CN115505041A

CN115505041A - anti-EphA 2 antibodies and uses thereof

Info

Publication number: CN115505041A
Application number: CN202110688952.7A
Authority: CN
Inventors: 徐勤枝; 高新; 钱尼良; 张建清; 王萌; 李宏杰; 杨翠马; 刘蕴会; 王富玉
Original assignee: Beijing Mianyifangzhou Medicine Technology Co ltd
Current assignee: Beijing Mianyifangzhou Medicine Technology Co ltd
Priority date: 2021-06-22
Filing date: 2021-06-22
Publication date: 2022-12-23

Abstract

The invention relates to the field of antibodies, in particular to an anti-EphA 2 antibody and application thereof. The present invention provides anti-EPH receptor A2 (EphA 2) antibodies and uses thereof. The present application also provides chimeric and humanized antibody forms of anti-EphA 2 antibodies. These antibodies have high affinity for human EphA2 protein and are promising antibody molecules for the prevention and/or treatment of EphA 2-associated diseases, such as EphA 2-associated tumors, osteoporosis, alzheimer's disease, and the like.

Description

anti-EphA 2 antibodies and uses thereof

Technical Field

The invention relates to the field of antibodies, in particular to an anti-EphA 2 antibody and application thereof.

Background

Tumorigenesis is the result of somatic gene mutation, and the tumor microenvironment, extracellular matrix and immune system play important roles in tumorigenesis and development. When cancerous cells escape immune surveillance, overcome the control of immune function, tumors can only progress or die the host. Therefore, improving the immune system of human body and adjusting immune microenvironment are important means for treating or preventing tumorigenesis and development. With the deep understanding of the tumor immune mechanism, the tumor immunotherapy technology is rapidly developed, and becomes a treatment mode which can be selected by many middle-and late-stage cancer patients, so that the life cycle and the quality of life of the patients are greatly improved.

Pancreatic cancer is one of the most challenging malignancies in tumor therapy, characterized by strong invasiveness, early metastasis, and high resistance to treatment. The average 5-year survival rate of patients is only 4%. Currently, surgical resection is the only viable treatment; however, 80% -85% of pancreatic cancer patients have an early stage and lost surgical opportunities. Poor prognosis of cancers such as pancreatic cancer has prompted researchers to search for new therapeutic targets.

EphA2 (Ephrin type-a receptor 2) is a tyrosine kinase receptor molecule identified in 1990, the ligand of which is EphrinA1.EphA2 protein is highly expressed in a plurality of tumors such as skin cancer, digestive tract tumor, prostatic cancer, ovarian cancer, breast cancer and the like, and is a molecular marker for poor prognosis of tumor patients. EphA2 functions in two modes: one is a ligand independent oncogene mode, and EphA2 promotes cell proliferation and migration by activating downstream PI3K, rho/Rac1 GTPase/MAPK signal pathways; the second mode is EphrinA1 ligand-dependent cancer suppressor gene mode, and after EphrinA1-EphA2 is combined, MAPK pathway can be inhibited in a feedback mode, and proliferation and migration of tumor cells are inhibited. Ye Hu et al teach that some pancreatic cancer patients develop targeted gemcitabine resistance, which is mediated by exosomes whose internalization is EphA 2-dependent, and which are transmitted between tumor cells. EphA2 overexpression is also involved in mediating therapeutic resistance to breast cancer, cervical cancer and melanoma. Recent studies have shown that inhibition of EphA2 can reduce the aggressiveness of pancreatic cancer and increase the sensitivity of melanoma to vemurafenib and breast cancer to tamoxifen, suggesting that EphA2 is a promising target for cancer immunotherapy.

In 2018, an EphA 2-activated antibody is prepared by Japanese scientists, has the characteristic of internalization, and in vitro experiments prove that the EphA2 antibody coupled with an immunotoxin can kill melanoma cells. At present, no therapeutic EphA2 antibody is on the market at home and abroad, and the research and development stage is in the preclinical stage. These data suggest that EphA2 may be a promising therapeutic target for the treatment of pancreatic cancer and other EphA2 positive tumors.

Alzheimer's Disease (AD) is a progressive degenerative disease of the nervous system. Clinically, it is characterized by general dementia such as dysmnesia, disappointment dysfunction, and personality and behavior changes. Current development directions for alzheimer's disease focus primarily on amyloid plaques and neurofibrillary tangles. Eph/Ephrin proteins are found to be involved in the brain learning and memory process, ephB2 protein is involved in regulating presynaptic terminal differentiation, soluble amyloid oligomer molecules can change the homeostasis of NMDA receptors, and EphB2 interacts with NMDA receptors, suggesting that Eph protein may be involved in the progression of Alzheimer's disease. In other researches, the levels of miR-214 and EphrinA2 in serum exosomes of osteoporosis patients are greatly up-regulated, and the exosomes interact with EphA2 receptors on osteoblast membranes through EphrinA2, so that osteoblast activity is inhibited, and osteoporosis is caused. These data suggest that EphA2 may also be a target for the treatment of alzheimer's disease and osteoporosis.

Therefore, it is of great practical significance to provide anti-EphA 2 antibodies and uses thereof.

Disclosure of Invention

In view of this, the present invention provides anti-EphA 2 antibodies and uses thereof. The antibody or antigen-binding portion thereof that specifically binds to EphA2 is capable of inducing an EphA 2-mediated anti-tumor immune response, and/or inhibiting tumor growth and alzheimer's disease, osteoporosis, and the like.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an antibody or antigen-binding portion thereof that specifically binds EphA2, comprising a heavy chain variable region;

the heavy chain variable region comprises any one or more of the HCDR1, HCDR2 or HCDR3 sequences:

(I) The amino acid sequence of the HCDR1 has an amino acid sequence shown as SEQ ID No.1, 7 or 13; or

The amino acid sequence of the HCDR2 has an amino acid sequence shown as SEQ ID No.2, 8 or 14; or

The amino acid sequence of the HCDR3 has an amino acid sequence shown as SEQ ID No.3, 9 or 15;

or

(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence described in (I) and has the same function with the amino acid sequence described in (I); or

(III) an amino acid sequence having 90% or more identity to the amino acid sequence described in (I) or (II).

