B7-h3-specific antibody and Anti-tumor use thereof

Abstract

The present invention provides an anti-B7-H3 (CD276) antibody or an antigen-binding fragment thereof, comprising CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NOs: 1-3, respectively, and CDR-H1, CDR-H2 and CDR-H3 as shown in SEQ ID NOs: 4-6, respectively, and a use thereof in treating tumors.

Description

B7-H3-specific antibodies and their anti-tumor applications TECHNICAL FIELD

[0001] The present invention is in the field of antibodies, and more specifically, the present invention relates to an antibody against B7-H3 (CD276), and to the use of said antibody in the treatment of tumors. TECHNICAL BACKGROUND

[0002] Monoclonal antibody is one of the most widely used targeted drugs, which shows good therapeutic effect and potential in the treatment of tumor, infectious disease and autoimmune disease. At present, the function of antibody can be improved by modifying the parent antibody through various related genetic engineering techniques, such as improving the antigen binding properties, regulating the Fc effector function and pharmacokinetic properties of antibody. The half-life of antibody is a key property of antibody pharmacokinetics, and prolonging the half-life of antibody can reduce the cost of treatment, reduce the frequency of infusion and reduce the side effects. Half-life modification is one of the key engineering of antibody Fc modification. At present, there are many mutation methods of antibody Fc fragment that have been confirmed to prolong the half-life of antibody.As T307A / E380A / N434A (AAA) mutant, M252Y / S254T / T256E (YTE) mutant, M428L / N434S (LS) mutant (Petkova S B, et al. Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model: potential application in humorally mediated autoimmune disease [J]. Int Immunol, 2006, 18(12): 1759-1769; Robbie G J, et al. A novel investigational Fc-modified humanized monoclonal antibody, motavizumab-YTE, has an extended half-life in healthy adults [J]. Antimicrob Agents Chemother, 2013, 57(12): 6147-6153; Ko S Y, et al. Enhanced neonatal Fc receptor function improves protection against primate SHIV infection [J]. Nature, 2014, 514(7524): 642-645), wherein the LS mutant is proved to enhance 11-fold binding to FcRn at pH 6.0, the HIV antibody VRC01 with LS mutant is improved 3.4-fold in half-life in human serum (Gaudinski M R, et al. Safety and pharmacokinetics of the Fc-modified HIV-1 human monoclonal antibody VRC01LS: A Phase 1 open-label clinical trial in healthy adults [J]. PLoS Med, 2018, 15(1): e1002493).ALXN1210 is a humanized anti-complement C5 IgG2 / 4 monoclonal antibody with LS mutation, which is used for paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. The half-life of the antibody is prolonged by 4 times, allowing the dose to be reduced from every 2 weeks to once every 8 weeks, while maintaining the therapeutic effect, greatly reducing the number of patient infusions (Kulasekararaj A G, et al. Ravulizumab (ALXN1210) vs eculizumab in C5-inhibitor-experienced adult patients with PNH: the 302 study [J]. Blood, 2019, 133(6): 540-549).

[0003] B7-H3 (CD276; Genbank Accession No: CAE47548.1) is a type I transmembrane protein belonging to the B7 immune co-stimulation and co-inhibition family of proteins. It is an important immune checkpoint member of the B7 family. There are two subtypes of B7-H3-2Ig and B7-H3-4Ig, of which B7-H3-4Ig is the main form present in human tissues.

[0004] B7-H3 is lowly expressed in most normal tissues, but overexpressed in various types of tumors, including melanoma, glioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, gastric cancer and colon cancer, etc. It is closely related to factors such as growth, metastasis, recurrence and poor prognosis of malignant tumors, and can also mediate immune escape. These characteristics show that B7-H3 is a very attractive target. Screening of high-affinity antibodies targeting B7-H3 can not only obtain therapeutic monoclonal antibodies, but also further develop new anti-tumor methods such as antibody drug conjugates, bispecific antibodies and CAR-T cells.

[0005] Although the exact function of B7-H3 and its receptor has yet to be elucidated, current research suggests that anti-B7-H3 monoclonal antibodies have good application prospects and are a very attractive target. SUMMARY

[0006] The objective of the present application is to develop antibodies targeting B7-H3 with good specificity and / or prolonged half-life and / or anti-tumor effect. The present inventors screened four high-affinity antibodies (A37, A48, A135, A172) by immunizing mice and using antibody phage display technology, and then constructed the full-length. Then, the four murine antibodies were detected for affinity and biological activity, and the antibody A172 with high affinity and obvious killing activity on esophageal squamous cell carcinoma cells was selected for humanization. Then, the humanized antibody was screened by ELISA, flow cytometry and SPR, and the humanized antibody A172-Hu3 was obtained.

[0007] Fc receptor (FcRn) is a key factor affecting the half-life of antibodies. By mutating the amino acids of the Fc segment of A172-Hu3, the affinity of the antibody to FcRn under pH 6.0 environment was improved, and the antibody after Fc modification was named A172-Hu3-LS. It was found that the A172-Hu3-LS antibody maintained the killing activity on esophageal squamous cell carcinoma cells in vitro while greatly improved the affinity to FcRn under pH 6.0 conditions. The half-life of the antibody was verified by using hFcRn transgenic mice, and it was found that the half-life of the A172-Hu3-LS antibody was significantly prolonged compared with A172-Hu3.

[0008] In one aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to B7-H3, the antibody or antigen-binding fragment thereof comprising complementarity determining regions (CDRs) as follows:

[0009] (a) a light chain CDR1 (CDR-L1) as set forth in SEQ ID NO: 1, a CDR-L2 as set forth in SEQ ID NO: 2, and a CDR-L3 as set forth in SEQ ID NO: 3; and

[0010] (b) a heavy chain CDR1 (CDR-H1) as set forth in SEQ ID NO: 4, a CDR-H2 as set forth in SEQ ID NO: 5, and a CDR-H3 as set forth in SEQ ID NO: 6.

[0011] In particular, the CDRs described herein are defined according to the Kabat numbering system.

[0012] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region (FR) from a human immunoglobulin in the VH and / or VL.

[0013] In certain embodiments, the VH of the antibody or antigen-binding fragment thereof of the present application comprises a framework region (FR) derived from a heavy chain variable region (VH) of a human immunoglobulin, and / or the VL of the antibody or antigen-binding fragment thereof comprises a framework region (FR) derived from a light chain variable region (VL) of a human immunoglobulin. Thus, in certain embodiments, the antibody or antigen-binding fragment thereof of the present application is humanized.

[0014] In one embodiment, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VL comprises SEQ ID NO: 7, 9, 15, 17, 19, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and the VH comprises SEQ ID NO: 8, 10, 16, 18, 20, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0015] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region as set forth in SEQ ID NO: 9 and / or a heavy chain variable region as set forth in SEQ ID NO: 10.

[0016] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof of the application comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having up to 50 conservative substitutions (e.g., up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 conservative substitutions; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions) compared to the wild-type sequence from which it is derived.

[0017] In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof of the application comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof having up to 50 conservative substitutions (e.g., up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 conservative substitutions; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions) compared to the wild-type sequence from which it is derived.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof of the application has a light chain constant region selected from, e.g., a kappa or lambda light chain constant region, preferably a kappa light chain constant region (e.g., a human kappa light chain constant region). In some embodiments, the light chain constant region of the antibody or antigen-binding fragment thereof of the application comprises amino acids 109-215 as set forth in SEQ ID NO: 11.

