Antibody or trop2 antigen binding fragment binding to trop2, Anti-trop2 car, and use

Abstract

Disclosed are an antibody or a Trop2 antigen binding fragment binding to Trop2, a nucleic acid for encoding the antibody or the Trop2 antigen binding fragment binding to Trop2, a vector and a host cell containing the nucleic acid, an anti-Trop2 CAR, and T cells, NK cells and macrophages containing the anti-Trop2 CAR, CIK cells, and other immune effector cells. Further disclosed are a method for generating the antibody or the antigen binding fragment, and a use of the substances in preparation of an anti-tumor drug, especially in preparation of an anti-tumor drug for the positive expression of Trop2.

Description

Antibodies or Trop2 antigen-binding fragments binding to Trop2 and anti-Trop2 CARs and their applications Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an antibody or a Trop2 antigen-binding fragment that binds to Trop2, as well as an anti-Trop2 CAR and their applications. Background Art

[0002] Globally, malignant tumors are the leading cause of disease-related death and a major obstacle to improving life expectancy. The U.S. National Cancer Institute provided the latest information on the global cancer burden in 2020. In 2020, there were approximately 19.3 million new cancer cases (18.1 million excluding non-melanoma skin cancer) and nearly 10 million cancer deaths (9.9 million excluding non-melanoma skin cancer) worldwide. The global cancer burden is projected to reach 28.4 million cases in 2040, a 47% increase from 2020. Malignant tumors pose a significant threat to human life and health. For most patients with malignant tumors, recurrence rates remain high, and 5-year survival rates remain low. Currently, the main treatments for malignant tumors include surgical resection, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. However, traditional treatments such as surgical resection, chemotherapy, and radiotherapy are generally only effective for patients with early-stage tumors and come with significant side effects. Targeted small molecule inhibitors have significant therapeutic effects only in patients with specific driver gene mutations. For example, gefitinib is only effective in lung cancer patients with EGFR mutations, while patients without EGFR mutations do not benefit from it. Cancer immunotherapies, such as monoclonal antibody checkpoint inhibitors like PDL1, PD1, and CTLA4, have significantly improved the five-year survival rate of melanoma patients, but remain ineffective in treating more aggressive solid tumors such as pancreatic and lung cancer. Therefore, there is an urgent need to develop new treatments to improve the safety and efficacy of treating malignant solid tumors.

[0003] CAR-T therapy is an emerging precision targeted therapy for malignant tumors. It uses genetic engineering to modify T cells to express a chimeric antigen receptor (CAR) containing components such as a scFv single-chain antibody and a costimulatory domain on their surface, enabling them to recognize specific tumor antigens and effectively kill antigen-positive tumor cells. Successful applications of CAR-T therapy have primarily focused on hematologic malignancies, including acute B-cell leukemia, large B-cell non-Hodgkin's lymphoma, and relapsed or refractory CD19-positive malignant lymphoma. However, CAR-T therapy has so far achieved few breakthroughs in solid tumors. This is primarily due to three major challenges in CAR-T therapy for solid tumors. The first is the selection of tumor antigens. Most solid tumor targets, such as Her2, mesothelin, Muc-1, GPC3, and CEA, are tumor-associated antigens (TAAs). These antigens are highly expressed relative to normal tissues, and the potential for off-target toxicity in normal tissues significantly limits the clinical application of CAR-T therapy for solid tumors. Therefore, selecting an antigen with a well-defined expression profile is crucial for CAR-T therapy. Second, the immunosuppressive tumor microenvironment. Solid tumors contain numerous inhibitory factors, such as TAM tumor-associated macrophages, Treg cells, and the negative regulatory cytokines they secrete, such as IL-10, which promote tumor invasion and migration. Furthermore, tumor cells overexpress CD47 and PDL1, suppressing immune cells. Third, CART cell infiltration into the tumor site. The dense extracellular matrix of solid tumors makes it difficult for CART cells to enter the tumor and function. Fourth, CART cells have a short survival time in vivo. These challenges in the application of CART in solid tumor treatment urgently need to be addressed.

[0004] Based on tumor antigen specificity, tumor antigens can be divided into tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). Tumor-specific antigens (TSAs) are expressed exclusively in tumor cells and not in any normal cells at any stage of development. They are antigens produced by the accumulation of gene mutations in cancer cells (such as Kras mutations) and are highly specific to each individual. Tumor-associated antigens (TAAs) are present in small amounts in normal cells but are highly expressed in tumor cells (CD19, CD20, CD38, and Her2).

[0005] Here, the present invention studies a tumor-associated antigen Trop2 that is specifically overexpressed in tumor tissues and lowly expressed in normal tissues. TROP2 belongs to the TACSTD family and was first identified and isolated as a membrane antigen by the monoclonal antibody GA733-1, so it is also called GA733-1. Trop2 is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene, also known as tumor-associated calcium signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), gastrointestinal tumor-associated antigen (GA733-1), and surface marker 1 (M1S1). Generally, lowercase letters + capitalized first letters are used to represent proteins or polypeptides (such as Trop2), and capital letters are used to represent genes or nucleotide fragments (such as TROP2). TROP2 is overexpressed in many malignant tumors and is an oncogene related to the occurrence, invasion and metastasis of malignant tumors. It is expressed at low levels in most normal tissues and is overexpressed in many malignant tumors.

[0006] Currently, companies conducting research targeting Trop2 include Immunomedics and Daiichi Sankyo, which have made rapid progress. Daiichi Sankyo's product, antibody-drug conjugate (ADC) Ds-1062, has entered Phase I clinical trials for the treatment of non-small cell lung cancer. S In July 2020, -1062 also reached a global development and commercialization license agreement with AstraZeneca for up to $6 billion. Immunomedics' antibody-drug conjugate Trodelvy was approved for the treatment of triple-negative breast cancer in April 2020 and has shown promising efficacy in patients with advanced NSCLC, improving overall survival to 9.5 months. In 2019, Everest Medicines (formerly Everest) entered into an exclusive licensing agreement with Immunomedics for the development, registration, and commercialization of its marketed antibody-drug conjugate IMMU-132 in Greater China, South Korea, and several Southeast Asian countries and regions. The transaction value reached $835 million. In April 2020, Everest Medicines received approval for IMMU-132's clinical trial application in China and plans to initiate clinical development programs in multiple solid tumor indications. In 2021, Henlius (formerly Henlius) received an exclusive license from Chiome to research, develop, manufacture, and commercialize antibodies targeting the human TROP2 target, investing over $100 million in its TROP-2 monoclonal antibody development program. Kelun Pharmaceutical's ADC drug SKB-264 was approved for clinical trials in 2019 for the treatment of solid tumors and has entered Phase II clinical trials. BioAtla Biotech's BAT8003 is in Phase I clinical trials for the treatment of gastric and breast cancer. Duoxi Biopharma's DAC-002 has also been approved for clinical trials. Currently, most drugs targeting Trop2 are antibody-drug conjugates.

[0007] Immunomedics' Trop2-targeting ADC, Trodelvy-Sacituzumab govitecan (IMMU-132), was approved for the treatment of triple-negative breast cancer in April 2020. It is the world's first approved antibody-drug conjugate targeting human trophoblast cell surface antigen 2 (Trop2). In March 2020, IMMU132 released updated clinical data for triple-negative breast cancer: as a third-line treatment, IMMU132 achieved a progression-free survival rate of 33.3%, a disease control rate of 45%, and 74% of patients experienced at least one tumor shrinkage. IMMU132 not only demonstrated remarkable efficacy in breast cancer but also improved the overall response rate in lung cancer patients. In August 2017, Rebecca Suk Heist et al. reported clinical trial results of IMMU-132 in 44 patients with non-small cell lung cancer, demonstrating an ORR of 19%, a clinical benefit rate (complete response + partial response + stable disease ≥ 4 months) of 43%, and a PFS of 5.2 months. In May 2017, Jhanelle E. Gray et al. reported clinical trial results of IMMU-132 in 50 patients with small cell lung cancer, demonstrating an ORR of 14%, a clinical benefit rate (complete response + partial response + stable disease ≥ 4 months) of 34%, and a PFS of 3.7 months. IMMU-132 has shown promising clinical efficacy in non-small cell lung cancer (NSCLC).

[0008] As mentioned above, many ADC drugs targeting Trop2 have entered clinical research or are already on the market. However, research on chimeric antigen receptor (CAR)-modified immune cells targeting Trop2 is still in the exploratory stage. The reason is that the use of CAR-T to treat solid tumors faces the three challenges mentioned above.

