, which can be single-stranded or double-stranded. Nucleic acid molecules can also be cDNA.

[0140] As used herein, the term "EphA2-related disease" includes diseases and / or conditions associated with the EphA2 signaling pathway. Exemplary EphA2-related diseases or conditions include tumors, cancers such as stomach cancer, pancreatic cancer, colorectal cancer, esophageal cancer, liver cancer, ovarian cancer, lung cancer, and bladder cancer, as well as metastatic cancers described above, and Alzheimer's disease and osteoporosis.

[0141] As used herein, the term "immune response" refers to the biological response within a vertebrate to an invading agent that protects the organism against such agents and the diseases they cause. An immune response is mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and any of these cells or by soluble macromolecules (including antibodies, cytokines, and complement) produced by the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or normal human cells or tissues in cases of autoimmunity or pathological inflammation. Immune responses include T cells (e.g., effector T cells) or Th cells (e.g., CD4+). + or CD8 + The activation or inhibition of T cells or the suppression of Treg cells.

[0142] As used in this article, the term "cancer" refers to a large class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Disordered cell division can form malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0143] As used herein, the term “treatment” means any type of intervention or method performed on a subject or the administration of an active agent thereto, wherein the aim is to reverse, alleviate, improve, suppress or relieve or prevent symptoms, complications, conditions or progression, development, severity or recurrence of disease.

[0144] As used in this article, the term "prevention" refers to administration to subjects who do not have the disease in order to prevent the disease from occurring or to minimize its effects (if any).

[0145] The inventors screened three hybridoma cell lines, whose supernatants contained antibodies capable of binding to EphA2-expressing cells. Therefore, this application provides three novel anti-EphA2 antibodies or their antigen-binding moieties that specifically bind to EphA2. The inventors also prepared chimeric and humanized antibody forms from murine anti-EphA2 antibodies using genetic engineering techniques. These antibodies can specifically bind to EphA2 expressed on the cell surface, thereby effectively inducing an EphA2-mediated immune response and playing a role in the prevention or treatment of EphA2-related diseases.

[0146] In a first aspect, this application provides an antibody that specifically binds to EphA2 or its antigen-binding 13E4 clone, which includes a heavy chain variable region comprising any one or more of the HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1 sequence is GYTFTSYWIQ (SEQ ID No. 1), the HCDR2 sequence is YINPSTGYN ENSQKFKD (SEQ ID No. 2), and the HCDR3 sequence is RGTWGFAY (SEQ ID No. 3).

[0147] In a preferred embodiment, the heavy chain variable region of the antibody or its antigen-binding portion described herein comprises the sequences HCDR1 shown in SEQ ID No. 1, HCDR2 shown in SEQ ID No. 2, and HCDR3 shown in SEQ ID No. 3.

[0148] In some embodiments, the antibody or its antigen-binding portion further comprises a light chain variable region comprising any one or more of the LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1 sequence is RASENINSYLT (SEQ ID No. 4), the LCDR2 sequence is NAKTLAE (SEQ ID No. 5), and the LCDR3 sequence is QHHYVTPLT (SEQ ID No. 6).

[0149] In a preferred embodiment, the light chain variable region of the antibody or its antigen-binding portion described herein includes the LCDR1 sequence shown in SEQ ID No. 4, the LCDR2 sequence shown in SEQ ID No. 5, and the LCDR3 sequence shown in SEQ ID No. 6.

[0150] In a preferred embodiment, the antibody or its antigen-binding portion described herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the HCDR1 sequence shown in SEQ ID No. 1, the HCDR2 sequence shown in SEQ ID No. 2, and the HCDR3 sequence shown in SEQ ID No. 3, and the light chain variable region comprises the LCDR1 sequence shown in SEQ ID No. 4, the LCDR2 sequence shown in SEQ ID No. 5, and the LCDR3 sequence shown in SEQ ID No. 6.

[0151] This application provides an antibody that specifically binds to EphA2 or its antigen-binding portion 1D6 clone, which includes a heavy chain variable region comprising any one or more of the HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1 sequence is GYTFTSYWIQ (SEQ ID No. 7), the HCDR2 sequence is IDPSDSYT (SEQ ID No. 8), and the HCDR3 sequence is ARGAY (SEQ ID No. 9).

[0152] In a preferred embodiment, the heavy chain variable region of the antibody or its antigen-binding portion described herein includes the HCDR1 sequence shown in SEQ ID No. 7, the HCDR2 sequence shown in SEQ ID No. 8, and the HCDR3 sequence shown in SEQ ID No. 9.

[0153] In some embodiments, the antibody or its antigen-binding portion further comprises a light chain variable region comprising any one or more of the LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1 sequence is RASENINSYLT (SEQ ID No. 10), the LCDR3 sequence is QHHYVTPLT (SEQ ID No. 12).

