Anti-human esm1 monoclonal antibody and use thereof

Abstract

The present invention belongs to the field of immunology and relates to the preparation and use of anti-human ESM1 monoclonal antibodies. The present invention contemplates monoclonal antibodies that specifically bind to human ESM1 and all compositions containing said antibodies or antibody fragments. Nucleic acid sequences encoding the antibodies or antibody fragments, as well as related uses, including diagnostic or therapeutic uses of said antibodies or antibody fragments. In particular, the present invention contemplates the use of these antibodies or antibody fragments in combination with other therapies.

Description

Technical Field

[0001] This invention belongs to the field of immunology, specifically relating to an anti-human ESM1 monoclonal antibody and its applications. Background Technology

[0002] ESM1 (endothelial cell specific molecule-1, also known as Endocan) is a proteoglycan composed of 165 amino acids and a sugar chain, with a protein molecular weight of approximately 21 kDa. It is primarily expressed in endothelial cells and is highly expressed in various tumor tissues, including breast cancer and colorectal cancer. In the TCGA database, ESM1 is upregulated in 21 human tumors, including invasive breast cancer. The tumor-promoting effects of ESM1 in the tumor microenvironment include: (1) ESM1 is associated with tumor metastasis. ESM1 promotes tumor proliferation and metastasis by activating the AKT / NF-κB / Cyclin D1 pathway. (2) ESM1 promotes VEGF A-induced tumor angiogenesis: ESM1 dose-dependently promotes the binding of VEGF A to VEGFR in endothelial cells. The absence of ESM1 reduces the sensitivity of endothelial cells to VEGF A, manifested as decreased VEGFR phosphorylation and weakened cell migration. This is because ESM1 enhances the bioavailability of VEGF A and the VEGF signaling response by reducing the binding of fibronectin to VEGF A; therefore, ESM1 has an enhancing effect on VEGF signaling. (3) Furthermore, ESM1 plays a role independent of VEGF in embryonic development and tumorigenesis. ko (3) Developmental defects in the vascular system of mouse embryos lead to death; overexpression of ESM1 increases tumorigenicity and radiotherapy tolerance. (4) ESM1 mediates tumor resistance to anti-VEGF antibody therapy.

[0003] Therefore, there is a need to develop anti-ESM1 antibodies with high affinity and good drug-like properties to enhance the therapeutic effect of tumor treatment. At the same time, this antibody can also be used in clinical practice to detect the ESM1 content in samples from tumor patients to determine whether anti-VEGF antibody treatment is effective. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the first objective of this invention is to provide an anti-ESM1 antibody or its antigen-binding fragment;

[0005] A second object of the present invention is to provide a nucleic acid encoding the aforementioned antibody or its antigen-binding fragment;

[0006] A third objective of this invention is to provide an expression vector for expressing the aforementioned nucleic acids;

[0007] A fourth objective of this invention is to provide a host cell containing the aforementioned nucleic acid or expression vector;

[0008] A fifth object of the present invention is to provide an immunoconjugate or pharmaceutical composition containing the aforementioned antibodies;

[0009] A sixth object of the present invention is to provide the use of the aforementioned antibodies;

[0010] The sixth objective of this invention is to provide a method for detecting ESM1 in a sample.

[0011] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0012] In a first aspect, the present invention provides an anti-ESM1 antibody, which or a fragment thereof can bind to human ESM1.

[0013] As a preferred technical solution of this application, the anti-ESM1 antibody or fragment thereof of the present invention binds to human ESM1 with high affinity, for example, with the following equilibrium dissociation constant (K). D Combined with ESM1, the K D Less than about 2 nM, preferably less than about 1 nM, more preferably less than or equal to about 0.7 nM or 0.6 nM. Most preferably 0.1-0.5 nM of K. D Incorporating ESM1. In some embodiments, ESM1 is human ESM1. In some embodiments, antibody affinity is determined using an indirect ELISA method.

[0014] As a preferred technical solution of this application, the antibody or fragment thereof of the present invention can improve the therapeutic effect when anti-VEGF antibody resistance is achieved, including the effects of inhibiting endothelial cell proliferation activity in vitro and inhibiting tumor growth, intratumoral angiogenesis, and tumor lung metastasis in vivo, such as enhancing the therapeutic effect on tumors after resistance to vesalicylate.

[0015] As a preferred embodiment of this application, the anti-ESM1 antibody of the present invention further comprises a human or mouse constant region.

[0016] As a preferred embodiment of the present invention, the anti-ESM1 antibody of the present invention is an antibody in the form of IgG1.

[0017] As a preferred embodiment of this application, the anti-ESM1 antibody is a monoclonal antibody or an antibody with monospecificity. The anti-ESM1 antibody of this invention also encompasses its antibody fragments, preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv) or (Fab')2, single-domain antibody, bispecific antibody (dAb), or linear antibody.

[0018] Preferably, the subject or individual is a non-human animal, such as a mammal, and preferably a human.

[0019] This invention protects an anti-human ESM1 monoclonal antibody or its antigen-binding fragment, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the variable region of the light chain includes light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein...

[0020] HCDR1 is selected from any of the amino acid sequences shown in SEQ ID NO.1-6, or sequences that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the same identity as any of the amino acid sequences in SEQ ID NO.1-6;

[0021] HCDR2 is selected from any of the amino acid sequences shown in SEQ ID NO. 7-11, or sequences that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the same identity as any of the amino acid sequences in SEQ ID NO. 7-11;

[0022] HCDR3 is selected from any of the amino acid sequences shown in SEQ ID NO.12-17, or sequences that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the same identity as any of the amino acid sequences in SEQ ID NO.12-17;

[0023] LCDR1 is selected from any of the amino acid sequences shown in SEQ ID NO.18-23, or sequences that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the same identity as any of the amino acid sequences in SEQ ID NO.18-23;

[0024] LCDR2 is selected from any of the amino acid sequences shown in (A1)-(A4), or sequences that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more identity with any of the amino acid sequences shown in (A1)-(A4): (A1) KVS; (A2) FTS; (A3) ETS; (A4) GAS;

[0025] LCDR3 is selected from any of the amino acid sequences shown in SEQ ID NO.24-28, or sequences that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the same identity as any of the amino acid sequences in SEQ ID NO.24-28;

[0026] Preferably, HCDR1, HCDR2 and HCDR3 are composed of amino acid sequences SEQ ID NO.1, SEQ ID NO.7 and SEQ ID NO.12, respectively, and LCDR1, LCDR2 and LCDR3 are composed of amino acid sequences SEQ ID NO.18, (A1)KVS and SEQ ID NO.24, respectively;

