Antibodies or Trop2 antigen-binding fragments binding to Trop2 and anti-Trop2 CAR and applications thereof

Abstract

The present invention discloses an antibody or a Trop2 antigen-binding fragment binding to Trop2, a nucleic acid encoding the antibody or the Trop2 antigen-binding fragment binding to Trop2, a vector and a host cell comprising the nucleic acid, and an anti-Trop2CAR, a T cell, a NK cell and a macrophage, CIK and other immune effector cells comprising the anti-Trop2CAR. The present invention also discloses a method for producing an antibody or an antigen-binding fragment, and the use of the above-mentioned substances in the preparation of anti-tumor drugs, especially in the preparation of anti-Trop2 positively expressed tumor drugs.

Description

Technical Field

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

[0002] Globally, malignant tumors are the leading cause of disease-related deaths and the main obstacle to improving human life expectancy. The National Cancer Center of the United States provides 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. It is estimated that the global cancer burden will reach 28.4 million cases in 2040, an increase of 47% from 2020. Malignant tumors are a great threat to human life and health. For most patients with malignant tumors, the recurrence rate remains high and the 5-year survival rate remains at a low level. At present, the main methods for treating malignant tumors include: surgical resection, radiotherapy, chemotherapy, targeted therapy and immunotherapy. Traditional treatment methods such as surgical resection, chemotherapy, radiotherapy, etc. are usually only effective for patients with early tumors and have large side effects. Small molecule inhibitor targeted drugs only have significant therapeutic effects on patients with specific driver gene mutations. For example, gefitinib is only suitable for lung cancer patients with EGFR mutations, while patients without EGFR mutations cannot benefit from it. In recent years, the popular cancer immunotherapy, such as monoclonal antibody immune checkpoint inhibitors such as PDL1, PD1, and CTLA4, has significantly improved the 5-year survival rate of melanoma patients, but the treatment effect is still not significant for more malignant solid tumors such as pancreatic cancer and lung cancer. Therefore, it is urgent to develop new treatment methods to improve the safety and effectiveness of the treatment of malignant solid tumors.

[0003] CAR-T therapy is an emerging precision targeted therapy for the treatment of malignant tumors. It transforms T cells through genetic engineering technology and expresses chimeric antigen receptors (CARs) including scFv single-chain antibodies, co-stimulatory domains and other elements on the surface of T cells, enabling them to recognize specific tumor antigens, thereby effectively killing antigen-positive tumor cells. The successful application of existing CAR-T therapy is mainly concentrated in the field of hematological tumors, including acute B lymphocytic leukemia, large B-cell non-Hodgkin's lymphoma and relapsed and refractory CD19-positive malignant lymphoma. However, CART therapy has rarely made breakthroughs in the field of solid tumors. This is mainly due to the fact that CART treatment of solid tumors currently faces challenges from three aspects: the first is the selection of tumor antigens. Most of the targets for solid tumor treatment, such as Her2, Mesothelin, Muc-1, GPC3, CEA, etc., are tumor-associated antigens (TAAs). Antigen proteins are highly expressed relative to normal tissues. Normal tissues may express low levels of antigens, and their potential off-target toxicity greatly limits the clinical application of CART therapy in solid tumors. Therefore, choosing an antigen with a good expression spectrum is crucial for CART treatment. The second is the immunosuppressive tumor microenvironment. There are many inhibitory factors in the microenvironment of solid tumors, such as TAM tumor-associated macrophages, Treg cells and negative regulatory cytokines such as IL-10 secreted by them, which promote tumor invasion and migration. In addition, tumor cells highly express CD47 and PDL1, which inhibit immune cells. The third is the infiltration of CART cells in the tumor site. The dense extracellular matrix of solid tumors makes it difficult for CART cells to enter the tumor and function. The fourth is that CART cells have a short maintenance time in the body. The problems of CART in the field of solid tumor treatment need to be solved urgently.

[0004] According to the specificity of tumor antigens, tumor antigens can be divided into tumor-associated antigens TAA and tumor-specific antigens TSA. Tumor-specific antigens (TSA) are "only" expressed in tumor cells, and do not exist in any normal cells at different developmental stages. They are antigens produced by the accumulation of gene mutations in cancer cells (such as Kras mutations) and are highly individual-specific; tumor-associated antigens (TAA) exist in normal cells in small amounts and are highly expressed in tumor cells (CD19, CD20, CD38, Her2).

[0005] Here, the present invention studies a tumor-associated antigen Trop2 that is specifically overexpressed in tumor tissues and low-expressed in normal tissues. TROP2 belongs to the TACSTD family and was first identified and separated by the monoclonal antibody GA733-1 as a membrane antigen, 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 ion 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 a low level 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 developed product, antibody-drug conjugate (ADC) Ds-1062, has entered Phase I clinical trials for the treatment of non-small cell lung cancer. S -1062 also reached a global development and commercialization license agreement with AstraZeneca worth up to US$6 billion in July 2020; Immunomedics' antibody-drug conjugate Trodelvy was approved for marketing in April 2020 for the treatment of triple-negative breast cancer, and it also has a good effect on patients with advanced NSCLC, with the overall survival period increased to 9.5 months; in 2019, Everest Pharmaceuticals reached an exclusive license agreement with Immunomedics, obtaining the development, registration and commercialization rights of its marketed product, antibody-drug conjugate IMMU-132, in Greater China, South Korea and some Southeast Asian countries and regions, with a transaction amount of up to US$835 million; in April 2020, Everest Pharmaceuticals obtained approval for the Chinese clinical trial application for IMMU-132, and plans to launch clinical research and development projects for multiple solid tumor indications. In 2021, Henlius obtained an exclusive license from Chiome to research, develop, produce and commercialize antibodies targeting the human TROP2 target, with a deployment of more than US$100 million for TROP-2 monoclonal antibodies. 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; Bio-Atla Biotech's BAT8003 has entered Phase I clinical trials for the treatment of gastric cancer and breast cancer; Duoxi Bio's DAC-002 has been approved to enter the clinical trial stage. Currently, most drugs targeting Trop2 are antibody-drug conjugates.

