Anti-human Semaphorin 4C antibody and its application

Abstract

The application discloses an anti-human Semaphorin 4C antibody and application thereof. The application obtains a series of anti-human SEMA4C antibodies by immunizing mice. The anti-human SEMA4C antibody can effectively combine with human SEMA4C protein, can be used for early screening of breast cancer, and part of the antibodies show good anti-TNBC subtype breast cancer curative effect in animal model tests.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy, specifically relating to an antibody based on human Semaphorin 4C protein and its application. Background Technology

[0002] Since the 1970s, the incidence of malignant tumors in my country has been on the rise, and cancer has become one of the major factors affecting the health of Chinese residents. In recent years, with the advancement and development of detection technology, the discovery of numerous molecular biomarkers, and the emergence of molecularly targeted drugs, cancer treatment has begun to move towards standardization, personalization, and precision. Achieving personalized treatment is an effective way to improve the current low cure rate and high mortality rate of cancer.

[0003] Tumor tissue is a complex ecological microenvironment composed of various cell types, including tumor cells and lymphocytes. Compared to normal tissue, tumor tissue is more acidic and has lower dissolved oxygen levels. Over the past 20 years, with in-depth research into tumors and immunology, immunotherapy has gradually become a mainstream approach to cancer treatment. Its principle is to regulate the body's own immune system to fight tumor cells through specific means, promoting the immune system's response to cancer cells to alleviate or cure the tumor. Immunotherapy primarily relies on various immune cells in the body, such as CD8+ T cells, NK cells, and NKT cells, all of which can kill tumor cells through specific pathways. However, the tumor microenvironment contains complex immunosuppressive molecules and mechanisms that mediate tumor cells' escape from immune cell attacks. These include hypoxic and low-pH environments and immunosuppressive receptors and ligands. These adverse factors suppress anti-tumor immune cells in the tumor environment. Therefore, antagonizing lymphocyte function-suppressing signals and normalizing their immune function has become an important direction in tumor immunotherapy. Since the beginning of the 21st century, with the discovery of a series of important immune checkpoint molecules, such as CTLA4, PD-1 / PD-L1, TIM3, LAG3, and TIGIT, and the approval of CTLA4 and PD-1 / PD-L1 antibody drugs, cancer immunotherapy has entered a new era. Meanwhile, PD-1 antibodies have become one of the best-selling biopharmaceutical drugs. In 2018, the Nobel Prize in Medicine and Physiology was awarded to Professor James P. Allison for discovering CTLA4 and Professor Tasuku Honjo for discovering PD-1, in recognition of their outstanding contributions to the field of anti-immune checkpoint therapy.

[0004] Based on current clinical data, anti-immune checkpoint drugs have shown promising efficacy in the clinical treatment of melanoma. For example, nivolumab (anti-PD-1) treatment for melanoma achieved an objective response rate (ORR) of 43.7%, while the ORR of nivolumab combined with ipilimumab (anti-CTLA4) reached as high as 57.6%. Anti-immune checkpoint drugs have also been applied to non-small cell lung cancer, renal cell carcinoma, prostate cancer, gastric cancer, and head and neck cancer. However, in some solid tumors, such as breast cancer, especially triple-negative breast cancer, the response rate of anti-immune checkpoint drugs is unsatisfactory. For instance, in a study by S. Adams et al. involving 170 patients with aggressive triple-negative breast cancer treated with anti-PD-1 antibodies, the overall response rate was only 4.7%. Therefore, discovering more effective immune checkpoint molecules or anti-tumor targets has become a crucial factor in cancer treatment.

[0005] Breast cancer is a heterogeneous disease encompassing subtypes with different biological characteristics. According to global cancer statistics, approximately 2.1 million new women are diagnosed with breast cancer each year, accounting for one-quarter of all female cancer cases. Triple-negative breast cancer (TNBC) is a highly malignant subtype of breast cancer, accounting for approximately 10%–20% of cases, and is associated with poor prognosis. The vast majority of TNBC cases are invasive ductal carcinomas (approximately 95%), and are negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2). Currently, there is no specific treatment for TNBC. Clinical treatment mainly involves chemotherapy, radiotherapy, and surgery, but the treatment effects are poor and the side effects are significant. Therefore, there is an urgent clinical need for a highly specific TNBC treatment drug with low toxicity.