[0027] Preferably, HCDR1, HCDR2 and HCDR3 are composed of amino acid sequences SEQ ID NO.2, SEQ ID NO.8 and SEQ ID NO.13, respectively, and LCDR1, LCDR2 and LCDR3 are composed of amino acid sequences SEQ ID NO.19, (A2)FTS and SEQ ID NO.25, respectively;

[0028] Preferably, HCDR1, HCDR2 and HCDR3 are composed of amino acid sequences SEQ ID NO.3, SEQ ID NO.9 and SEQ ID NO.14, respectively, and LCDR1, LCDR2 and LCDR3 are composed of amino acid sequences SEQ ID NO.20, (A3)ETS and SEQ ID NO.26, respectively;

[0029] Preferably, HCDR1, HCDR2 and HCDR3 are composed of amino acid sequences SEQ ID NO.4, SEQ ID NO.10 and SEQ ID NO.15, respectively, and LCDR1, LCDR2 and LCDR3 are composed of amino acid sequences SEQ ID NO.21, (A1)KVS and SEQ ID NO.27, respectively;

[0030] Preferably, HCDR1, HCDR2 and HCDR3 are composed of amino acid sequences SEQ ID NO.5, SEQ ID NO.10 and SEQ ID NO.16, respectively, and LCDR1, LCDR2 and LCDR3 are composed of amino acid sequences SEQ ID NO.22, (A4)GAS and SEQ ID NO.28, respectively;

[0031] Preferably, HCDR1, HCDR2 and HCDR3 are composed of amino acid sequences SEQ ID NO.6, SEQ ID NO.11 and SEQ ID NO.17, respectively, and LCDR1, LCDR2 and LCDR3 are composed of amino acid sequences SEQ ID NO.23, (A2)FTS and SEQ ID NO.25, respectively.

[0032] The present invention also protects an anti-human ESM1 monoclonal antibody or its antigen-binding fragment, wherein the heavy chain variable region is selected from any of the amino acid sequences shown in SEQ ID NO.29-34, or sequences having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of identity with any of the amino acid sequences in SEQ ID NO.29-34;

[0033] The light chain variable region is selected from any of the amino acid sequences shown in SEQ ID NO.35-40, or sequences that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the same identity as any of the amino acid sequences in SEQ ID NO.35-40;

[0034] Preferably, the heavy chain variable region is as shown in the amino acid sequence SEQ ID NO.29, and the light chain variable region is as shown in the amino acid sequence SEQ ID NO.35;

[0035] Preferably, the heavy chain variable region is as shown in the amino acid sequence SEQ ID NO.30, and the light chain variable region is as shown in the amino acid sequence SEQ ID NO.36;

[0036] Preferably, the heavy chain variable region is as shown in the amino acid sequence SEQ ID NO.31, and the light chain variable region is as shown in the amino acid sequence SEQ ID NO.37;

[0037] Preferably, the heavy chain variable region is as shown in the amino acid sequence SEQ ID NO.32, and the light chain variable region is as shown in the amino acid sequence SEQ ID NO.38;

[0038] Preferably, the heavy chain variable region is as shown in the amino acid sequence SEQ ID NO.33, and the light chain variable region is as shown in the amino acid sequence SEQ ID NO.39;

[0039] Preferably, the heavy chain variable region is as shown in amino acid sequence SEQ ID NO.34, and the light chain variable region is as shown in amino acid sequence SEQ ID NO.40.

[0040] Secondly, the present invention also protects nucleic acids encoding the anti-ESM1 antibody or its antigen-binding fragment described above.

[0041] Thirdly, the present invention also protects expression vectors comprising the nucleic acids described above.

[0042] As a preferred technical solution of this application, the expression vector is pTT5 or pCDH vector.

[0043] Fourthly, the present invention also protects host cells comprising the nucleic acids or vectors described above.

[0044] As a preferred embodiment of this application, the host cell is either prokaryotic or eukaryotic.

[0045] Preferably, the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing antibodies or their antigen-binding fragments.

[0046] More preferably, the host cell is a 293 cell or a CHO cell.

[0047] Fifthly, the present invention also protects a method for preparing an anti-ESM1 antibody or an antigen-binding fragment thereof, the method comprising culturing the aforementioned host cells under conditions suitable for expressing nucleic acids of the aforementioned anti-ESM1 antibody or antigen-binding fragment thereof, optionally isolating the antibody or antigen-binding fragment thereof, and optionally further comprising recovering the anti-ESM1 antibody or antigen-binding fragment thereof from the host cells.

[0048] In a sixth aspect, the present invention also protects a conjugate comprising the anti-ESM1 antibody described above or its antigen-binding fragment and other substances.

[0049] As a preferred embodiment of this application, the other substances are cytotoxic agents.

[0050] In a seventh aspect, the present invention also protects a pharmaceutical composition comprising the anti-ESM1 antibody described above or its antigen-binding fragment or the immunoconjugate described above, and optionally pharmaceutical excipients.

[0051] Eighthly, the present invention also protects the use of an effective amount of the aforementioned anti-ESM1 antibody or its antigen-binding fragment, or the aforementioned immunoconjugate, in the preparation of a medicament for treating a subject or individual with a tumor disease for the purpose of inhibiting angiogenesis in a subject.

[0052] The present invention also protects the use of an effective amount of the aforementioned anti-ESM1 antibody or its antigen-binding fragment, or the aforementioned immune conjugate, in combination with a second therapeutic agent in the preparation of a medicament for the treatment of a subject or individual with a tumor disease for the purpose of inhibiting angiogenesis in a subject.

[0053] As a preferred technical solution of this application, the second therapeutic drug is bevacizumab.

[0054] As a preferred technical solution of this application, the tumor is cancer.

[0055] Preferably, the cancer is selected from any one of gastrointestinal cancer, colon cancer, metastatic breast cancer, lung cancer, or non-small cell lung cancer.

[0056] Ninthly, the present invention also protects a method for detecting ESM1 in a sample, the method comprising:

[0057] (a) Contact the sample with any of the anti-ESM1 antibodies or their antigen-binding fragments described above; and

[0058] (b) Detect the formation of a complex between an anti-ESM1 antibody or its antigen-binding fragment and ESM1; optionally, the anti-ESM1 antibody is detectably labeled.