In some embodiments of the invention, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region;

(I-1), wherein the amino acid sequence of the HCDR1 has an amino acid sequence shown as SEQ ID No. 1; or

The amino acid sequence of the HCDR2 has an amino acid sequence shown as SEQ ID No. 2; or

The amino acid sequence of the HCDR3 has an amino acid sequence shown as SEQ ID No. 3;

or

(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 the amino acid sequence has the same function with the amino acid sequence described in (I-1); or

(III-1) an amino acid sequence having 90% or more identity to the amino acid sequence described in (I-1) or (II-1); or

(I-2), the amino acid sequence of the HCDR1 has an amino acid sequence shown as SEQ ID No. 7; or

The amino acid sequence of the HCDR2 has an amino acid sequence shown as SEQ ID No. 8; or

The amino acid sequence of the HCDR3 has an amino acid sequence shown as SEQ ID No. 9;

or

(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 the amino acid sequence has the same function with the amino acid sequence described in (I-2); or

(III-2) an amino acid sequence having 90% or more identity to the amino acid sequence described in (I-2) or (II-2); or

(I-3), the amino acid sequence of the HCDR1 has an amino acid sequence shown as SEQ ID No. 13; or

The amino acid sequence of the HCDR2 has an amino acid sequence shown as SEQ ID No. 14; or

The amino acid sequence of the HCDR3 has an amino acid sequence shown as SEQ ID No. 15;

or

(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 the amino acid sequence has the same function with the amino acid sequence described in (I-3); or

(III-3) and an amino acid sequence having 90% or more identity to the amino acid sequence described in (I-3) or (II-3).

In some embodiments of the invention, the antibody, or antigen-binding portion thereof, further comprises a light chain variable region;

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

(I) The amino acid sequence of the LCDR1 has an amino acid sequence shown as SEQ ID No.4, 10 or 16; or

The amino acid sequence of the LCDR2 has an amino acid sequence shown as SEQ ID No.5, 11 or 17; or

The amino acid sequence of the LCDR3 has an amino acid sequence shown as SEQ ID No.6, 12 or 18;

or

(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence described in (I), and the amino acid sequence has the same function with the amino acid sequence described in (I); or

(III) an amino acid sequence having 90% or more identity to the amino acid sequence of (I) or (II).

(I-1), wherein the amino acid sequence of the LCDR1 has an amino acid sequence shown as SEQ ID No. 4; or

The amino acid sequence of the LCDR2 has an amino acid sequence shown as SEQ ID No. 5; or

The amino acid sequence of the LCDR3 has an amino acid sequence shown as SEQ ID No. 6;

or

(I-2), the amino acid sequence of the LCDR1 has an amino acid sequence shown as SEQ ID No. 10; or

The amino acid sequence of the LCDR2 has an amino acid sequence shown as SEQ ID No. 11; or

The amino acid sequence of the LCDR3 has an amino acid sequence shown as SEQ ID No. 12;

or

(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), which has the same function as the amino acid sequence described in (I-2); or

(I-3) the amino acid sequence of the LCDR1 has an amino acid sequence shown as SEQ ID No. 16; or

The amino acid sequence of the LCDR2 has an amino acid sequence shown as SEQ ID No. 17; or

The amino acid sequence of the LCDR3 has an amino acid sequence shown as SEQ ID No. 18;

or

In some embodiments of the invention, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region and a light chain variable region;

(I) The amino acid sequence of the HCDR1 has an amino acid sequence shown as SEQ ID No. 1; or

or

(III) an amino acid sequence having 90% or more identity to the amino acid sequence of (I) or (II);

(IV) the amino acid sequence of the LCDR1 has an amino acid sequence shown as SEQ ID No. 4; or

or

(V) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence described In (IV), and the amino acid sequence has the same function with the amino acid sequence described In (IV); or

(VI) an amino acid sequence having 90% or more identity to the amino acid sequence described In (IV) or (V).

(I) The amino acid sequence of the HCDR1 has an amino acid sequence shown as SEQ ID No. 7; or

or

(IV) the amino acid sequence of the LCDR1 has an amino acid sequence shown as SEQ ID No. 10; or

or

(I) The amino acid sequence of the HCDR1 has an amino acid sequence shown as SEQ ID No. 13; or

or

(IV) the amino acid sequence of the LCDR1 has an amino acid sequence shown as SEQ ID No. 16; or

or

In some embodiments of the invention, the antigen binding moiety is selected from the group consisting of: fab fragments, fab 'fragments, F (ab') 2 fragments, scFv fragments, fd fragments or single domain antibodies.

In some embodiments of the invention, the antibody is murine, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No.19, and/or the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 22; or

The antibody is a chimeric antibody, and optionally, the chimeric antibody comprises a heavy chain with an amino acid sequence shown as SEQ ID No.20 and/or a light chain with an amino acid sequence shown as SEQ ID No. 23; or

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

In some embodiments of the invention, the EphA2 is primate EphA2; preferably, the primate EphA2 is selected from human EphA2 or monkey EphA2.

In some embodiments of the invention, more of the one or more amino acids are substituted, deleted or added is 2, 3, 4 or 5.

In some embodiments of the invention, the antibody, or antigen-binding portion thereof, further comprises a constant region, wherein the constant region of the heavy chain of the antibody, or antigen-binding portion thereof, is any one of human IgG1, igG2, igG3, or IgG 4; the constant region of the light chain of the antibody, or antigen-binding portion thereof, is of the kappa or lambda type.

The invention also provides application of the antibody or the antigen binding part thereof in preparing a medicament for preventing and/or treating EphA2 related diseases.

In some embodiments of the invention, the EphA 2-associated disease is one or more of a tumor, 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.

The antibody or antigen-binding portion thereof specifically binding to EphA2 provided by the invention can induce EphA 2-mediated anti-tumor immune response, and/or inhibit tumor growth, alzheimer's disease, osteoporosis and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below.