[0006] Semaphorins (SEMAs) were initially identified as axonal signaling molecules, but subsequent studies have shown that SEMAs can be divided into more than 20 different types and play a role in a range of physiological processes, including neurogenesis, angiogenesis, bone resorption, and immune regulation. SEMA molecules are also involved in the development of human diseases, including tumorigenesis and neuroregenerative diseases. Semaphorin 4C (SEMA4C) belongs to the type IV membrane-associated SEMA family, which also includes SEMA4D and SEMA4A. Members of this family often bind to the Plexins family to transduce signals, participate in immune response regulation, and play an important role in tumor immunotherapy. Human SEMA4C consists of 883 amino acids and possesses various post-translational modifications, including glycosylation and phosphorylation. Recent studies have shown that SEMA4C protein expression is low in healthy individuals, but significantly elevated expression can be detected in various tumors, such as breast cancer (including TNBC subtypes), cervical cancer, esophageal cancer, gastric cancer, colorectal cancer, bladder cancer, and ovarian cancer. Furthermore, SEMA4C exists as a secreted factor, promoting lymphangiogenesis and lymphatic metastasis of tumor cells. SEMA4C may be an effective target in the treatment of the aforementioned cancers, particularly breast cancer. Simultaneously, high serum SEMA4C levels may be a biomarker for breast and cervical cancer. The discovery of antibodies specifically targeting SEMA4C has significant social and clinical value, both for the treatment of cancer, particularly breast cancer, and for early screening of breast cancer. Summary of the Invention

[0007] To address the problems in existing technologies, this invention prepares a series of human SEMA4C antibodies by immunizing mice. These SEMA4C antibodies can effectively bind to the human SEMA4C protein and can be used for early screening of breast cancer. Furthermore, some antibodies have demonstrated good anti-breast cancer efficacy in animal model experiments.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] In a first aspect, an antibody or antibody fragment targeting SEMA4C is provided, comprising a heavy chain variable region (VH) and / or a light chain variable region (VL) of one of the antibodies shown in the table below, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region and the CDR1, CDR2, and CDR3 of the light chain variable region are shown in the table below (CDR region delineation uses the IMGT database):

[0010] Table 1

[0011]

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019] Preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in Table 2 below:

[0020] Table 2

[0021]

[0022]

[0023]

[0024] ;

[0025] Preferably, the antibody or antibody fragment targeting SEMA4C is a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, single-chain antibody, bispecific antibody, or multispecific antibody.

[0026] Preferably, the antibody is a full-length antibody comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is selected from hIgG1, hIgG2, hIgG3 or hIgG4; and / or, the light chain constant region is selected from the κ chain or the λ chain;

[0027] More preferably, the heavy chain constant region is hIgG1 or hIgG4;

[0028] More preferably, the light chain constant region is selected from the κ chain; even more preferably, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:198 or SEQ ID NO:199, and the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:200 or SEQ ID NO:201.

[0029] In a second aspect, an antibody or antibody fragment described in the first aspect is provided with a label;

[0030] Preferably, the label is a fluorescent label, an enzyme label, or a radioactive label.

[0031] Thirdly, the present invention provides a nucleic acid encoding the antibody or antibody fragment described in the first aspect.

[0032] Fourthly, the present invention provides a recombinant expression vector comprising the nucleic acid as described in the third aspect;

[0033] Preferably, the recombinant expression vector comprises a eukaryotic cell expression vector and / or a prokaryotic cell expression vector.

[0034] Fifthly, the present invention provides a host cell comprising the recombinant expression vector as described in the fourth aspect;

[0035] Preferably, the host cell of the transformant is a prokaryotic cell and / or a eukaryotic cell;

[0036] More preferably, the prokaryotic cells are E. coli TG1 or BL21 cells, and the eukaryotic cells are HEK293 cells or CHO cells.

[0037] In a sixth aspect, the present invention provides a composition comprising any one of the antibodies or antibody fragments described in the first aspect and a pharmaceutically acceptable carrier.

[0038] In a seventh aspect, the present invention provides a kit comprising any one of the antibodies or antibody fragments described in the first aspect.

[0039] Eighthly, the present invention provides the use of the antibody or antibody fragment described in any one of the first aspects, or any one of the compositions described in the sixth aspect, in the preparation of medicaments for treating breast cancer (including TNBC subtype), cervical cancer, esophageal cancer, gastric cancer, colorectal cancer, bladder cancer, and ovarian cancer.

[0040] In a ninth aspect, the present invention provides the use of the antibody or antibody fragment of any one of the first aspects, any one of the compositions of the sixth aspect, and any one of the kits of the seventh aspect in the preparation of reagents for detecting SEMA4C protein in a sample.