[0154] In a preferred embodiment, the light chain variable region of the antibody or its antigen-binding portion described herein includes the LCDR1 sequence shown in SEQ ID No. 10, the LCDR2 sequence shown in SEQ ID No. 11, and the LCDR3 sequence shown in SEQ ID No. 12.

[0155] This application provides an antibody that specifically binds to EphA2 or its antigen-binding 6D5 clone, which includes a heavy chain variable region comprising any one or more of the HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1 sequence is GFNIKDTY (SEQ ID No. 13), the HCDR2 sequence is VDPANGKI (SEQ ID No. 14), and the HCDR3 sequence is AKHYGVTYAMDY (SEQ ID No. 15).

[0156] In a preferred embodiment, the heavy chain variable region of the antibody or its antigen-binding portion described herein includes the sequences HCDR1 shown in SEQ ID No. 13, HCDR2 shown in SEQ ID No. 14, and HCDR3 shown in SEQ ID No. 15.

[0157] In some embodiments, the antibody or its antigen-binding portion further comprises a light chain variable region comprising any one or more of the LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1 sequence is QGISNY (SEQ ID No. 16), the LCDR2 sequence is YTS (SEQ ID No. 17), and the LCDR3 sequence is QHGDTLPT (SEQ ID No. 18).

[0158] In a preferred embodiment, the light chain variable region of the antibody or its antigen-binding portion described herein includes the LCDR1 sequence shown in SEQ ID No. 16, the LCDR2 sequence shown in SEQ ID No. 17, and the LCDR3 sequence shown in SEQ ID No. 18.

[0159] In some more specific embodiments, the antibodies disclosed herein may be anti-human EphA2 monoclonal antibodies. The type and subtype of anti-EphA2 antibodies may be determined by any means known in the art. Typically, antibody type and subtype are determined using antibodies specific to a particular antibody type and subtype. Anti-EphA2 antibody isotypes can be determined using ELISA assays, for example, using mouse Ig-adsorbed anti-human Ig to identify human Ig.

[0160] In some embodiments, the antibody described herein is a murine antibody, preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 19, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 22.

[0161] In a preferred embodiment, the murine antibody described herein comprises the heavy chain variable region shown in SEQ ID No. 20 and the light chain variable region shown in SEQ ID No. 23.

[0162] In some embodiments, the antibodies described herein are chimeric antibodies. The chimeric antibodies described herein comprise a variable region of a murine antibody (including a heavy chain variable region VH and / or a light chain variable region VL) and a constant region of a human antibody.

[0163] In a preferred embodiment, the chimeric antibody described herein comprises a variable region (including a heavy chain variable region and a light chain variable region) of a murine antibody and a constant region of a human antibody.

[0164] Preferably, the chimeric antibody comprises the heavy chain shown in SEQ ID No. 21 and / or the light chain shown in SEQ ID No. 24.

[0165] In a preferred embodiment, the chimeric antibody described herein comprises the heavy chain shown in SEQ ID No. 21 and the light chain shown in SEQ ID No. 24.

[0166] In some embodiments, the antibodies described herein are humanized antibodies. The humanized antibodies described herein comprise the CDR region of a murine antibody (including any one or more of HCDR1, HCDR2, and HCDR3 and / or any one or more of LCDR1, LCDR2, and LCDR3), the framework region of a human antibody variable region (including any one or more of FR1, FR2, FR3, and FR4), and optionally, the constant region of the human antibody.

[0167] In a preferred embodiment, the humanized antibody described herein comprises the CDR region of a murine antibody (including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3), the framework region of a human antibody variable region (including FR1, FR2, FR3, and FR4), and optionally the constant region of a human antibody.

[0168] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 19, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 22.

[0169] In a preferred embodiment, the humanized antibody described herein comprises the heavy chain variable region shown in SEQ ID No. 20 and the light chain variable region shown in SEQ ID No. 23.

[0170] The antibodies described herein may also contain constant regions of mouse or human antibodies. Mouse antibody constant regions include the heavy chain constant regions of mouse IgG1, IgG2a, IgG2b, or IgG3, as well as the κ or λ type light chain constant regions. Human antibody constant regions include the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4, as well as the κ or λ type light chain constant regions.

[0171] In some implementations, the EphA2 described herein is a primate EphA2. Preferably, the primate EphA2 described herein is selected from human EphA2 or monkey EphA2.

[0172] In some implementations, the antigen-binding portion described herein is selected from: Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, Fd fragment, or single-domain antibody.

[0173] As used in this article, the term "Fab fragment" includes both the light chain and the CH1 and variable regions of the heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0174] As used herein, the term "Fab' segment" comprises a portion or fragment of a light chain and a heavy chain, the portion or fragment containing a VH domain and a CH1 domain, as well as a region between the CH1 and CH2 domains, such that interchain disulfide bonds can be formed between the two heavy chains of two Fab' segments to form an F(ab')2 molecule.