FIG. 1 shows FACS screening of anti-human EphA2 hybridoma antibodies;

FIG. 2 shows FACS screening for competitive binding of candidate clonal antibodies to ligand;

figure 3 shows FACS identification of anti-EphA 2 chimeric antibody clones; (A) The 13E4 and 1D6 antibodies are combined with human colon cancer cells HCT 116; (B) the 6D5 antibody binds to human colon cancer cell HCT 116;

FIG. 4 (A) shows the result of identifying the binding of the humanized 13E4 antibody against EphA2 to HepG2, a human hepatoma cell; FIG. 4 (B) shows the result of identifying that an anti-EphA 2 humanized 13E4 antibody binds to human lung cancer cell HCC 827; FIG. 4 (C) shows the result of identification of the binding of the anti-EphA 2 humanized 13E4 antibody to human breast cancer cell MDA-MB-231;

FIG. 5 (A) shows the identification of 11D6 binding activity to human/murine EphA2 protein; FIG. 5 (B) shows the identification of binding activity of 6D5 to human/murine EphA2 protein;

fig. 6 shows the effect of anti-EphA 2 antibody 6D5 on osteoclast-associated gene activity.

Detailed Description

The invention discloses an anti-EphA 2 antibody and application thereof, and can be realized by appropriately improving process parameters by one skilled in the art with reference to the content. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate variations and combinations of the methods and applications described herein may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Definition of

As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by a disulfide bond, and multimers thereof (e.g., igM). Each heavy chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region (abbreviated CH). The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region (abbreviated CL). The light chain constant region comprises one domain (CL 1). The VH and VL regions can be further subdivided into hypervariable regions known as Complementarity Determining Regions (CDRs) into which conserved regions known as Framework Regions (FRs) are interspersed. In some embodiments, both the light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus.

As used herein, the term "antigen-binding portion" of an antibody refers to a portion or segment of an intact antibody molecule that is responsible for binding to an antigen. The antigen-binding portion may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. The antigen-binding portion of the antibody may be prepared from the intact antibody molecule using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques, among others. Non-limiting examples of antigen-binding moieties include: fab fragment, F (ab') ₂ Fragments, fd fragments, fv fragments, single-chain Fv (scFv) molecules, single-domain antibodies, dAb fragments, and amino groups derived from hypervariable regions of mimobodiesAcid residues constitute the smallest recognition unit (e.g., an isolated CDR). The term "antigen-binding portion" also includes other engineered molecules, such as diabodies, triabodies, tetrabodies, minibodies, and the like. For example, the Fd fragment herein refers to an antibody fragment consisting of VH and CH1 domains; the Fv fragment consists of the VL and VH domains in a single arm of the antibody; dAb fragments (Ward et al, nature 1989, 341, 544-546) consist of VH domains.

It is well known to those skilled in the art that the complementarity determining regions (CDRs, usually CDR1, CDR2 and CDR 3) are the regions of the variable region that have the greatest impact on the affinity and specificity of an antibody. There are two common definitions of CDR Sequences for VH or VL, namely Kabat definition and Chothia definition, see, for example, 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. For a given antibody variable region sequence, can according to Kabat definition or Chothia definition to determine VH and VL sequence in CDR region sequence. In embodiments of the present application, the CDR sequences are defined using Kabat. Herein, CDR1, CDR2 and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2 and HCDR3, respectively; CDR1, CDR2 and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2 and LCDR3, respectively.

The CDR region sequences in the variable region sequences can be analyzed in a variety of ways for a given antibody variable region sequence, such as can be determined using the online software Abysis (http:// www.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, e.g., binding of an antibody to an epitope of an antigen, e.g., the ability of an antibody to bind to a specific antigen with an affinity that is at least two times greater than its affinity for a non-specific antigen. It will be appreciated, however, that an antibody is capable of specifically binding two or more antigens associated with its sequence. For example, an antibody of the present application can specifically bind to human EphA2.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations in a small number of individuals. The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies, so long as they exhibit the desired biological activity (see, U.S. Pat. No.4,816,567; and Morrison et al, proc. Natl. Acad. Sci. USA 81.

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

As used herein, the term "chimeric antibody" refers to an antibody comprising segments from two or more different antibodies. In some embodiments, one or more CDRs are derived from a mouse anti-EphA 2 antibody. In other embodiments, all CDRs are derived from a mouse anti-EphA 2 antibody. In some embodiments, the CDRs from more than one mouse anti-EphA 2 antibody are combined in a chimeric antibody. For example, a chimeric antibody can comprise a CDR1 from the light chain in a first mouse anti-EphA 2 antibody, a CDR2 from the light chain in a second mouse anti-EphA 2 antibody, and a CDR3 from the light chain in a third mouse anti-EphA 2 antibody, and the CDRs from the heavy chain can be derived from one or more other anti-EphA 2 antibodies. Furthermore, the framework regions may be from the same anti-EphA 2 antibody or from one or more different individuals.

As used herein, the term "humanized antibody" refers to a CDR-grafted antibody, specifically to an antibody produced by grafting mouse CDR region sequences into a human antibody variable region framework. The aim is to overcome the strong immune side effects induced by chimeric antibodies in humans due to the large number of protein components carried by other species such as mice.

As used herein, the term "nucleic acid molecule" may refer to DNA molecules as well as RNA molecules, which may be single-stranded or double-stranded. The nucleic acid molecule may also be a cDNA.

As used herein, the term "EphA 2-associated disease" includes diseases and/or disorders associated with the EphA2 signaling pathway. Exemplary EphA 2-associated diseases or disorders include tumors, cancers such as gastric, pancreatic, intestinal, esophageal, liver, ovarian, lung, and bladder cancers, as well as metastatic cancers of the above, as well as senile dementia and osteoporosis.

As used herein, the term "immune response" refers to a biological response in a vertebrate against an external agent that protects an organism against such agent and the disease caused by it. The 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 soluble macromolecules produced by the liver, including antibodies, cytokines, and complements, which results 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 the case of autoimmune or pathological inflammation. The immune response includes T cells (e.g., effector T cells) or Th cells (e.g., CD 4) ⁺ Or CD8 ⁺ T cells) or suppression of Treg cells.

As used herein, the term "cancer" refers to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Dysregulated 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 the bloodstream.