[0019] In some embodiments, the constant region is altered, e.g., mutated, to modify the properties of the anti-B7-H3 antibody molecule (e.g., to alter one or more of the following properties: Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function). Functional changes can be made by replacing at least one amino acid residue in the constant region of the antibody with a different residue, e.g., to change the affinity of the antibody for an effector ligand (e.g., FcR or complement Clq), thereby altering effector function (e.g., decreasing it). The Fc region of an antibody mediates several important effector functions, e.g., ADCC, phagocytosis (ADCP), CDC, etc.

[0020] In certain embodiments, the antibody or antigen-binding fragment thereof of the present application has a heavy chain constant region selected from, e.g., a heavy chain constant region of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE; in particular, a heavy chain constant region of IgGl, IgG2, IgG3, and IgG4. In some embodiments, the heavy chain constant region comprises or consists of an Fc region.

[0021] In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 14, or a conservative substitution of at most 20 amino acids thereof (e.g., a conservative substitution of at most 20, at most 15, at most 10, or at most 5 amino acids; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions), or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0022] In some embodiments, the Fc comprises at least one, preferably two, mutations in the positions corresponding to positions 311 and 317, with reference to the numbering of the sequence set forth in SEQ ID NO: 14. In some embodiments, the Fc comprises a mutation in the positions corresponding to positions 311 and 317, with reference to the numbering of the sequence set forth in SEQ ID NO: 14. In some embodiments, the Fc comprises at least one, preferably two, of the following mutations: 311L and 317S, with reference to the numbering of the sequence set forth in SEQ ID NO: 14. In some embodiments, the Fc comprises 311L and 317S, with reference to the numbering of the sequence set forth in SEQ ID NO: 14.

[0023] As used herein, a position corresponding to position X as set forth in SEQ ID NO: 14 refers to a position corresponding to position X of SEQ ID NO: 14 as identified when aligning a polypeptide of interest to the sequence set forth in SEQ ID NO: 14 for maximum identity or homology (where aligning conserved amino acids) in accordance with sequence alignment means known in the art, including, for example, using manual alignment and by using available alignment programs such as BLASTP or GAP algorithm and other methods known to one of skill in the art.

[0024] In certain embodiments, an antibody or antigen-binding fragment thereof of the present application comprises:

[0025] (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 12 or 13, or a substitution, deletion, or addition of one or several amino acids, or any combination thereof (e.g., a substitution, deletion, or addition of up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids, or any combination thereof; e.g., a substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof) or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; and

[0026] (b) a light chain comprising the amino acid sequence set forth in SEQ ID NO: 11, or a substitution, deletion, or addition of one or several amino acids, or any combination thereof (e.g., a substitution, deletion, or addition of up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids, or any combination thereof; e.g., a substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof) or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0027] In certain embodiments, the substitution is a conservative substitution.

[0028] In certain embodiments, the antibody of the present application is a humanized antibody. In certain embodiments, the antibody of the present application is a monoclonal antibody. In certain embodiments, the antibody of the present application or antigen-binding fragment thereof is selected from the group consisting of a scFv, a Fab, a Fab', a F(ab')2, a Fv fragment, a disulfide linked Fv (dsFv), a diabody.

[0029] Preparation of antibodies

[0030] The antibody of the present application can be prepared in various methods known in the art, for example, by obtaining a DNA molecule encoding the heavy and light chain genes of the antibody of the present application through genetic engineering recombination techniques. The resulting DNA molecule is inserted into an expression vector, and then the host cell is transfected. Then, the transfected host cell is cultured under specific conditions, and the antibody of the present application is expressed.

[0031] Antigen-binding fragments of the antibody of the present application can be obtained by hydrolyzing an intact antibody molecule (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). In addition, these antigen-binding fragments can also be directly produced from a recombinant host cell (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab' fragments can be directly obtained from a host cell; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can also be directly isolated from a recombinant host cell culture solution. Those of ordinary skill in the art are fully aware of other techniques for preparing these antigen-binding fragments.

[0032] In another aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody of the present application or antigen-binding fragment thereof. In certain embodiments, the nucleotide sequence is codon-optimized according to codon degeneracy. In certain embodiments, the nucleotide sequence is codon-optimized.

[0033] In certain embodiments, the VL of the antibody or antigen-binding fragment thereof of the present application is encoded by the nucleic acid sequence set forth in any one of SEQ ID NO: 21, 23, 29, 31, or 33, and / or the VH of the antibody or antigen-binding fragment thereof of the present application is encoded by the nucleic acid sequence set forth in any one of SEQ ID NO: 22, 24, 30, 32, or 34.

[0034] In certain embodiments, the Fc region of the antibody or antigen-binding fragment thereof of the present application is encoded by the nucleic acid sequence set forth in SEQ ID NO: 28.

[0035] In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof of the present application is encoded by the nucleic acid sequence set forth in SEQ ID NO: 25, and / or the heavy chain of the antibody or antigen-binding fragment thereof of the present application is encoded by the nucleic acid sequence set forth in SEQ ID NO: 26 or 27.

[0036] In certain embodiments, the isolated nucleic acid molecule of the present application comprises: (i) a first nucleic acid and a second nucleic acid that encode the heavy chain variable region and the light chain variable region, respectively, of the antibody or antigen-binding fragment thereof of the present application, or (ii) a first nucleic acid that encodes the heavy chain variable region and the heavy chain constant region, respectively, of the antibody or antigen-binding fragment thereof of the present application, and a second nucleic acid that encodes the light chain variable region and the light chain constant region, or (iii) a first nucleic acid and a second nucleic acid that encode the heavy chain and the light chain, respectively, of the antibody or antigen-binding fragment thereof of the present application. In certain embodiments, the first nucleic acid and the second nucleic acid comprise nucleic acids that are degenerate to or substantially identical to the first nucleic acid and the second nucleic acid of any one of (i)-(iii) above. In certain embodiments, the degenerate or substantially identical sequence means a sequence that has at least about 85%, 90%, 95%, 99% or more sequence identity compared to the nucleic acid molecule described in (i)-(iii), or a sequence with one or more nucleotide substitutions, or differs by no more than 3, 6, 15, 30, or 45 nucleotides.

[0037] In another aspect, a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the present application is provided. In certain embodiments, the vector of the present application is, e.g., a plasmid, a cosmid, a phage, a lentivirus, etc. In certain embodiments, the vector is capable of expressing the antibody or antigen-binding fragment thereof of the present application in vivo in a subject (e.g., a mammal, e.g., a human).

[0038] In another aspect, there is provided a host cell comprising the isolated nucleic acid molecule of the application or the vector of the application. The host cell can be a eukaryotic cell (e.g. mammalian cell, insect cell, yeast cell) or a prokaryotic cell (e.g. E. coli). Suitable eukaryotic cells include, but are not limited to, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain embodiments, the host cell of the application is a mammalian cell, e.g. CHO (e.g. CHO-K1, CHO-S, CHO DXB11, CHO DG44).

[0039] In another aspect, there is provided a method of producing an antibody or antigen binding fragment thereof of the application, comprising culturing a host cell of the application under conditions permitting expression of the antibody or antigen binding fragment thereof, and recovering the antibody or antigen binding fragment thereof from the cultured host cell culture.

[0040] Uses and methods of treatment

[0041] In another aspect of the application, there is provided an antibody or antigen binding fragment thereof of the application for use in the treatment of a tumor (in particular a tumor that uses B7-H3 as a marker or therapeutic target).

[0042] In another aspect of the application, there is provided the use of an antibody or antigen binding fragment thereof, nucleic acid, vector or host cell of the application in the manufacture of a medicament for the treatment of a tumor.

[0043] The tumor described herein, in particular a tumor that uses B7-H3 as a marker or therapeutic target, includes hematological cancers such as leukemia and solid cancers, e.g. esophageal cancer, neuroblastoma, melanoma, glioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, gastric cancer, colon cancer, colorectal cancer, large intestine cancer, cervical cancer, medullary thyroid cancer, liver cancer.