[0059] This invention also covers any combination of any embodiments described herein. Any combination of any embodiments described herein is applicable to any and all anti-ESM1 antibodies or fragments thereof, methods, and uses of the invention described herein.

[0060] Beneficial effects

[0061] The anti-ESM1 antibody and its application provided by this invention have the following advantages compared with the prior art:

[0062] (1) The antibody or antigen-binding fragment of the present invention binds to ESM1 with high affinity and binds to ESM1 with the following equilibrium dissociation constant (KD), wherein the KD is less than 2nM;

[0063] (2) When resistance to anti-VEGF antibodies is encountered, the antibody of the present invention can inhibit the proliferation function of endothelial cells HUVEC alone or in combination with anti-VEGF antibodies, and is superior to the known anti-VEGF antibody bevacizumab.

[0064] (3) When combined with Bevacizumab in the treatment of tumors resistant to anti-VEGF antibodies in vivo, it can significantly enhance the inhibition of tumor growth, angiogenesis and lung metastasis by Bevacizumab. Attached Figure Description

[0065] Figure 1 The image shows the serum titer determination of mice immunized with anti-ESM1 monoclonal antibody. Negative represents serum from mice not immunized with ESM1 antigen under the same conditions. Mouse 1-10 represent serum from 10 mice immunized with ESM1 antigen. The ratio of absorbance of immunized serum to negative serum (P / N) ≥ 2.1 was used as the cutoff point for determining titer, where P is the absorbance value of immunized mouse serum and N is the absorbance value of non-immunized mouse serum. It can be seen that the serum titers of mice 1-10 all met the immunization requirements, and their spleens could be extracted for subsequent hybridoma fusion.

[0066] Figure 2This is a diagram for identifying the subtypes of anti-ESM1 monoclonal antibodies. According to the instructions of the ProteinTech Mouse Monoclonal Antibody Subtype Identification Kit (catalog number PK20003), all six monoclonal antibodies were identified as having IgG1 heavy chains and κ light chains.

[0067] Figure 3 The image shows the antibody gene acquisition process. Using cDNA derived from reverse transcription of RNA extracted from various cells as a template, and primers for the variable regions of the antibody heavy and light chains were used to retrieve the genes of different antibodies, resulting in an agarose gel electrophoresis image.

[0068] Figure 4 For the SDS-PAGE electrophoresis identification of anti-ESM1 monoclonal antibody, lanes 1, 3, 5, 7, 9, and 11 show the reducing electrophoresis patterns of antibodies 1-2B7, 1-6D11, 4-2F1, 4-2F4, 4-3B4, and 4-3C2, respectively. The heavy chains are all around 50 kDa, and the light chains are around 25 kDa. Lanes 2, 4, 6, 8, 10, and 12 show the non-reducing electrophoresis patterns of antibodies 1-2B7, 1-6D11, 4-2F1, 4-2F4, 4-3B4, and 4-3C2, respectively. The total molecular weight of the antibodies is around 150 kDa, consistent with the antibody band distribution characteristics.

[0069] Figure 5 The results show the binding of anti-ESM1 monoclonal antibodies to ESM1 secreted by tumor cells. Negative represents the PBST control group without antibody, and positive represents the anti-ESM1 polyclonal antibody control group (Abcam, catalog number ab103590). The ratio of absorbance (P / N) between each experimental group or the positive control group and the negative PBST group was ≥2.1 as the critical point for determining potency, where P is the absorbance value of each experimental group or the positive control group, and N is the absorbance value of the negative PBST group. It can be seen that monoclonal antibodies 1-2B7, 1-6D11, 4-2F1, 4-2F4, 4-3B4, and 4-3C2 can all bind to native ESM1. 1-6D11 has a weaker binding affinity to the native antigen than the positive control. 4-2F1 and 4-3C2 have comparable binding affinity to the native antigen compared to the positive control. 1-2B7, 4-2F4, and 4-3B4 have stronger binding affinity than the positive control.

[0070] Figure 6 The curve is the fitting curve for the affinity constant of the anti-ESM1 monoclonal antibody.

[0071] Figure 7 The anti-ESM1 monoclonal antibody 1-2B7 inhibited the proliferation of bevacizumab-resistant HUVECs. 7a shows the comparison results of cell proliferation ability detected by MTT assay after HUVECs were incubated with the supernatant of ESM1-overexpressing cells or the supernatant of sensitive cells under the same culture conditions following ESM1-overexpressing cell overexpression using lentiviral stable transfection of sensitive cells. Figure 7 b shows the comparison of HUVEC cells cultured under the same conditions, using the MTT assay to detect cell proliferation. It is evident that overexpression of ESM1 enhances the proliferation-promoting ability of endothelial cells. 7c shows the results of inhibiting HUVEC cell proliferation using a combination or separate clones of bevacizumab and / or anti-ESM1 monoclonal antibodies 1-2B7.

[0072] Figure 8 The figures show the tumor growth curves in mice. This depicts the volume growth of subcutaneous xenografts in nude mice after subcutaneous inoculation with bevacizumab-resistant MDA-MB-231 cells and subsequent treatment with different conditions.

[0073] Figure 9 The images shown are tumor images of mice from different groups.

[0074] Figure 10 HE staining images of tumors in the bevacizumab monotherapy group and the bevacizumab combined with anti-ESM1 monotherapy group.

[0075] Figure 11 Representative fields of CD31 and lectin staining in tumors from the bevacizumab monotherapy group and the bevacizumab combined with anti-ESM1 monotherapy group.

[0076] Figure 12 The dosing regimens were for the bevacizumab monotherapy group and the bevacizumab in combination with anti-ESM1 monotherapy group. Specific Implementation

[0077] The present invention will be further described in detail below with reference to the embodiments. Reagents or instruments used without a specified manufacturer are considered to be conventional products that can be purchased on the market.

[0078] This invention protects an anti-human ESM1 monoclonal antibody or its antigen-binding fragment, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the variable region of the light chain includes light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein...