As used herein, the term "treatment" refers to any type of intervention or method performed on a subject or administration of an active agent thereto, wherein the objective is to reverse, alleviate, ameliorate, inhibit or alleviate or prevent symptoms, complications, conditions, or progression, development, severity, or recurrence associated with the disease.

As used herein, the term "prevention" refers to administration to a subject not suffering from a disease, to prevent the disease from occurring or to minimize its effects (if any).

The present inventors screened three hybridoma cells, and the antibody in the supernatant was able to bind to EphA 2-expressing cells. Accordingly, the present application provides three novel anti-EphA 2 antibodies, or antigen-binding portions thereof, that specifically bind EphA2. The present inventors have also prepared chimeric antibodies and humanized antibody forms from murine anti-EphA 2 antibodies by genetic engineering means, which are capable of specifically binding to EphA2 expressed on the cell surface, thereby effectively inducing an EphA 2-mediated immune response and playing a role in the prevention or treatment of EphA 2-related diseases.

In a first aspect, the present application provides an antibody or antigen-binding portion thereof 13E4 clone that specifically binds EphA2 comprising a heavy chain variable region comprising any one or more of the HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1 sequence is gytftssywiq (SEQ ID No. 1), the HCDR2 sequence is YINPSTGYN ENSQKFKD (SEQ ID No. 2), and the HCDR3 sequence is twrggfay (SEQ ID No. 3).

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

In some embodiments, the antibody, or antigen-binding portion thereof, further comprises a light chain variable region comprising any one or more of 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).

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

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

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

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

In some embodiments, the antibody, or antigen-binding portion thereof, further comprises a light chain variable region comprising any one or more of LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1 sequence is RASENINSYLT (SEQ ID No. 10) the NAKTLAE (SEQ ID No. 11) and the LCDR3 sequence is QHHYVTPLT (SEQ ID No. 12).

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

The present application provides an antibody or antigen-binding portion 6D5 clone thereof that specifically binds EphA2 comprising a heavy chain variable region comprising any one or more of 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 akhyggvtyamdy (SEQ ID No. 15).

In a preferred embodiment, the heavy chain variable region of the antibodies described herein, or antigen-binding portions thereof, comprises the HCDR1 shown in SEQ ID No.13, the HCDR2 shown in SEQ ID No.14 and the HCDR3 sequence shown in SEQ ID No. 15.

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

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

In some more specific embodiments, the antibodies disclosed herein can be anti-human EphA2 monoclonal antibodies. The type and subtype of anti-EphA 2 antibody can be determined by any means known in the art. Generally, antibody types and subtypes can be determined using antibodies specific for a particular antibody type and subtype. anti-EphA 2 antibody isotypes can be determined using ELISA assays, e.g., human igs can be identified using anti-human igs adsorbed by murine igs.

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

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

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

In preferred embodiments, the chimeric antibodies described herein comprise the variable regions of a murine antibody (including the heavy chain variable region and the light chain variable region) and the constant regions of a human antibody.

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

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

In some embodiments, the antibodies described herein are humanized antibodies. The humanized antibodies described herein comprise CDR regions of a murine antibody (including any one or more of HCDR1, HCDR2 and HCDR3 and/or any one or more of LCDR1, LCDR2 and LCDR 3), framework regions of a human antibody variable region (including any one or more of FR1, FR2, FR3 and FR 4), and optionally a human antibody constant region.

In a preferred embodiment, the humanized antibody described herein comprises the CDR regions of a murine antibody (including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR 3), the framework regions of a human antibody variable region (including FR1, FR2, FR3 and FR 4), and optionally the constant regions of a human antibody.

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

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

The antibodies described herein may also comprise murine or human antibody constant regions. Murine antibody constant regions include murine IgG1, igG2a, igG2b, or IgG3 heavy chain constant regions, as well as kappa or lambda type light chain constant regions, and the like. The human antibody constant region includes a heavy chain constant region of human IgG1, igG2, igG3 or IgG4, a light chain constant region of kappa or lambda type, and the like.

In some embodiments, ephA2 described herein is primate EphA2. Preferably, the primate EphA2 described herein is selected from human EphA2 or monkey EphA2.

In some embodiments, the antigen binding moiety described herein is selected from the group consisting of: fab fragment, fab 'fragment, F (ab') ₂ A fragment, fv fragment, scFv fragment, fd fragment or single domain antibody.

The term "Fab fragment" as used herein encompasses the light chain as well as the CH1 and variable regions of the heavy chain. The heavy chain of a Fab molecule is unable to form a disulfide bond with another heavy chain molecule.

The term "Fab' fragment" as used herein contains the light chain as well as the part or fragment of the heavy chain containing the VH domain and CH1 domain and the region between the CH1 and CH2 domains such that there are 2An interchain disulfide bond may form between the two heavy chains of an Fab 'fragment to form F (ab') ₂ A molecule.

The term "F (ab') ₂ A fragment "contains two light chains and two heavy chains, the heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. F (ab') ₂ The fragment thus consists of two Fab' fragments linked together by a disulfide bond between the two heavy chains.

The term "Fv fragment" as used herein encompasses the variable regions from the heavy and light chains, but lacks the constant regions.

The term "single chain Fv" or "scFv", as used herein, refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present as a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

The term "single domain antibody" as used herein refers to an antigen-binding portion comprising one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Originally, a naturally light chain-deficient antibody was found in the peripheral blood of alpacas, which, although comprising only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, did not adhere to each other as readily as artificially engineered single chain antibody fragments (scFv) and even aggregated into a mass. More importantly, the structure of the VHH which is cloned and expressed independently has the structural stability which is equivalent to that of the original heavy chain antibody and the binding activity with the antigen, and is the minimum unit which is known to be combined with the target antigen. VHH crystals have a molecular weight of only 15kDa and are therefore also called Nanobodies (Nb)

The method of making an anti-EphA 2 monoclonal antibody disclosed herein can comprise: culturing the host cell under expression conditions such that the anti-EphA 2 monoclonal antibody is expressed; the expressed anti-EphA 2 monoclonal antibody was isolated and purified. Using the above procedure, a crude anti-EphA 2 monoclonal antibody can be obtained. The anti-EphA 2 monoclonal antibody is then purified to a substantially homogeneous substance by a purification method, including EphA 2-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, e.g., as a single band on SDS-PAGE electrophoresis.