[0175] As used herein, the term "F(ab')2 fragment" comprises two light chains and two heavy chains, the heavy chains containing a portion of a constant region between the CH1 and CH2 domains, such that interchain disulfide bonds are formed between the two heavy chains. The F(ab')2 fragment is thus composed of two Fab' fragments, which are linked together by disulfide bonds between the two heavy chains.

[0176] The term "Fv fragment" as used in this paper includes the variable region from the heavy chain and the light chain, but lacks the constant region.

[0177] As used herein, the term "single-chain Fv" or "scFv" refers to an antibody fragment containing both VH and VL domains of the antibody, wherein these domains exist as a single polypeptide chain. Typically, Fv polypeptides also include a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0178] The term "single-domain antibody" as used in this article refers to an antigen-binding portion containing a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Originally discovered in alpaca peripheral blood, this naturally occurring antibody lacking a light chain, while containing only a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, does not readily adhere to itself or aggregate like artificially engineered single-chain antibody fragments (scFv). More importantly, the VHH structure, when cloned and expressed independently, possesses structural stability and antigen-binding activity comparable to the original heavy chain antibody, making it the smallest known unit capable of binding target antigens. VHH crystals have a molecular weight of only 15 kDa, hence they are also called nanobodies (Nb).

[0179] The method for preparing anti-EphA2 monoclonal antibodies disclosed herein may include: culturing host cells under expression conditions to express anti-EphA2 monoclonal antibodies; and isolating and purifying the expressed anti-EphA2 monoclonal antibodies. Using the above method, crude anti-EphA2 monoclonal antibodies can be obtained. Then, the anti-EphA2 monoclonal antibodies are purified to a substantially homogeneous substance, for example, as a single band on SDS-PAGE electrophoresis, by purification methods including EphA2-based affinity purification, non-denaturing gel purification, HPLC or RP-HPLC, size exclusion, purification on a protein A column, or any combination of these techniques.

[0180] This application provides the use of the antibody or its antigen-binding portion as described in the first aspect, and the host cell as described in the second aspect, in the preparation of a medicament for the prevention and / or treatment of EphA2-related diseases.

[0181] As used herein, the term "individual" refers to a mammal, including but not limited to primates, cattle, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice. Preferably, the mammal is a non-human primate or a human. A particularly preferred mammal is a human. The terms "individual" and "subject" are used interchangeably herein.

[0182] "Treatment" refers to both therapeutic procedures and preventative or preventative measures aimed at preventing or alleviating a target pathological state or symptom. Individuals requiring treatment include those who already possess the described symptom, as well as those who are likely to develop the symptom or wish to prevent it. Therefore, the individuals to be treated in this article have already been diagnosed with the symptom or are predisposed to or susceptible to it.

[0183] In any embodiment, the EphA2-related diseases are tumors, Alzheimer's disease, and osteoporosis.

[0184] In some implementations, the tumors described herein are primary or metastatic cancers. In specific implementations, the tumors are selected from lung cancer such as non-small cell lung cancer, colorectal cancer, bladder cancer, hematopoietic system cancers such as leukemia, breast cancer, gastric cancer, esophageal cancer, B-cell non-Hodgkin lymphoma, Hodgkin lymphoma, anaplastic large cell lymphoma, head and neck cancers such as squamous cell carcinoma of the head and neck, malignant glioma, renal cancer, melanoma, prostate cancer, bone cancer, giant cell tumor of bone, pancreatic cancer, ovarian cancer, sarcoma, liver cancer, squamous cell carcinoma of the skin, thyroid cancer, cervical cancer, nasopharyngeal carcinoma, endometrial cancer, or metastatic cancers of the above tumors.

[0185] It should be understood that the features, characteristics, components or steps described in a particular aspect, embodiment or example of this application may be applied to any other aspect, embodiment or example described herein, unless there is any contradiction.

[0186] This invention discloses antibodies that specifically bind to EphA2 in mammals (humans, primates, etc.). This invention provides applications of such proteins in treatment and other fields, such as their use in cancer therapy. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention. Those skilled in the art can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply this invention.

[0187] The raw materials and reagents involved in the anti-EphA2 antibody and its application provided by this invention are all commercially available.

[0188] The following embodiments are used to illustrate this application, but are not intended to limit the scope of this application. Any modifications or substitutions made to the methods, steps, or conditions of this application without departing from the spirit and substance of this application are within the scope of this application.

[0189] Unless otherwise specified, the chemical reagents used in the examples are all commercially available conventional reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0190] The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods for inserting genes encoding proteins into vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications, for example see Sambrook, J., Fritsch, E.F., and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0191] In this invention, the sequence information is recorded as follows:

[0192] 13E4 VH Mouse Source Sequence

[0193] Protein sequence

[0194] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4(117aa)

[0195] QVQLLESGAELAKPGASVKMSCKASGYTFTSYWIQWVKQRPGQGLEWIGYINPSTGYNENSQKFKDKATLTADKSSSTAYMQLSSSLTSEDSAVYYCARRGTWGFAYWGQGTLVTVSA

[0196] The 13E4 HCDR1 sequence is GYTFTSYWIQ (SEQ ID No. 1).