[0044] In certain embodiments, the subject is a mammal, including non-human mammals and humans. In certain embodiments, the subject is a human.

[0045] In another aspect, the present application provides a method of treating a tumor in a subject, the method comprising administering to a subject in need thereof an effective amount of an antibody or antigen binding fragment thereof described herein. The "treatment" method described herein refers to the process of reaching the tumor tissue after the antibody or antigen binding fragment thereof or a pharmaceutical composition comprising the same is introduced into the body by intravenous injection or local injection at the lesion, etc., and then exerting the therapeutic effect.

[0046] The antibody or antigen-binding fragment thereof of the present application can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present application should be sterile and stable under the conditions of production and storage, and can be prepared into injections.

[0047] In addition, the antibody or antigen-binding fragment thereof of the present application can be present in a pharmaceutical composition in unit dosage form for ease of administration.

[0048] The pharmaceutical composition of the present application can include a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody or antigen-binding fragment thereof of the present application. The "prophylactically effective amount" refers to an amount sufficient to prevent, deter, or delay the onset of a disease. The "therapeutically effective amount" refers to an amount sufficient to cure, or at least partially arrest the disease and its complications in an individual already suffering from a disease, for example, 0.1 mg / ml to 5000 mg / ml.

[0049] In the present application, the subject can be a mammal (including non-human mammals and humans), for example, a human.

[0050] In another aspect, the present application provides a method of detecting the presence or level of B7-H3 in a sample, which comprises the steps of using the antibody or antigen-binding fragment thereof of the present application, and detecting the complex formed between the antibody or antigen-binding fragment thereof of the present application and B7-H3. In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present application is further labeled with a detectable label. In another preferred embodiment, the method further comprises detecting the antibody or antigen-binding fragment thereof of the present application using a reagent labeled with a detectable label. The method can be used for diagnostic purposes, or non-diagnostic purposes (for example, for B7-H3 pathway research, drug screening, histochemical analysis, etc.). In certain embodiments, the sample for diagnostic purposes can be a tissue or cell sample (for example, a biopsy sample), and the sample for non-diagnostic purposes is a cell sample, for example, a cell line or an ex vivo cell culture.

[0051] In another aspect, there is provided use of the antibody or antigen-binding fragment thereof of the present application in the manufacture of a kit for detecting the presence or level of B7-H3 in a sample. In another aspect, the present application provides a diagnostic or therapeutic kit comprising one or more of the following: the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, multispecific antibody, conjugate, or pharmaceutical composition of the present application. Optionally, the diagnostic or therapeutic kit further comprises instructions for use.

[0052] The main component of the "kit" described herein is the antibody or antigen-binding fragment thereof described herein, which can further include other required reagents, such as labeled fluorescein, radioisotope, peroxidase, alkaline phosphatase, buffer, non-antibodies described herein, substrates of enzymatic reactions such as diaminobenzidine (DAB), etc. and corresponding supports such as enzyme-labeled plates, magnetic beads, etc. The kit can be used for diagnosing the in vivo distribution of tumor tissues, pathological tissue sections, etc., or for analyzing and identifying cells, proteins, etc., or for affinity purification of cells or protein molecules containing B7-H3 protein domains.

[0053] The "diagnostic" method described herein refers to qualitative and quantitative detection of substances reflecting the health status of the human body, such as human body fluids, blood, tissues, etc. Common experimental techniques include immunohistochemistry, immunocytochemistry, enzyme-linked immunoassay, etc.

[0054] Definitions of terms

[0055] In this document, unless otherwise indicated, the scientific and technical terms used in this document have the meanings commonly understood by one of ordinary skill in the art. And the cell culture, biochemical, nucleic acid chemistry, immunology laboratory, etc. The operation steps used herein are the conventional steps widely used in the corresponding field. At the same time, in order to better understand the present application, the definitions and explanations of related terms are provided as follows.

[0056] As used in this application and in the appended claims, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, the meaning of the singular form "one" includes "one or more."

[0057] As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally comprised of two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define a different class of antibodies as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 3 or more amino acids. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant domains are not involved directly in binding of an antibody to an antigen, but exhibit various effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) form the antigen binding site.

[0058] In the present context, the CDRs comprised by the antibodies of the application or antigen binding fragments thereof can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs comprised by the antibodies of the application or antigen binding fragments thereof are preferably determined by the Kabat, Chothia, or AbM or IMGT numbering system.

[0059] As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in a variable region of an antibody other than the CDR residues as defined above.

[0060] The term "antibody" is not limited by any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotype, e.g., an IgG (e.g., IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibody.

[0061] As used herein, the term "antigen binding fragment" of an antibody refers to a polypeptide fragment of an antibody, e.g., a polypeptide fragment of a full length antibody, that retains the ability to specifically bind to the same antigen bound by the full length antibody, and / or competes with the full length antibody for specific binding to the antigen, which is also referred to as an "antigen binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single chain antibodies (e.g., scFv), diabodies, linear antibodies, nanobodies (e.g., technology from Ablynx), domain antibodies (e.g., technology from Domantis), and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding capacity of the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0062] As used herein, the term "full length antibody" means an antibody that is composed of two "full length heavy chains" and two "full length light chains". Wherein, a "full length heavy chain" refers to a polypeptide chain that is composed of, in the direction from N-terminus to C-terminus, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and, optionally, a heavy chain constant region CH4 domain when the full length antibody is of IgE isotype. Preferably, a "full length heavy chain" is a polypeptide chain that is composed of, in the direction from N-terminus to C-terminus, VH, CH1, HR, CH2 and CH3. A "full length light chain" is a polypeptide chain that is composed of, in the direction from N-terminus to C-terminus, a light chain variable region (VL) and a light chain constant region (CL). The two pairs of full length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HR of the two full length heavy chains. The full length antibody of the present application can be from a single species, e.g., human; and can also be a humanized antibody. The full length antibody of the present application contains two antigen binding sites formed by a pair of VH and VL, respectively, which specifically recognize / bind to the same antigen.

[0063] As used herein, the term "Fd fragment" means an antibody fragment consisting of VH and CHI domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341 :544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CHI domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region; the term "Fab' fragment" means a fragment obtained after reduction of the disulfide bond connecting the two heavy chain fragments in an F(ab')2 fragment, consisting of one complete light chain and a Fd fragment (consisting of VH and CHI domains) of a heavy chain.

[0064] As used herein, the term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. An Fv fragment is generally considered to be the smallest antibody fragment that is capable of forming a complete antigen binding site. It is generally believed that the six CDRs confer the antigen binding specificity of an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding an antigen, although its affinity can be lower than that of the complete binding site.

[0065] As used herein, the term "Fc fragment" means an antibody fragment formed by disulfide bond binding of the second, third constant regions of the first heavy chain with the second, third constant regions of the second heavy chain of an antibody. The Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding.

[0066] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4may be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present application are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond can also exist between the VH and VL of the scFv. As used herein, the term "di-scFv" refers to an antibody fragment formed by the linkage of two scFv.

[0067] As used herein, the term "diabodies" means that the VH and VL domains of a single polypeptide chain are expressed, but using a linker that is too short to allow for pairing between the two domains of a single chain, thereby forcing the domains to pair with the complement domains of another chain and creating two antigen binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).

[0068] Each of the above-mentioned antibody fragments is maintained by the ability to specifically bind the same antigen bound by the full-length antibody from which the fragment was derived, and / or competes with the full-length antibody for specific binding to the antigen.

[0069] Antigen binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those of skill in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage procedures) and screened for specificity in the same manner as is done for whole antibodies.