[0007] Immunomedics' Trop2-targeted ADC drug Trodelvy-Sacituzumab govitecan (IMMU-132) was approved for marketing in April 2020 for the treatment of triple-negative breast cancer. It is the world's first approved antibody-drug conjugate targeting human trophoblast cell surface antigen 2 (Trop2). In March 2020, IMMU132 updated its clinical data for triple-negative breast cancer: as a third-line treatment, IMMU132 had 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 has amazing efficacy in breast cancer, but also improves the overall remission rate of lung cancer patients. In August 2017, Rebecca Suk Heist et al. reported the clinical trial results of IMMU-132 in the treatment of 44 patients with non-small cell lung cancer, with an ORR rate 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 the clinical trial results of IMMU-132 in the treatment of 50 patients with small cell lung cancer, with an ORR rate 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 showed good clinical effects on 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 has the three challenges mentioned above. Summary of the invention

[0009] In order to solve the first problem of CART treatment of 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:

[0010] a1) a heavy chain variable region comprising VH CDR1 of SEQ ID NO.3, VH CDR2 of SEQ ID NO.4 and VHCDR3 of SEQ ID NO.5; a light chain variable region comprising VL CDR1 of SEQ ID NO.6, VL CDR2 of SEQ ID NO.7 and VL CDR3 of SEQ ID NO.8; or

[0011] a2) a heavy chain variable region comprising VH CDR1 of SEQ ID NO.9, VH CDR2 of SEQ ID NO.10 and VHCDR3 of SEQ ID NO.11; a light chain variable region comprising VL CDR1 of SEQ ID NO.12, VL CDR2 of SEQ ID NO.13 and VL CDR3 of SEQ ID NO.14; or

[0012] a3) a heavy chain variable region comprising VH CDR1 of SEQ ID NO.15, VH CDR2 of SEQ ID NO.16 and VHCDR3 of SEQ ID NO.11; a light chain variable region comprising VL CDR1 of SEQ ID NO.12, VL CDR2 of SEQ ID NO.13 and VL CDR3 of SEQ ID NO.14;

[0013] The antibody binding to Trop2 is a polyclonal antibody or an anti-Trop2 monoclonal antibody;

[0014] The Trop2 antigen binding fragment is:

[0015] b1) single chain Fv; or

[0016] b2) disulfide-linked Fv; or

[0017] b3) Fab fragment; or

[0018] b4) F(ab') fragment; or

[0019] b5) a monovalent fragment consisting of a VL domain, a VH domain, a CL domain and a CH1 domain; or

[0020] b6) F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; or

[0021] b7) Fv fragment consisting of the VL domain and VH domain of a single antibody arm. The sequence of SEQ ID NO.7 is LysAla Ser, which is less than 4 amino acid residues but is still included in the sequence list.

[0022] In some embodiments, 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.

[0023] In some embodiments, the antibody or Trop2 antigen-binding fragment that binds Trop2 is a single chain antibody.

[0024] In some embodiments, the antibody that binds Trop2 is a human antibody, a mouse antibody, or a camelid antibody.

[0025] In some embodiments, the antibody or Trop2 antigen-binding fragment that binds Trop2 is a fully human antibody or a fully human antigen-binding fragment.

[0026] In some embodiments, the heavy chain variable region and the light chain variable region are connected by a (G4S)3 linker.

[0027] 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.17; the amino acid sequence of the heavy chain constant region CH2 of the antibody that binds to Trop2 is shown as SEQ ID NO.18; the amino acid sequence of the heavy chain constant region CH3 of the antibody that binds to Trop2 is shown as SEQ ID NO.19.

[0028] 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.20.

[0029] In a second aspect, the present invention also provides related nucleic acids, which encode the aforementioned antibody or Trop2 antigen-binding fragment that binds to Trop2.

[0030] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a heavy chain variable region comprising SEQ ID NO.21 and a nucleotide sequence encoding a light chain variable region comprising SEQ ID NO.22.

[0031] In a third aspect, the present invention also provides a related vector comprising the aforementioned nucleic acid.

[0032] In some embodiments, the vector is a recombinant lentiviral vector; the recombinant lentiviral vector contains the nucleotide sequence of Trop2scFv; the anti-Trop2 scFv is the Trop2 antigen binding fragment described above; and the Trop2 antigen binding fragment is a single-chain Fv.

[0033] In a fourth aspect, the present invention also provides related host cells, which contain the aforementioned nucleic acid or the aforementioned vector.

[0034] 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.

[0035] 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.

[0036] In some embodiments, the structure of the anti-Trop2 CAR is CD8 leader-anti-Trop2 scFv-CD8Hinge-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.

[0037] 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.

[0038] In some embodiments, the CAR structure includes but is not limited to CD8 leader-anti-Trop2 scFv-CD8Hinge-CD8 TM-costimulatory domain-intracellular signal peptide structure.

[0039] 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.

[0040] 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.

[0041] In an eighth aspect, the present invention also provides the use of the above-described antibody or Trop2 antigen-binding fragment binding to Trop2, or the above-described nucleic acid, or the above-described vector, or the above-described host cell, or the above-described anti-Trop2 CAR, or the above-described anti-Trop2 CAR-T or anti-Trop2 CAR-NK or anti-Trop2CAR-M in the preparation of anti-tumor drugs.

[0042] In some embodiments, the tumor expresses Trop2.