[0197] The 13E4 HCDR2 sequence is YINPSTGYNENSQKFKD (SEQ ID No. 2).

[0198] The 13E4 HCDR3 sequence is RGTWGFAY (SEQ ID No. 3).

[0199] 13E4 VL mouse-derived sequence (Vκ)

[0200] Protein sequence

[0201] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4(107aa)

[0202] DIVMTQTPASLSASVGETVTITCRASENINSYLTWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYVTPLTFGAGTKLELK

[0203] The 13E4 LCDR1 sequence is RASENINSYLT (SEQ ID No. 4).

[0204] The 13E4 LCDR2 sequence is NAKTLAE (SEQ ID No. 5).

[0205] The 13E4 LCDR3 sequence is QHHYVTPLT (SEQ ID No. 6).

[0206] 1D6 VH Mouse Source Sequence

[0207] DVQLLESGAELVNPGASVKISCKASGYTFTSYWLNWVKQRPGQGLEWIGDIDPSDSYTNKNQKFKDKATLTVDKSSSTAYMQLSSSLTSEDSAVYYCARGAYWGQGTLVTVSA

[0208] The 1D6 HCDR1 sequence is GYTFTSYW (SEQ ID No. 7).

[0209] The 1D6 HCDR2 sequence is IDPSDSYT (SEQ ID No. 8).

[0210] The 1D6 HCDR3 sequence is ARGAY (SEQ ID No. 9).

[0211] 1D6 VL mouse-derived sequence (Vκ)

[0212] DVVMTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVCKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK

[0213] The 1D6 LCDR1 sequence is QSLLDSDGKTY (SEQ ID No. 10).

[0214] The 1D6 LCDR2 sequence is LVC (SEQ ID No. 11).

[0215] The 1D6 LCDR3 sequence is WQGTHFPQT (SEQ ID No. 12).

[0216] 6D5 VH Mouse Source Sequence

[0217] EVQLLESGAELVKPGASVKLSCTASGFNIKDTYIHWVKLRPEQGLEWIGRVDPANGKIKYDPKFQGKATITADTSSNTAYLHLSSLTSEDAAVFYCAKHYGVTYAMDYWGQGASVTVSS

[0218] The 6D5 HCDR1 sequence is GFNIKDTY (SEQ ID No. 13).

[0219] The 6D5 HCDR2 sequence is VDPANGKI (SEQ ID No. 14).

[0220] The 6D5 HCDR3 sequence is AKHYGVTYAMDY (SEQ ID No. 15).

[0221] 6D5 VL mouse-derived sequence (Vκ)

[0222] DIQMTQTTSSLSASLGDRVTISCRASQGISNYLNWYQRKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQHGDTLPTFGGGTKLEMKKGDTPGISK

[0223] The 6D5 LCDR1 sequence is QGISNY (SEQ ID No. 16).

[0224] The 6D5 LCDR2 sequence is YTS (SEQ ID No. 17).

[0225] The 6D5 LCDR3 sequence is QHGDTLPT (SEQ ID No. 18).

[0226] 13E4 VH Humanization Sequence

[0227] Protein sequence

[0228] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIQWVKQAPGQGLEWIGYINPSTGYNENSQKFKDRVTLTADKSTSTVYMELSSLRSEDTAVYYCARRGTWGFAYWGQGTLVTVSS

[0229] The sequence of SEQ ID No. 19 is GYTFTSYWIQ (SEQ ID No. 19).

[0230] The sequence of SEQ ID No. 20 is YINPSTGYNENSQKFKD (SEQ ID No. 20).

[0231] The sequence SEQ ID No. 21 is RGTWGFAY (SEQ ID No. 21).

[0232] 13E4 VL Humanization Sequence

[0233] Protein sequence

[0234] DIQMTQSPSSSLSASVGDRVTITCRASENINSYLTWYQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYVTPLTFGGGTKVEIK

[0235] The sequence SEQ ID No. 22 is RASENINSYLT (SEQ ID No. 22).

[0236] The sequence of SEQ ID No. 23 is NAKTLAE (SEQ ID No. 23).

[0237] The sequence of SEQ ID No. 24 is QHHYVTPLT (SEQ ID No. 24).

[0238] The present invention will be further illustrated below with reference to the embodiments:

[0239] Example 1. Construction of EphA2 overexpressing cell lines

[0240] The following steps were taken to construct a high-expression cell line for human EphA2 using a stable cell line construction platform:

[0241] Construction of plasmid pEnter-EphA2-puromycin: Using EphA2 cDNA (General Biosystems (Anhui) Co., Ltd.) as a template, a fragment of approximately 3000 bp was amplified using primers and recovered by gel extraction; the pENTER plasmid was digested with KpnI / HindIII enzymes, and a large fragment of 7450 bp was recovered by gel extraction; the above-obtained fragments were ligated into the large fragment of the pENTER plasmid recovered by gel extraction with KpnI / HindIII enzymes using recombination technology, and positive clones were selected for sequencing. The plasmids with correct sequencing results were named pEnter-EphA2-puromycin.