[0070] In this document, the term "antibody" includes not only intact antibodies, but also antigen binding fragments of antibodies, unless the context indicates otherwise.

[0071] The terms "monoclonal antibody," "monoclonal," "mAb," as used herein have the same meaning and are used interchangeably to refer to an antibody from a population of highly homogenous antibody molecules (i.e., a population of identical antibody molecules except for possible naturally occurring mutations that can arise during production). A monoclonal antibody has high specificity for a single epitope on an antigen. Polyclonal antibodies are in contrast to monoclonal antibodies and generally comprise at least 2 or more different antibodies, which typically recognize different epitopes on an antigen. Moreover, the modifier "monoclonal" is not to be construed as a requirement for using any particular method for making the antibody.

[0072] To prepare humanized antibodies, murine CDR regions can be inserted into human framework sequences using methods known in the art (see U.S. Patent No. 5,225,539 to Winter; U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370 to Queen et al.; and Lo, Benny, K.C., editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can also be utilized that are capable of producing a complete repertoire of human antibodies upon immunization and that are incapable of producing endogenous immunoglobulins (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258; Lonberg et al. (1994) Nature 368(6474):856-859; WO 02 / 43478). Other methods of antibody humanization include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991, 222:581-597; Vaughan et al., 1996, Nature Biotech 14:309).

[0073] As used herein, the term "degree of humanization" is a measure used to evaluate the number of non-human derived amino acid residues in a humanized antibody. The degree of humanization of a humanized antibody can be predicted, for example, by IMGT website Domain Gap Align to compare the homology of the variable region sequence to a human V domain.

[0074] As used herein, the term "specifically binds" refers to a nonrandom binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed in terms of the dissociation equilibrium constant (KD) of the interaction. In the present context, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the dissociation equilibrium constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. In certain embodiments, an antibody that specifically binds to (or an antibody specific for) an antigen refers to an antibody that binds to the antigen with a dissociation constant (KD) of less than about 10 -8M, for example, less than about 10 -8 M, 10 -9 M, 10 -10 M, or 10 -11 M or less. In certain embodiments, an antibody or antigen-binding fragment thereof of the present application is considered to specifically bind TSLP when KD≤ 10 x 10 -8 M. In certain embodiments, an antibody or antigen-binding fragment thereof of the present application is considered to specifically bind TSLP when KD≤ 10 x 10

[0075] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 : 186-187). The ratio kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kon, and kdis values can be measured using any effective method. In certain embodiments, the dissociation constant can be measured using bioluminescence interferometry (e.g., ForteBio Octet method). In addition to this, the dissociation constant can be measured using surface plasmon resonance technology (e.g., Biacore) or Kinexa.

[0076] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of the inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, and directs the expression of elements of genetic material that it carries in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 -derived artificial chromosomes (PAC); bacteriophages, such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can contain a replication origin.

[0077] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as E. coli or Bacillus subtilis, a fungal cell such as a yeast cell or Aspergillus, an insect cell such as S2 Drosophila cell or Sf9, or an animal cell such as fibroblast, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, HEK 293 cell, or human cell.

[0078] As used herein, the term "identity" is used in reference to the match between two polypeptide sequences or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., if a position in each of two DNA molecules is occupied by adenine, or if a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is the number of matching positions shared by the two sequences divided by the number of positions compared times 100. For example, if two sequences have 6 of their 10 positions matched, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of 6 positions are matched). Typically, the comparison is made over the length of the sequences being compared, after aligning the two sequences to produce maximum identity. Such alignment can be achieved, for example, by the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, using a computer program such as the Align program (DNAstar, Inc.). In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a gap length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)).

[0079] As used herein, a sequence having a certain percent identity retains an important biological activity of the sequence with which it is compared or from which it is derived, such as antibody binding specificity. A sequence having one or several substitutions, deletions, or additions of amino acids, or any combination thereof, retains an important biological activity of the sequence with which it is compared or from which it is derived, such as antibody binding specificity.

[0080] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of an amino acid residue for an amino acid residue with similar side chains, e.g., substitutions that take place within a family of amino acid residues that have similar physico-chemical or functional properties, e.g., size, shape, charge, chemical properties including ability to form covalent or hydrogen bonds, etc. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a conservative substitution is one in which the replaced amino acid residue is replaced with another amino acid residue from the same side chain family. Methods of identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0081] The nomenclature used herein to refer to the twenty conventional amino acids follows conventional usage. See, e.g., Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this document, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in this document, amino acids are generally referred to by their commonly accepted single and three letter abbreviations well known in the art. For example, alanine can be referred to as A or Ala; arginine can be referred to as R or Arg; glycine can be referred to as G or Gly; glutamine can be referred to as Q or Gin.

[0082] As used herein, the term "treatment" refers to the methods performed with the intent to obtain a beneficial or desired clinical result. For purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of the state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Further, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0083] As used herein, the term "subject" refers to a mammal, such as a primate, e.g., a non-human primate or a human. In certain embodiments, the subject (e.g., a human) has a tumor (particularly a tumor that uses B7-H3 as a marker), or is at risk of having a tumor (particularly a tumor that uses B7-H3 as a marker).

[0084] As used herein, the term "effective amount" refers to an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., a solid tumor such as esophageal cancer) refers to an amount that is sufficient to prevent, arrest, or delay the onset of the disease (e.g., a solid tumor such as esophageal cancer); an effective amount for treating a disease refers to an amount that is sufficient to cure or at least partially arrest the disease and its complications in an already afflicted patient. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic purposes will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, body weight, and sex, the mode of administration of the drug, and other therapies being administered to the patient, etc.

[0085] As used herein, the term "immune cell" includes cells that have hematopoietic origin and play a role in the immune response, such as lymphocytes, e.g., B cells and T cells; natural killer cells; myeloid cells, e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0086] As used herein, the term "immune response" refers to the action of immune cells (e.g., lymphocytes, antigen presenting cells, phagocytes, or granulocytes) and soluble macromolecules produced by immune cells or the liver (including antibodies, cytokines, and complement) that result in the selective damage, destruction of, or clearance from the body of invading pathogens, cells or tissues infected with pathogens, cancer cells, or normal human cells or tissues in the context of autoimmunity or pathological inflammation. In this context, the term "antigen-specific T cell response" refers to an immune response produced by a T cell that results upon stimulation of that T cell by the antigen to which that T cell is specific. Non-limiting examples of responses produced by T cells upon antigen-specific stimulation include proliferation of the T cell and production of cytokines (e.g., IL-2).

[0087] As used herein, the term "effector function" refers to those biological activities attributable to an antibody Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) and which vary with the antibody isotype.

[0088] The term "pharmaceutically acceptable" means moieties, fragments or compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human as appropriate. Specific examples of materials that can serve as pharmaceutically acceptable carriers or components thereof include sugars (e.g., lactose), starches, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, and the like.

[0089] Advantages of the Invention

[0090] Compared with the prior art, the technical scheme of the present application has the following advantages:

[0091] (1) The antibodies of the present application can specifically recognize / bind B7-H3 with high affinity;

[0092] (2) Some of the antibodies of the present application are humanized antibodies, which can be safely administered to subjects without eliciting an immunogenic response;

[0093] (3) Some of the antibodies of the present application are Fc engineered antibodies, which have significantly improved affinity to FcRn at pH 6.0 and significantly prolonged half-life.

[0094] Therefore, the antibodies of the present application show good anti-tumor effect and have important clinical value.