[0009] Summary of the Invention

[0010] In order to solve the first problem of CART therapy for solid tumors and find tumor-associated antigens with a wide expression spectrum, the present invention provides an antibody or a Trop2 antigen-binding fragment that binds to Trop2, comprising:

[0011] a1) a heavy chain variable region comprising VH CDR1 of SEQ ID NO. 3, VH CDR2 of SEQ ID NO. 4, and VH CDR3 of SEQ ID NO. 5; a light chain variable region comprising VL CDR1 of SEQ ID NO. 6, VL CDR2 of the Lys Ala Ser tripeptide sequence, and VL CDR3 of SEQ ID NO. 7; or

[0012] a2) a heavy chain variable region comprising VH CDR1 of SEQ ID NO.8, VH CDR2 of SEQ ID NO.9, and VH CDR3 of SEQ ID NO.10; a light chain variable region comprising VL CDR1 of SEQ ID NO.11, VL CDR2 of SEQ ID NO.12, and VL CDR3 of SEQ ID NO.13; or

[0013] a3) a heavy chain variable region comprising the VH CDR1 of SEQ ID NO.14, the VH CDR2 of SEQ ID NO.15, and the VH CDR3 of SEQ ID NO.10; a light chain variable region comprising the VL CDR1 of SEQ ID NO.11, the VL CDR2 of SEQ ID NO.12, and the VL CDR3 of SEQ ID NO.13;

[0014] The antibody that binds to Trop2 is a polyclonal antibody or an anti-Trop2 monoclonal antibody;

[0015] The Trop2 antigen-binding fragment is:

[0016] b1) single-chain Fv; or

[0017] b2) disulfide-linked Fv; or

[0018] b3) Fab fragment; or

[0019] b4) F(ab') fragment; or

[0020] b5) a monovalent fragment consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; or

[0021] b6) F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; or

[0022] b7) An Fv fragment consisting of the VL and VH domains of a single antibody arm.

[0023] In some embodiments, the antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO.1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO.2.

[0024] In some embodiments, the antibody or Trop2 antigen-binding fragment that binds Trop2 is a single-chain antibody.

[0025] In some embodiments, the antibody that binds to Trop2 is a human antibody, a murine antibody, or a camelid antibody.

[0026] In some embodiments, the antibody or the antigen-binding fragment that binds to Trop2 is a fully human antibody or a fully human antigen-binding fragment.

[0027] In some embodiments, the heavy chain variable region and the light chain variable region are connected by a (G4S)3 linker.

[0028] In some embodiments, the amino acid sequence of the heavy chain variable region VH of the antibody that binds to Trop2 is shown as SEQ ID NO.1; the amino acid sequence of the heavy chain constant region CH1 of the antibody that binds to Trop2 is shown as SEQ ID NO.16; the amino acid sequence of the heavy chain constant region CH2 of the antibody that binds to Trop2 is shown as SEQ ID NO.17; and the amino acid sequence of the heavy chain constant region CH3 of the antibody that binds to Trop2 is shown as SEQ ID NO.18.

[0029] In some embodiments, the amino acid sequence of the light chain variable region VL of the antibody that binds to Trop2 is shown as SEQ ID NO.2; the amino acid sequence of the light chain constant region CL of the antibody that binds to Trop2 is shown as SEQ ID NO.19.

[0030] In a second aspect, the present invention also provides related nucleic acids encoding the aforementioned antibodies or Trop2 antigen-binding fragments.

[0031] In some embodiments, the nucleic acid comprises the nucleotide sequence encoding the heavy chain variable region comprised by SEQ ID NO. 20 and the nucleotide sequence encoding the light chain variable region comprised by SEQ ID NO. 21.

[0032] In a third aspect, the present invention also provides a related vector comprising the aforementioned nucleic acid.

[0033] In some embodiments, the vector is a recombinant lentiviral vector; the recombinant lentiviral vector contains the nucleotide sequence of Trop2scFv; the anti-Trop2scFv is the Trop2 antigen-binding fragment described above; and the Trop2 antigen-binding fragment is a single-chain Fv.

[0034] In a fourth aspect, the present invention also provides related host cells, which contain the aforementioned nucleic acid or the aforementioned vector.

[0035] In a fifth aspect, the present invention also provides a method for producing an antibody or an antigen-binding fragment, which comprises culturing the aforementioned host cell and recovering the antibody or antigen-binding fragment from the culture.

[0036] In a sixth aspect, the present invention further provides an anti-Trop2 CAR, comprising an anti-Trop2 scFv; the anti-Trop2 scFv is the Trop2 antigen-binding fragment according to claim 1; the Trop2 antigen-binding fragment is a single-chain Fv.

[0037] In some embodiments, the structure of the anti-Trop2 CAR is CD8leader-anti-Trop2scFv-CD8Hinge-CD8TM-costimulatory domain-intracellular signal peptide, including CD8leader membrane receptor signal peptide, anti-Trop2scFv, CD8Hinge chimeric receptor hinge region, CD8TM chimeric receptor transmembrane region, co-stimulatory domain and intracellular signal peptide in series.

[0038] In some embodiments, the costimulatory domain includes but is not limited to one or more selected from CD28, OX40, and 4-1BB. Further, the intracellular signal peptide is CD3ζ.

[0039] In some embodiments, the CAR structure includes but is not limited to CD8leader-anti-Trop2scFv-CD8Hinge-CD8TM-costimulatory domain-intracellular signal peptide structure.

[0040] In a seventh aspect, the present invention also provides related anti-Trop2 CAR-T or anti-Trop2 CAR-NK or anti-Trop2CAR-M, on which a chimeric antigen receptor is modified, and the chimeric antigen receptor is the anti-Trop2 CAR described above.

[0041] In some embodiments, the T cells in the anti-Trop2 CAR-T include but are not limited to one or more of αβT cells, γδT cells, NKT cells, MAIT cells, and CIK cells.

[0042] In an eighth aspect, the present invention also provides the use of the aforementioned Trop2-binding antibody or Trop2 antigen-binding fragment, or the aforementioned nucleic acid, or the aforementioned vector, or the aforementioned host cell, or the aforementioned anti-Trop2 CAR, or the aforementioned anti-Trop2 CAR-T or anti-Trop2 CAR-NK or anti-Trop2 CAR-M in the preparation of anti-tumor drugs.

[0043] In some embodiments, the tumor expresses Trop2.

[0044] In some embodiments, the tumor is a solid tumor that is positive for Trop2 expression.

[0045] In some embodiments, the tumor expresses Trop2; the tumor includes but is not limited to one or more of pancreatic cancer, gastric cancer, colorectal cancer, breast cancer, prostate cancer, lung cancer, and oral squamous cell carcinoma.

[0046] In some embodiments, the breast cancer comprises triple-negative breast cancer, and the lung cancer comprises small cell lung cancer and non-small cell lung cancer.

[0047] As used herein, the term "antibody that binds to Trop2" should be understood to mean antibodies from other sources, such as human antibodies, humanized antibodies, murine antibodies, or antibodies from other sources, unless specifically indicated as antibodies from a certain source in a specific implementation.

[0048] As used herein, the T cells in the term "anti-Trop2 CAR-T" should be understood as T cells in a broad sense, unless specifically specified as a certain type of T cell in the specific implementation, including but not limited to αβT cells and / or γδT cells, and also including some atypical T cells, such as natural killer T (NKT) cells, mucosal associated invariant T (Mucosal Associated Invariant T, MAIT) cells, cytokine-induced killer cells (Cytokine-Induced Killer, CIK), etc. These atypical T cells are also used as effector types of T lymphocytes. The source of T cells is also not limited to peripheral blood, including cells induced by differentiation and transformation such as umbilical cord blood, stem cells, IPSC, cell lines, and other cell types differentiated from T cells. αβT and γδT cells are classifications of typical T cells based on different TCR types.

[0049] As used herein, the term "host cell" should be understood in a broad sense and can be a prokaryotic cell or a eukaryotic cell; it can be any suitable host cell known in the art, for example, a mammalian host cell, a bacterial host cell, a yeast host cell, an insect host cell, etc.

[0050] As used herein, the term "fully human antibody" refers to an antibody whose constant and variable regions are all derived from humans (0% murine, 100% human). Fully human antibodies can be obtained through phage display, yeast, ribosome display, and single-cell PCR fully human monoclonal antibody technology. The anti-Trop2 antibody in the specific embodiments herein is a fully human antibody obtained by screening from a fully human scFv antibody library using phage display technology.

[0051] As used herein, the term "monoclonal antibody" or "mAb" or "IgG" is composed of a heavy chain and a light chain. The light chain consists of the variable region (VL) of the light chain and the constant region (CL) of the light chain; the heavy chain consists of the variable region (VH) of the heavy chain, the constant region 1 (CH1) domain of the heavy chain, the constant region 2 (CH2) of the heavy chain, and the constant region 3 (CH3) of the heavy chain, which can specifically bind to the antigen. It is a heterotetrameric glycoprotein of approximately 150,000 daltons with identical structural features. Each light chain is linked to the heavy chain by a single covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region. Each light chain has a variable region (VL) at one end and a constant region at the other end. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0052] As used herein, the term "variable region" or "variable region" or "V region" refers to a variable region with varying amino acid composition and sequence. Both the heavy and light chain variable regions include three complementarity determining regions (CDR1, CDR2, CDR3) and four framework regions (FR1, FR2, FR3, FR4).

[0053] As used herein, the term "complementarity determining region" or "CDR" refers to a region of the genome that is highly variable and participates in the formation of the antigen binding site.

[0054] As used herein, the term "constant region" or "constant region" or "C region" refers to a constant region whose amino acid composition and sequence are relatively stable. The constant regions of the heavy chain and light chain are called CH and CL, respectively.

[0055] As used herein, the term "antibody heavy chain" consists of the heavy chain variable region (VH), the heavy chain constant region 1 (CH1) domain, the heavy chain constant region 2 (CH2), and the heavy chain constant region 3 (CH3) domain, which can bind to the antigen.