The application provides the use of the antibody or antigen-binding portion thereof of the first aspect, and the host cell of the second aspect, in the preparation of a medicament for preventing and/or treating an EphA 2-associated disease.

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

"treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathological state or disorder. Those in need of treatment include those already with the disorder, as well as those who will develop the disorder or for whom the disorder is to be prevented. Thus, the subject to be treated herein has been diagnosed as suffering from, or predisposed to, or susceptible to the disorder.

In any embodiment, the EphA 2-associated disease is a tumor, alzheimer's disease, and osteoporosis.

In some embodiments, a tumor described herein is a primary cancer or a metastatic cancer. In particular embodiments, the tumor is selected from lung cancer such as non-small cell lung cancer, colorectal cancer, bladder cancer, hematopoietic cancers such as leukemia, breast cancer, gastric cancer, esophageal cancer, B-lymphocyte type non-hodgkin lymphoma, anaplastic cell lymphoma, head and neck cancer such as head and neck squamous cell carcinoma, glioblastoma, 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 cancer, endometrial cancer, or metastases of the foregoing tumors.

It should be understood that features, characteristics, components or steps described in a particular aspect, embodiment or example of the present application may be applied to any other aspect, embodiment or example described herein unless incompatible therewith.

Antibodies that specifically bind to mammalian (human, primate, etc.) EphA2 are disclosed, as are uses of such proteins in therapy, e.g., in the treatment of cancer. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention. Those skilled in the art can implement and use the invention by making modifications, or appropriate alterations and combinations, of the methods and applications described herein without departing from the spirit, scope, and content of the invention.

In the anti-EphA 2 antibody and the application thereof, related raw materials and reagents can be purchased from the market.

The following examples are intended to illustrate the present application but are not intended to limit the scope of the present application. Modifications or substitutions to methods, steps or conditions disclosed herein may be made without departing from the spirit and scope of the present application.

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

The examples do not include detailed descriptions of conventional methods such as those for constructing 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 of ordinary skill in the art and are described in numerous publications, see, for example, sambrook, j., fritsch, ef.and maniis, t. (1989) Molecular Cloning: a Laboratory Manual,2nd edition, cold spring Harbor Laboratory Press.

In the present invention, the sequence information is described as follows:

13E4 VH mouse Sequence

Protein sequence

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

QVQLLESGAELAKPGASVKMSCKASGYTFTSYWIQWVKQRPGQGLEWIGYINPSTGYNENSQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARRGTWGFAYWGQGTLVTVSA

13E4 HCDR1 with sequence GYTFTSYWIQ (SEQ ID No. 1)

13E4 HCDR2 has a sequence YINPSTDYNSSQKFKD (SEQ ID No. 2)

13E4 HCDR3 sequence RGTWGFAY (SEQ ID No. 3)

13E4 VL mouse Sequence (V kappa)

Protein sequence

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

DIVMTQTPASLSASVGETVTITCRASENINSYLTWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYVTPLTFGAGTKLELK

13E4 LCDR1 is RASENNSYLT (SEQ ID No. 4),

13E4 LCDR2 has sequence NAKTLAE (SEQ ID No. 5),

13E4 LCDR3 has the sequence QHHYVTPLT (SEQ ID No. 6)

1D6 VH murine Sequence

DVQLLESGAELVNPGASVKISCKASGYTFTSYWLNWVKQRPGQGLEWIGDIDPSDSYTNKNQKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGAYWGQGTLVTVSA

1D6 HCDR1 sequence GYTFTSYW (SEQ ID No. 7)

1D6 HCDR2 sequence is IDPSDSYT (SEQ ID No. 8)

1D6 HCDR3 sequence is ARGAY (SEQ ID No. 9)

1D6 VL mouse Sequence (V kappa)

DVVMTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVCKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK

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

1D6 LCDR2 sequence is LVC (SEQ ID No. 11)

1D6 LCDR3 sequence is WQGTHFQT (SEQ ID No. 12)

6D5 VH murine Sequence

EVQLLESGAELVKPGASVKLSCTASGFNIKDTYIHWVKLRPEQGLEWIGRVDPANGKIKYDPKFQGKATITADTSSNTAYLHLSSLTSEDAAVFYCAKHYGVTYAMDYWGQGASVTVSS

The sequence of 6D5 HCDR1 is GFNIKDTY (SEQ ID No. 13)

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

The 6D5 HCDR3 sequence is AKHYGVTEAMDY (SEQ ID No. 15)

6D5 VL murine Sequence (V κ)

DIQMTQTTSSLSASLGDRVTISCRASQGISNYLNWYQRKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQHGDTLPTFGGGTKLEMKKGDTPGISK

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

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

The sequence of 6D5 LCDR3 is QHGDTLPT (SEQ ID No. 18)

13E4 VH humanized Sequence

Protein sequence

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIQWVKQAPGQGLEWIGYINPSTGYNENSQKFKDRVTLTADKSTSTVYMELSSLRSEDTAVYYCARRGTWGFAYWGQGTLVTVSS

SEQ ID No.19 sequence is GYTFTSYWIQ (SEQ ID No. 19)

SEQ ID No.20 sequence is YINPSTGNYNENSQKFKD (SEQ ID No. 20)

SEQ ID No.21 sequence RGTWGFAY (SEQ ID No. 21)

13E4 VL humanised Sequence

Protein sequence

DIQMTQSPSSLSASVGDRVTITCRASENINSYLTWYQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYVTPLTFGGGTKVEIK

SEQ ID No.22 sequence RASENNSYLT (SEQ ID No. 22)

SEQ ID No.23 sequence is NAKTLAE (SEQ ID No. 23)

SEQ ID No.24 sequence is QHHYVTPLT (SEQ ID No. 24)

The invention is further illustrated by the following examples:

example 1 construction of EphA 2-highly expressed cell line

A high-expression cell strain of human EphA2 is constructed by a stable cell strain construction platform, and the specific steps are as follows:

construction of the plasmid pEnter-EphA 2-puromycin: ephA2 cDNA (general biological System (Anhui) Co., ltd.) was used as a template, and a fragment of about 3000bp was obtained by amplification with primers and recovered by gel; the pENTER plasmid is cut by KpnI/HindIII enzyme, and the large fragment 7450bp is recovered by glue; the fragments obtained above were ligated with a plasmid pENTER large fragment digested with KpnI/HindIII recovered from gel by recombinant technique, and the plasmid with correct sequencing was selected and sequenced, and named pEnter-EphA2-puromycin.