[0095] Abbreviations CDR Complementarity Determining Region in an immunoglobulin variable region FR Antibody framework region: amino acid residues in an antibody variable region other than CDR residues VH Antibody heavy chain variable region VL Antibody light chain variable region IgG Immunoglobulin G Kabat Immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991) mAb Monoclonal antibody EC50 Concentration that produces 50% efficacy or binding ELISA Enzyme-linked immunosorbent assay PCR Polymerase chain reaction HRP Horseradish peroxidase Fc IgG antibody Fc fragment KD Dissociation equilibrium constant CDR-H1 Complementarity Determining Region 1 in an immunoglobulin heavy chain variable region CDR-H2 Complementarity Determining Region 2 in an immunoglobulin heavy chain variable region CDR-H3 Complementarity Determining Region 3 in an immunoglobulin heavy chain variable region CDR-L1 Complementarity Determining Region 1 in an immunoglobulin light chain variable region CDR-L2 Complementarity Determining Region 2 in an immunoglobulin light chain variable region CDR-L3 Complementarity Determining Region 3 in an immunoglobulin light chain variable region BRIEF DESCRIPTION OF DRAWINGS

[0096] Figure 1: ELISA identifies Fab antibody binding affinity to B7-H3 antibody. (A): OD450 values of 16 Fab antibodies binding to B7-H3-2Ig. (B): OD450 values of 16 Fab antibodies binding to B7-H3-4Ig. MGA271 is a positive control.

[0097] Figure 2: SDS-PAGE identification results. SDS-PAGE results show that the antibody purity is >90%.

[0098] Figure 3: Monoclonal antibody binding ability to esophageal squamous cell carcinoma cells. Flow cytometry results show that 4 antibodies all bind to esophageal squamous cell carcinoma cell lines.

[0099] Figure 4: Detection of the EC50 values of monoclonal antibodies binding to B7-H3 protein and esophageal squamous cell carcinoma cells. (A): ELISA was used to determine the binding curves of four antibodies to B7-H3-2Ig and B7-H3-4Ig. (B): Flow cytometry was used to detect the binding curves of four antibodies to esophageal squamous cell carcinoma cell line KYSE150.

[0100] Figure 5: Detection of the ADCC activity of four murine antibodies. LDH method was used to detect the killing activity of A37, A48, A135, and A172 four murine antibodies on esophageal squamous cell carcinoma cell line KYSE150. (*p<0.05, **p<0.01, ***p<0.001).

[0101] Figure 6: Identification results of SDS-PAGE.

[0102] Figure 7: ELISA detection of the EC50 values of humanized A172 antibody binding to different forms of proteins. (A): ELISA was used to analyze the binding of monoclonal antibodies to human B7-H3-2Ig and B7-H3-4Ig. (B): Flow cytometry was used to detect the binding ability of monoclonal antibodies to esophageal squamous cell carcinoma cell line KYSE150.

[0103] Figure 8: Affinity detection of A172 humanized antibody. SPR was used to detect the sensorgrams of A172-Mu, A172-Hu1, A172-Hu2, A172-Hu3, and A172-Hu5 binding to B7-H3-4Ig protein.

[0104] Figure 9: In vitro cytotoxicity detection of A172 humanized antibody. (A, B): LDH method was used to detect the in vitro killing effect mediated by A172 humanized antibody. Human peripheral blood PBMC was used as effector cells, and esophageal squamous cell carcinoma cell lines KYSE150 and KYSE450 were used as target cells. The ratio of effector cells to target cells was set to 20:1. (*p<0.05, **p<0.01, ***p<0.001).

[0105] Figure 10: A172-Hu3-LS has strong affinity to FcRn. (A): Binding curves of A172-Hu3 and A172-Hu3-LS antibodies to FcRn at pH 6.0. (B): Binding curves of A172-Hu3 and A172-Hu3-LS antibodies to FcRn at pH 7.4.

[0106] Figure 11: A172-Hu3-LS has enhanced affinity to FcRn at pH 6.0. (A): SPR was used to detect the sensorgrams of A172-Hu3 binding to FcRn protein at pH 6.0. (B): SPR was used to detect the sensorgrams of A172-Hu3-LS binding to FcRn protein at pH 6.0.

[0107] Figure 12: A172-Hu3-LS has strong ADCC activity in vitro. (A, B): Fc-engineered antibody A172-Hu3-LS mediated ADCC in vitro killing effect was detected by LDH method. Human peripheral blood PBMC was effector cell, esophageal squamous cell carcinoma cell KYSE150 and KYSE450 were target cells. The ratio of effector cells to target cells was set to 20: 1. (* p < 0.05, ** p < 0.01)

[0108] Figure 13: Antibody half-life curve before and after Fc engineering. Example

[0109] The present application will now be described with reference to the following examples which illustrate the application (but do not limit the application) by way of example only. Unless otherwise specifically indicated, the molecular biology and immunological techniques utilized in the present application are in accordance with standard methods, as described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc., 1995. Those skilled in the art will appreciate that the examples describe the application by way of example only, and are not intended to limit the scope of the application as claimed.

[0110] Example 1: Mouse immunization and library screening of lead antibody by phage display technology

[0111] 1.1) Mouse immunization

[0112] The mice were immunized with B7-H3 2Ig protein (purchased from Beijing Bypass Biotech Co., Ltd., Cat. #B73-H52E2) with a purity of more than 95% and no endotoxin. Three 6-8 week old female Balb / c mice were selected, and multiple point subcutaneous injection was performed on the back, 100 μg of protein was injected per mouse each time, a total of 3 times, with an interval of 2 weeks each time. After the last booster immunization, blood was collected to measure the serum antibody titer, which reached 80,000, and impact immunization was performed by intraperitoneal injection, and the mouse spleen was taken on the 3rd day.

[0113] 1.2) Library screening of lead antibody by phage display library

[0114] The RNA was extracted from the spleen cells of the immunized mice and, after reverse transcription, the mouse immune antibody library was constructed by the method of group primer amplification, and the library capacity of the antibody library was determined, and the correct insertion rate of the antibody gene was verified by monoclonal sequencing analysis. The immune tube, magnetic bead screening instrument, solid phase and liquid phase cross screening method were used to screen the mouse immune library, and the specific Fab antibody was enriched. After antibody sequencing and ELISA primary screening, positive clones combined with the antigen were obtained (see Figures 1A and 1B). Finally, four lead antibodies (A37, A48, A135, A172) with high primary screening OD value, specific sequence and good diversity were selected, and the full-length antibody was constructed, and the vector used was pCDNA3.1.

[0115] 1.3) Identification of the purity of the four antibodies

[0116] The A37, A48, A135, and A172 light and heavy chain plasmids were transfected into HEK293 cells, and the supernatant was collected after 5 days of transfection and further purified by Protein A affinity chromatography to obtain the antibody protein to be tested. The obtained antibodies were identified for molecular weight size and purity by 10% SDS-PAGE gel protein electrophoresis and Coomassie blue staining, as shown in Figure 2. The non-reduced-SDS-PAG results showed that the sizes of the four proteins were about 150 kDa, and the reduced-SDS-PAGE showed that the sizes of the heavy and light chains of the four proteins were 55 kDa and 25 kDa. There was no obvious impurity band, and the buffer for protein preservation was 1×PBS (pH 7.4).

[0117] 1.4) Flow cytometry detection of the binding ability of murine monoclonal antibodies to B7-H3-expressing cells

[0118] (1) Esophageal squamous cell carcinoma cells KYSE150 and KYSE450 were collected and centrifuged, resuspended and counted in a PBS solution containing 2% FBS, and the density was adjusted to a cell suspension of 2×10 6 cells / mL. The cell suspension was added to the EP tube at a volume of 200 μL per tube, centrifuged at 2000 rpm for 5 min, and the supernatant was discarded.

[0119] (2) Dilute the antibody with buffer and add it to the cells, resuspend the cells, and the final concentration of the antibody is 5 μg / mL. Another EP tube with 200 μL of buffer was used as a blank control. Incubate the EP tube on ice for 1 h.