[0056] As used herein, the term "antibody light chain" is composed of a light chain variable region (VH) and a light chain constant region (CL) domain, and is capable of binding to an antigen. Depending on the antigenic specificity of the immunoglobulin light chain C region, it can be divided into κ or λ types. The difference in antigenic specificity that determines the Ig type is determined by the amino acid composition, arrangement, and spatial configuration of the constant region CL of the light chain, which is divided into κ or λ types. The anti-Trop2 monoclonal antibody light chain in the present invention is of the κ type.

[0057] As used herein, the term "single-chain fragment variable" (scFv) consists of the variable region of the light chain (VL), the variable region of the heavy chain (VH) and a linker, and can bind to an antigen.

[0058] As used herein, the term "Fc region" (fragment crystallizable, Fc) is composed of the IgG constant region CH2, CH3 domains and hinge region.

[0059] As used herein, the terms "treat," "therapy," and "treatment" are used interchangeably. The term "treat" includes controlling the progression of a disease, disorder, or condition and associated symptoms, preferably reducing the disease, disorder, or condition or alleviating the effects of one or more symptoms of the disease, disorder, or condition. The term includes curing the disease or completely eliminating symptoms. The term includes symptom relief. The term also includes, but is not limited to, non-curative palliative treatment. The term "treat" includes administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising a recombinant protein or fusion protein of the present invention to prevent or delay, alleviate or relieve the progression of a disease, disorder, or condition or the effects of one or more symptoms of the disease, disorder, or condition.

[0060] As used herein, the term "NCG mouse" (NOD / ShiLtJGpt-Prkdc em26Il2rg em26 / Gpt, strain type KnoCL out, strain number T001475, background NOD / ShiltJGpt) lacks mature T, B, and NK cells and is an important carrier for humanized mice, xenotransplantation, and immune reconstitution; it is of great significance for the study of human hematopoietic stem cells, tumor occurrence and treatment, immunodeficiency diseases, and in vivo immune mechanisms; due to the lack of animal models, most of the current CAR therapy field uses immunodeficient mice for in vivo experiments.

[0061] Through flow cytometry analysis of multiple solid tumor cancer cell lines, the present invention discovered that Trop2 is overexpressed in multiple malignant solid tumor cell lines, making it a novel target for solid tumor treatment. The disclosed Trop2-binding antibodies, anti-Trop2 scFv, and other Trop2 antigen-binding fragments can specifically bind to Trop2 and exert a cell-killing effect.

[0062] Compared with ADC drugs targeting Trop2, the advantages of using anti-Trop2 CAR to modify immune cells in the present invention are: T cells are used as living drugs, and after receiving antigen stimulation, the cells can proliferate in vivo, so their persistence and sustained action time are longer; CART cells can release various pro-apoptotic cytokines such as granzymes, perforins, IFN-γ after receiving target cell stimulation, so their anti-tumor effect is better; there are multiple chemokine receptors on the surface of T cells, such as CCR2, CCR4, etc., which can have corresponding chemotactic effects on tumor tissues, so the targeting is better; in addition, the human body's own T cells are used for genetic engineering modification and then returned to the original donor, and the body will not produce an immune rejection reaction. If there is no antigen stimulation in the body, the CART cells will not function. ADC drugs are coupled with highly active cytotoxic drugs. If toxic drugs are released in non-tumor sites, serious toxic side effects may occur. Therefore, the safety of anti-Trop2 CAR modified immune cells is relatively high; and compared with CART cells, ADC drugs have a short half-life in vivo and patients are prone to relapse. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0064] FIG1 is a diagram showing the screening of anti-Trop2 phage antibodies by phage enzyme-linked immunosorbent assay (Phage ELISA), and FIG1 is the OD450 reading result.

[0065] FIG2 is an SDS-PAGE electrophoresis diagram of the purified T3 monoclonal antibody, wherein the two lanes are: M-Marker and T3-anti-Trop2 monoclonal antibody.

[0066] Figure 3 shows the affinity of the Trop2 antibody to the antigen as determined by enzyme-linked immunosorbent assay (ELISA). NC is a negative control, which is an unrelated antibody that does not bind to the Trop2-His antigen.

[0067] Figure 4 shows flow cytometry analysis of antibody binding to cell surface antigens. T3 represents cells co-incubated with the T3 monoclonal antibody. Blank represents cells without any antibody added, serving as a blank control.

[0068] FIG5 is a schematic diagram of the structures of CARs for anti-Trop2 CAR-T cells and anti-CD19 CAR-T cells in a specific embodiment. a is an anti-Trop2 CAR; b is an anti-CD19 CAR.

[0069] Figure 6 shows the transduction efficiency of D7 and D17 CAR-T cells, which was detected using FITC-Protein L. SSC-H in the coordinates represents the side scatter channel; CAR-FITC-H represents the FITC channel.

[0070] Figure 7 shows the LDH killing of anti-Trop2 CAR-T cells against different solid tumor cell lines in vitro.

[0071] Figure 8 shows the cytokine release levels of anti-Trop2 CAR-T cells co-cultured with different solid tumor cell lines in vitro. Among them, T-Blank refers to blank anti-Trop2 CAR-T cells without target cells; T-A549 refers to co-incubation of anti-Trop2 CAR-T with A549 (Trop2 negative) cells; T-MDA-MB-231 refers to co-incubation of anti-Trop2 CAR-T with MDA-MB-231 (Trop2 positive) cells; T-Bxpc3 refers to co-incubation of anti-Trop2 CAR-T with Bxpc3 (Trop2 positive) cells; C-Blank refers to blank anti-CD19 CAR-T cells without target cells; C-A549 refers to co-incubation of anti-CD19 CAR-T with A549 (Trop2 negative) cells; C-MDA-MB-231 refers to co-incubation of anti-CD19 CAR-T with MDA-MB-231 (Trop2 positive) cells; C-Bxpc3 refers to co-incubation of anti-CD19 CAR-T cells were co-incubated with Bxpc3 (Trop2-positive) cells at a ratio of 5:1 effector cells to target cells. After 24 hours of co-incubation, the supernatant was collected and cytokine levels were measured by flow cytometry using a CBA kit.

[0072] Figure 9 shows that anti-Trop2 CAR-T cells specifically kill MDA-MB-231 triple-negative breast cancer cells in vivo, significantly inhibiting tumor growth. Figure a is the tumor volume curve, and figure b is the mouse weight change curve.

[0073] Figure 10 shows that anti-Trop2 CAR-T cells specifically kill H1975 lung cancer cells in vivo and significantly inhibit tumor growth. (a) is the tumor volume curve, and (b) is the mouse weight change curve. DETAILED DESCRIPTION

[0074] In order to promote understanding of the present invention, the present invention will be described below with reference to certain embodiments and using specific language. However, it should be understood that these specific embodiments are not intended to limit the scope of the present invention. Any changes and further modifications in the described embodiments, as well as any further applications of the present invention, are generally contemplated by those skilled in the art.

[0075] The nucleotide sequence and amino acid sequence information of the present invention are shown in the attached table.

[0076] Explanation of the attached table

[0077] Table 1 Amino acid sequences of light chain and heavy chain variable regions of T3 positive clones obtained by phage display screening

[0078] Table 2 Amino acid sequences of the CDR regions (complementarity determining regions) of the heavy and light chain variable regions of the T3 monoclonal antibody

[0079] Table 3 Amino acid sequence of the constant region of T3 monoclonal antibody

[0080] Table 4 Nucleotide sequences of heavy and light chain variable regions of T3 clones

[0081] Table 5 Nucleotide sequences of heavy chain and light chain expression vectors

[0082] Table 6 Anti-Trop2 CAR amino acid sequence

[0083] Table 7 Anti-Trop2 CAR nucleotide sequences

[0084] Example 1 Screening of anti-Trop2 phage antibodies

[0085] The phage antibody library is constructed by amplifying the antibody heavy chain variable region (VH) and light chain variable region (VL) genes from healthy human B lymphocytes using PCR technology, and expressing the scFv segment on the phage surface. The fully human scFv phage antibody library of the present invention is from Shanghai Youkadi Biopharmaceutical Co., Ltd., and the phage display technology is used to screen the fully human scFv antibody library for anti-Trop2 antibodies; the extracellular segment protein of human trophoblast cell surface antigen 2 (Trop2) with a His tag, namely Human TROP-2 / TACSTD2 Protein, His Tag, is from ACRO (Product No. TR2-H5223) and is used for screening anti-Trop2 specific antibodies; Escherichia coli Tg1 (E. coli Tg1, from manufacturer Lucigen, Product No. 60502-2); trypsin (Product No. and specification T1426-250 mg), from Sigma-Aldrich; M13KO7 Helper Phage (M13KO7 helper phage), from NEB, Product No. N0315S; Mouse Anti-M13 Antibody (HRP) was from NBbiolab, catalog number S004H-250ul; TMB was from eBioscience; and 96-well ELISA plate was from Costar (catalog number 3590).

[0086] 2×YT medium recipe: 16g / L tryptone, 10g / L yeast extract, 5g / L sodium chloride. Place some double-distilled water in a beaker, weigh the peptone, yeast extract, and sodium chloride in the above proportions, and stir until fully dissolved and clear. Place the weighed agar powder in a conical flask, add the culture medium, seal with parafilm, and sterilize in an autoclave at 121°C for 20 minutes. Once cooled to below 55°C, add the appropriate antibiotics.