[0242] 293T cells (from the Kyowa Cell Bank) were seeded into T25 culture flasks, with 2 × 10⁶ cells seeded per flask. 6 On the second day, the 293T cell culture medium was replaced with 4 mL of Opti-MEM (Thermofisher Scientific Cat. 31985070). 5 μg of the EphA2 plasmid pEnter-EphA2-puromycin was added to the Opti-MEM, bringing the final volume to 500 μL. Separately, 500 μL of Opti-MEM was prepared, and 7 μL of transfection reagent PEI (3 μg / mL) was added. The mixture was thoroughly combined and incubated at room temperature for 20 min before being added to the previously cultured 4 mL of 293T cells. On the third day, the cell culture medium was replaced with 5 mL of LDMEM high-glucose medium. On the fourth day, 2 μg / mL puromycin was added for selection. After 2-3 days, a large number of cells died. The medium was replaced with fresh medium until the cells reached stable growth. Single-clone selection was then performed, followed by expansion culture and cryopreservation.

[0243] The cell line stably expressing the target gene constructed in this application is named 293T-EphA2 cell. The protein sequences used are derived from publicly available databases.

[0244] Example 2. Preparation of hybridomas producing anti-EphA2 antibodies

[0245] The preparation method is as follows:

[0246] 1. Use 8-10 week old Balb / c mice and subcutaneously inject EphA2 protein (50 μg / dose / mouse). The human EphA2 protein sequence (XP_016856026.1) has an extracellular region of Glu28-Arg328, a C-terminus containing His and Fc tags, and a molecular weight of 66 kDa. Repeat this dose 3 times over 3-8 weeks. 4 days before fusion, administer EphA2 protein intraperitoneally to mice for 3 consecutive days.

[0247] 2. One day before fusion, macrophages from the peritoneal cavity of Kunming rats were used as a feeder layer and seeded into 96-well plates.

[0248] 3. Spleens from immunized mice were fused with non-secretory myeloma SP2 / 0 cell lines. The cells were added to 96-well plates pre-coated with a feeder layer, and the fused cells were subjected to HAT selection (Galfre and Milstein, Methods Enzymol 1981; 73:3-46).

[0249] 4. A group of hybridoma cells secreting anti-EphA2 specific antibodies were recovered. Initial screening was performed using enzyme-linked immunosorbent assay (ELISA) to determine the titer of anti-EphA2 antibodies secreted by the hybridomas.

[0250] Anti-EphA2 hybridoma rescreening (FACS)

[0251] The second screening method 1 is as follows:

[0252] Collect 293T-EphA2 cells, wash once with PBS buffer, and then... 5 Seed cells per well into a 96-well deep plate, add 50 μL of hybridoma supernatant to each well, and incubate for 1 hour.

[0253] Add 400 μl of FACS buffer to each well and wash twice. Add APC anti-mouse IgG Fc secondary antibody (Biolegend, Cat405308), incubate for 1 hour, and then perform flow cytometry analysis.

[0254] The results are shown in Table 1. Figure 1 As shown, five candidate antibodies were obtained, with clone numbers 1D6, 6H3, 6D5, 13A6, and 13E4.

[0255] Table 1 Screening of Anti-human EphA2 Hybridoma Antibodies

[0256]

[0257]

[0258] Second screening method 2 is as follows:

[0259] Collect 293T-EphA2EphrinA1 cells, wash once with PBS buffer, and then... 5 Cells were seeded per well into 96-well deep-well plates, and 50 μL of hybridoma supernatant was added to each well. Recombinant EphrinA1-hFc ligand protein was also added to conduct ligand competitive binding. Positive control and blank / secondary antibody control were set up without hybridoma supernatant. The cells were incubated for 1 hour.

[0260] Add 400 μl of FACS buffer to each well and wash twice. Add APC anti-human IgG Fc secondary antibody (Biolegend, Cat405308), incubate for 1 hour, and then perform flow cytometry analysis.

[0261] Table 2. Screening results of ligand competitive binding of candidate clonal antibodies.

[0262] Initial hybridoma clone number 293T-EphA2 cell binding activity Secondary antibody 1,988 EphrinA1 protein 227,994 13E4+EphrinA1 protein 22,614 1D6+EphrinA1 protein 189,106 6D5+EphrinA1 protein 166,373 6H3+EphrinA1 protein 171,080

[0263] The results are shown in Table 2. Figure 2As shown, the hybridoma cell 13E4, the antibody in the supernatant of this clone can specifically block the binding of ligand 293T-EphA2 to EphrinA1 protein, indicating that the anti-EphA2 antibody secreted by this hybridoma cell is a blocking antibody, while 1D6, 6H3 and 6D5 are affinity non-blocking antibodies.