[0120] (3) Centrifuge at 2000 rpm for 5 min, discard the supernatant, and wash twice with PBS.

[0121] (4) Use FITC-labeled IgG secondary antibody, dilute with buffer at 1:200. Resuspend the cells with 200 μL of secondary antibody dilution per tube. Incubate on ice for 1 h in the dark. Centrifuge and wash twice with PBS.

[0122] (5) Resuspend the cells with 200 μL buffer, filter through a 300 mesh screen, and then detect the fluorescence intensity by flow cytometry.

[0123] The biological activity of the antibody binding to the cell surface B7-H3 was detected by flow cytometry, as shown in Figure 3. The results showed that A37, A48, A135, and A172 all had strong binding capacity to esophageal squamous cell carcinoma cells KYSE150 and KYSE450, and A37, A48, and A172 were superior to the positive control MGA271.

[0124] Example 2: ELISA and flow cytometry detection of the EC50 value of the murine monoclonal antibody binding to B7-H3 protein and esophageal squamous cell carcinoma cells

[0125] 1) ELISA detection of the binding of murine monoclonal antibody to B7-H3 protein and calculation of EC50 value

[0126] 1) Antigen coating: B7-H3-2Ig and B7-H3-4Ig proteins were diluted to a concentration of 2 μg / mL using PBS (pH 7.4), with a coating amount of 50 μL / well, and placed in a 4°C refrigerator for 12 h.

[0127] 2) Blocking: The plate was washed 3 times with PBST, then 3% BSA-PBST blocking solution was added to the wells, and incubated at 37°C for 1 h. The plate was washed 3 times.

[0128] 6) Add the sample to be tested: Add the antibody diluted with PBS, 100 μL per well, with a concentration of 20 μg / mL in the first well, and then 1:3 gradient dilution for a total of 8 wells. Incubate at 37°C for 2 h.

[0129] 7) Secondary antibody incubation: Use HRP-conjugated IgG secondary antibody, dilute the secondary antibody (1:5000) with 0.1% BSA-PBST, 100 μL / well, and incubate at 37°C for 1 h.

[0130] 8) Color development: Wash the plate 3 times, add TMB color developing solution, 100 μL / well, and incubate at 37°C in the dark for 15 min.

[0131] 9) Termination: Add an equal volume (100 μL / well) of 1M hydrochloric acid solution to terminate the reaction, and the reaction solution in the well changes from blue to yellow.

[0132] 10) Read the results: Measure the OD450 value with a microplate reader within 15 min.

[0133] The ELISA experiment results show that the EC50 values of A172 antibody binding with B7-H3-2lg and B7-H3-4lg are 15.45 ± 1.798 ng / mL and 25.97 ± 3.148 ng / mL, respectively. Compared with other antibodies, A172 has higher affinity for specific binding with B7-H3, and is superior to the positive control MGA271 (see Figure 4A, Table 1).

[0134] 2) Flow cytometry detection of EC50 values of mouse monoclonal antibody binding with esophageal squamous cell carcinoma cells

[0135] 1) Esophageal squamous cell carcinoma cells KYSE150 were collected and centrifuged, resuspended with buffer (2% FBS in PBS solution) for counting and preparation into a cell suspension with a density of 2 x 10 6 The cell suspension was added to an EP tube at a volume of 200 μL per tube, centrifuged at 2000 rpm for 5 min, and the supernatant was discarded.

[0136] 2) The antibody to be tested was diluted with buffer to different concentration gradients, starting at 30 μg / ml, diluted 1:3, and 200 μL of each concentration was added to the resuspended cells in the EP tube to make the final concentration of the antibody 30, 10, 3, 1, 0.33, 0.11, 0.037, 0.012 μg / mL, and another EP tube with 200 μL of buffer was added as a blank control.

[0137] 3) The EP tube was incubated on ice for 1 h, centrifuged at 2000 rpm for 5 min, and the supernatant was discarded.

[0138] 4) FITC-labeled IgG secondary antibody was used, diluted 1:200 with buffer. 200 μL of the secondary antibody diluent was used to resuspend the cells. Incubate on ice for 1 h in the dark. Centrifuge and wash twice with PBS.

[0139] 5) Add 200 μL of buffer to resuspend the cells, filter through a 300-mesh sieve, and then detect the fluorescence intensity by flow cytometry.

[0140] Table 1: EC50 values of mouse monoclonal antibodies binding with B7-H3 (average ± standard deviation)

[0141] The results show that the EC50 values of A172 binding with esophageal squamous cell carcinoma cells are 300.2 ± 13.68 ng / mL. Compared with other antibodies, A172 has the highest affinity for specific binding with esophageal squamous cell carcinoma cells (see Figure 4B, Table 1). A172 was selected for the next step of humanization.

[0142] Example 3: LDH method for detecting the ADCC effect of mouse antibody A172 on esophageal squamous cell carcinoma cells in vitro

[0143] 1. Collect 20 mL blood from healthy donors by venipuncture, dilute the collected blood with PBS at equal proportion. Prepare 5 15 mL centrifuge tubes, add 4 mL lymphocyte separation medium into each 15 mL centrifuge tube, then slowly add 8 mL diluted blood, keep the blood on the upper layer of the separation medium, centrifuge at 1500 rpm for 22 min. Transfer the white membrane layer to a new centrifuge tube, wash twice with PBS, resuspend the cells with complete 1640 medium containing 200 ng / mL IL2, place in a dish for culture for 24 h, use the next day.

[0144] 2. Collect esophageal squamous carcinoma KYSE150 and KYSE450 cells (target cells) in logarithmic growth phase, centrifuge at 800 rpm for 3 min to discard the supernatant. Adjust the cell density to 1×10 5 6 cells / mL with culture solution, 100 μL per well, culture overnight, and wait for cell adhesion.

[0145] 3. Dilute the antibody to 10 μg / mL with complete 1640 medium. Add the antibody to the 96-well plate, 50 μL per well, incubate at 37°C in a 5% CO2 incubator for 30 min.

[0146] 4. Collect PBMC cells, centrifuge, discard the supernatant, wash twice with PBS. Adjust the cell density to 2×10 6 6 cells / mL (containing 200 ng / mL IL2) with culture medium, mix and add to the above 96-well plate, 100 μL per well (effector to target ratio of 20:1). Set up medium background wells, volume correction wells, target cell lysis wells, target cell spontaneous release wells, and effector cell spontaneous release wells. Incubate at 37°C in a 5% CO2 incubator for 18 h.

[0147] 5. Add 25 μL lysis solution in the detection kit to the target cell lysis wells and volume correction wells 45 min before detection.

[0148] 6. Centrifuge the 96-well plate at 300 g for 3 min, and then transfer 50 μL supernatant to another 96-well plate, add 50 μL LDH detection reagent to each well, incubate at room temperature for 20 min in the dark. Add 50 μL stop solution in the detection kit to each well. Detect OD490 value by a microplate reader.

[0149] 7. Calculate: according to the formula: cell killing toxicity = [(experimental wells-medium background wells)-(target cell spontaneous release wells-medium background wells)-(effector cell spontaneous release wells-medium background wells)] / [(target cell lysis wells-medium correction wells)-(target cell spontaneous release wells-medium background wells)]×100%, calculate the cell killing toxicity.

[0150] The results showed that the ADCC activity of A172 and A48 was higher, and had obvious toxic effect on esophageal squamous cell carcinoma cells (see Figure 5).