[0041] The beneficial effects of this invention are:

[0042] By using antibodies, the concentration of SEMA4C in a sample can be specifically and accurately detected, thereby aiding in the diagnosis of specific cancers, such as breast cancer.

[0043] This invention prepares SEMA4C antibody by immunizing mice, and demonstrates good anti-TNBC subtype breast cancer efficacy in animal model experiments. Attached Figure Description

[0044] Figure 1 SDS-PAGE analysis of purified human SEMA4C protein;

[0045] Figure 2 Partial heavy chain PCR amplification results;

[0046] Figure 3 Partial light chain PCR amplification results;

[0047] Figure 4 .ScFv overlapping amplification results;

[0048] Figure 5 Efficacy studies of SEMA4C antibody in a mouse TNBC tumor model;

[0049] Figure 6 The effect of different heavy chain constant regions on the antitumor activity of SEMA4C antibody was tested. Detailed Implementation

[0050] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0051] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0052] Definitions and Explanations:

[0053] In this invention, unless otherwise stated, the term "antibody fragment" generally refers to an antigen-binding antibody fragment, which may include a portion of a complete antibody, typically an antigen-binding region or a variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv or scFv, biantibodies, linear antibodies, single-chain antibody molecules, etc.

[0054] The terms "complementarity-determining region" or "CDR amino acid sequence" or "CDR sequence" refer to the amino acid sequence in an antibody responsible for antigen binding. Generally, the light chain variable region and heavy chain variable region of an antibody or antibody fragment each contain three CDR sequences. The resulting CDR sequences may vary slightly when using different tools for CDR sequence delimitation; the CDR sequences defined in this patent are uniformly determined using IMGT (https: / / www.imgt.org / ).

[0055] In this invention, the antibody or antibody fragment is capable of binding to the SEMA4C protein (antigen), particularly the amino acid sequence shown in SEQ ID NO: 197. Preferably, the dissociation constant (K0) between the antibody or antibody fragment of this invention and the SEMA4C protein is [not specified]. D The value is above 100 nM. The term "dissociation constant" indicates the tightness of the antibody-antigen binding and can be measured using techniques such as ForteBio. The calculation formula is K. D =k off / k on , where k off It is the dissociation rate constant, k on It is the binding rate constant.

[0056] The term "pharmaceutically acceptable" indicates that the composition can be administered to a subject without producing adverse physiological reactions that would prevent the administration of the composition. For example, "pharmaceutically acceptable carrier" refers to a carrier that is useful in the preparation of a generally safe, non-toxic, and desirable pharmaceutical composition. Preferably, examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and non-aqueous mediators, such as non-volatile oils, may also be used.

[0057] The compositions of the present invention can also be administered in combination with each other or in combination with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Therefore, the compositions may also contain a chemotherapeutic agent.

[0058] Typically, the antibody or antibody fragment is administered in a therapeutically effective amount, i.e., an amount sufficient to achieve the desired therapeutic and / or preventative effect, for example, an amount that causes prevention or relief of symptoms associated with the treated disease, such as a disease related to the SEMA4C protein. The therapeutically effective amount of the composition administered to the subject will depend on the type and severity of the disease, as well as on individual characteristics such as general health status, age, sex, weight, and tolerance to the drug; it will also depend on the severity and type of the disease, factors that a person skilled in the art will be able to determine the appropriate dosage based on.

[0059] The kit for detecting SEMA4C protein in a sample provided by this invention contains the aforementioned antibody or antibody fragment. The sample can be a sample from a cancer patient (breast cancer, cervical cancer, esophageal cancer, gastric cancer, colorectal cancer, bladder cancer, and ovarian cancer, preferably breast cancer), such as tumor biopsy tissue or peripheral blood. The kit may also include reagents commonly used for detecting SEMA4C protein, such as coating solutions.