[0079] HCDR1 is selected from any of the amino acid sequences shown in SEQ ID NO.1-6, or sequences that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the same identity as any of the amino acid sequences in SEQ ID NO.1-6;

[0080] HCDR2 is selected from any of the amino acid sequences shown in SEQ ID NO. 7-11, or sequences that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the same identity as any of the amino acid sequences in SEQ ID NO. 7-11;

[0081] HCDR3 is selected from any of the amino acid sequences shown in SEQ ID NO.12-17, or sequences that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the same identity as any of the amino acid sequences in SEQ ID NO.12-17;

[0082] LCDR1 is selected from any of the amino acid sequences shown in SEQ ID NO.18-23, or sequences that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the same identity as any of the amino acid sequences in SEQ ID NO.18-23;

[0083] LCDR2 is selected from any of the amino acid sequences shown in (A1)-(A4), or sequences that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more identity with any of the amino acid sequences shown in (A1)-(A4): (A1) KVS; (A2) FTS; (A3) ETS; (A4) GAS;

[0084] LCDR3 is selected from any of the amino acid sequences shown in SEQ ID NO.24-28, or sequences that have at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the same identity as any of the amino acid sequences in SEQ ID NO.24-28.

[0085] Preferably, the monoclonal antibodies are named 1-2B7, 4-2F1, 1-6D11, 4-2F4, 4-3C2 and 4-3B4, respectively; the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 of 1-2B7, 4-2F1, 1-6D11, 4-2F4, 4-3C2 and 4-3B4 are shown in Table A.

[0086] Table A: HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of each antibody

[0087]

[0088] Preferably, the heavy chain variable region HL and light chain variable region VL of 1-2B7, 4-2F1, 1-6D11, 4-2F4, 4-3C2 and 4-3B4 are shown in Table B.

[0089] Table B shows the heavy chain and light chain variable region sequences of each antibody.

[0090]

[0091]

[0092] This invention also protects nucleic acids encoding the anti-ESM1 antibody or its antigen-binding fragment described above.

[0093] The present invention also protects expression vectors comprising the nucleic acids described above.

[0094] Preferably, the expression vector is a pTT5 or pCDH vector.

[0095] The present invention also protects host cells comprising the nucleic acids or vectors described above.

[0096] Preferably, the host cell is prokaryotic or eukaryotic.

[0097] More preferably, the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing antibodies or their antigen-binding fragments.

[0098] Most preferably, the host cell is a 293 cell or a CHO cell.

[0099] The present invention also protects a method for preparing an anti-ESM1 antibody or an antigen-binding fragment thereof, the method comprising culturing the host cells described above under conditions suitable for expressing nucleic acids of the anti-ESM1 antibody or an antigen-binding fragment thereof as described above, optionally isolating the antibody or an antigen-binding fragment thereof, and optionally further comprising recovering the anti-ESM1 antibody or an antigen-binding fragment thereof from the host cells.

[0100] The present invention also protects a conjugate comprising the anti-ESM1 antibody described above or its antigen-binding fragment and other substances.

[0101] Preferably, the other substance is a cytotoxic agent.

[0102] The present invention also protects a pharmaceutical composition comprising the anti-ESM1 antibody described above or its antigen-binding fragment or the immunoconjugate described above, and optionally pharmaceutical excipients.

[0103] The present invention also protects the use of an effective amount of the aforementioned anti-ESM1 antibody or its antigen-binding fragment, or the aforementioned immunoconjugate, in the preparation of a medicament for treating a subject or individual with a tumor disease for the purpose of inhibiting angiogenesis in a subject, preferably, the tumor being cancer, preferably selected from any one of gastrointestinal cancer, colon cancer, metastatic breast cancer, lung cancer, or non-small cell lung cancer.

[0104] This invention also protects a method for detecting ESM1 in a sample, the method comprising:

[0105] (a) Contact the sample with any of the anti-ESM1 antibodies or their antigen-binding fragments described above; and

[0106] (b) Detect the formation of a complex between an anti-ESM1 antibody or its antigen-binding fragment and ESM1; optionally, the anti-ESM1 antibody is detectably labeled.

[0107] Example 1: Preparation of hybridoma cells

[0108] Hybridoma technology refers to the fusion of two cell types using specific techniques, resulting in a fused cell that possesses the main characteristics of both cell types. In this invention, the parental sources of the hybridoma cells are mouse spleen cells and mouse myeloma cells immunized with human ESM1 antigen through a specific procedure. The resulting hybridoma cells possess both the antibody-secreting properties derived from splenic B lymphocytes and the immortalization properties of myeloma cells. Under HAT culture medium selection, only hybridoma cells possessing both of these characteristics can survive and form clones. In this experiment, mice were immunized with hESM1 protein, and then mouse spleen cells and myeloma cells were fused to obtain hybridoma cells capable of expressing positive antibodies.

[0109] 1) Laboratory animals and immunization information

[0110] Mouse information: 6-8 week old Balb / c female mice were purchased from the Animal Experiment Center of Yangzhou University. The immunogen was hESM1, purified by fermentation of 293F cells. The immunization regimen was 50 μg / (mouse·dose), administered every two weeks via subcutaneous five-point injection and intraperitoneal injection, for a total of three times. A booster immunization was administered via tail vein injection three days before fusion.

[0111] 2) Antibody titer determination

[0112] Fourteen days after the second booster immunization, blood was collected from the posterior ocular venous plexus of mice to determine serum titer (200 μl / mouse). The collected blood was allowed to stand at room temperature for 2-3 hours, then centrifuged at 4500 rpm for 5 minutes at 4°C. Serum from unimmunized mice served as a negative control. The specific procedures are as follows: Antigen coating: The antigen hESM1 was diluted to a 1 μg / ml solution with coating buffer. 100 μl was added to each well and incubated at 37°C for 2 hours or overnight at 4°C. The plate was then washed 5 times with PBST for 5 minutes each time. Blocking: 200 μl of PBST containing 3% BSA was used for blocking. The plate was incubated at 37°C for 2 hours or overnight at 4°C. The plate was then washed 5 times with PBST for 5 minutes each time. Primary antibody incubation: Serum was diluted in eight gradients (1:10000, 1:20000, 1:40000, 1:80000, 1:160000, 1:320000, 1:640000, 1:1280000) using PBST containing 1% BSA, 100 μl per well. The plates were incubated at 37°C for 1.5 h, followed by 5 washes with PBST for 5 min each. Secondary antibody was Goat Anti-Mouse IgG H&L (HRP), diluted 1:25000 using PBST containing 1% BSA. The plates were incubated at 37°C for 1 h, followed by 5 washes with PBST for 5 min each. TMB chromogenic buffer was added, 100 μl per well, and the plates were incubated at 37°C in the dark for 20 min. Finally, 2M H2SO4 was added, 100 μl per well. Results: The absorbance of each well was measured using an ELISA reader at a wavelength of 450 nm. The ratio of the absorbance of immune serum to negative serum (P / N) ≥ 2.1 was used as the cutoff point for determining the titer, where P is the absorbance value of immunized mouse serum and N is the absorbance value of non-immunized mouse serum.