[0087] 2×YTA agar culture plates: 10g / L tryptone, 5g / L yeast powder, 8g / L sodium chloride, 15g / L agar powder. To pour solid culture medium onto plates, place some double-distilled water in a beaker, weigh the peptone, yeast powder, and sodium chloride in the above proportions, and pour them in. Stir until they are dissolved and clear. Place the weighed agar powder into a conical flask, pour in the culture medium, seal with sealing film, and sterilize with high-pressure steam. Cool the high-pressure steam sterilization to about 55°C (tolerable by the back of the hand) before adding antibiotics to prevent the antibiotics from being inactivated by excessive temperature. Add ampicillin at a ratio of 1:1000, such as adding 400μl ampicillin to 400ml of culture medium. Too high a temperature will cause the antibiotic activity to decrease or become inactivated, and most antibiotics are not resistant to high temperatures. When pouring onto plates, avoid bubbles and stack multiple plates together to prevent the plate lids from being too cold and condensing too many water droplets. After the agar is completely solidified, turn it upside down and place it at room temperature for one day to avoid storing it at 4°C too early (if it is cultured at 37°C too early, water droplets will often cause the bacterial colonies to fuse.) Place it on the clean bench overnight and then put it in the refrigerator. Make sure the refrigerator is sterile.

[0088] Ampicillin preparation (Ampicillin, from Sangon Biotechnology, product number A610028-0025): 100 mg / ml, 100 mg of ampicillin was dissolved in 1 M sterile water, sterilized by filtration through a 0.22 μm microporous filter, and stored at 4°C until use.

[0089] Preparation of PEG / NaCl solution: 20% PEG8000, 2.5M NaCl stoCL, the specific formula is: NaCl 73.1g, PEG8000 (from Sangon Biotechnology, A100159-0500) 100g, double-distilled water 500ml. After preparation, autoclave the solution at 100kPa, 121°C, 20min, and mix well.

[0090] Phage display technology involves inserting the DNA sequence of a foreign protein or polypeptide into the appropriate location of the structural gene for a bacteriophage coat protein, allowing the foreign gene to be expressed along with the coat protein. Simultaneously, the foreign protein is displayed on the phage surface as the phage reassembles. The present invention utilizes phage display technology for solid-phase screening of anti-Trop2 antibodies.

[0091] The screening procedure was as follows: 10 μg / ml Trop2-His protein (0.2 μg / ul) was coated onto a 96-well ELISA plate, which was sealed with a sealing film and incubated at 4°C overnight. The antigen solution on the plate was removed and washed three times with PBS (using a pipette to remove the plate; pouring the plate may cause contamination). The plate was then blocked with MPBS for 1 hour at room temperature and aspirated. Simultaneously, 100 μl of the phage antibody library was blocked with 100 μl of MPBS (2% skim milk) at room temperature for 1 hour. 200 μl of the blocked phage library was added to the wells containing the antigen and incubated at room temperature for 1 hour. Remove the phage solution and wash the ELSIA plate 14 times with PBST (0.1% Tween), then twice with PBS. Add 250μl of trypsin (100ul + 900ul of 1xPBS) and incubate at room temperature for 15 minutes. Aspirate the eluate and transfer it to a 1.5ml EP tube. Add 250ul of trypsin and digest for 15 minutes. During the second digestion, blow thoroughly and aspirate thoroughly. The digested volume, totaling 500ul, is transferred to an EP tube. Take 10ul of the eluted phage and perform a 10-fold serial dilution with 2YT (10^2, 10^4, 10^6, 10^7, 10^8, and 10^9 dilutions). After the 10^7, 10^8, and 10^9 dilutions, take 100ul of the phage and add 200ul of TG1 (OD600 = 0.5) to measure the titer. Take another 250 μl of eluted phage and infect 3 ml of TG1 E. coli at OD600 = 0.5. Add Helper phage and mix thoroughly. Incubate at 37°C in a water bath for 30 min. Dilute the solution serially to 10^2, 10^4, and 10^5. Take 200 μl of the solution and spread it on a 75 mm2 YTA plate to measure the output titer. Centrifuge the remaining solution at 3500 rpm for 5 min. Discard the supernatant and spread it on a 150 mm2 YT plate (for phage amplification). Label the outputs 10^2, 10^4, and 10^5. Incubate in a 37°C incubator overnight. Store the remaining eluted phage at 4°C. Scrape the plate with 2YTA medium. Add approximately 150 μl of the scraped bacteria to 25 ml of 2YT Amp 2% glucose medium and shake at 220 rpm for 30 min to reach logarithmic growth phase. Preserve the remaining scraped bacteria in 50% glycerol and freeze at -20°C. After shaking for 30 minutes until the logarithmic phase, add 100ul Helper phage (1x10 12pfu / ml), add helper phage, incubate at 37°C in a water bath for 30 minutes, then shake at 37°C at 220 rpm for 1 hour. Change to 50 ml of 2YT Canadensis dual-antibody medium and place in a 125 ml Erlenmeyer flask. Incubate overnight at 30°C at 220 rpm. Divide the 50 ml medium from the 125 ml Erlenmeyer flask into two centrifuge tubes, centrifuge at 8000 g for 10 minutes, and retain the supernatant. Add PEG-NaCl to 1 / 3 or 1 / 4 of the supernatant volume to precipitate the phage. Mix thoroughly and let stand on ice for 1 hour. Centrifuge at 10,000 rpm at 4°C for 10 minutes. Circle the white precipitate with a marker and discard the supernatant. Be careful not to pour out the precipitate, as it floats easily. Centrifuge again at 10,000 rpm for 1 minute. Resuspend the precipitate in 400 μl of 1× PBS, rinse thoroughly, and transfer it to a 1.5 ml EP tube. Centrifuge at 12,000 g for 5 minutes. Discard the precipitate and other impurities. Combine the supernatants from both tubes and transfer them to a 1.5 ml EP tube. This serves as the input for the first round of screening. 200 μl of the supernatant will be used for the next round of screening, and the remaining input will be stored in a refrigerator at 4°C. Repeat these screening steps for a total of four rounds of "adsorption-elution-amplification" to enrich and screen. Then, perform phage ELISA screening to identify positive single clones.

[0092] Phage ELISA Procedure: The output phage antibody library from the fourth round of screening was serially diluted and infected with 200 μl of logarithmic-phase Ecoli.Tg1 cells. The cells were then coated on 2YTA plates (Amp concentration: 100 mg / ml) and incubated overnight at 37°C. To each well of two 96-well deep-well plates, 300 μl of 2YTGA (1% Glucose, 100 mg / ml Ampicillin) was added to prepare 60 ml of 2YTAG. Ninety-two colonies were randomly selected from the plates screened in the final round and added to the plates, along with two TG1 controls and two 2YTA controls. The cells were pipetted and shaken at 37°C for 3.5-4 hours. The cells were then cultured in 96-well cell culture plates. 100 μl of cells from the 96-well plates were added to 100 μl of 50% glycerol and stored at -20°C. Add 50µl of Helper phage to 3.3ml of 2YT antibody-free medium and mix thoroughly. Then, add 30µl of medium with Helper phage to each well of a deep-well plate, reaching below the liquid surface and mixing thoroughly. Incubate at 37°C for 30 minutes. Incubate at 37°C at 220rpm for 1 hour. Add 400µl of 2YT AMP+KANA+ medium (ampicariin) to each well of the deep-well plate, for a final volume of 600µl per well. Incubate at 30°C at 220rpm overnight. Antigen coating: Coat one 96-well ELISA plate with Trop2-His and another 96-well ELISA plate with bovine serum albumin (BSA, for detecting nonspecific binding) at 1µg / ml. Incubate at 4°C overnight. ELISA assay: The next day, remove the 96-well plate containing the phage and let it rest for the bacteria to settle. Remove the supernatant. A 96-well ELISA plate coated with antigen overnight at 4°C was blocked with 200 μl of 2% skim milk per well for 1 hour and washed three times with PBST. During milk blocking, 120 μl of Phage supernatant was added to 120 μl of 2% skim milk and blocked using a dilution plate. After blocking, the corresponding supernatant was added to the deep-well plate and the 96-well ELISA plate, one for each well, for 1.5 hours. The plate was then washed six times with PBST. Anti-M13-HRP (anti-M13 phage) was added at a 1:5000 dilution in 2% skim milk, 100 μl per well, and incubated for 1 hour. TMB (100 μl / well) was added for 10 minutes. Development was stopped with 2 M H₂SO₄ (100 μl / well). The OD450 nm was read on a microplate reader; a negative standard was defined as an OD450 < 0.2. The positive clones shown by the Phage ELISA results were selected and sent to Sangon Biotech Co., Ltd. for sequencing to identify the diversity of the positive clone sequences.

[0093] Enzyme-linked immunosorbent assay (ELISA) screening of anti-Trop2 phage antibodies yielded five positive clones (Figure 1): anti-Trop2 positive clone 1 (T1), anti-Trop2 positive clone 2 (T2), anti-Trop2 positive clone 3 (T3), anti-Trop2 positive clone 4 (T4), and anti-Trop2 positive clone 5 (T5). All five positive clones showed specific binding to the Trop2 antigen, with T3 exhibiting the strongest binding activity. The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the T3 monoclonal clone are shown in Table 1 (T3-VH SEQ ID NO. 1 and T3-VL SEQ ID NO. 2), and the nucleotide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the T3 monoclonal clone are shown in Table 4 (T3-VH SEQ ID NO. 20 and T3-VL SEQ ID NO. 21). The T3 monoclonal clone with the highest binding activity was selected for subsequent experiments.