[0264] Example 3. Binding activity of anti-EphA2 chimeric antibody to human EphA2 positive cells (FACS assay)

[0265] Preparation of anti-EphA2 chimeric antibody

[0266] cDNA was obtained from 13E4, 1D6, and 6D5 hybridoma cells. The cDNA was amplified by PCR and sequenced using antibody primers stored in our laboratory. The nucleotide sequences encoding VH-13E4, VH-1D6, and VH-6D5 by the heavy chain of the mouse anti-EphA2 antibody and VL-13E4, VL-1D6, and VL-6D5 by the corresponding light chain of the antibody were finally obtained. The light and heavy chain encoding nucleic acid sequences of the anti-EphA2 antibody were constructed into plasmids pUC57EphA2VH-13E4, pUC57EphA2VL-13E4, pUC57EphA2VH-1D6, pUC57EphA2VL-1D6, pUC57EphA2VH-6D5, and pUC57EphA2VL-6D5 (General Biosystems (Anhui) Co., Ltd.) using total synthesis. Using the corresponding pUC57EphA2VH and pUC57EphA2VL as templates, anti-EphA2 VH and anti-EphA2 antibodies were amplified using a Gold Mix PCR kit (TSINGKE Corporation) according to the kit's instructions. The VL fragment was amplified, with a product size of approximately 0.4 kb. Simultaneously, the vector plasmids pQKX1 and pQKX2 (General Biosystems (Anhui) Co., Ltd.) were digested with the restriction endonuclease SapI (NEB, R0569S). The resulting PCR amplification products and digested vectors were then recombined and ligated using the BM Seamless Cloning Kit (Bomaide Company) according to the kit's instructions to obtain the heavy chain expression vectors pQK EphA2 H-13E4, pQK EphA2 L-13E4, pQK EphA2 H-1D6, pQK EphA2 L-1D6, pQK EphA2 H-6D5, and pQK EphA2 L-6D5.

[0267] 293Fv cells (Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were diluted to 1.5 × 10⁻⁶. 6Cells / mL, final volume 200mL, cultured at 37℃ in a shaker for 24h. Dilute paired plasmids pQK EphA2 H and pQK EphA2 L with 10% of the transfection volume of fresh medium to a concentration of 1μg / mL, calculated based on the transfection cell volume. Add 200 μl of 3 mg / mL PEI to the diluted plasmid at 1 / 1000 of the cell volume, vortex immediately for 10 seconds, and incubate at room temperature for 15 minutes. Add the plasmid / PEI mixture dropwise to the cell culture medium, gently shaking the culture flask while adding. Incubate on a shaker. After 7 days, collect the culture supernatant. Use a Mabsure Lx 5 mL purification column to capture antibodies from the culture supernatant at a flow rate of 3 mL / min. Equilibrate the purification column with 5 column volumes of 20 mM PB + 150 mM NaCl, pH 7.4 equilibration buffer. After column equilibration is stable, load the antibody. After loading, elute with 20 mM PB + 150 mM NaCl, pH 7.4. After elution, elute with 50 mM citrate, pH 3.0 elution buffer. Collect the eluted antibody and neutralize with 1 M Tris-HCl, pH 9.0.

[0268] Collect one T75 cell culture flask of human colon cancer cell line HCT116, wash once with FACS buffer, resuspend in FACS buffer for counting, and adjust the cell concentration to 1×10⁻⁶. 6 Dilute the antibody to a final concentration of 10 μg / ml. Take 10 μL of each diluted antibody and add 10 μg of each antibody to 100 μL of the cell sample after adjusting the concentration. Mix well and incubate at room temperature for 1 hour.

[0269] Each well was washed twice with 400 μl of FACS buffer. The humanized antibody group was then incubated with APC anti-human IgG Fc secondary antibody for 1 hour, followed by flow cytometry analysis. Results are shown in Table 3. Figure 3 The results in Table 5 indicate that anti-chimeric 1D6, 6D5, and 13E4 all specifically bind to the tumor cell line HCT116.

[0270] Table 3 Cellular activity of anti-EphA2 chimeric antibodies 13E4, 1D6, and 6D5

[0271]

[0272] Example 4. Binding activity of anti-EphA2 humanized antibody 13E4 to human EphA2 positive cells (FACS assay)

[0273] Preparation of anti-EphA2 humanized antibody 13E4 clone

[0274] The sequence optimization services for the VH and VL nucleotide sequences encoded by the anti-EphA2 humanized antibody were provided by Shanghai Aopumai Biotechnology Co., Ltd. The VH3 and VL12 encoding nucleic acid sequences of the anti-EphA2 antibody were constructed into plasmids pUC57 EphA2 VH3 and pUC57 EphA2 VL1 through total synthesis. Using pUC57 EphA2 VH3 and pUC57 EphA2 VL1 as templates, the anti-EphA2 VH3 and EphA2 VL1 fragments were amplified using a Gold Mix PCR kit according to the kit's instructions. The amplification product size was approximately 0.4 kb. Simultaneously, the vector plasmids pQKX1 and pQKX2 were digested with the restriction endonuclease SapI (NEB, R0569S). The resulting PCR amplification products and digested vectors were then recombined and ligated using a BM Seamless Cloning Kit (Biomed) according to the kit's instructions to obtain the heavy chain expression vectors pQK EphA2 H3 and pQK EphA2 L1.