[0151] Example 4: Humanization of murine A172 antibody and purification of humanized antibody thereof

[0152] Using the IMGT database (http: / / www.imgt.org / IMGTrepertoire / Proteins / ), the complementarity determining regions (CDRs, as shown in SEQ ID NOs: 1-6) in the light and heavy chain variable regions of the murine antibody were retained, the framework regions (FRs) were aligned with human framework regions, and the four human antibody framework regions with the highest homology ranking were determined to replace the murine antibody framework regions. The light and heavy chain constant regions of the antibody were replaced with a human light chain constant region (к, Uniprot P01834, as shown in amino acids 109-215 of SEQ ID NO: 11) and an IgG1 heavy chain constant region (Uniprot P01857, as shown in amino acids 123-452 of SEQ ID NO: 12). The replaced sequences were then codon-optimized and synthesized by Shanghai Generay Biotech Co., Ltd., and were ligated to the pTT5 vector to construct plasmids, which were named A172-Hu1, A172-Hu2, A172-Hu3, and A172-Hu5. The antibody type was IgG1к.

[0153] We used HEK293 cells to express A172 and its humanized antibodies, and purified the antibodies by Protein A affinity chromatography. The purity of the obtained antibodies was identified by 10% SDS-PAGE gel electrophoresis and Coomassie blue staining, as shown in Figure 6. The results showed that the antibody purity was >90%, and there was no obvious impurity band. The protein was stored in 1x PBS (pH 7.4) buffer.

[0154] Example 5: ELISA and flow cytometry detection of the affinity of humanized A172 antibody to B7-H3 protein and esophageal squamous cell carcinoma cells

[0155] 5.1) ELISA was used to detect the binding of humanized monoclonal antibody to B7-H3 and calculate the EC50 value.

[0156] The method was the same as in Example 2.1).

[0157] 5.2) Flow cytometry was used to detect the binding of humanized monoclonal antibody to esophageal squamous cell carcinoma cells and calculate the EC50 value.

[0158] The method was the same as in Example 2.2).

[0159] Table 2: EC50 values (mean ± standard deviation) of humanized A172 monoclonal antibody binding to B7-H3

[0160] ELISA and flow cytometry results showed that A172-Hu3 had higher affinity to B7-H3 than A172-Mu, A172-Hu1, A172-Hu2, and A172-Hu5 (see FIGS. 7A and 7B, and Table 2).

[0161] Example 6: Surface Plasmon Resonance (SPR) detection of the affinity of A172 antibodies after humanization to bind to B7-H3

[0162] (1) Capture A172-Mu, A172-Hu1, A172-Hu2, A172-Hu3, and A172-Hu5 antibodies on the surface of a Protein A chip. Dilute the antibodies to 1 μg / mL with HBS-EP+ buffer, and set the flow rate to 10 μL / min.

[0163] (2) Set 10 different concentration gradients for B7-H3 antigen, and dilute the antigen to 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100, and 200 nM with 1x HBS-EP+ buffer. Set the flow rate of B7-H3 antigen to 30 μL / min, and the binding time to 300 s.

[0164] (3) Regenerate the chip according to the regeneration procedure using Glycine 1.5 as the regeneration buffer.

[0165] (4) Analyze the results using GraphPad Prism 8 software, perform binding mode fitting, and calculate the binding kinetic parameters.

[0166] Table 3: KD values of MGA271, A172-Mu, A172-Hu1, A172-Hu2, A172-Hu3, and A172-Hu5 binding to B7-H3-4Ig protein Note: Ka is the association constant, Kd is the dissociation constant, and KD is the equilibrium dissociation constant

[0167] The results show that after humanization, the affinity does not change significantly, and the affinity to B7-H3 protein is better than that of the positive control MGA271 (see FIG. 8 and Table 3).

[0168] Example 7: Detection of the ADCC effect of humanized A172 antibodies on esophageal squamous cell carcinoma cells in vitro by LDH method

[0169] The method is the same as in Example 3.

[0170] The results show that the ADCC effect mediated by humanized antibodies A172-Hu1, A172-Hu2, and A172-Hu3 is better than A172-Mu, and has obvious killing activity on tumor cells (see FIG. 9A and FIG. 9B). Based on the above results, we found that the affinity and the ability to mediate ADCC of A172-Hu2 and A172-Hu3 are basically equivalent, but the expression amount per unit volume of A172-Hu3 antibody is higher than that of A172-Hu2, and after comprehensive consideration, A172-Hu3 is selected for the next step experiment.

[0171] The half-life of an antibody depends largely on the transport of FcRn to IgG, FcRn can effectively intercept IgG degradation and return it to the blood circulation, thereby prolonging the half-life of IgG in serum. The binding of FcRn and IgG is pH-dependent, they bind at pH 6.0 and dissociate at pH 7.4. Therefore, improving the affinity of FcRn to IgG at pH 6.0 is an effective way to prolong the half-life of antibodies. At present, it is mainly achieved by mutating the amino acids of the Fc segment. In order to not affect the ADCC activity of the antibody itself, we choose the mutation type M311L / N317S, and the mutated antibody is named A172-Hu3-LS (the light chain and heavy chain are shown in SEQ ID NO: 11 and 13, respectively).

[0172] Example 8: ELISA method for detecting the affinity of A172-Hu3-LS to FcRn (pH 6.0 / pH 7.4)

[0173] The affinity of A172-Hu3-LS antibody to FcRn and IgG at different pH was verified by ELISA.

[0174] 1. Antigen coating: Dilute human FcRn protein to a concentration of 2 μg / mL using PBS (pH 7.4), and the coating amount is 50 μL / well. The coating condition is 4°C for 12 h.

[0175] 2. Blocking: Wash the plate with PBST 3 times, 200 μL / well, and the blocking solution is 3% BSA-PBST. The blocking condition is 37°C for 1 h, and the plate is washed 3 times.

[0176] 3. Add the sample to be tested: Add the antibody diluted with 0.1% BSA-PBST (pH 6.0 / pH 7.4), 100 μL per well, the first well is 100 μg / mL, the second well is diluted 1:9, then 1:3 gradient dilution, and the last well is diluted 1:9. Incubate at 37°C for 1 h.

[0177] 4. Secondary antibody incubation: HRP goat anti-human secondary antibody was used, the secondary antibody was diluted with 0.1% BSA-PBST (pH 6.0 / pH 7.4) (1:5000), 100 μL / well, 37°C incubation for 1 h.

[0178] 5. After color development and termination, the OD450 value was measured with a microplate reader.

[0179] The results showed that the affinity of A172-Hu3-LS to FcRn at pH 6.0 was significantly improved, and the affinity of A172-Hu3-LS to FcRn at pH 7.4 was slightly increased compared with the control antibody A172-Hu3 (see Figures 10A and 10B).

[0180] Example 9: Surface plasmon resonance (SPR) detection of the affinity of A172-Hu3-LS to FcRn at pH 6.0

[0181] 1. His antibody was coupled to the surface of a CM5 chip. The antibody was diluted to 20 μg / mL with HBS-EP+ (pH 6.0) buffer, and the flow rate was set to 10 μL / min.

[0182] 2. The His-tagged FcRn protein (2 μg / mL) was captured using the His antibody-coupled CM5 chip. A172-Hu3, A172-Hu3-LS antibodies were diluted to 0, 7.8, 15.625, 31.25, 62.5, 125, 250, 500 nM with buffer 1x HBS-EP+ (pH 6.0). The flow rate of the antibody was set to 30 μL / min, the binding time was 60 s, and the dissociation time was 120 s.

[0183] 3. Chip regeneration. The chip was regenerated according to the regeneration procedure using Glycine 1.5 as the regeneration buffer.

[0184] 4. Result analysis. The results were analyzed with GraphPad Prism 8 software, the binding mode was fitted, and the binding kinetic parameters were calculated.