[0113] 3) Hybridoma fusion

[0114] A mouse with a serum titer of 1.28 million or higher was euthanized by cervical dislocation and immersed in 75% ethanol for 3 minutes. In a biosafety cabinet, the mouse's abdominal cavity was opened using sterile surgical scissors, and the spleen was removed and placed in a large dish containing DMEM. A small incision was made at one end of the spleen, and 10 ml of DMEM was injected into the spleen using a syringe at the other end. This injection was repeated several times until the spleen turned white. The treated spleen was placed in a 200-mesh sieve and crushed with the syringe plunger until it was completely crushed, yielding a cell suspension. The suspension was centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were washed twice with DMEM medium and resuspended for counting. SP2 / 0-Ag14 cells in the logarithmic growth phase were taken, and a cell suspension was prepared. The suspension was centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were washed twice with DMEM medium and resuspended for counting. Spleen cells and SP2 / 0 cells were mixed at a 1:1 ratio and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and 1 ml of PEG1450 was added dropwise along the tube wall. After adding all the ingredients, gently shake for 30 seconds, then add DMEM medium to stop the reaction. Let stand at room temperature for 2 minutes, then incubate at 37°C for 8 minutes. Centrifuge at 1000 rpm for 10 minutes, resuspend in 1×HAT complete medium, and evenly spread the suspension into 96-well plates containing feeder cells. Incubate at 37°C with 5% CO2. Cell clusters can be observed under a microscope on the fourth day. Replace half of the medium with HT medium around days 12-14. When most cloning wells have grown to about 1 / 3 of their capacity, completely replace the medium with HT medium, and finally replace with normal complete medium.

[0115] 4) Identification of positive clones and subcloning

[0116] When most cloning wells have grown to approximately 1 / 3 of their cell volume, the medium can be completely replaced with 1x HT medium. After 24 hours, 150 μl of cell culture supernatant is transferred to a 0.5 ml EP tube, labeled, and fresh HT medium is added simultaneously. Hybridoma-positive wells are identified by serum titer, using immunized mouse serum as a positive control and non-immunized mouse serum as a negative control, following the procedure for serum titer ELISA. Wells with a P / N ratio ≥ 2.1 are considered positive wells, and cells from wells with high positive values ​​are subcloned and expanded for culture.

[0117] 5) Cell cryopreservation

[0118] If the monoclonal cells grow to a good condition and have a viability greater than 90%, remove the supernatant, collect the cell suspension and centrifuge at 1000 rpm for 5 min, resuspend the cell pellet in cryopreservation solution, aliquot into cryovials, place them in a gradient cooling box and store at -80°C, and store in liquid nitrogen after 48 hours.

[0119] Example 2: Preparation, purification, and identification of monoclonal antibodies

[0120] 1) Preparation of monoclonal antibodies

[0121] Preparing ascites fluid from mice is an economical and efficient method for large-scale antibody production, making it the ideal choice for rapid antibody preparation in the laboratory. The main procedures are as follows: Five 8-week-old female Balb / c mice are sensitized by intraperitoneal injection of 500 μl of liquid paraffin. Approximately one million well-developed monoclonal hybridoma cells are injected intraperitoneally into one mouse. The mice's condition is observed daily; ascites fluid is collected when the abdomen becomes significantly enlarged and the limbs are weak and immobile. The peritoneum is gently lifted with forceps, and negative pressure is applied using a sterile syringe. The peritoneal cavity is flushed with 3-4 ml of physiological saline and collected to ensure sufficient antibody collection. The collected ascites fluid is centrifuged at 8000 rpm, 4°C for 15 min. The resulting sample separates into three layers; the middle layer is used for antibody purification.

[0122] 2) Antibody purification

[0123] Antibody purification utilizes a Protein A column, which specifically binds to the Fc fragments of human or mammalian antibodies, especially IgG, thereby specifically separating them. When eluted with acidic elution buffer, the antibody structure changes, causing it to dissociate from Protein A and be eluted. The specific procedure is as follows: After fixing the column, rinse with water at a rate of 1 ml / min for 8-10 column volumes using 20% ​​ethanol for column preservation. Rinse with Protein A column equilibration buffer to maintain a stable baseline. Adjust the flow rate to 0.5 ml / min and load the sample. After loading, replace the equilibration buffer, adjust the flow rate to 1 ml / min, and rinse for at least 1 column volume until the baseline stabilizes. Replace the elution buffer, observe the absorbance changes, and collect the elution buffer when the detector shows a peak. After sample elution, neutralize the elution sample to pH 7 with neutralization buffer, then rinse with 5 column volumes of elution buffer to fully elute contaminating proteins. Rinse with water for 5-10 column volumes to remove salts from the column, and then rinse with 20% ethanol until the column is completely filled with ethanol.

[0124] 3) Antibody affinity assay

[0125] Affinity is one of the important characteristics of antibodies, representing their ability to bind to antigens. This experiment used the indirect ELISA detection method established by Beatty to determine the affinity of the prepared antibodies. The specific procedure is as follows: Human ESM1 antigen was diluted to 1 μg / ml and 2 μg / ml using antigen coating buffer, and 100 μl was added to each well of the ELISA plate. Coating was performed overnight at 4°C. The next day, the liquid in the wells was discarded, and the plate was washed 5 times with PBST, 5 min each time. 200 μl of 3% BSA-PBST solution was prepared and added to each well of the ELISA plate. Blocking was performed overnight at 4°C or incubated at 37°C for 2 h. The liquid in the wells was discarded, and the plate was washed 5 times with PBST, 5 min each time. The antibody to be tested was serially diluted with 1% BSA-PBST antibody diluent to final concentrations of 1 mg / ml, 0.1 mg / ml, 0.01 mg / ml, 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, 1 ng / ml, 0.1 ng / ml, 0.01 ng / ml, 1 pg / ml, 0.1 pg / ml, and 0. 100 μl of each solution was added sequentially to the wells of an ELISA plate and incubated at 37°C for 2 h. After 2 h, the liquid in the wells was discarded, and the plate was washed 5 times with PBST for 5 min each time. Goat anti-Mouse IgG HRP was diluted with 1% BSA-PBST antibody diluent at a dilution ratio of 1:20000. 100 μl of the diluted secondary antibody solution was added to each well of the ELISA plate. The plate was incubated at 37°C for 1.5 h. The liquid in the wells was discarded, and the plate was washed 5 times with PBST for 5 min each time. Add 100 μl of TMB chromogenic solution to each well under light-protected conditions. Incubate at 37°C for 20 min. After the chromogenic reaction, stop the reaction by adding 100 μl of 1 M H2SO4 sulfuric acid stop solution to each well. Measure the absorbance at 450 nm using a microplate reader. Use Bio-Red's Microplate Manager 6 software to plot the OD450 and concentration of each antibody as a quarter curve. Obtain the antibody concentration (EC50) corresponding to half of the maximum OD450 value from the derived formula. Calculate the affinity constant K_affinity for each antibody using the Beatty formula: K_affinity = (n-1) / 2(n[Ab']t - [Ab]t), where n[Ab']t is the EC50 corresponding to the high antigen concentration and [Ab]t is the EC50 corresponding to the low antigen concentration. In this experiment, n = 2.