293T cells (Synergestic cell Bank) were seeded in T25 flasks at 2X 10 cells per flask ⁶ . The next day 293T cell culture medium was changed to 4mL Opti-MEM (Thermofeisher Scientific Cat.31985070). The plasmid pEnter-EphA2-puromycin of EphA2 (5. Mu.g) was added to Opti-MEM to a final volume of 500. Mu.L, and 500. Mu.L of Opti-MEM was prepared and added with 7. Mu.L of PEI (3. Mu.g/mL) as a transfection reagent, and after mixing and allowing to stand at room temperature for 20min, the mixture was added to the above-mentioned cultured 4mL of 293T cells. On the third day, the cell culture solution was changed to 5mL of DMEM high-sugar medium. On the fourth day, 2. Mu.g/mL puromycin was added for screening. After 2-3 days, the cells die in a large amount, the fresh culture medium is replaced until the cells grow stably, and then the monoclonal screening, the amplification culture and the frozen seed preservation are carried out.

The cell line stably expressing the gene of interest constructed herein was designated as 293T-EphA2 cell. The protein sequences used were derived from publicly published databases.

Example 2 preparation of hybridomas producing anti-EphA 2 antibodies

The preparation method comprises the following steps:

1. balb/c mice were harvested for 8-10 weeks and injected subcutaneously with EphA2 protein (50. Mu.g/dose/mouse). The extracellular Glu28-Arg328 region of human EphA2 protein sequence (XP-016856026.1) has His and Fc tag at C terminal and has 66kDa molecular weight. This dose was repeated 3 times over a 3-8 week period. Mice were given an intraperitoneal injection of EphA2 protein for 3 consecutive days 4 days before fusion.

2. Macrophages in abdominal cavities of Kunming mice are taken as trophoblasts 1 day before fusion and inoculated in a 96-well plate.

3. Spleens from immunized mice were taken and fused with a non-secretory myeloma SP2/0 cell line, and the cells were added to a 96-well plate previously plated with trophoblasts, and the fused cells were subjected to HAT selection (Galfre and Milstein, methods Enzymol 1981.

4. A panel of hybridoma cells secreting anti-EphA 2 specific antibodies is recovered. Primary screening the titers of anti-EphA 2 antibodies secreted by the hybridomas were determined using enzyme-linked immunosorbent assay (ELISA).

anti-EphA 2 hybridoma rescreening (FACS)

The rescreening method 1 is as follows:

293T-EphA2 cells were collected, washed once with PBS buffer, and washed at 2X 10 ⁵ One/well of the cells was plated in a 96-well deep-well plate, 50. Mu.L of hybridoma supernatant was added to each well, and incubated for 1 hour.

Two washes were performed by adding 400 μ l FACS buffer per well, and an APC anti-mouse IgG Fc secondary antibody (Biolegend, cat 405308) was added and detected by flow cytometry after 1 hour incubation.

As a result, 5 candidate antibodies were obtained, clone numbers 1D6, 6H3, 6D5, 13A6 and 13E4, as shown in Table 1 and FIG. 1.

TABLE 1 anti-human EphA2 hybridoma antibody screening

The rescreening method 2 is as follows:

293T-EphA2EphrinA1 cells were collected, washed once with PBS buffer, and washed at 2X 10 ⁵ And (3) paving the cells per well in a 96-well deep-well plate, adding 50 mu L of hybridoma supernatant into each well, simultaneously adding the recombinant EphrinA1-hFc ligand protein, carrying out ligand competitive binding, setting a positive control without adding the hybridoma supernatant, a blank control and a secondary antibody control, and incubating for 1h.

Each well was washed twice with 400. Mu.l FACS buffer, and an APC anti-human IgG Fc secondary antibody (Biolegend, cat 405308) was added and incubated for 1 hour before detection by flow cytometry.

TABLE 2 competitive binding screening results for candidate monoclonal antibody ligands

Initial hybridoma clone number	293T-EphA2 cell binding Activity
		Second 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

The results are shown in Table 2, FIG. 2, and show that the antibody in the supernatant of hybridoma 13E4, which specifically blocks the binding of the ligand 293T-EphA2 to EphrinA1 protein, indicates that the anti-EphA 2 antibody secreted by the hybridoma is a blocking antibody, and 1D6, 6H3 and 6D5 are affinity non-blocking antibodies.

Example 3 binding Activity of anti-EphA 2 chimeric antibody with human EphA2 Positive cells (FACS detection)

Preparation of anti-EphA 2 chimeric antibody

13E4, 1D6 and 6D5 hybridoma cell cDNA is obtained, and the nucleotide sequences of the heavy chain codes of VH-13E4, VH-1D6 and VH-6D5 of the murine anti-EphA 2 antibody and the corresponding light chain codes of VL-13E4, VL-1D6 and VL-6D5 of the antibody are finally obtained by PCR amplification sequencing by using antibody primers preserved in the laboratory. Light and heavy chain encoding nucleic acid sequences of the anti-EphA 2 antibody were constructed by total synthesis into plasmids pUC57EphA2 VH-13E4, pUC57EphA2 VL-13E4, pUC57EphA2 VH-1D6, pUC57EphA2 VL-1D6, and pUC57EphA2 VH-6D5, pUC57EphA2 VL-6D5 (general purpose biosystems (Anhui) Co., ltd.), using the corresponding pUC57EphA2 VH and pUC57EphA2 VL as templates, and using a gold-brand Mix PCR kit (TSINGKE Co., ltd.) according to the kit instructions, the anti-EphA 2 VH and EphA2 VL fragments were amplified to a size of about 0.4kb; meanwhile, vector plasmids pQKX1 and pQKX2 (general biological System (Anhui) Co., ltd.) were digested with restriction enzymes SapI (NEB, R0569S), and the resulting PCR amplification products and digested vectors were recombinantly ligated with a BM seamless cloning kit (Bomeide Co., ltd.) according to the instructions of the kit to obtain heavy chain expression vectors pQK EphA 2H-13E 4, pQK EphA 2L-13E 4, pQK EphA 2H-1D 6, pQK EphA 2L-1D 6, pQK EphA 2H-6D 5, and pQK EphA 2L-6D 5.