[0094] Example 2 Identification of anti-Trop2 antibodies

[0095] The source of Trop2 antigen was the same as in Example 1, the plasmid DNA miniprep kit was from Corning; the E. coli Top10 competent cell was from Tiangen Biochemical Technology Co., Ltd.; the HRP-labeled anti-Fc antibody was from Bethyl; TMB was from eBioscience; the SDS-PAGE gel rapid preparation kit was from Beyotime; and the Protein A protein purification prepacked column was from Yeasen.

[0096] DMEM was from Gibco; Mouse anti-human IgG-Fc (HRP) was from Bethyl; PE anti-human Fc antibody was from Biolegend; BxPC-3 pancreatic cancer cell line, MDA-MB-231 triple-negative breast cancer cell line, and H1975 non-small cell lung cancer cell line were from Shanghai Academy of Sciences; the heavy chain expression vector PUT-VH and the light chain expression vector PUT-VL were from Eucardi, and the nucleotide sequences are shown in Table 5 (the full sequence of the heavy chain vector PUT-VH is shown in SEQ ID NO. 22, and the full sequence of the light chain vector PUT-VL is shown in SEQ ID NO. 23).

[0097] The heavy chain variable region (VH) segment of the positive T3 monoclonal scFv fragment was constructed into the heavy chain expression vector PUT-VH (nucleotide sequence see Table 5) to obtain the T3 VH heavy chain plasmid. The light chain variable region (VL) segment of the scFv fragment from the T3 monoclonal clone was constructed into the light chain expression vector PUT-VL (nucleotide sequence see Table 5) to obtain the T3 VL light chain plasmid. The T3 VH and T3VL plasmids were separately transformed into E. coli Top10 (TOP10 E. coli) using calcium transformation. Single clones were selected and expanded for plasmid extraction. The extracted plasmids (T3 VH and T3 VL plasmids added in a 1:1 ratio) were co-transfected into 293T cells using the calcium chloride transfection method. The supernatant was collected and purified using a Protein A column. Characterization of monoclonal antibodies primarily involves determining their molecular weight and their antigen binding activity at the protein and cellular levels. The molecular weight of the monoclonal antibody was determined by SDS-PAGE, and the binding of the antigen and antibody was detected by ELISA and flow cytometry.

[0098] SDS-PAGE gel electrophoresis identification steps: 1) Prepare the gel; a) Plate washing: Scrub two glass plates and then dry them with alcohol. b) Plate assembly: Stack the two glass plates neatly, clamp them on both sides, and secure them to the base. Add deionized water to test for leaks. Insert the supplied comb and draw a line on the bottom edge of the comb to indicate the gel placement position (concentrated gel). c) Casting gels: Prepare 1.5mm gels using a rapid PAGE gel preparation kit: 1. Take equal volumes of 4 mL each of lower gel solution and lower gel buffer and mix thoroughly. 2. Add 80 μL of modified coagulant (ammonium sulfate) to the mixed solution from step 1 and mix thoroughly. 3. Pour the entire mixed solution from step 2 onto the glass plate, keeping it within the marked line, leaving enough space for the stacking gel. After a few minutes (5 minutes), add an appropriate amount of water. Add 2 mL of water to press the separating gel downward. A broken line appears between the water and gel, indicating solidification. 4. Pour off the upper layer of water and blot dry with filter paper. 5. Take equal volumes of 1 mL each of upper gel solution and colored upper gel buffer and mix thoroughly. Shake well before use. 6. Add 20 μL of modified coagulant to the mixed solution from step 5 and mix thoroughly. 7. Pour the mixed solution from step 6 onto the glass plate. 8. After the upper gel has solidified (10 to 15 minutes), remove the comb and prepare for electrophoresis. 9) Sample loading for electrophoresis: Add 10 µl of protein to 10 µl of 2× loading buffer at a 1:1 ratio. Boil in a 100°C water bath for 10 minutes, centrifuge at 12,000 x g for 1 minute, and load 20 µl of sample onto the SDS-PAGE gel. Run the stacking gel at 90V. Run the separating gel at 120V for approximately 2 hours. 2) Stain with Coomassie Brilliant Blue for 30 minutes and begin destaining: a. After electrophoresis, remove the gel and place it in an appropriate amount of Coomassie Brilliant Blue staining solution, ensuring that the solution fully covers the gel. b. Place the gel on a horizontal or side-to-side shaker and gently rock it. Stain for 1 hour at room temperature. c. Pour off the staining solution. The staining solution can be recycled and reused at least 2-3 times. d. Add an appropriate amount of destaining solution, ensuring that the solution fully covers the gel. The destaining solution formula is: 40% ethanol, 10% acetic acid, and 50% distilled water. e. Place the gel on a horizontal or side-to-side shaker and shake gently. Destain at room temperature for 4-24 hours. Change the destaining solution 2-4 times until the blue background is essentially gone and the protein bands are stained as expected. Protein bands should appear after 1-2 hours of destaining. f. After destaining, store the gel in water for subsequent imaging.

[0099] ELISA assay to determine antibody affinity for the Trop2 antigen: 1) Coat the Trop2-His antigen at a concentration of 1 μg / ml in a 96-well ELISA plate, seal the plate with sealing film, and incubate at 4°C overnight. 2) After overnight coating, wash the plate once with 0.05% PBST and block with skim milk for 1 hour, during which time the primary antibody is diluted. 3) Dilute the primary antibody in 2% skim milk in a 3.3-fold series, starting at 5 μg / ml, for a total of 12 primary antibody concentrations. 4) After blocking, add the primary antibody at 100 μl / well and incubate for 1 hour. 5) Wash the plate six times with 0.05% PBST. 6) Add the secondary antibody (mouse anti-human IgG-Fc (HRP)) at a 1:1000 dilution in 2% skim milk at 100 μl / well and incubate for 1 hour. 7) Wash the plate six times with 0.05% PBST. 8) Add 100 μl of TMB colorimetric solution to each well and allow to develop for 10 minutes. Terminate the reaction if the color becomes too dark. 9) Add 100 μl of 2 M sulfuric acid to each well to terminate the colorimetric reaction. 10) Measure the colorimetric reaction using a microplate reader (450 nm).

[0100] Flow cytometry was used to screen the binding of the obtained T3 anti-Trop2 monoclonal antibody to cell surface Trop2. The detection steps were as follows: 1. Dilute the antibody to a final concentration of 5 μg / ml with pre-cooled 1× PBS and a final volume of 200 μl. 2. Digest the cells to be tested and adjust the density of the cells to be tested to 1×10 6 cells / ml, add 1ml of cell suspension to each tube (depending on the cell amount), centrifuge at 4℃ and discard the supernatant. 3. Wash once with 1mL pre-cooled 1XPBS, 1500rpm for 5min. 4. Add 200uL of diluted primary antibody (T3 monoclonal antibody) to each tube to resuspend the cells and place on ice for 60min. 5. Centrifuge and discard the supernatant, wash once with 1mL pre-cooled 1×PBS, 1200rpm for 5min. 6. Dilute the secondary antibody with pre-cooled 1×PBS containing 5% BSA, add 1ul secondary antibody anti-Fc (PE) to each tube to resuspend the cells and place on ice for 30min. 8. Wash once with 1mL pre-cooled 1XPBS, 1500rpm for 5min. 9. Resuspend the cells with 200ul pre-cooled 1XPBS and detect on the instrument.

[0101] The supernatant obtained by purification using an AKTA Protein A purification column was analyzed by SDS-PAGE electrophoresis. The SDS-PAGE electrophoresis results (Figure 2) showed that the molecular weights of the heavy and light chains of the T3 monoclonal antibody in the supernatant were consistent with expectations (Figure 2). After gradient dilution of the T3 monoclonal antibody, ELISA was used to detect binding of the antibody to the Trop2 antigen. The results are shown in Figure 3, demonstrating that the screened antibody specifically binds to the Trop2 antigen with high activity (Figure 3). NC is a negative antibody control, and the negative antibody is an anti-human CD7 antibody from Eucardi, with the antibody number PUT644, which can be obtained using the method described in patent CN113603778A.

[0102] The binding of the antibody to the cell surface Trop2 antigen was then identified by flow cytometry. In Figure 4, the Blank group is a blank cell without antibody, and the T3 group shows the binding of T3 monoclonal antibody to the cells. It shows that Trop2 antibody has strong binding to three solid tumor cell lines: triple-negative breast cancer MDA-MB-231, non-small cell lung cancer, H1975 pancreatic cancer Bxpc3. The binding strength can be characterized by the median fluorescence intensity (MFI). The MFI of each group obtained by Flowjo analysis are: H1975 binding to T3 MFI is 65580 (621), MDA-MB-231 binding to T3 MFI is 3150 (938), Bxpc3 binding to T3 MFI is 174408 (557), and the values ​​in brackets are the MFI corresponding to the blank cells.

[0103] Pancreatic cancer Bxpc3, triple-negative breast cancer MDA-MB-231, and non-small cell lung cancer H1975 all had strong binding ( FIG. 4 ), indicating that the Trop2 antibodies screened by the present invention can bind to Trop2 antigens expressed on the cell surface with high binding strength.