[0043] In some embodiments, the tumor is a solid tumor that is positive for Trop2 expression.

[0044] In some embodiments, the tumor can express 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.

[0045] 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.

[0046] 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 during implementation.

[0047] As used herein, the T cells in the term "anti-Trop2 CAR-T" should be understood as T cells in a broad sense, unless they are specifically specified as a certain type of T cells in the specific implementation, including but not limited to αβT cells and / or γδT cells, and also include 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 of 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.

[0048] 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.

[0049] As used herein, the term "fully human antibody" means that both the constant region and the variable region are derived from humans (0% mouse component, 100% human). Fully human antibodies can be obtained through display technologies such as phage, yeast, ribosome, and single cell PCR fully human monoclonal antibody technology. The anti-Trop2 antibody in the specific embodiment of this invention is a fully human antibody obtained by screening from a fully human scFv antibody library using phage display technology.

[0050] As used herein, the term "monoclonal antibody" or "mAb" or "IgG" is composed of a heavy chain and a light chain, wherein the light chain is composed of a variable region (VL) of the light chain and a constant region (CL) of the light chain; the heavy chain is composed of a variable region (VH) of the heavy chain, a constant region 1 (CH1) domain of the heavy chain, a constant region 2 (CH2) of the heavy chain, and a constant region 3 (CH3) of the heavy chain, and can specifically bind to an antigen. It is a heterotetrameric glycoprotein of about 150,000 daltons with the same structural characteristics, each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy chain 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 chain and the heavy chain.

[0051] As used herein, the term "variable region" or "variable region" or "V region" refers to a variable amino acid composition and sequence. Both the heavy chain and light chain variable regions include three complementarity determining regions (CDR1, CDR2, CDR3) and four framework regions (FR1, FR2, FR3, FR4).

[0052] As used herein, the term "complementarity determining region" or "CDR" refers to regions that are highly variable and participate in the formation of the antigen binding site.

[0053] As used herein, the term "constant region" or "constant region" or "C region", the constant region, the amino acid composition and sequence are relatively stable, the constant regions of the heavy chain and light chain are called CH and CL, respectively.

[0054] 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.

[0055] 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 can bind 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 κ type.

[0056] As used herein, the term "single-chain fragment variable" (scFv) consists of a light chain variable region (VL), a heavy chain variable region (VH) and a linker, and can bind to an antigen.

[0057] As used herein, the term "Fc region" (fragment crystallizable, Fc) is composed of the IgG constant region CH2, CH3 domains and hinge region.

[0058] As used herein, the terms "treat", "therapy" and "treatment" can be used interchangeably. The term "treat" includes controlling the progression of a disease, disorder, condition and associated symptoms, preferably reducing the disease, disorder, condition or alleviating the effects of one or more symptoms of the disease, disorder, condition. This term includes curing the disease or completely eliminating symptoms. This term includes symptom relief. This term also includes, but is not limited to, non-curative palliative treatment. The term "treatment" 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, condition or the effects of one or more symptoms of a disease, disorder, condition.

[0059] As used in this article, 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 reconstruction; 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 in vivo experiments in the field of CAR therapy are currently conducted using immunodeficient mice.

[0060] The present invention, through flow cytometry identification of multiple solid tumor cancer cell lines, found that Trop2 is overexpressed in multiple malignant solid tumor cell lines and can be used as a new target for treating solid tumors. The antibodies, anti-Trop2 scFv and other Trop2 antigen binding fragments disclosed in the present invention can specifically bind to Trop2 to exert a cell-killing effect.

[0061] 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 live drugs, and the cells can proliferate in vivo after receiving antigen stimulation, so their persistence and sustained action time are longer; CART cells can release various pro-apoptotic cytokines such as granzymes, perforins, IFN-γ, etc. after receiving target cell stimulation, so their anti-tumor effect is better; there are various 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, human T cells are used to genetically modify them and then return them to the original donor, and the body will not produce an immune rejection reaction. If there is no antigen stimulation in the body, CART cells will not function, and ADC drugs are coupled with highly active cytotoxic drugs. If toxic drugs are released in non-tumor sites, serious toxic side effects may occur, so anti-Trop2 CAR modified immune cells are relatively safe; 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

[0062] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0063] Figure 1 The anti-Trop2 phage antibodies were screened by phage ELISA. Figure 1 The results are OD450 readings.

[0064] Figure 2 This is the SDS-PAGE electrophoresis diagram of the purified T3 monoclonal antibody. The two lanes are: M-Marker, T3-anti-Trop2 monoclonal antibody.

[0065] Figure 3 It is an enzyme-linked immunosorbent assay (ELISA) to identify the affinity of Trop2 antibody to antigen. Among them, NC is a negative control, which is an irrelevant antibody that does not bind to Trop2-His antigen.

[0066] Figure 4 It is a flow cytometry test to identify the binding of antibodies to cell surface antigens. Among them, T3: T3 monoclonal antibody is co-incubated with the cells to be tested. Blank: The cells to be tested are not added with any antibodies, which serves as a blank control.

[0067] Figure 5The figure is a schematic diagram of the structure of CAR of anti-Trop2 CAR-T cell and anti-CD19 CAR-T cell in a specific embodiment. a is anti-Trop2 CAR; b is anti-CD19 CAR.

[0068] Figure 6 It is the transduction efficiency of D7 and D17 CAR-T cells, and the transduction efficiency is detected using FITC-Protein L. The SSC-H in the coordinates represents the side scatter channel; CAR-FITC-H represents the FITC channel.

[0069] Figure 7 The figure shows the LDH killing effect of anti-Trop2 CAR-T cells on different solid tumor cell lines in vitro.