[0275] Dilute 293Fv cells to 1.5 × 10⁻⁶. 6 Cells / mL, final volume 200mL, cultured at 37℃ in a shaker for 24h. Dilute plasmids pQK EphA2 H3 and pQK EphA2 L1 with 10% of the transfection volume of fresh medium to a concentration of 1μg / mL, calculated based on the transfection cell volume. Add 200 μl of 3 mg / mL PEI to the diluted plasmid at 1 / 1000 of the cell volume, vortex immediately for 10 seconds, and incubate at room temperature for 15 minutes. Add the plasmid / PEI mixture to the cell culture medium and incubate on a shaker. After 7 days, collect the culture supernatant. Use a Mabsure Lx 5 mL purification column to capture antibodies from the culture supernatant at a flow rate of 3 mL / min. Equilibrate the purification column with 20 mM PB + 150 mM NaCl, pH 7.4. After the column is stable, load the antibody. After loading, rinse with 20 mM PB + 150 mM NaCl, pH 7.4. After rinsing, elute with 50 mM citrate, pH 3.0. Collect the eluted antibody and neutralize it with 1 M Tris-HCl, pH 9.0.

[0276] Collect one T75 cell culture flask containing the human hepatocellular carcinoma cell line HepG2, the non-small cell lung cancer cell line HCC827, and the breast cancer cell line MDA-MB-231. Wash once with FACS buffer, resuspend in FACS buffer for counting, and adjust the cell concentration to 1×10⁻⁶. 6 Dilute the antibody to a final concentration of 10 μg / ml, add 100 μL of cell sample, and mix well. Use the EphrinA1-Fc recombinant protein ligand as a positive control and incubate at room temperature for 1 hour.

[0277] Each well was washed twice with 400 μl of FACS buffer, then incubated with APC anti-human IgG Fc secondary antibody for 1 h, and analyzed by flow cytometry. The results are shown in Table 4 and Figure 4. The results in Table 4 show that both the anti-EphA2 humanized antibody and the control ligand protein EphrinA1-Fc specifically bound to each tumor cell line, and the peak pattern of the anti-EphA2 humanized antibody was basically the same as that of the recombinant ligand protein.

[0278] Table 4. Binding activity of humanized 13E4 antibody to human EphA2 positive cells.

[0279]

[0280]

[0281] Example 5. Detection of the binding activity of anti-EphA2 antibodies 1D6 and 6D5 to mouse EphA2 protein (Fortebio)

[0282] Human EphA2-his-Fc protein was prepared by the patent applicant, while mouse EphA2-his protein was purchased from Beijing SinoBiological Co., Ltd. (Cat: 50586-M08H). Protein binding activity was detected using a ForteBio molecular interaction analyzer. The antigen was tagged with His, and a Ni-NTA sensor was used to immobilize the antigen at a working concentration of 10 μg / mL.

[0283] After washing, mouse anti-human EphA2 antibodies 1D6 and 6D5 were bound at a working concentration of 100 nM. Binding / dissociation curves were observed, and protein binding activity was analyzed using the instrument's built-in program. The results are shown in Table 5 and Figure 5. The results indicate that both 1D6 and 6D5 antibodies bound to human EphA2 protein, while 1D6 antibody did not bind to mouse EphA2 protein, and 6D5 antibody bound mouse EphA2 protein with a KD value of 6.00E-08.

[0284] Table 5. Detection of binding activity between anti-human EphA2 antibody and mouse antigen

[0285]

[0286] Example 6. Detection of the inhibitory activity of anti-EphA2 antibody 6D5 on osteoclast-related genes.

[0287] Mouse osteoclasts were purchased from Procell (Cat: CP-M088) and routinely cultured on complete osteoclast medium (Procell Cat NO. CM-M088).

[0288] Collect cells from a T75 culture flask, discard the culture medium, wash the cells once with PBS, add a certain amount of trypsin and digest at 37°C. Once the cells begin to loosen, add serum to immediately stop the digestion. Gently pipette the cells and collect them into a 15ml centrifuge tube. Centrifuge and discard the supernatant. Adjust the cell density to 1.5 x 10⁻⁶ cells / mL using complete osteoclast culture medium. 5 / ml, inoculate osteoclasts into 6-well plates, 2ml per well.