Diluting 293Fv cells (cell resource center of institute of basic medicine of Chinese academy of medical science) to 1.5 × 10 ⁶ cells/mL, final volume 200ml,37 degrees C shake culture 24h. The plasmids pQK EphA 2H and pQK EphA 2L were diluted with fresh medium at 10% of the transfection volume, calculated on the transfected cell volume, to a concentration of 1. Mu.g/mL. Adding 200 μ l of PEI to the diluted plasmid in an amount of 1/1000 of the cell volume, vortexing immediately for 10 seconds, standing at room temperature for 15 minutes, adding the plasmid/PEI mixture dropwise to the cell culture medium, shaking gently the flask while adding dropwise, shaking, collecting the culture supernatant after 7 days, capturing the antibody from the culture supernatant using a Mab sure Lx 5mL purification column at a flow rate of 3mL/min, equilibrating the purification column with 5 column volumes of 20mM PB 150mM NaCl, pH7.4 equilibration solution, and loading the column after equilibration and stabilizationAfter the sample loading is finished, 20mM PB +150mM NaCl, pH7.4 is selected for elution, 50mM citric acid, pH3.0 eluent is used for elution after the elution is finished, and the eluted antibody is collected by neutralizing with 1M Tris-Hcl, pH 9.0.

Collecting human colon cancer cell line HCT116 from a T75 cell culture flask, washing with FACS buffer solution once, re-suspending and counting with FACS buffer solution, and adjusting cell concentration to 1 × 10 ⁶ (ii) a The antibody was diluted to a final concentration of 10. Mu.g/ml, and 10. Mu.l of each diluted antibody 10. Mu.g was added to 100. Mu.l of each cell sample adjusted in the above concentration, mixed well, and incubated at room temperature for 1 hour.

Each well was washed twice with 400. Mu.l FACS buffer, and the humanized antibody group was incubated for 1 hour with APC anti-human IgG Fc secondary antibody and examined by flow cytometry, the results are shown in Table 3 and FIG. 3. The results in table 5 show that anti-chimeric 1D6, 6D5 and 13E4 tumor cell line HCT116 binds specifically.

TABLE 3 cell binding Activity of anti-EphA 2 chimeric antibodies 13E4, 1D6 and 6D5

Example 4 binding Activity of anti-EphA 2 humanized antibody 13E4 with human EphA2 Positive cells (FACS detection)

Preparation of clone of anti-EphA 2 humanized antibody 13E4

The nucleotide sequences encoding VH and VL for the anti-EphA 2 humanized antibodies were provided by the aboveground biotechnology limited of shanghai for sequence optimization services. Wherein the nucleic acid sequences encoding VH3 and VL12 of the anti-EphA 2 antibody are constructed into plasmids pUC57EphA2 VH3 and pUC57EphA2 VL1 by total synthesis, the fragments of anti-EphA 2 VH3 and EphA2 VL1 are amplified by using a gold-brand Mix PCR kit with pUC57EphA2 VH3 and pUC57EphA2 VL1 as templates, according to the instructions of the kit, and the size of the amplified product is about 0.4kb; meanwhile, vector plasmids pQKX1 and pQKX2 are digested by restriction enzymes SapI (NEB, R0569S), and the obtained PCR amplification product and the digested vector are recombined and connected by a BM seamless cloning kit (Bomaide company) according to the instruction of the kit to obtain heavy chain expression vectors pQK EphA 2H 3 and pQK EphA 2L 1.

293Fv cells were diluted to 1.5X10 ⁶ cells/mL, final volume 200ml,37 degrees C shake culture 24h. The plasmids pQK EphA 2H 3 and pQK EphA 2L 1 were diluted with fresh medium at a transfection volume of 10%, calculated on the volume of transfected cells, to a concentration of 1. Mu.g/mL. Adding 200 mu l of PEI (3 mg/mL) into the diluted plasmid according to 1/1000 of the cell volume, immediately vortex and shake for 10 seconds, standing at room temperature for 15 minutes, dropwise adding the plasmid/PEI mixture into a cell culture medium, placing the cell culture medium into a shaking table for culture, collecting a culture supernatant after 7 days, capturing antibodies from the culture supernatant by using a Mab sure Lx 5mL purification column, setting the flow rate to be 3mL/min, balancing the purification column by using 20mM PB +150mM NaCl and pH7.4 balance liquid, loading after the column is balanced and stabilized, selecting 20mM PB +150mM NaCl, rinsing at pH7.4 after the loading is finished, eluting by using 50mM citric acid pH3.0 eluent, collecting and neutralizing and eluting antibodies by using Tris-HCl and pH 9.0.

Collecting human liver cancer cell line HepG2, non-small cell lung cancer cell line HCC827 and breast cancer cell line MDA-MB-231 in a T75 cell culture flask, washing with FACS buffer solution once, re-suspending and counting with FACS buffer solution, adjusting cell concentration to 1 × 10 ⁶ (ii) a The antibody was diluted to a final concentration of 10. Mu.g/ml and the cell sample was 100. Mu.L, and mixed well. And (3) taking the ligand EphrinA1-Fc recombinant protein as a positive control, and incubating at room temperature for 1h.

Each well was washed twice with 400 μ l FACS buffer, and APC anti-human IgG Fc secondary antibody was added, incubated for 1h, and detected by flow cytometry, the results are shown in table 4 and fig. 4. The results in table 4 indicate that both the anti-EphA 2 humanized antibody and the control ligand protein EphrinA1-Fc specifically bind to each tumor cell line, and the peak pattern of the anti-EphA 2 humanized antibody is substantially the same as that of the ligand recombinant protein.