[0070] Figure 8 It is the level of cytokine release when anti-Trop2 CAR-T cells are co-incubated 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. The co-incubation ratio was effector cells: target cells = 5:1. After 24 hours of co-incubation, the supernatant was collected and the cytokine content in the supernatant was detected by flow cytometry using a CBA kit.

[0071] Fig. 9 Anti-Trop2 CAR-T cells specifically kill MDA-MB-231 triple-negative breast cancer cells in vivo and significantly inhibit tumor growth. Among them, a is the tumor volume curve, and b is the mouse weight change curve.

[0072] Fig.10 Anti-Trop2 CAR-T cells specifically kill H1975 lung cancer cells in vivo and significantly inhibit tumor growth. Among them, a is the tumor volume curve, and b is the mouse weight change curve. DETAILED DESCRIPTION

[0073] In order to promote understanding of the present invention, some embodiments will be referred to below, and specific language will be used to describe the present invention. 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 expected by those skilled in the art.

[0074] The nucleotide sequence and amino acid sequence information of the present invention are shown in the attached table.

[0075] Explanation of the attached table

[0076] Table 1 Amino acid sequences of light chain and heavy chain variable regions of T3 positive clones obtained by phage display screening

[0077] sequence name serial number T3-VH SEQ ID NO.1 T3-VL SEQ ID NO.2

[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

[0079]

[0080]

[0081] Table 3 Amino acid sequence of the constant region of T3 monoclonal antibody

[0082] sequence name serial number T3-CH1 SEQ ID NO.17 T3-CH2 SEQ ID NO.18 T3-CH3 SEQ ID NO.19 T3-CL SEQ ID NO.20

[0083] Table 4 Nucleotide sequences of heavy chain and light chain variable regions of T3 clone

[0084] sequence name serial number T3-VH SEQ ID NO.21 T3-VL SEQ ID NO.22

[0085] Table 5 Nucleotide sequences of heavy chain and light chain expression vectors

[0086] sequence name serial number Heavy chain vector PUT-VH full sequence SEQ ID NO.23 Light chain vector PUT-VL full sequence SEQ ID NO.24

[0087] Table 6 Anti-Trop2 CAR amino acid sequence

[0088] sequence name serial number CD8 leader SEQ ID NO.25 T3 anti-Trop2 scFv SEQ ID NO.26 CD8 Hinge SEQ ID NO.27 CD8 TM transmembrane region SEQ ID NO.28 41BB co-stimulatory domain SEQ ID NO.29 CD3ζ SEQ ID NO.30

[0089] Table 7 Anti-Trop2 CAR nucleotide sequences

[0090]

[0091]

[0092] Example 1 Screening of anti-Trop2 phage antibodies

[0093] 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 comes from Shanghai Youkadi Biopharmaceutical Co., Ltd., and the phage display technology is used to screen the anti-Trop2 antibody of the fully human scFv antibody library; the extracellular segment protein of human trophoblast cell surface antigen 2 (Trop2) with His tag, namely Human TROP-2 / TACSTD2 Protein, His Tag, comes from ACRO (Product No. TR2-H5223), which is used for screening anti-Trop2 specific antibodies; Escherichia coli Tg1 (E.coli Tg1, from manufacturer Lucigen, Product No. 60502-2); trypsin (T1426-250mg), from Sigma-Aldrich; M13KO7 Helper Phage (M13KO7 helper phage), from NEB, Product No. N0315S; Mouse Anti-M13 antibody (HRP), from NBbiolab, catalog number S004H-250ul; TMB from eBioscience; 96-well ELISA plate from Costar (catalog number 3590).

[0094] 2×YT medium formula: 16g / L tryptone, 10g / L yeast powder, 5g / L sodium chloride; put some double distilled water in a beaker, weigh the above proportion of peptone, yeast powder, and sodium chloride and pour them in, stir until they are dissolved and clear. Put the weighed agar powder into a conical flask, pour in the culture solution, seal it with a sealing film, and sterilize it in an autoclave at 121 degrees Celsius for 20 minutes. Add the corresponding antibiotics after cooling to below 55 degrees Celsius.

[0095] 2×YTA agar culture plate: 10g / L tryptone, 5g / L yeast powder, 8g / L sodium chloride, 15g / L agar powder. Pour the solid culture medium into the plate, put some double distilled water in the beaker, weigh the peptone, yeast powder and sodium chloride in the above proportion and pour them in, stir until they are dissolved repeatedly and clear. Put the weighed agar powder into a conical flask, pour in the culture solution, seal it with a sealing film, and sterilize it with high pressure steam. After high pressure steam sterilization, cool it to about 55°C (tolerable on the back of the hand) and then add antibiotics to prevent the antibiotics from being inactivated by excessive temperature. Add ampicillin at a ratio of 1:1000, such as 400μl ampicillin to 400ml culture medium. Too high a temperature will cause the antibiotic activity to decrease or inactivate, and most antibiotics are not resistant to high temperatures. When pouring the plate, avoid bubbles and stack multiple plates together to prevent the plate cover from being too cold and condensing too many water droplets. After the agar is completely solidified, turn it upside down and leave 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.

[0096] Preparation of ampicillin (Ampicillin, from Sangon Biotechnology, product number A610028-0025): 100 mg / ml, 100 mg ampicillin was dissolved in 1 m sterile water, filtered and sterilized with a 0.22 um microporous filter membrane, and stored at 4°C for use.

[0097] 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, high pressure steam sterilization, 100kPa, 121°C, 20min, mix well.

[0098] Phage display technology is a biotechnology that inserts the DNA sequence of foreign proteins or polypeptides into the appropriate position of the phage coat protein structural gene, so that the foreign gene is expressed along with the coat protein expression, and at the same time, the foreign protein is displayed on the phage surface along with the reassembly of the phage. The present invention uses phage display technology to perform solid phase screening of anti-Trop2 antibodies.