[0104] An increasing number of researchers are focusing on antibody production. Hybridoma antibody technology is now relatively well-established, and many mouse monoclonal antibody drugs have been clinically used. However, the immunogenicity of mouse monoclonal antibody drugs significantly limits their development prospects. The antibodies screened in this invention are fully human antibodies obtained through phage display technology. Compared with traditional antibodies obtained by immunizing animals, fully human antibodies have lower immunogenicity and are a major future development direction.

[0105] Single-chain variable fragments (scFvs) are composed of a heavy chain variable region (VH) and a light chain variable region (VL) linked by a peptide chain. The generation of tumor-specific scFvs and their affinity are fundamental to the safety and effectiveness of CAR-T cell therapy. Currently, the two main technologies for constructing scFvs are hybridoma cell lines and phage display, corresponding to antibody types ranging from 100% mouse to 100% human. Mouse antibodies, including mouse monoclonal antibodies, refer to monoclonal antibodies in which both the constant and variable region sequences are derived from mouse genes, with a 100% mouse component; their production relies on hybridoma technology. Mice are exposed to a specific antigen, and then antibody-producing B lymphocytes are screened and extracted from them. These cells are then fused with mouse myeloma cells that cannot produce antibodies, and hybrid fusion cells that do not undergo apoptosis, can proliferate indefinitely, and can secrete specific antibodies are screened. Mouse antibodies have two main advantages: 1) As first-generation monoclonal antibodies, basic research and preparation techniques are relatively complete and thorough, and the cost is relatively low; 2) The maturity of hybridoma technology ensures that mouse antibodies can be of identical quality across batches and are easier to replicate. However, they also have significant limitations. On the one hand, mouse monoclonal antibodies have strong immunogenicity and are prone to triggering HAMA reactions (i.e., mouse antibodies themselves are recognized as antigens by the human immune system, causing unnecessary immune reactions). This can accelerate the clearance of antibodies from the body, shorten their half-life, and thus affect their efficacy, or even cause serious immune diseases. On the other hand, the constant region of mouse antibodies differs from that of humans, and the subsequent immune response they trigger is therefore weaker. These two shortcomings have led to the widespread elimination of mouse antibodies in clinical applications, but they are still widely used in laboratory settings due to their low cost. It also led to the emergence of the second and third generation humanized monoclonal antibodies.

[0106] Human antibodies can be further divided into three categories based on the human proportion and location. The constant region of human-mouse chimeric monoclonal antibodies comes from humans, and the variable region comes from mice (mouse components ~30%); the constant region of humanized monoclonal antibodies comes from humans, and the variable region is a combination of human and mouse sequences (mouse components ~10%); the constant region and variable region of fully human monoclonal antibodies are both from humans (mouse components 0%). Compared with mouse antibodies, human antibodies are generally superior. This is mainly reflected in 1) fully human antibodies effectively weaken the human anti-mouse antibody response, reducing immunogenicity while also reducing toxic side effects; 2) their property of not being easily self-recognized by the immune system greatly prolongs their retention time in the body.

[0107] The single-chain variable fragment (scFv) obtained through phage display technology in this invention is 100% human, with 0% murine content. The characteristics, advantages, and disadvantages of human and murine antibodies are further amplified in CAR-T therapy. The advantages of human antibodies in CAR T cell immunotherapy are mainly reflected in the following three aspects:

[0108] First, the efficacy of human antibodies is more stable: the extracellular scFv used to construct CARs in current clinical trials is mostly mouse-derived monoclonal antibodies. However, the HLA antigen T cell-mediated immune response caused by the immune epitopes of the mouse scFv that specifically bind to the antigen in the CAR structure will, on the one hand, hinder the interaction between CAR and the target antigen to inhibit the function of CAR-T cells, and on the other hand, it will also affect the activation and expansion of cells, and even lead to the inactivation of CAR-T cells in the patient's body. Humanized scFv can achieve more stable efficacy by reducing the immunogenicity of CAR.

[0109] Second, human antibodies can effectively prolong the time to recurrence: after CAR-T treatment, factors such as CAR-T exhaustion and changes in the tumor microenvironment may lead to disease recurrence. The HAMA stimulated by the high immunogenicity of mouse antibodies will shorten their half-life in the human body, thereby possibly shortening the time to disease recurrence.

[0110] Third, the effect of secondary treatment with mouse antibodies is weakened: For tumors targeting CD19, after the first treatment with mouse CAR-T and relapse, the efficacy of mouse CAR-T is weakened. High immunogenicity may induce the production of anti-mouse antigen antibodies in the body, manifesting as drug resistance. At this time, secondary treatment with humanized, fully human CAR-T can effectively alleviate the symptoms.

[0111] The following examples will demonstrate the preparation process of the chimeric antigen receptor (anti-Trop2 CAR) prepared by the Trop2 antibody screened by the present invention and the T cell modified with the chimeric antigen receptor (anti-Trop2 CAR-T) from the aspects of CAR and CAR-T, the LDH killing and cytokine release effects on different target cells, and the in vivo animal experiments to prove that the anti-Trop2 CAR-T specifically kills non-small cell lung cancer and triple-negative breast cancer cell xenograft tumors in vivo. However, this does not mean that the Trop2 antibody screened by the present invention can only be used to prepare chimeric antigen receptors and T cells modified with the chimeric antigen receptor. It itself has great biological value as an antibody or Trop2 antigen-binding fragment that binds to Trop2, as well as the nucleic acid encoding it, the vector containing the nucleic acid, and the host cell containing the nucleic acid or vector. Due to space limitations, the research data on CAR and CAR-T are highlighted here.

[0112] Example 3 Construction of anti-Trop2 CAR and lentiviral packaging

[0113] The lentiviral backbone plasmid vector was PSB1819, from Eucardi; pPac-R, pPac-GP, and pEnv-G were all from Eucardi; DMEM medium was from Gibco; and TOP10 competent cells were from Tiangen Biotechnology Co., Ltd.

[0114] The scFv segment of the anti-Trop2 positive clone 3 obtained by screening in Example 1 was seamlessly cloned into the lentiviral backbone plasmid vector PSB1819 (from Eucardi) to obtain the target plasmid, which was transformed into TOP10 Escherichia coli. After sequencing and identification, the culture was expanded and the plasmid was extracted. After the plasmid extraction was completed, the target plasmid and the lentiviral packaging plasmid pPac-R (from Eucardi), the lentiviral packaging plasmid pPac-GP (from Eucardi) and the lentiviral packaging plasmid pEnv-G (from Eucardi) were used to transfect 293T cells.

[0115] Plate the 293T cells to be transfected in a 10cm culture dish. When the cell density reaches approximately 70%, proceed with lentiviral packaging. The steps are as follows: Remove the 293T cells and observe their status. For a total of 10 dishes of 293T cells, if the cells are in good condition, with a confluence between 80% and 90%, discard the culture medium and slowly add 9mL of 4% DMEM complete medium. Add 2500μl of CaCl2, three packaging plasmids (80μg pPac-R, 100μg pPac-GP, 60μg pEnv-G), and 220μg of the target plasmid Trop2 CAR to a 15mL centrifuge tube. Immediately add bacterial endotoxin test water to 5000μl, add 5000μl of HBS, and continue shaking for approximately 20 seconds after addition. Remove the 293T cells from the incubator and add 1mL of the mixed transfection reagent to each dish of cells. Gently shake to mix thoroughly and place in the incubator. After 6 hours of transfection, the culture supernatant was discarded and 10 mL of 4% DMEM complete medium was added. After 24 hours of transfection, 10 mL of 4% DMEM complete medium was added. The viral supernatant was collected at 24 hours, 48 ​​hours, and 72 hours of culture and stored at 4 ° C. After the viral supernatant was collected, the viral supernatant was filtered using a 0.45 μm filter. After filtering out the cell debris, polyethylene glycol 8000 (PEG8000) was added to concentrate the virus. The virus was incubated at 4 ° C for 12 hours, then centrifuged at 3000 rpm for 15 minutes. After centrifugation, the supernatant was discarded and an appropriate amount of sterile PBS was added to dissolve the viral precipitate. The dissolved virus was packaged and stored in a -80 ° C refrigerator to obtain a recombinant lentiviral vector. The recombinant lentiviral vector contains an anti-Trop2 CAR encoding nucleic acid fragment.

[0116] The construction of CARs for anti-Trop2 CAR-T cells and anti-CD19 CAR-T cells is shown in Figure 5. The coding fragment of the anti-Trop2 CAR is the coding fragment of CD8 leader-anti-Trop2 scFv-CD8 Hinge-CD8 TM-41BB costimulatory domain-CD3ζ intracellular signal peptide. The amino acid sequence and nucleotide sequence of each fragment are shown in Tables 6 and 7. Among them, the anti-Trop2 scFv is derived from the T3 clone and is therefore represented as T3 anti-Trop2 scFv in Tables 6 and 7.

[0117] Example 4 Preparation and Characterization of Anti-Trop2 CAR-T Cells

[0118] CD4 / CD8 magnetic beads, CD3 antibody, and CD28 antibody were from Miltenyi Biotec; IL-2 was from Peprotech; A-IMV culture medium was from Gibco; and FITC-Protein L protein was from Biolegend.