[0126] In the experiments described above for affinity determination, the affinities of antibodies 1-2B7, 4-2F1, 1-6D11, 4-2F4, and 4-3C2 to human ESM1 are shown in Table C.

[0127] Table C shows the affinity (equilibrium dissociation constant) for the anti-ESM1 monoclonal antibody.

[0128]

[0129] 4) Antibody subtype identification

[0130] The antibody subtype identification kit was purchased from Proteintech (catalog number PK20003). The enzyme-labeled strips in the kit were equilibrated at room temperature for 30 minutes. The hybridoma supernatant to be tested was diluted 1:100 with PBST. 50 μl of the sample to be tested was added to each well. 50 μl of diluted anti-mouse IgM+IgG was added to each well. The plate was gently tapped on both sides for 1 minute to mix thoroughly. The plate was sealed and incubated at room temperature for 1 hour. The liquid in the wells was discarded, and the plate was washed three times with PBST for 5 minutes each time, then patted dry on absorbent paper. 100 μl of freshly prepared chromogenic solution was added to each well. The plate was incubated at room temperature in the dark for 10-20 minutes, and then 100 μl of stop solution was added to each well. The OD450 was measured using a microplate reader for interpretation, or the results were directly interpreted visually based on the plate settings. 4) Antibody-tumor cell supernatant ESM1 assay

[0131] The inventors constructed a tumor cell line, MDA-MB-231S-ovESM1, that secretes ESM1-overexpressing tumor cells. The supernatant was collected and coated onto an ELISA plate, and the prepared antibody was used to detect the binding of the monoclonal antibody to the secretory natural ESM1 protein. The specific procedures are as follows: MDA-MB-231S-ovESM1 cells, in good condition and stably secreting ESM1, were cultured until the cell confluence reached 80-90%. The cell culture supernatant was collected. 50 μl each of the collected cell supernatant and antigen coating solution were added to the ELISA plate and mixed thoroughly. Coating was performed overnight at 4°C. The liquid in the wells was discarded, and the cells were washed five times with PBST for 5 min each time. The plate was then blocked. 200 μl of 3% BSA-PBST solution was prepared and added to each well of the ELISA plate. The plate was incubated at 37°C for 2 h. The liquid in the wells was discarded, and the cells were washed five times with PBST for 5 min each time. Add 100 μl of the prepared monoclonal antibody (final concentration 10 μg / ml, diluent: 1% BSA-PBST) to each well of the ELISA plate. Incubate at 37°C for 2 h. Discard the liquid in the wells and wash 5 times with PBST, 5 min each time. Dilute Goat anti-Mouse IgG HRP with 1% BSA-PBST antibody diluent at a dilution ratio of 1:20000. Add 100 μl of the diluted secondary antibody solution to each well of the ELISA plate. Incubate at 37°C for 1.5 h. Discard the liquid in the wells and wash 5 times with PBST, 5 min each time. Add 100 μl of TMB chromogenic solution to each well under light-protected conditions. React at 37°C for 20 min. After the chromogenic reaction is complete, stop the reaction by adding 100 μl of 1M H2SO4 sulfuric acid stop solution to each well. Measure the absorbance at 490 nm.

[0132] Example 3: Acquisition of the variable region sequence of a monoclonal antibody

[0133] Antibodies secreted by different hybridoma cell lines may recognize different antigenic epitopes, and antibodies recognizing the same antigenic epitope may also have different variable region sequences. Therefore, obtaining the antibody variable region gene based on subtype identification allows for antibody identification at the gene level and provides a preliminary foundation for subsequent antibody expression and modification.

[0134] 1) Extraction of total RNA from cells

[0135] This invention employs the classic Trizol method for total RNA extraction from cells. The specific procedure is as follows: Collect hybridoma cells in good growth condition, wash twice with PBS, then add 1 ml of Trizol and mix thoroughly to completely lyse the cells. The mixture will become viscous at this point. Add 200 μl of chloroform to the mixture and mix thoroughly until it reaches a strawberry smoothie consistency. Incubate on ice for 5 min. Centrifuge at 12000 rpm, 4°C for 10 min. The centrifuged sample will separate into three layers: an upper aqueous phase, a lower organic phase, and the genomic DNA in the middle. Transfer the upper aqueous phase to a 1.5 ml nuclease-free EP tube, add an equal volume of isopropanol and mix to precipitate RNA. Incubate the mixture at -20°C for 10 min. Centrifuge at 12000 rpm, 4°C for 10 min. Discard the supernatant. Resuspend the precipitate in 1 ml of 75% ethanol to wash away the isopropanol on the surface of the precipitate. Centrifuge at 12000 rpm, 4°C for 5 min. Repeat the washing process twice. After the final washing, discard as much supernatant as possible and air dry in a fume hood. Dissolve the RNA precipitate in 30 μl of nuclease-free water and incubate at 4 °C for 5 min. Measure the RNA concentration using Quick Drop.

[0136] 2) Obtain the first strand of cDNA using total RNA as a template.

[0137] a) RNA template denaturation

[0138] Prepare the following mixture in a nuclease-free centrifuge tube:

[0139]

[0140] The reaction products can be used immediately for PCR reactions or stored at -20°C.

[0141] 3) PCR amplification of antibody variable region gene

[0142] The variable region gene sequence of an antibody encodes the variable region amino acid sequence, therefore the variable region gene sequence is different for each antibody strain. The constant region of the antibody structure has the same sequence within the same species; therefore, theoretically, primers designed based on the constant region gene sequence can specifically amplify the variable region gene sequence of the antibody.