[0185] Table 4. KD values of A172-Hu3 and A172-Hu3-LS binding to FcRn protein at pH 6.0 Note: Ka is the association constant, Kd is the dissociation constant, and KD is the equilibrium dissociation constant

[0186] The results showed that the KD values of A172-Hu3 and A172-Hu3-LS binding to FcRn at pH 6.0 were 22.2 nM and 1.27 nM, respectively (see Figures 11A, 11B, and Table 4). Compared with A172-Hu3, the affinity of A172-Hu3-LS to FcRn was significantly improved.

[0187] Example 10: LDH method to detect the ADCC effect of A172-Hu3-LS antibody on esophageal squamous cell carcinoma cells

[0188] To confirm whether the ADCC activity of A172-Hu3-LS antibody is affected after mutation of the Fc segment, we performed an LDH experiment. The method is the same as Example 3.

[0189] The results show that the ability of A172-Hu3 and Fc-engineered antibody A172-Hu3-LS to mediate ADCC is basically equivalent, and there is obvious killing activity on esophageal squamous cell carcinoma KYSE150 and KYSE450 cells (Figures 12A and 12B). This shows that the LS mutation of the Fc segment of A172-Hu3-LS antibody does not affect the ADCC activity of the antibody.

[0190] Example 11: Detection of antibody half-life

[0191] We detected the half-life extension of A172-Hu-LS antibody by a human FcRn transgenic mouse in vivo pharmacokinetic model.

[0192] (1) B-hFCRN mice, female, 5-6 weeks old, purchased from Bao Saiguang Biotechnology Co., Ltd. Jiangsu. Randomly divided into 2 groups, 6 in each group.

[0193] (2) On day 0, A172-Hu3 and A172-Hu3-LS antibodies were injected intraperitoneally into the two groups of mice, respectively, at a dose of 5 mg / kg.

[0194] (3) At 15 min, 24 h, 4 d, 7 d, 10 d, 14 d, 18 d, 22 d, 26 d, 30 d, 35 d, 39 d, 45 d and 50 d, the mouse blood was collected through the tail vein, the serum was separated and stored. After the last blood collection was completed, the antibody concentration in the mouse serum was detected by Elisa method using B7-H3 protein coated 96-well plate, and the serum pharmacokinetic curve was drawn (Figure 13). The half-life was calculated using Phoenix winnonlin 8.1 software.

[0195] The results show that the serum half-lives of A172-Hu3 and A172-Hu3-LS are 114 h and 189 h, respectively, and the half-life of A172-Hu3-LS is significantly prolonged. In contrast, the antibody concentration in the serum of A172-Hu3-LS decreases slowly and still maintains a high value at day 50, about 100 times the concentration of A172-Hu3.

[0196] Sequence information

[0197] While the specific embodiments of the application have been described in detail, those skilled in the art will appreciate that various modifications and alterations to the details can be made within the scope of the application as disclosed in the teachings of the present application. The scope of the application is indicated by the appended claims and any equivalents thereof.

Claims

1. An antibody against B7-H3 or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a complementarity-determining region (CDR): (a) The light chain CDR1 (CDR-L1) shown in SEQ ID NO:1, CDR-L2 shown in SEQ ID NO:2, and CDR-L3 shown in SEQ ID NO:3; and (b) Heavy chain CDR1 (CDR-H1) shown in SEQ ID NO:4, CDR-H2 shown in SEQ ID NO:5, and CDR-H3 shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment comprises: a light chain variable region (VL) comprising a sequence of SEQ ID NO:7, 9, 15, 17, 19 or having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or a heavy chain variable region (VH) comprising SEQ ID NO:8, 10, 16, 18, 20 or having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is humanized, preferably comprising the light chain variable region shown in SEQ ID NO:9 and / or the heavy chain variable region shown in SEQ ID NO:

10.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein, The antibody or its antigen-binding fragment further comprises: (a) The heavy chain constant region (CH) of human immunoglobulins or a variant thereof; and / or (b) The light chain constant region (CL) of human immunoglobulins or a variant thereof. The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the wild-type sequence from which it originates; or, the variant has one or more amino acid substitutions, deletions, or additions, or any combination thereof, compared with the wild-type sequence from which it originates; preferably, the substitutions are conservative substitutions.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the Fc segment of the heavy chain constant region or a variant thereof comprises the amino acid sequence shown in SEQ ID NO:14, or contains amino acid L (311L) at position 311 corresponding to position 311 of SEQ ID NO:14, and contains amino acid S (317S) at position 317 corresponding to position 317 of SEQ ID NO:

14.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody comprises: The antibody comprises the light chain shown in SEQ ID NO:11 and / or the heavy chain shown in SEQ ID NO:12 or 13. Preferably, the antibody comprises the light chain shown in SEQ ID NO:11 and the heavy chain shown in SEQ ID NO:

13.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein, The antibody or its antigen-binding fragment is selected from scFv, Fab, Fab', F(ab')2, Fv fragment, disulfide-linked Fv(dsFv) and biantibody.

8. An isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-7.

9. A vector comprising the isolated nucleic acid of claim 8; preferably, the vector is a cloning vector or an expression vector.

10. A host cell comprising the isolated nucleic acid of claim 8 or the vector of claim 9.

11. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-7, comprising culturing the host cell of claim 10 under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

12. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-7, and a pharmaceutically acceptable carrier and / or excipient, preferably, the pharmaceutical composition being used to treat tumors, particularly hematologic malignancies such as leukemia and solid tumors, such as esophageal cancer, neuroblastoma, melanoma, glioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, gastric cancer, colon cancer, colorectal cancer, colorectal cancer, cervical cancer, medullary thyroid carcinoma, and liver cancer.

13. A kit comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-7, an isolated nucleic acid as described in claim 8, a vector as described in claim 9, a host cell as described in claim 10, and / or a pharmaceutical composition as described in claim 12, and optionally including instructions for use. Preferably, the kit is used for the diagnosis or treatment of tumors, particularly hematologic malignancies such as leukemia and solid tumors, such as esophageal cancer, neuroblastoma, melanoma, glioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, gastric cancer, colon cancer, colorectal cancer, colorectal cancer, cervical cancer, medullary thyroid carcinoma, and liver cancer.

14. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-7, and / or the nucleic acid according to claim 8, and / or the vector according to claim 9, and / or the host cell according to claim 10 in the preparation of a medicament or kit for treating or diagnosing tumors; Preferably, the tumor is selected from hematologic cancers such as leukemia and solid tumors, such as esophageal cancer, neuroblastoma, melanoma, glioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, gastric cancer, colon cancer, colorectal cancer, colorectal cancer, cervical cancer, medullary thyroid carcinoma, and liver cancer.

15. A method for treating a tumor in a subject, the method comprising administering to a subject in need an effective amount of the antibody or antigen-binding fragment of any one of claims 1-7, wherein the subject is a mammal; preferably, the subject is a human; Preferably, the tumor is selected from hematologic cancers such as leukemia and solid tumors, such as esophageal cancer, neuroblastoma, melanoma, glioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, gastric cancer, colon cancer, colorectal cancer, colorectal cancer, cervical cancer, medullary thyroid carcinoma, and liver cancer.

16. A method for diagnosing a tumor in a subject, the method comprising contacting a biological sample from the subject with an antibody or an antigen-binding fragment thereof as described in any one of claims 1-7, and detecting the formation of an antigen-antibody complex, wherein the subject is a mammal; preferably, the subject is a human; Preferably, the tumor is selected from hematologic cancers such as leukemia and solid tumors, such as esophageal cancer, neuroblastoma, melanoma, glioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, gastric cancer, colon cancer, colorectal cancer, colorectal cancer, cervical cancer, medullary thyroid carcinoma, and liver cancer.

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