[0119] Isolation of CD4 from human peripheral blood mononuclear cells using CD4 and CD8 magnetic beads + CD8 + T cells were activated for 24 hours in a T25 flask coated with CD3 and CD28 antibodies. After full activation, the recombinant lentiviral vector prepared in Example 2 was added at an MOI of 120. After 48 hours of infection, the cells were washed twice with PBS and resuspended in fresh complete medium. The transfection efficiency of the lentiviral-transfected T cells was then measured using flow cytometry using FITC-Protein L protein. Protein L is an immunoglobulin-binding protein that specifically binds to immunoglobulin light chains.

[0120] The construction of CARs for anti-Trop2 CAR-T cells and anti-CD19 CAR-T cells is shown in Figure 5. After lentiviral transfection of T cells, the cells were co-incubated with ProteinL-FITC on days 7 (D7) and 17 (D17), respectively, and the infection efficiency was measured by flow cytometry. The infection efficiency was above 40% in both cases (Figure 6). The day of T cell activation was designated as day 0 (D0), and transfection was performed 24 hours after activation. The day of lentiviral transfection of T cells was designated as day 1 (D1).

[0121] Example 5 Detection of LDH Killing and Cytokine Release of Anti-Trop2 CAR-T Cells on Different Target Cells

[0122] Source of the LDH killing kit: CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega, catalog number REF: G1782; A-IMV medium from Gibco; A549 (Trop2 negative) from the cell bank of the Chinese Academy of Sciences in Shanghai; CD19-overexpressing myeloid leukemia cell line K562-CD19 (from Shanghai Youkadi).

[0123] The lactate dehydrogenase (LDH) release method detects the killing efficiency of effector cells (here, anti-Trop2 CAR-T cells) on target cells. Perform LDH killing assay according to the kit instructions: 1) Collect target cell suspension: Discard the culture supernatant of adherent cells and use 10ml of saline to soak the bottom of each dish and discard the saline. Add 1ml of trypsin to digest the cells. The digestion time is determined according to the characteristics of the cell line. Under a microscope, observe that the cells are not attached to the wall, are suspended and have a rounded morphology. Add 3mL of complete culture medium to terminate the digestion. Centrifuge at 1500rpm for 5min, discard the supernatant, add 5mL of PBS to resuspend, centrifuge at 1500rpm for 5min, and discard the supernatant. 2) Target cell plating: Resuspend the target cells separately with 1mL of AIM-V killing medium (AIM-V + 4% FBS). Count using a hemocytometer. 1x104 target cells were added to each well of a 96-well plate. According to the required cell amount, a target cell suspension was prepared using a killing medium. After preparation, the target cells were plated in a 96-well cell culture plate, centrifuged at 250g for 5 minutes, and cultured in a 37°C carbon dioxide incubator for 2 hours to promote target cell adhesion. 3) The effector cell suspension was collected, the T cells were mixed evenly, and counted using a hemocytometer. According to the amount of effector cells required for killing, the effector cells were taken, centrifuged at 1500rpm for 5 minutes, and the effector cells were resuspended in 1mL AIM-V killing medium (AIM-V + 4% FBS), and counted with trypan blue staining. 4) 1x10 target cells were added to each well of a 96-well plate. 4 When the E:T ratio (effector cell: target cell) is set to 2.5:1, 2.5x10 effector cells are added to each well. 4 When E:T=5:1, effector cells 5x10 4 / well; when E:T=10:1, effector cells 1x10 5 / well, the volume is 50ul / well. According to the system calculation, when E:T=10:1, the effector cells are 1x10 5Prepare 1 volume of cells per well and perform a two-fold serial dilution in killing medium to prepare effector cell suspensions with an E:T ratio of 5:1 and 2.5:1. 5) Allow target cells to adhere for 2 hours. After preparing the effector cell suspension, plate the plate. Set up effector cell blank, target cell blank, maximum target cell lysis, and medium blank groups (these groups are required for calculating killing efficiency). After plating, seal the 96-well plate with sealing film and centrifuge at 250g, 3 steps up and 1 step down for 5 minutes. Remove the sealing film and incubate in a 37°C incubator for 24 hours. 6) 45 minutes before the LDH killing assay, add 10 μL of lysis buffer to each well with maximum target cell lysis and continue incubation in a 37°C incubator. 7) Remove the substrate from -20°C, allow it to cool to room temperature, and gently shake it upside down to dissolve the substrate. 8) Remove the 96-well plate, seal it with sealing film, and centrifuge it at 250g, 3 steps up and 1 step down for 5 minutes. Transfer 50 μL of supernatant from each well to a new 96-well plate. Add 50 μL of substrate to each well and incubate in the dark for 10-15 minutes. Measure the absorbance at 490 nm using a microplate reader. 9) Calculate the killing efficiency using the following formula: % cytotoxicity = killing in the experimental group / maximum killing × 100% = (experimental well - effector cell blank well - target cell blank well + culture medium background well) / (maximum target cell lysis well - culture medium volume correction well - target cell well + culture medium background well) × 100%.

[0124] Cytokine Detection Steps: After incubation of effector T cells with target cells, collect the supernatant into a 1.5mL EP tube for cytokine detection. a. Sample Processing: Centrifuge the cell suspension at 1500 rpm for 3 minutes, collect the supernatant into a new 1.5mL EP tube. b. Standard Preparation: Dilute the lyophilized standard powder with 2mL of Standard Diluent and incubate at room temperature for 15 minutes. Then, perform a 2-fold serial dilution of the standard (10 steps from the highest concentration to blank dilution). c. Add 50μL of standard or sample to a new EP tube, followed by 50μL of magnetic beads and 50μL of detection antibody, vortex to mix thoroughly, and incubate at room temperature for 3 hours. d. Wash twice with Wash Buffer, discard the supernatant, resuspend in 200μL of Wash Buffer, and analyze for cytokines IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF.

[0125] LDH killing results showed that anti-CD19 CAR-T cells could only kill K562 cells expressing CD19, but not the other five cells. Compared with the control group of anti-CD19 CAR-T cells, anti-Trop2 CAR-T cells showed high specificity and a dose-dependent effect on four solid tumor cell lines that highly express Trop2: pancreatic cancer cell line Bxpc3, bladder cancer cell line T24, and non-small cell lung cancer cell lines PC-9 and H1975. However, anti-Trop2 CAR-T cells showed no killing effect on the Trop2-negative cell line A549, or the CD19-overexpressing myeloid leukemia cell line K562-CD19 (Figure 7).

[0126] In addition, the cytokines released by anti-Trop2 CAR-T cells after co-incubation with different solid tumor cell lines in vitro were detected, and the results showed:

[0127] Compared to the control group of anti-CD19 CAR-T cells, anti-Trop2 CAR-T cells released large amounts of IFN-r, TNF-α, and IL-2 after 24 hours of co-incubation with solid tumor cell lines that highly express Trop2 (pancreatic cancer cell line Bxpc3 and triple-negative breast cancer cell line MDA-MB-231). In contrast, the Trop2-negative cell line A549 released cytokines at lower levels (Figure 8).

[0128] These in vitro results demonstrate that anti-Trop2 CAR-T cells can be activated by Trop2-positive target cells in vitro, producing a series of cytokines associated with immune activation. They can also undergo specific proliferation, achieving a potent anti-tumor effect against target cells. To investigate whether anti-Trop2 CAR-T cells can exert similar anti-tumor activity in vivo, we conducted in vivo mouse experiments. We established a mouse model of human pancreatic cancer to investigate the anti-tumor effector function of anti-Trop2 CAR-T cells in vivo.

[0129] Example 6 In vivo animal experiment on anti-Trop2 CAR-T cells

[0130] Tumorigenic cell preparation: MDA-MB-231 (triple-negative breast cancer cell line) and H1975 (non-small cell lung cancer cell line) were obtained from the Cell Bank of the Chinese Academy of Sciences in Shanghai;

[0131] Animal preparation: Six-week-old female NCG mice (NOD / ShiLtJGpt-Prkdc em26Il2rg em26 / Gpt, from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., strain type KnoCL out, strain number T001475, background NOD / ShiltJGpt) weighing 20-25 g were purchased one week prior to vaccination. Animal husbandry and experimental procedures were strictly carried out in accordance with SPF standards. Animal experiments were approved by the Ethics Committee of East China Normal University and were conducted in strict accordance with the guidelines of the Animal Research Committee. Mice were maintained in a specific-pathogen-free (SPF) environment at 26°C with sterile feed and water. Mice were allowed to acclimate to the environment for 7 days before the start of relevant experiments.