[0143] a) Primer selection

[0144] Primers targeting the variable region of the antibody heavy chain:

[0145]

[0146] Primers targeting the variable region of the antibody light chain:

[0147]

[0148]

[0149] b) Target gene amplification

[0150] This PCR was performed using Hieff 2×PCR Master Mix (with dye) to amplify the target gene. Therefore, the system preparation and program settings were designed in accordance with the product instructions.

[0151]

[0152] After the reaction is complete, the product is stored at 4°C.

[0153] 4) Agarose gel electrophoresis. Weigh 0.6g of agarose powder and boil it in 60ml of 1×TAE solution until fully dissolved. Add 6μl of 10000×TS-GelRed nucleic acid dye, mix thoroughly, and pour into a gel tank with comb teeth inserted. Allow it to solidify. Remove the comb teeth. Add 100bp DNA marker and different samples to the sample wells respectively. Electrophoresis at 120V for 40min. After electrophoresis, remove the gel and place it in a gel imaging system. Observe the bands and their positions under UV light.

[0154] 5) PCR Product Recovery. Using the Kangwei Century Agarose Gel Extraction Kit (Catalog No.: CW2302M), select positive PCR product bands and cut them out, removing as much excess gel as possible. Place the gel in a 2ml EP tube and weigh (by weight reduction). Add an equal volume of PG buffer to the tube and incubate at 50°C to dissolve. Gently invert the centrifuge tube during this process to ensure complete dissolution of the gel. Column Equilibration. Add 200μl of PS buffer to the adsorption column, centrifuge at 13000rpm for 1 minute, and discard the liquid in the collection tube. After the completely dissolved mixture has cooled to room temperature, add it to the adsorption column. Incubate at room temperature for 2 minutes, centrifuge at 13000rpm for 1 minute, and discard the liquid in the collection tube. Add 450μl of buffer PW (confirm that anhydrous ethanol has been added before use) to the adsorption column, centrifuge at 13000rpm for 1 minute, and discard the liquid in the collection tube. Wash twice with PW. Centrifuge at 13000rpm for 1 minute to remove ethanol from the adsorption column. Discard the waste liquid in the collection tube. Place the adsorption column in a ventilated area to air dry. After the ethanol has fully evaporated, place the adsorption column in a new 1.5 ml EP tube, add 30 μl of ddH2O to the center of the adsorption column, and incubate at room temperature for 2 min. Centrifuge at 13000 rpm for 1 min to obtain a DNA solution containing the target fragment.

[0155] 5) Sequencing and identification

[0156] The recovered DNA solution was sent to the company for sequencing. The sequencing results were functionally identified on the IMGT website to distinguish the coding sequence of the antibody variable region. The IMGT partitioning principle was used to partition the variable region sequence of the amino acids encoded by the antibody gene.

[0157] Example 4: Assay of Monoclonal Antibody Activity

[0158] Using the 1-2B7 monoclonal antibody with an affinity of 3.7E-10M for the antigen ESM1 as the in vitro and in vivo activity validation data, its mitigation / overcoming effect on bevacizumab resistance by antagonizing ESM1 was tested, mainly including the following examples:

[0159] 1) Bevacizumab-resistant breast cancer cells secrete ESM1, promoting endothelial cell proliferation. The applicant of this invention constructed ESM1-overexpressing cell lines and knockdown cell lines using gene overexpression / knockout technology. Subsequently, the MTT assay was used to investigate the ability of ESM1 to promote HUVEC proliferation. The specific procedures were as follows: One flask each of MDA-MB-231S, MDA-MB-231R, 231S-ovESM1, and 231R-shESM1 cells in good growth condition were prepared. After digestion with 0.25% trypsin and resuspension, the cells were counted using a hemocytometer, and the same number of cells were seeded into 6-well plates, approximately 5 × 10⁶ cells / well. 5Collect culture supernatant from each well after 24 hours; pre-plate HUVECs into 96-well plates at 5 × 10⁶ cells / well. 3 Experiments were conducted 24 hours after each well containing 1 sample. The HUVEC culture medium was removed, and 200 μL of conditioned culture supernatant was added to each well. Each experimental group was replicated 5 times, and cultured at 37°C for 36 hours. 20 μL of MTT was added to each well, and the culture was continued at 37°C for 4 hours. The supernatant was aspirated with a 1 ml syringe, and 100 μL of dimethyl sulfoxide was added to each well to dissolve formazan. The mixture was shaken on a decolorizing shaker for 10 minutes, and the absorbance at 490 nm was measured using a microplate reader.

[0160] 2) Inhibition of HUVEC proliferation under ESM1-promoted bevacizumab resistance conditions by 1-2B7. HUVECs were pre-plated in 96-well plates at 5 × 10⁻⁶ wells. 3 Cells per well, experiments were conducted after 24 hours. MDA-MB-231-R cells (bevacizumab-resistant cells) in good growth condition were selected, and the culture supernatant after 24 hours of culture was collected and filtered through a 0.45 μm filter membrane for later use. After removing the culture medium from HUVECs, the HUVEC cells were divided into 6 groups: control (with an equal volume of PBS), 250 μg / ml Bevacizumab, 250 μg / ml Bevacizumab + 100 μg / ml 1-2B7, 250 μg / ml Bevacizumab + 200 μg / ml 1-2B7, 100 μg / ml 1-2B7, and 200 μg / ml 1-2B7. 200 μL of conditioned culture supernatant was added to each well, with 4 replicates. The cells were cultured at 37°C for 36 h. 20 μL of MTT was added to each well, and the cells were cultured at 37°C for another 4 h. The supernatant was aspirated using a 1 ml syringe, and 100 μL of dimethyl sulfoxide was added to each well to dissolve the formazan. The cells were then shaken on a decolorizing shaker for 10 min, and the absorbance at 490 nm was measured using a microplate reader.

[0161] 3) Animal experimental modeling and dosing regimen, the specific procedures are as follows:

[0162] MDA-MB-231R cells in good growth condition were prepared into a cell suspension for inoculation. Following the ATCC-recommended inoculation rate of 10 million cells / mouse for MDA-MB-231 subcutaneous xenografts, 200 μl of the cell suspension was injected subcutaneously into the right axilla of each mouse using a 1 ml syringe. The mice were then returned to their cages for further rearing. Tumor volume V = 0.52 * major axis * minor axis * minor axis was calculated to be between 100-200 mm. 3 The patients were randomly assigned to groups and started medication, with the following dosing regimen: Figure 12 As shown, the largest tumor volume in the experimental group was greater than 1000 mm. 3 At that time, the administration was stopped and the mice in each group were sacrificed.