[0060] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0061] Example 1: SEMA4C protein expression

[0062] The SEMA4C gene (uniprot Q9C0C4, 1-664, SEQ ID NO: 197) was synthesized, and an HIS*6 tag was added to the C-terminus of the gene. This was then ligated into the PCDNA3.4 vector using NotI / XhoI. After successful vector sequencing, plasmids were extracted using a large-scale plasmid extraction kit (Tiangen Biotech) according to the manufacturer's instructions. HEK293 cells were counted, and the cell density was adjusted to 1-1.2E6. The extracted plasmid was mixed with PEI transfection reagent in transfection buffer. After incubation at room temperature for 10 minutes, the plasmid / transfection reagent mixture was added dropwise to the density-adjusted HEK293 cells. Shake flasks containing transfected cells were placed in a CO2 incubator at 37°C, 80% humidity, and 0.5% CO2 concentration for 6 days of expression. After 6 days, the cell expression supernatant was separated by centrifugation. The supernatant was filtered through a membrane and purified using an AKTA 100 filter. The NTA column was equilibrated with PBS buffer, and the sample was loaded. The column was then washed with PBS containing 10 mM imidazole, and finally eluted with PBS containing 250 mM imidazole at pH 7.4. The second step of purification was SEC column chromatography, replacing the SEMA4C purification buffer with PBS at pH 7.4. The purified SEMA4C protein exhibited high purity, as shown in the SDS-PAGE chromatogram. Figure 1 As shown.

[0063] Example 2. Construction of ScFv phage display antibody library and antibody screening

[0064] The SEMA4C purified in Example 1 was emulsified with Freund's adjuvant and used to immunize 4-6 week old BALB / c normal mice or humanized mice. After 3 immunizations, an immunization shock was performed 14 days later. Three days later, the spleen of the mice was harvested and RNA was extracted using an RNA extraction kit according to the instructions.

[0065] First-strand cDNA was synthesized according to the instructions of the RNA reverse transcription kit (Kangwei Century, CAT: CW2569M-CWBio). The antibody gene was amplified using the cDNA as a template, yielding heavy chain and light chain fragments of the antibody. Figure 2 , 3 The heavy chain and light chain fragments were spliced ​​together to obtain the ScFv fragment with restriction enzyme sites at both ends. Figure 4 ).

[0066] The phage vector, single-chain antibody fragment, and pcomb3X vector were digested with SFII, mixed in an appropriate ratio, and ligated using T4 ligase to obtain the ligation product. Competent cells were prepared using *E. coli* TG1 host bacteria. The ligation product was electroporated and transformed into competent cells, followed by resuscitation to construct the original antibody library. The original antibody library was cryopreserved, and its volume was measured. A portion of the un-cryopreserved original antibody library was cultured for 1 hour, then infected with M13K07, centrifuged, and transferred to 2YT-AK (100 μg / ml Amp, 20 μg / ml Kana) medium. Expression was performed overnight at 30°C and 220 rpm. The supernatant was collected and precipitated with PEG / NaCl. The supernatant was discarded, the precipitate was resuspended in PBS, filtered to remove bacteria, and the phage display antibody library was obtained. A small amount of glycerol was added, and the library was stored at -80°C.

[0067] The SEMA4C protein prepared in Example 1 was coated in 96-well plates. After washing and blocking, serially diluted phage display antibody libraries were added and incubated at room temperature for 1 hour. After washing three times, horseradish peroxidase-conjugated goat anti-mouse Fc antibody (Biolegend, 405306) was added and incubated at room temperature for 1 hour. After three washes, tetramethylbenzidine (TMB, Biolegend, 421101) was added for color development, and the color development was stopped with 1M HCl. The absorbance was read at 450 nm using a microplate reader. After multiple rounds of screening, phage enrichment products that bound to the target molecule were obtained. After screening, 96 single clones were randomly selected from the products of the second and third rounds of screening for phage display, coated with SEMA4C antigen, and detected by single-clone ELISA. Positive clones were sequenced, and sequence analysis yielded several ScFv antibody sequences. Some of the selected ScFv were used to construct full-length antibodies, which were then recombinantly expressed in HEK293 cells, and the affinity was verified again by ELISA.

[0068] Clones obtained by screening using the scFv phage display library include antibodies numbered 1001, 1006, 1016, 1024, 1002, 1009, 1028, 1029, 1033, 1040, 1041, 1044, 1046, and 1047. The amino acid sequences of their heavy chain variable regions and light chain variable regions are shown in Table 2. The light chain constant region is selected from the κ chain, and the heavy chain constant region is hIgG1.