[0099] The screening steps are as follows: 10 μg / ml Trop2-His protein (0.2 ug / ul) was coated on a 96-well ELISA plate, the plate was sealed with a sealing film, and the plate was incubated at 4°C overnight. The antigen solution on the ELISA plate was removed, and the plate was washed with PBS for 3 times (sucked out with a gun, and there may be contamination when pouring out), and MPBS was added to block at room temperature for 1 hour and then sucked out. At the same time, 100 ul of phage antibody library was blocked with 100 ul MPBS (2% skim milk) at room temperature for 1 hour; 200 ul 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, wash the ELSIA plate 14 times with PBST (0.1% Tween), then wash twice with PBS; add 250μl trypsin (100ul+900ul 1xPBS) and place at room temperature for 15 minutes, then aspirate the eluate, transfer to a 1.5ml EP tube, and add 250ul trypsin to digest for 15min. During the second digestion, blow evenly and aspirate cleanly. The digested volume is 500ul in total and transferred to the EP tube. Take 10ul of the eluted phage and dilute it 10-fold with 2YT, 10^2, 10^4, 10^6, 10^7, 10^8, 10^9 times, take 100ul of the phage after 10^7, 10^8, 10^9 dilution, add 200ul TG1 with OD600=0.5, and detect the titer. Take another 250ul of eluted phage to infect 3ml of TG1 E. coli with OD600=0.5, add Helper phage and mix well, place in a 37℃ water bath for 30min, dilute the bacterial solution by 10^2, 10^4, 10^5 times, take 200ul to apply on 75mm2YTA plate, and detect the titer of output. Centrifuge the remaining bacterial solution at 3500rpm for 5min, discard part of the supernatant and apply on 150mm 2YT plate (to amplify phage), mark Output 10^2, 10^4, 10^5, and culture in a 37℃ incubator overnight. The remaining eluted phage is stored at 4℃. Scrape the plate with 2YTA medium, take about 150ul of scraped bacteria and add it to 25ml 2YT Amp 2% glucose medium, shake at 220r for 30min to reach the logarithmic growth phase. The remaining scraped bacteria are preserved with 50% glycerol and frozen in a -20℃ refrigerator. After shaking for 30 minutes until the logarithmic phase, add 100ul Helper phage (1x1012 pfu / ml), add helper phage, incubate at 37℃ water bath for 30 minutes, then shake at 37℃ 220r for 1 hour. 3500rpm, 10min, change to 50ml 2YT Canabiquinone dual-antibody culture medium, place in a 125ml conical flask, and culture overnight at 30℃, 220r. Divide the 50ml culture medium in the 125ml conical flask into two centrifuge tubes, centrifuge 25ml per tube, 8000g, 10min, and retain the supernatant.Add PEG-NaCl to 1 / 3 or 1 / 4 of the supernatant volume after centrifugation to precipitate phages, mix thoroughly, and place on ice for 1 hour. Centrifuge at 10000rpm, 4℃, for 10 minutes, circle the white precipitate after centrifugation with a marker pen, discard the supernatant, note that the precipitate is easy to float and cannot be poured out, centrifuge again at 10000rpm, centrifuge for 1 minute, resuspend the precipitate with 400ul 1×PBS, rinse it, transfer it to a 1.5ml EP tube, centrifuge at 12000g for 5 minutes, discard the precipitate as other impurities, combine the supernatants of the two tubes and transfer them to a 1.5ml EP tube, this is the input obtained in the first round of screening, 200ul is used for the next round of screening, and the remaining input is stored in a 4℃ refrigerator. Repeat the above screening steps, and perform four rounds of "adsorption-elution-amplification" for enrichment screening, and then perform phage ELISA screening to identify positive monoclonal clones.

[0100] Phage ELISA (phage ELISA) steps: Output phage antibody library obtained in the 4th round of screening, after gradient dilution, infect 200ul logarithmic phase Ecoli.Tg1, apply 2YTA plate (Amp concentration 100mg / ml), and culture at 37℃ overnight. Add 300ul 2YTGA (1% Glucose, 100mg / ml ampicillin) to each well of 2 96 deep-well plates, prepare 60ml 2YTAG, randomly select 92 clones from the plate of the last round of screening and add them to the deep-well plate, and the remaining 2 TG1 controls and 2 2YTA controls, blow with a gun, shake at 37℃ for 3.5-4h. Use 96-well cell culture plates to preserve bacteria, take 100ul bacteria from the 96 deep-well plates and add 100ul 50% glycerol to preserve bacteria, and store at -20℃. Take 50ul Helper phage and add it to 3.3ml 2YT antibody-free culture medium and mix well. Then add 30ul of culture medium with helper phage to each well of a deep-well plate, extend it below the liquid surface, and mix well. Place it in a 37℃ incubator for 30 minutes. 37℃220rpm for 1 hour. Add 400ul 2YT AMP+KANA+ culture medium (ampicillin KANA double antibody) to each well of the deep-well plate, with a final volume of 600ul per well, and culture overnight at 30℃220r. Antigen coating, a 96-well ELISA coated antigen Trop2-His, a 96-well ELISA plate coated antigen bovine serum albumin (BSA, used to detect non-specific binding), 1ug / ml, 4℃ overnight. ELISA identification: The next day, take out the 96 deep-well plate with phage culture and let it stand, let the bacteria precipitate, and take the supernatant. The 96-well ELISA plate coated with antigen overnight at 4°C was blocked with 200ul 2% skim milk per well for 1h and washed 3 times with PBST. During the milk blocking of the ELISA plate, 120ul of Phage supernatant was added with 120ul 2% skim milk, and the phage supernatant was blocked with a dilution plate. After blocking, the corresponding supernatant was added as the primary antibody. The deep-well plate and the 96-well ELISA plate were added with the supernatant corresponding to each other, 100ul / well, incubated for 1.5h, and washed 6 times with PBST. Anti M13-HRP (anti-M13 phage) was added with 2% skim milk at a dilution of 1:5000, 100ul / well, and incubated for 1h. TMB 100ul / well was used for color development for 10 minutes. 2M H2SO4 100ul / well was used to stop the color development, and the microplate reader read OD450nm. The negative standard was 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.