TABLE 4 binding Activity of humanized 13E4 antibody and human EphA2 Positive cells

Example 5 detection of binding Activity of anti-EphA 2 antibodies 1D6, 6D5 with mouse EphA2 protein (Fortebio)

Human EphA2-his-Fc protein was prepared by the patent application unit, and mouse EphA2-his protein was purchased from SinoBiological, cat:50586-M08H, biotech, inc., beijing Yizhi. The protein binding activity is detected by adopting a fortebio molecular interaction instrument. The tag carried by the antigen is His, a Ni-NTA sensor is selected to fix the antigen, and the working concentration is 10 mug/ml L

After washing, the murine anti-human EphA2 antibody 1D6, 6D5 antibody was bound at a working concentration of 100nM. The binding/dissociation curves were observed and the protein binding activity was analyzed using the own instrument program. The results are shown in Table 5 and FIG. 5. The results showed that both 1D6 and 6D5 antibodies bound to the human EphA2 protein, that 1D6 antibody did not bind to the murine EphA2 protein, and that 6D5 antibody bound to the murine EphA2 protein, with a KD value of 6.00E-08.

TABLE 5 detection of binding Activity of anti-human EphA2 antibodies with mouse antigens

Example 6 detection of osteoclast-associated Gene Activity inhibited by anti-EphA 2 antibody 6D5

Mouse osteoclasts were purchased from Procel, cat: CP-M088, and routinely cultured in osteoclast complete medium (Procell Cat NO. CM-M088).

Collecting cells in a T75 culture flask, removing a culture medium in a culture dish, adding PBS to wash the cells once, adding a certain amount of pancreatin to digest at 37 ℃, adding serum to stop digesting in time after the cells become loose, gently blowing and beating the cells, collecting the cells in a 15ml centrifugal tube, centrifuging and removing a supernatant. Osteoclast complete medium with adjusted cell density of 1.5x10 ⁵ Osteoclasts were seeded into 6-well plates at 2ml per well.

A murine anti-EphA 2-6D5 antibody was added to a final concentration of 10. Mu.g/ml, while an isotype control antibody was set as a negative control. After 24h antibody reaction, the supernatant was discarded, 1ml Trizol was added, cells were lysed, RNA was isolated and purified by chloroform/isopropanol, and RNA was dissolved in RNase-free water. Mu.g of the DNA was taken and reverse transcribed into cDNA using random primers/reverse transcriptase. The expression abundances of the genes Trap, ctsk, mmp9 and Clc7 related to osteoclastic activity were obtained by PCR amplification using cDNA as a template, and the results are shown in table 6 and fig. 6. The results in table 6 indicate that the anti-EphA 2-6D5 antibody can decrease the expression abundance of the osteoclast activity gene and inhibit the function of osteoclasts, compared with the control antibody.

TABLE 6 Effect of anti-EphA 2 antibodies on osteoclast-associated Gene Activity

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Sequence listing

<110> Beijing immune Canoe medicine science and technology Co., ltd

<120> anti-EphA 2 antibody and use thereof

<130> MP2036248

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<213> Artificial Sequence (Artificial Sequence)

<400> 24

Gln His His Tyr Val Thr Pro Leu Thr

1 5

Claims

1. An antibody or antigen-binding portion thereof that specifically binds EphA2, comprising a heavy chain variable region;

or

2. The antibody or antigen binding portion thereof of claim 1, comprising a heavy chain variable region;

or

(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), which has the same function as the amino acid sequence described in (I-1); or

(I-2) the amino acid sequence of the HCDR1 has an amino acid sequence shown as SEQ ID No. 7; or

or

(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), which is functionally identical to the amino acid sequence described in (I-3); or

3. The antibody, or antigen-binding portion thereof, of claim 1 or 2, further comprising a light chain variable region;

or

4. The antibody, or antigen-binding portion thereof, of claim 2, wherein the light chain variable region comprises any one or more of the LCDR1, LCDR2 or LCDR3 sequences:

or

(I-3), wherein the amino acid sequence of the LCDR1 has an amino acid sequence shown as SEQ ID No. 16; or

or

(III-3) an amino acid sequence having 90% or more identity to the amino acid sequence described in (I-3) or (II-3).

5. The antibody, or antigen binding portion thereof, of any one of claims 1 to 4, comprising a heavy chain variable region and a light chain variable region;

or

(VI) an amino acid sequence having 90% or more identity to the amino acid sequence of (IV) or (V).

6. The antibody, or antigen binding portion thereof, of any one of claims 1 to 4, comprising a heavy chain variable region and a light chain variable region;

or

7. The antibody, or antigen binding portion thereof, of any one of claims 1 to 4, comprising a heavy chain variable region and a light chain variable region;

or

8. The antibody or antigen binding portion thereof of any one of claims 1 to 7, wherein the antigen binding portion is selected from the group consisting of: fab fragments, fab 'fragments, F (ab') 2 fragments, scFv fragments, fd fragments or single domain antibodies.

9. The antibody or antigen-binding portion thereof of any one of claims 1 to 8, wherein the antibody is murine, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No.19, and/or the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 22; or

10. The antibody or antigen-binding portion thereof of any one of claims 1 to 9, wherein said EphA2 is primate EphA2; preferably, the primate EphA2 is selected from human EphA2 or monkey EphA2.

11. The antibody or antigen binding portion thereof of any one of claims 1 to 10, wherein the plurality of one or more amino acids substitutions, deletions or additions is 2, 3, 4 or 5.

12. The antibody or antigen-binding portion thereof of any one of claims 1-11, further comprising a constant region, wherein the constant region of the heavy chain of the antibody or antigen-binding portion thereof is any one of human IgG1, igG2, igG3, or IgG 4; the constant region of the light chain of the antibody, or antigen-binding portion thereof, is of the kappa or lambda type.

13. Use of the antibody or antigen-binding portion thereof of any one of claims 1 to 12 in the preparation of a medicament for the prevention and/or treatment of an EphA 2-associated disease.

14. The use of claim 13, wherein said EphA 2-associated disease is one or more of a tumor, 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 metastasis of the above tumors.

15. A medicament comprising the antibody or antigen-binding portion thereof according to any one of claims 1 to 12.