[0069] Example 3. Screening for anti-SEMA4C antibodies using hybridoma cell method

[0070] The SEMA4C protein prepared in Example 1 was used as an immunogen and emulsified with an equal volume of Freund's complete adjuvant (Sigma-Alderich, F5881) for initial immunization. Ten 6-week-old BALB / c, C57, and SJL mice (Jiangsu Huafukang) were prepared, and each animal was subcutaneously injected with 50 μg of immunogen (excluding adjuvant mass, the same below). The immunogen was emulsified with Freund's incomplete adjuvant (Sigma-Alderich, F5506) for subsequent booster immunization. Two weeks after the initial immunization, each animal received an intraperitoneal injection of 25 μg of immunogen for the first booster immunization; two weeks later, each animal received a subcutaneous injection of 25 μg of immunogen for the second booster immunization. Four to five weeks later, the final immunization was performed with an intraperitoneal injection of 25 μg of immunogen. After immunization, mouse B cells were isolated and mixed with SP2 / 0 cells (Chinese Academy of Sciences Cell Bank, TCM18), and fused according to the BTX electroporator operation manual. After culturing, the fusion cells were screened using enzyme-linked immunosorbent assay (ELISA) to identify hybridoma cells that secreted antibodies capable of binding to SEMA4C. Subcloning was performed using limiting dilution, and positive hybridoma monoclonal cell lines were obtained by screening using the same ELISA method as in Example 2.

[0071] Hybridoma monoclonal cell lines were expanded and cultured in serum-free medium. The culture medium was collected and purified using a protein A column to obtain mouse-derived anti-human SEMA4C monoclonal antibodies for subsequent experiments.

[0072] Apart from the 14 antibodies obtained by phage display screening mentioned in Example 2 (numbered 1001, 1006, 1016, 1024, 1002, 1009, 1028, 1029, 1033, 1040, 1041, 1044, 1046, 1047), all other antibodies mentioned in this invention were obtained by screening using hybridoma cell methods. The amino acid sequences of their heavy chain variable region and light chain variable region are shown in Table 2. The light chain constant region is selected from the κ chain (SEQ ID NO: 198), and the heavy chain constant region is hIgG1 (SEQ ID NO: 200).

[0073] Example 4. In vitro affinity assay of antibody

[0074] To further confirm that the screened antibodies possess high affinity for SEMA4C, this invention used a Fortebio Octet 96E to determine the affinity of the screened antibodies. In this experiment, an AMC sensor was used to capture the antibody to be detected, and SEMA4C was used as the analyte for analysis. The specific steps are as follows: The antibody to be detected was diluted to a concentration of 10 μg / ml with PBST (0.02%) buffer for capture by the AMC sensor. SEMA4C was diluted to 100 nm with PBST (0.02%) buffer, then serially diluted 1:1, and a 0 concentration was set. Eight AMC sensors were placed in the first column of the sensor holder, and the detection conditions were set in Data Acquisition 11.0 software. The main information is as follows: 1) Pre-wetting: Baseline, 60s; 2) Capturing the antibody: 150s-300s; 3) Equilibration: Baseline 2, 60s; 4) Binding: Association, 150s-300s; 5) Dissociation: Dissociation, 450s-1000s; 6) Sensor regeneration: 30s. After the experiment, affinity analysis was performed using Fortebio software. Some representative antibody affinity data are shown in Table 3.

[0075] Table 3 Antibody Affinity

[0076]

[0077]

[0078] Example 5. Animal efficacy experiment of anti-SEMA4C antibody

[0079] To further verify the anti-tumor effect of anti-SEMA4C antibodies in animal models, this invention selected six screened anti-SEMA4C antibodies for animal experiments. In this experiment, MDA-MB-231(ATCC) tumor cells belonging to TNBC were subcutaneously inoculated into donor mice. After the tumors grew, tumors with a volume of approximately 500–1000 mm³ were removed under aseptic conditions. 3 The tumors were cut into pieces approximately 2mm × 2mm × 2mm in size and inoculated subcutaneously into the right flank of female NCG-hIL15 mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) using a cannula. One tumor piece was inoculated into each mouse. Three days after tumor inoculation, PBMCs (human peripheral blood mononuclear cells) from healthy adult donors were inoculated into the experimental mice at a seeding density of 2 × 10⁻⁶ cells / mL. 6 / mouse. When the tumor grew to 60mm 3Animals were divided into seven groups (n=6 per group) for treatment, including one negative control group and six SEMA4C antibody treatment groups. The dosage was 10 mg / kg, administered twice weekly for three weeks via intraperitoneal injection. Animal weight and tumor volume were assessed.