[0101] Five positive clones were identified by ELISA screening of anti-Trop2 phage antibodies ( Figure 1), respectively: anti-Trop2 positive clone 1 (T1), anti-Trop2 positive clone 2 (T2), anti-Trop2 positive clone 3 (T3), anti-Trop2 positive clone 4 (T4), anti-Trop2 positive clone 5 (T5). These 5 positive clones all showed specific binding to Trop2 antigen, among which T3 had stronger binding activity. The amino acid sequences of the heavy chain variable region VH and light chain variable region VL of the T3 monoclonal 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 are shown in Table 4 (T3-VH SEQ ID NO.21 and T3-VL SEQ ID NO.22). In the present invention, the T3 monoclonal clone with higher binding activity was selected for subsequent experiments.

[0102] Example 2 Identification of anti-Trop2 antibodies

[0103] The source of Trop2 antigen was the same as in Example 1, the plasmid DNA minipreparation 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.

[0104] DMEM was from Gibco; Mouse anti-human IgG-Fc (HRP) was from Bethyl; PE anti-human Fcantibody 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 as shown in SEQ ID NO.23, and the full sequence of the light chain vector PUT-VL as shown in SEQ ID NO.24).

[0105] The heavy chain variable region VH segment of the positive T3 monoclonal scFv segment was constructed into the heavy chain expression vector PUT-VH (nucleotide sequence is shown in Table 5) to obtain the T3 VH heavy chain plasmid; the light chain variable region VL segment of the scFv in the T3 monoclonal was constructed into the light chain expression vector PUT-VL (nucleotide sequence is shown in Table 5) to obtain the T3 VL light chain plasmid. The T3 VH plasmid and the T3 VL plasmid were respectively transferred into E.coli.Top10 (TOP10 Escherichia coli) by calcium transformation, and the monoclonal was selected for expansion and culture and then the plasmid was extracted. The extracted plasmid (T3 VH plasmid and T3 VL plasmid were added at a ratio of 1:1) was co-transfected into 293T cells using the calcium chloride transfection method, and the supernatant was collected and purified by Protein A protein purification column. The identification of monoclonal antibodies mainly requires the identification of the molecular weight of the monoclonal antibody and its binding activity with the antigen at the protein level and the cell level. SDS-PAGE was used to determine whether the molecular weight of the monoclonal antibody was correct, and ELISA and flow cytometry were used to detect the binding of the antigen and antibody.

[0106] SDS-PAGE gel electrophoresis identification steps: 1) Prepare the gel; a) Wash the plate: Scrub the two glass plates and then wipe them dry with alcohol. b) Match the plates: Stack the two glass plates neatly, clamp them on both sides with clips, and fix the two glass plates on the base. Add deionized water to check for leakage. Insert the matching comb and draw a line on the bottom edge of the comb to indicate the gel filling position (concentrated gel). c) Glue pouring, use PAGE gel rapid preparation kit to prepare gel preparation process 1.5mm gel: 1. Take equal volumes of lower gel solution and lower gel buffer, 4 mL each, and mix well; 2. Add 80 μL of improved coagulant (ammonium sulfate) to the mixed solution in step 1 and mix well; 3. Inject all the mixed solution in step 2 into the glass plate for gel preparation, not too far from the drawn line position, leaving space for concentrated gel, wait for a few minutes and add appropriate amount of water, add 2 ml of water to press the separation gel downward, and a broken line appears between the water and the gel, indicating that it has solidified; 4. Pour off the upper layer of water and absorb with filter paper; 5. Take equal volumes of upper gel solution and colored upper gel buffer, 1 mL each, and mix well; shake well before use. 6. Add 20 μL of improved coagulant to the mixed solution in step 5 and mix well; 7. Inject the mixed solution in step 6 into the glass plate for gel preparation. 8. After the upper gel solidifies (10 to 15 minutes), remove the comb teeth and use it for electrophoresis. 9) Loading electrophoresis: 10ul protein is added with 10ul 2× loading buffer at a ratio of 1:1, boiled in 100℃ water bath for 10min, centrifuged at 12000 for 1min, and 20ul is loaded on the SDS-PAGE gel. Run at 90v on the concentrated gel. Run at 120v on the separation gel, and the electrophoresis time is about 2h. 2) Coomassie brilliant blue staining for 30min, start decolorization: a. After the electrophoresis is completed, take the gel and put it into an appropriate amount of Coomassie brilliant blue staining solution to ensure that the staining solution can fully cover the gel. b. Place it on a horizontal shaker or a side-swing shaker and shake it slowly, and stain it at room temperature for 1 hour. c. Pour out the staining solution. The staining solution can be recycled and reused at least 2-3 times. d. Add an appropriate amount of decolorizing solution to ensure that the decolorizing solution can fully cover the gel. The decolorizing solution formula is: 40% ethanol, 10% acetic acid, and 50% distilled water. e. Place the gel on a horizontal shaker or a side-swing shaker and shake slowly for 4-24 hours at room temperature. Change the destaining solution 2-4 times during this period until the blue background is almost completely removed and the protein band staining effect reaches the expected effect. The protein band will appear after 1-2 hours of destaining. f. After destaining, store the gel in water for subsequent photography.