[0163] 4) Immunohistochemical staining of tumor tissue.

[0164] a) Tumor HE staining. Immediately after ex vivo tissue removal, immerse in 4% paraformaldehyde fixative for 2-3 days, followed by overnight rinsing with running water for 24 hours. Immerse sequentially in 70%, 80%, 90%, and 95% ethanol for 10 minutes each, and finally immerse twice in anhydrous ethanol for 10 minutes each time. Then, immerse the tissue in a 1:1 ethanol:xylene mixture for 30 minutes, followed by two 30-minute immersions in xylene. Next, immerse the tissue in a 1:1 xylene:paraffin mixture for 30 minutes, then immerse twice in paraffin for 30 minutes each time. After tissue infiltration, melt the paraffin, allow to cool, and section using a microtome. Dewax and place the sections in a 60°C oven for two hours. After dewaxing, the samples were immersed twice in xylene for 15 minutes each time, followed by immersion in anhydrous ethanol, 95%, 85%, 75%, and ultrapure water for 5 minutes each, and finally in PBS for 5 minutes. Hematoxylin solution was added, and staining was performed for 5 minutes. Excess staining solution was rinsed with tap water, followed by one wash with ultrapure water. Differentiation solution was added for a few seconds, followed by rinsing with tap water and then one wash with ultrapure water. Eosin solution was added, and staining was performed for 3 minutes. Excess staining solution was rinsed with tap water, followed by one wash with ultrapure water. The samples were then immersed in 70%, 80%, and 95% ethanol for 10 seconds each, followed by treatment with anhydrous ethanol for 10 minutes. Finally, the samples were immersed twice in xylene for 10 minutes each, air-dried in a fume hood, mounted with neutral resin, scanned, and statistically analyzed.

[0165] b) CD31 and Lectin staining. Three live tumor-bearing mice were randomly selected from each group. A 0.1 mg / ml DL488-Lectin solution was injected via the tail vein. Half an hour later, the mice were anesthetized, and their hearts were perfused with 4% paraformaldehyde fixative. After perfusion, tumor tissue was collected from the mice, and tissue sections were obtained using the same method as in section a) of Example 4. Antigen retrieval was then performed. The sections were immersed in sodium citrate buffer and placed in a microwave oven at a gentle boil for 5 minutes. After removal, they were allowed to cool naturally to room temperature and washed three times with PBS for 5 minutes each time. Endogenous peroxidase blocking was performed by placing the sections horizontally in a humidified chamber and immersing them in 3% H2O2 for 30 minutes in the dark. Blocking was performed by washing the sections three times with PBS for 5 minutes each time, then uniformly covering the tissue with 3% BSA solution and incubating at room temperature for 30 minutes. Incubation with the primary antibody was performed by washing the sections three times with PBS for 5 minutes each time, diluting the anti-CD31 protein primary antibody according to the antibody manufacturer's instructions, covering the tissue location on the sections, and incubating overnight at 4°C. After incubating with the second antibody, the slides were rinsed three times with PBS for 5 minutes each time. The Cy3-labeled secondary antibody was diluted according to the manufacturer's instructions and used to cover the tissue sites on the slides. The slides were incubated at room temperature for 50 minutes. The cell nuclei were stained with DAPI, dehydrated, mounted, scanned, and statistically analyzed.

[0166] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. An anti-human ESMl monoclonal antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain, characterized in that, The heavy chain variable region comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2 and HCDR3 consist of the amino acid sequences of SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 12, respectively, and the LCDR1, LCDR2 and LCDR3 consist of the amino acid sequences of SEQ ID NO. 18, KVS and SEQ ID NO. 24, respectively.

2. The anti-human ESMl monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that, The heavy chain variable region is as shown in the amino acid sequence of SEQ ID NO. 29, and the light chain variable region is as shown in the amino acid sequence of SEQ ID NO.

35.

3. A nucleic acid encoding the anti-ESMl antibody or antigen-binding fragment thereof of any one of claims 1-2.

4. An expression vector comprising the nucleic acid of claim 3.

5. The carrier of claim 4, wherein, The expression vector is a pTT5 or pCDH vector.

6. A host cell comprising the nucleic acid of claim 3 or the vector of claim 4.

7. The host cell of claim 6, wherein, The host cell is prokaryotic or eukaryotic.

8. The host cell of claim 7, wherein, The host cell is selected from an E. coli cell, a yeast cell or a mammalian cell.

9. The host cell of claim 8, wherein, The host cell is a 293 cell or a CHO cell.

10. A method of producing an anti-ESMl antibody or antigen-binding fragment thereof, the method comprising culturing the host cell of claim 6 under conditions suitable for expression of a nucleic acid encoding the anti-ESMl antibody or antigen-binding fragment thereof of any one of claims 1-2, and isolating the antibody or antigen-binding fragment thereof.

11. The method of claim 10, wherein, The method further comprises recovering the anti-ESMl antibody or antigen-binding fragment thereof from the host cell.

12. A pharmaceutical composition comprising the anti-ESMl antibody or antigen-binding fragment thereof of any one of claims 1-2 and optionally a pharmaceutically acceptable excipient.

13. Use of an effective amount of the anti-ESMl antibody or antigen-binding fragment thereof of any one of claims 1-2 in the manufacture of a medicament for treating a neoplastic disease in a subject or individual in a subject for the purpose of inhibiting angiogenesis, the neoplasm being a cancer selected from any one of a gastrointestinal cancer, a colon cancer, a metastatic breast cancer, a lung cancer or a non-small cell lung cancer.

14. Use according to claim 13, characterized in that, The use is use of an effective amount of the anti-ESMl antibody or antigen-binding fragment thereof of any one of claims 1-2 in combination with a second therapeutic agent in the manufacture of a medicament for treating a neoplastic disease in a subject or individual in a subject for the purpose of inhibiting angiogenesis.

15. Use according to claim 14, characterized in that, The second therapeutic agent is bevacizumab.

16. Use of the anti-human ESMl monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-2 in the manufacture of a kit for detecting ESMl in a sample for a non-diagnostic purpose, the use comprising: (a) contacting a sample with the anti-ESMl antibody or antigen-binding fragment thereof of any one of claims 1-2; and (b) detecting the formation of a complex between the anti-ESMl antibody or antigen-binding fragment thereof and ESMl.

17. Use according to claim 16, characterized in that, The anti-ESMl antibody is detectably labeled.

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