[0080] The tumor volume was measured twice a week using calipers, and the mice were weighed using an electronic balance. The major and minor axes of the tumor were measured, and the volume was calculated using the formula: Tumor volume TV (mm²). 3 = 0.5 × major axis (mm) × minor axis (mm) 2 Tumor growth inhibition rate (TGI) TV % = (1-T / C) × 100%, T / C = RTV of treatment group / RTV of control group.

[0081] At the end of the experiment, the animals were euthanized, and the tumor growth inhibition rate was calculated and statistically analyzed. Specific data are shown in Table 4.

[0082] Table 4. Tumor inhibition rate and statistical difference analysis on day 25 (mixed linear model)

[0083] 60A4 16.12 0.083 60H7 30.50 0.001 93B5 30.15 0.001 21E12-2 30.03 0.006 68A12-2 28.59 0.001 1001 22.64 0.026

[0084] In this animal efficacy experiment, all antibodies showed good activity against TNBC tumor cells, and the tumor volume was significantly reduced compared to the negative control group, with a statistically significant difference (p<0.05). Figure 5 In this experiment, the animals gained weight normally, and the safety and tolerability were good; no drug-related toxic side effects were observed.

[0085] Example 6. Test on the effect of different heavy chain constant regions on the antitumor activity of SEMA4C antibody

[0086] To further verify the effect of using different heavy chain constant regions on the anti-tumor effect of anti-SEMA4C antibodies in animal models, this invention selected the 1001 antibody and simultaneously constructed antibodies with mouse IgG2a and human IgG1 as their heavy chain constant regions, labeled as 1001-mIgG2a and 1001-hIgG1, respectively. The animal model experiment in Example 6 was repeated, and the results are as follows: Figure 6 As shown in the figure. In this experiment, no significant difference in antitumor activity was found between 1001-mIgG2a and 1001-hIgG1. This is because mIgG2a only has a very weak ADCC effect (antibody-dependent cell-mediated cytotoxicity) on human PBMCs, while hIgG1 has a strong ADCC effect. Therefore, we predict that the ADCC effect will not have a significant impact on the antitumor activity of SEMA4C. Thus, whether hIgG1 with a high ADCC effect or hIgG4 with a low ADCC effect is used, similar antitumor effects can be achieved.

[0087] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antibody or antibody fragment targeting Semaphorin 4C, characterized in that, The amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or antibody fragment are shown in SEQ ID NO:101 and SEQ ID NO:102, respectively.

2. The antibody or antibody fragment targeting Semaphorin 4C according to claim 1, characterized in that, The antibody or antibody fragment is a full-length antibody, Fab, Fab', F(ab')2, Fv, or scFv.

3. The antibody or antibody fragment according to claim 2, characterized in that, The antibody is a full-length antibody comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is selected from hIgG1, hIgG2, hIgG3 or hIgG4; and / or, the light chain constant region is selected from the κ chain or the λ chain.

4. The antibody or antibody fragment according to claim 3, characterized in that, The heavy chain constant region is hIgG1 or hIgG4; the light chain constant region is selected from the κ chain.

5. The antibody or antibody fragment according to claim 4, characterized in that, The amino acid sequence of the light chain constant region is shown in SEQ ID NO:198 or SEQ ID NO:199, and the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:200 or SEQ ID NO:

201.

6. An antibody or antibody fragment of any one of claims 1-5, labeled.

7. The antibody or antibody fragment according to claim 6, characterized in that, The label is a fluorescent label, an enzyme label, or a radioactive label.

8. A nucleic acid encoding an antibody or antibody fragment according to any one of claims 1-5.

9. A recombinant expression vector comprising the nucleic acid as described in claim 8.

10. A host cell comprising the recombinant expression vector as described in claim 9.

11. The host cell according to claim 10, characterized in that, The host cell is a prokaryotic cell and / or a eukaryotic cell.

12. The host cell according to claim 11, characterized in that, The prokaryotic cells are E. coli TG1 or BL21 cells, wherein the eukaryotic cells are HEK293 cells or CHO cells.

13. A composition, characterized in that, The composition contains the antibody or antibody fragment as described in any one of claims 1-5 and a pharmaceutically acceptable carrier.

14. A reagent kit, characterized in that, The kit contains the antibody or antibody fragment as described in any one of claims 1-5.

15. The use of the antibody or antibody fragment according to any one of claims 1-5, or the composition according to claim 13, in the preparation of a medicament for treating breast cancer.

16. Use of the antibody or antibody fragment of any one of claims 1-5, the composition of claim 13, or the kit of claim 14 in the preparation of a reagent for detecting Semaphorin 4C protein in a sample.

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