[0107] ELISA identification of antibody and Trop2 antigen affinity steps: 1) Coat Trop2-His antigen at a concentration of 1ug / ml on a 96-well ELISA plate, seal the plate with a sealing film, and incubate at 4°C overnight. 2) After the ELISA plate is coated with the antigen overnight, wash once with 0.05% PBST, block with skim milk powder for 1h, and dilute the primary antibody during this period. 3) Use 2% skim milk to dilute the primary antibody 3.3 times, with a starting concentration of 5ug / ml, for a total of 12 primary antibody concentrations. 4) After the ELISA plate is blocked, add the primary antibody, 100ul / well, and incubate for 1 hour after the primary antibody is added. 5) Wash 6 times with 0.05% PBST. 6) Use 2% skim milk to add the secondary antibody Mouse anti-human IgG-Fc (HRP) at a ratio of 1:1000, 100ul / well, and incubate for 1h. 7) Wash 6 times with 0.05% PBST. 8) 100ul / well TMB color development, add 100ul, color development for 10 minutes, stop if the color is too dark. 9) 100ul sulfuric acid (2M) per well, stop the color development reaction. 10) Measure the photometry with an enzyme reader (450nm).

[0108] Flow cytometry was used to screen the binding of the obtained T3 anti-Trop2 monoclonal antibody to Trop2 on the cell surface. The detection steps were as follows: 1. Dilute the antibody with pre-cooled 1×PBS to a final concentration of 5ug / ml and a final volume of 200ul. 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 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 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 machine.

[0109] The supernatant obtained by AKTA Protein A purification column was detected by SDS-PAGE electrophoresis. SDS-PAGE electrophoresis results ( Figure 2 ) showed that the molecular weight of the heavy and light chains of the T3 monoclonal antibody in the supernatant was consistent with the expected size ( Figure 2 ). After gradient dilution of T3 monoclonal antibody, ELISA was used to detect the binding of antibody to Trop2 antigen. The results are as follows Figure 3 As shown, the screened antibodies specifically bind to Trop2 antigen with high activity ( Figure 3 ), NC is a negative antibody control, the negative antibody is an anti-human CD7 antibody from Eucardi, the antibody number is PUT644, and it can be obtained by the method described in patent CN113603778A.

[0110] The binding of the antibody to the cell surface Trop2 antigen was then identified by flow cytometry. Figure 4 The Blank group is blank cells without antibody, and the T3 group shows the binding of T3 monoclonal antibody to 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 is: the MFI of H1975 binding to T3 is 65580 (621), the MFI of MDA-MB-231 binding to T3 is 3150 (938), and the MFI of Bxpc3 binding to T3 is 174408 (557). The values ​​in brackets are the MFIs corresponding to the corresponding Blank cells.

[0111] Pancreatic cancer Bxpc3, triple-negative breast cancer MDA-MB-231, and non-small cell lung cancer H1975 all have strong binding ( Figure 4 ), indicating that the Trop2 antibody screened by the present invention can bind to the Trop2 antigen expressed on the cell surface with a high binding strength.

[0112] More and more researchers are focusing on the preparation of antibodies. At present, hybridoma antibody technology has been relatively complete, and many mouse monoclonal antibody drugs have been used in clinical practice. However, mouse monoclonal antibody drugs have immunogenicity, which greatly limits the development prospects. The antibodies obtained by the screening of the present invention are fully human antibodies obtained by phage display technology. Compared with traditional antibodies obtained by immunizing animals, fully human antibodies have lower immunogenicity and are the main development direction in the future.

[0113] The single-chain variable fragment (scFv) is composed of the heavy chain variable region (VH) and the light chain variable region (VL) connected together by a peptide chain. The production of tumor-specific scFv and its affinity are the basis for the safety and effectiveness of CAR-T cell therapy. At present, there are two main technologies for constructing scFv: hybridoma cell line and phage display, and the corresponding antibody types are 100% mouse antibodies to 100% human antibodies. Mouse antibodies, whose mouse monoclonal antibodies refer to monoclonal antibodies whose constant region and variable region sequences are all derived from mouse genes, and whose mouse components reach 100%; the preparation of mouse monoclonal antibodies relies on hybridoma technology. That is, mice are exposed to specific antigens, and then the B lymphocytes that produce antibodies in their bodies are screened and extracted; then these cells are fused with mouse myeloma cells that cannot produce antibodies, and then mouse hybrid fusion cells that will not die, can proliferate indefinitely, and can secrete specific antibodies are screened; mouse antibodies have two main advantages: 1) As the first generation of monoclonal antibodies, basic research and preparation technology have been relatively complete and thorough, and the cost is relatively low; 2) The maturity of hybridoma technology allows mouse antibodies to ensure that the quality of different batches is exactly the same, and it is easier to replicate. However, it also has great limitations. On the one hand, mouse monoclonal antibodies have strong immunogenicity and are prone to induce HAMA reactions (that is, mouse antibodies themselves are regarded as antigens by the human immune system, causing unnecessary immune reactions), which can accelerate the clearance of antibodies from the human body, shorten the half-life and thus affect the efficacy, and even cause serious immune diseases; on the other hand, the constant region of mouse antibodies is different from the human constant region sequence, so the subsequent immune response triggered is weaker. The above two shortcomings have led to the elimination of mouse antibodies in clinical applications, but they are still widely used in laboratory environments due to their low cost. It also led to the emergence of the second and third generation humanized monoclonal antibodies.

[0114] Human antibodies can be further divided into three categories according to 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 better. 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 characteristics of not being easily self-recognized by the immune system greatly prolong the retention time in the body.