[0289] Mouse anti-EphA2-6D5 antibody was added to a final concentration of 10 μg / ml, with an isotype control antibody used as a negative control. After 24 hours of antibody treatment, the supernatant was discarded, and 1 ml of Trizol was added to lyse the cells. RNA was purified by chloroform / isopropanol separation and dissolved in RNase-free water. 1 μg of RNA was taken and reverse transcribed into cDNA using random primers / reverse transcriptase. PCR amplification was performed using the cDNA as a template to obtain the expression abundance of genes related to osteoclast activity: Trap, Ctsk, Mmp9, and Clc7. The results are shown in Table 6. Figure 6 Table 6 shows that, compared with the control antibody, the anti-EphA2-6D5 antibody reduced the expression abundance of osteoclast-active genes and inhibited osteoclast function.

[0290] Table 6. Effects of anti-EphA2 antibody on osteoclast-related gene activity

[0291]

[0292] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> Beijing ImmunoArk Pharmaceutical Technology Co., Ltd. <120> Anti-EphA2 antibodies and their applications <130> MP2036248 <160> twenty four <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <400> 1 Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Gln 1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <400> 2 Tyr Ile Asn Pro Ser Thr Gly Tyr Asn Glu Asn Ser Gln Lys Phe Lys 1 5 10 15 Asp <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <400> 3 Arg Gly Thr Trp Gly Phe Ala Tyr 1 5 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <400> 4 Arg Ala Ser Glu Asn Ile Asn Ser Tyr Leu Thr 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <400> 5 Asn Ala Lys Thr Leu Ala Glu 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <400> 6 Gln His His Tyr Val Thr Pro Leu Thr 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <400> 7 Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <400> 8 Ile Asp Pro Ser Asp Ser Tyr Thr 1 5 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <400> 9 Ala Arg Gly Ala Tyr 1 5 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <400> 10 Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr 1 5 10 <210> 11 <211> 3 <212> PRT <213> Artificial Sequence <400> 11 Leu Val Cys 1 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <400> 12 Trp Gln Gly Thr His Phe Pro Gln Thr 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <400> 13 Gly Phe Asn Ile Lys Asp Thr Tyr 1 5 <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <400> 14 Val Asp Pro Ala Asn Gly Lys Ile 1 5 <210> 15 <211> 12 <212> PRT <213> Artificial Sequence <400> 15 Ala Lys His Tyr Gly Val Thr Tyr Ala Met Asp Tyr 1 5 10 <210> 16 <211> 6 <212> PRT <213> Artificial Sequence <400> 16 Gln Gly Ile Ser Asn Tyr 1 5 <210> 17 <211> 3 <212> PRT <213> Artificial Sequence <400> 17 Tyr Thr Ser 1 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <400> 18 Gln His Gly Asp Thr Leu Pro Thr 1 5 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <400> 19 Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Gln 1 5 10 <210> 20 <211> 17 <212> PRT <213> Artificial Sequence <400> 20 Tyr Ile Asn Pro Ser Thr Gly Tyr Asn Glu Asn Ser Gln Lys Phe Lys 1 5 10 15 Asp <210> twenty one <211> 8 <212> PRT <213> Artificial Sequence <400> twenty one Arg Gly Thr Trp Gly Phe Ala Tyr 1 5 <210> twenty two <211> 11 <212> PRT <213> Artificial Sequence <400> twenty two Arg Ala Ser Glu Asn Ile Asn Ser Tyr Leu Thr 1 5 10 <210> twenty three <211> 7 <212> PRT <213> Artificial Sequence <400> twenty three Asn Ala Lys Thr Leu Ala Glu 1 5 <210> twenty four <211> 9 <212> PRT <213> Artificial Sequence <400> twenty four Gln His His Tyr Val Thr Pro Leu Thr 1 5

Claims

1. An antibody that specifically binds to EphA2, characterized in that, It includes variable regions for heavy chains and variable regions for light chains; The heavy chain variable region includes HCDR1, HCDR2, and HCDR3: The amino acid sequence of HCDR1 is shown in SEQ ID No. 1; The amino acid sequence of HCDR2 is shown in SEQ ID No. 2; The amino acid sequence of HCDR3 is shown in SEQ ID No. 3; The light chain variable region includes LCDR1, LCDR2, and LCDR3: The amino acid sequence of LCDR1 is shown in SEQ ID No. 4; The amino acid sequence of LCDR2 is shown in SEQ ID No. 5; The amino acid sequence of LCDR3 is shown in SEQ ID No.

6.

2. The antibody as described in claim 1, characterized in that, The antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

3. The antibody as described in claim 1, characterized in that, The EphA2 mentioned is a primate EphA2.

4. The antibody as described in claim 3, characterized in that, The primate EphA2 is selected from human EphA2 or monkey EphA2.

5. The antibody as described in claim 4, characterized in that, It also includes a constant region, wherein the constant region of the heavy chain of the antibody is any one of human IgG1, IgG2, IgG3 or IgG4; and the constant region of the light chain of the antibody is κ or λ type.

6. A drug, characterized in that, Includes the antibody as described in any one of claims 1 to 5.

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