[0132] NCG mouse subcutaneous transplant tumor modeling and in vivo experimental steps: 1) Treatment and collection of adherent target cells: Take Bxpc3 cells with a confluence of 80%-90%, add 3ml of trypsin to each T75 flask, digest for 6-8 minutes, add 6ml of RPMI1640 complete medium to each flask to terminate digestion, 1500rpm, increase 9 to 9, centrifuge for 5 minutes, discard the supernatant, then resuspend and mix with RPMI1640 basal medium, 1500rpm, increase 9 to 9, centrifuge for 5 minutes, discard the supernatant, and repeat the washing once. 2) Inoculation: After rinsing twice, count with trypan blue staining, determine cell viability (above 95%), and adjust the cell density to 5.0×10 7 / ml, use 75% alcohol to disinfect the skin under the armpit of the NCG mouse forelimb, mix the cell suspension, and inject 100ul of cell suspension subcutaneously with an insulin needle. Then slowly withdraw the needle and continue to raise the mice at SPF level. 3) Mouse grouping: mice with tumors up to 100mm 3 Then they were randomly divided into groups. The bedding of mice was changed regularly, and the general condition and feeding activity of mice were observed. The weight and tumor volume of mice were measured every two days. 4) Preparation of CAR-T cells: Use AIMV complete medium (5% FBS + 1000IU / ml IL-2), culture in a 37-degree Celsius, 5% carbon dioxide incubator, observe cell growth every 2 days, take pictures under a microscope, count, and replenish fluids. T cells were expanded and cultured in vitro to the required cell amount for reinfusion, and sterility, mycoplasma, and CAR transduction efficiency were tested before reinfusion. CAR-T cell processing: Take the CAR-T cells to be reinfused, 1500rpm, rise 9 and drop 9, centrifuge for 5 minutes, discard the supernatant, rinse twice with AIMV basal medium, count with trypan blue, measure cell viability, and then use basal medium to adjust the T cell density to 1.0×10 8 / ml for reinfusion. 5) CAR-T cell reinfusion: mice with tumors up to 100mm 3CAR-T cell infusion was started, and the CAR-T cell infusion regimen was 1×10 7 Cell volume / time, a total of 4 times of transfusion, with CAR-T transfusion performed every 4 days (Figure 10). Transfusion procedure: Use 75% alcohol to disinfect the tail of NCG mice, mix the cell suspension, and inject 100ul of CAR-T cell suspension through the tail vein of the mouse using an insulin needle. Then slowly withdraw the needle, disinfect the wound and stop bleeding for 20 seconds, and continue to maintain SPF level.

[0133] The in vivo experimental method of anti-Trop2 CAR-T cells in triple-negative breast cancer cell line MDA-MB-231 was as follows: 6-week-old NCG mice were injected with 5×10 6 Target cells MDA-MB-231, 5 days after target cell inoculation, the tumor volume is about 100mm 3 The tumor-bearing mice were randomly divided into three groups of 6. The experimental mice were treated with 4 CAR-T cell transfusions (1×10 7 / only / times), with an interval of 3 days in between.

[0134] The in vivo experimental method of anti-Trop2 CAR-T cells in lung cancer cell line H1975 was as follows: 6-8 week old NCG mice were injected with 5×10 6 Target cells H1975, 9 days after inoculation of target cells, the tumor volume was about 100mm 3 The tumor-bearing mice were randomly divided into three groups of 5. The experimental mice were treated with CAR-T cell transfusion for 4 times (1×10 7 / only / times), with an interval of 3 days in between.

[0135] During treatment, we continuously observed and recorded the tumor size, body weight, and activity status of the mice, and statistically analyzed the tumor size of the mice. The experimental results showed that the anti-Trop2 CAR-T cell group significantly inhibited tumor growth compared to the anti-CD19 CAR-T cell group and the untreated group. Figure 9 shows the in vivo specific killing of MDA-MB-231 triple-negative breast cancer cells by anti-Trop2 CAR-T cells, significantly inhibiting tumor growth. (a) shows the tumor volume curve, and (b) shows the mouse weight change curve. Compared with the control group and the CD19 CAR-T treatment group, the tumor volume of mice treated with anti-Trop2 CAR-T cells was significantly reduced, indicating that anti-Trop2 CAR-T cells can significantly inhibit the growth of MDA-MB-231 triple-negative breast cancer xenograft tumors. Figure 10 shows the in vivo specific killing of H1975 lung cancer cells by anti-Trop2 CAR-T cells, significantly inhibiting tumor growth. (a) shows the tumor volume curve, and (b) shows the mouse weight change curve. Compared with the control group and CD19 CART treatment group, the tumor volume of mice treated with anti-Trop2 CAR-T cells was significantly reduced, indicating that anti-Trop2 CAR-T cells can significantly inhibit the growth of H1975 lung cancer xenograft tumors. There was no significant decrease in the weight of mice treated with anti-Trop2 CAR-T cells, indicating that anti-Trop2 CAR-T cells are tolerated in mice.

[0136] Significant anti-tumor effects were achieved in mouse models of lung cancer cell line H1975 and triple-negative breast cancer cell line MDA-MB-231 ( Figures 9 and 10 ).

[0137] In vivo results from the NCG mouse animal model showed that anti-Trop2 CAR-T cells could specifically kill non-small cell lung cancer and triple-negative breast cancer cell xenograft tumors in vivo.

[0138] All publications and patent applications cited in this specification are incorporated herein by reference, as if each independent publication or patent application is specifically and individually indicated to be incorporated by reference. In addition, any theory, mechanism, proof or discovery described herein is intended to further enhance understanding of the present invention, and is not intended to limit the present invention to such theory, mechanism, proof or discovery in any way. Although the present invention has been shown and described in detail in the accompanying drawings and the preceding description, the present invention should be considered to be illustrative rather than restrictive.

Claims

1. An antibody or a Trop2 antigen-binding fragment that binds to Trop2, characterized in that: Include: a1) a heavy chain variable region comprising VH CDR1 of SEQ ID NO.3, VH CDR2 of SEQ ID NO.4 and VH CDR3 of SEQ ID NO.5; a light chain variable region comprising VL CDR1 of SEQ ID NO.6, VL CDR2 of a Lys Ala Ser tripeptide sequence and VL CDR3 of SEQ ID NO.7; or a2) a heavy chain variable region comprising VH CDR1 of SEQ ID NO.8, VH CDR2 of SEQ ID NO.9 and VH CDR3 of SEQ ID NO.10; a light chain variable region comprising VL CDR1 of SEQ ID NO.11, VL CDR2 of SEQ ID NO.12 and VL CDR3 of SEQ ID NO.13; or a3) a heavy chain variable region comprising VH CDR1 of SEQ ID NO.14, VH CDR2 of SEQ ID NO.15 and VH CDR3 of SEQ ID NO.10; a light chain variable region comprising VL CDR1 of SEQ ID NO.11, VL CDR2 of SEQ ID NO.12 and VL CDR3 of SEQ ID NO.13; The antibody binding to Trop2 is a polyclonal antibody or an anti-Trop2 monoclonal antibody; The Trop2 antigen binding fragment is: b1) single chain Fv; or b2) disulfide-linked Fv; or b3) Fab fragment; or b4) F(ab') fragment; or b5) a monovalent fragment consisting of a VL domain, a VH domain, a CL domain and a CH1 domain; or b6) F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; or b7) An Fv fragment consisting of the VL domain and VH domain of a single antibody arm.

2. The antibody or Trop2 antigen-binding fragment binding to Trop2 according to claim 1, characterized in that: It comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO.1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO.

2.

3. The antibody or the Trop2 antigen-binding fragment binding to Trop2 according to claim 1, characterized in that: The antibody or Trop2 antigen-binding fragment that binds to Trop2 is a fully human antibody or a fully human antigen-binding fragment.

4. The antibody or the Trop2 antigen-binding fragment binding to Trop2 according to claim 1, characterized in that: The Trop2-binding antibody or Trop2 antigen-binding fragment is a single-chain antibody.

5. A nucleic acid, characterized in that Encodes the antibody or Trop2 antigen-binding fragment binding to Trop2 according to any one of claims 1 to 4.

6. The nucleic acid according to claim 5, characterized in that It comprises the nucleotide sequence SEQ ID NO.20 encoding the heavy chain variable region and the nucleotide sequence SEQ ID NO.21 encoding the light chain variable region.

7. A carrier, characterized in that Comprising the nucleic acid according to any one of claims 5 or 6.

8. A host cell, characterized in that Comprising the nucleic acid according to any one of claims 5 or 6 or the vector according to claim 7.

9. A method for producing an antibody or antigen-binding fragment, characterized in that The host cell according to claim 8 is cultured and the antibody or antigen-binding fragment is recovered from the culture.

10. An anti-Trop2 CAR, characterized in that It comprises an anti-Trop2 scFv; the anti-Trop2 scFv is the Trop2 antigen binding fragment according to claim 1; the Trop2 antigen binding fragment is a single-chain Fv.

11. The anti-Trop2 CAR according to claim 10, characterized in that The structure of the anti-Trop2 CAR is CD8 leader-anti-Trop2 scFv-CD8 Hinge-CD8 TM-co-stimulatory domain-intracellular signal peptide, including a CD8 leader membrane receptor signal peptide, an anti-Trop2 scFv, a CD8 Hinge chimeric receptor hinge region, a CD8 TM chimeric receptor transmembrane region, a co-stimulatory domain and an intracellular signal peptide connected in series in sequence; the anti-Trop2 scFv is the Trop2 antigen binding fragment according to claim 1; the Trop2 antigen binding fragment is a single-chain Fv.

12. Anti-Trop2 CAR-T or anti-Trop2 CAR-NK or anti-Trop2 CAR-M, characterized in that: A chimeric antigen receptor is modified thereon, and the chimeric antigen receptor is the anti-Trop2 CAR according to claim 10.

13. Use of the Trop2-binding antibody or Trop2 antigen-binding fragment according to claim 1, or the nucleic acid according to claim 5, or the vector according to claim 7, or the host cell according to claim 8, or the anti-Trop2 CAR according to claim 10, or the anti-Trop2 CAR-T or anti-Trop2 CAR-NK or anti-Trop2 CAR-M according to claim 12 in the preparation of anti-tumor drugs.

14. The use according to claim 13, characterized in that The tumor can express Trop2.

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