[0115] The single-chain variable fragment scFv obtained by phage display technology screening in this invention is 100% human antibody, with 0% mouse component. The characteristics and advantages and disadvantages of human and mouse antibodies are further magnified in CAR-T therapy. The advantages of human antibodies in the application of CART cell immunotherapy are mainly reflected in the following three aspects:

[0116] First, the efficacy of human antibodies is more stable: the extracellular scFv used to construct CARs in current clinical trials is mostly mouse 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 hinder the interaction between CAR and the target antigen to inhibit the function of CAR-T cells. On the other hand, it will also affect the activation and expansion of cells, and even cause the inactivation of CAR-T cells in the patient's body. Humanized scFv can achieve a more stable efficacy by reducing the immunogenicity of CAR.

[0117] Second, human antibodies can effectively prolong the time of 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 its half-life in the human body, thereby possibly shortening the time of disease recurrence.

[0118] 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 when it is used again. High immunogenicity may induce the production of anti-mouse antigen antibodies in the body, which manifests as drug resistance. At this time, the use of humanized, fully human CAR-T for secondary treatment can effectively alleviate the symptoms.

[0119] 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 (anti-Trop2 CAR-T) modified with the chimeric antigen receptor 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 the anti-Trop2CAR-T specific killing of 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 the vector. Due to space limitations, the research data on CAR and CAR-T are mainly cited here.

[0120] Example 3 Construction of anti-Trop2 CAR and packaging of lentivirus

[0121] 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.

[0122] 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, expanded and cultured after sequencing and identification, 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.

[0123] Use a 10cm culture dish to plate the 293T cells to be transfected. When the cell density reaches about 70%, perform lentiviral packaging. The steps are as follows: Take out the 293T cells and observe the cell status. A total of 10 dishes of 293T cells, if the cell status is good and the cell confluence is between 80%-90%, discard the culture medium and slowly add 9mL of 4% DMEM complete culture medium. Add 2500ul CaCl2, 3 packaging plasmids (80ug pPac-R, 100g pPac-GP, 60g pEnv-G) and 220ug target plasmid Trop2 CAR to a 15mL centrifuge tube, then add bacterial endotoxin test water to 5000ul, add 5000ul HBS, and continue to shake for about 20s after adding. Take out the 293T cells from the incubator, add 1mL of the mixed transfection reagent to each dish of cells, shake gently to mix evenly, 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 virus supernatant was collected at 24 hours, 48 ​​hours and 72 hours of culture and stored at 4 ° C. After the virus supernatant was collected, the virus supernatant was filtered using a 0.45um filter, and polyethylene glycol 8000 (PEG8000) was added to concentrate the virus after filtering out the cell residue, incubated at 4 ° C for 12 hours, and 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 virus 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.

[0124] Construction of CARs for anti-Trop2 CAR-T cells and anti-CD19 CAR-T cells Figure 5The coding fragment of anti-Trop2 CAR is the coding fragment of CD8 leader-anti-Trop2 scFv-CD8 Hinge-CD8 TM-41BB co-stimulatory domain-CD3ζ intracellular signal peptide, and Tables 6 and 7 show the amino acid sequence and nucleotide sequence of each fragment. Among them, since anti-Trop2 scFv is derived from T3 clone, it is represented as T3 anti-Trop2 scFv in Tables 6 and 7.

[0125] Example 4 Preparation and characterization of anti-Trop2 CAR-T cells

[0126] CD4 / CD8 magnetic beads, CD3 antibody, and CD28 antibody were all from Miltenyi Biotec; IL-2 was from Peprotech; A-IMV culture medium was from Gibco; and FITC-Protein L protein was from Biolegend.

[0127] Isolation of CD4 from human peripheral blood mononuclear cells using CD4 and CD8 magnetic beads + CD8 + T cells were placed in a T25 bottle coated with CD3 antibody and CD28 antibody for activation for 24 hours. After the T cells were fully activated, the recombinant lentiviral vector prepared in Example 2 was added at MOI = 120. After 48 hours of infection, the cells were washed with PBS twice, replaced with new complete medium and resuspended, and then the transfection efficiency of lentiviral transfected T cells was detected by flow cytometry using FITC-Protein L protein. Protein L is an immunoglobulin binding protein that can specifically bind to immunoglobulin light chains.

[0128] Construction of CARs for anti-Trop2 CAR-T cells and anti-CD19 CAR-T cells Figure 5 After lentivirus transfection of T cells, the infection efficiency was detected by flow cytometry after co-incubation with ProteinL-FITC on the 7th day (D7) and the 17th day (D17). The infection efficiency was above 40% ( Figure 6 The day of T cell activation was day 0 (D0), transfection was performed 24 hours after activation, and the day of lentivirus transfection of T cells was day 1 (D1).

[0129] Example 5 Detection of LDH killing and cytokine release of anti-Trop2 CAR-T cells on different target cells

[0130] Source of LDH killing kit: CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega, catalog number REF: G1782; A-IMV culture medium comes from Gibco; A549 (Trop2 negative) comes from the cell bank of the Chinese Academy of Sciences in Shanghai; myeloid leukemia cell line K562-CD19 overexpressing CD19 (from Shanghai Youkadi).

[0131] 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 detection according to the instructions of the kit: 1) Collect the suspension of target cells: discard the culture supernatant of adherent cells, use 10ml of saline per dish to soak the bottom of the dish, and then 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 the microscope, observe that the cells are not attached to the wall, suspended and round in shape. Add 3mL of complete culture medium to terminate the digestion. Centrifuge at 1500rpm for 5min, discard the supernatant, add 5mL PBS to resuspend, centrifuge at 1500rpm for 5min, and discard the supernatant. 2) Target cell plating: Resuspend the target cells separately with 1mL AIM-V killing medium (AIM-V+4% FBS). Count using the hemocytometer method. 1x104 target cells are added to each well of a 96-well plate. According to the required cell amount, a target cell suspension is prepared using a killing medium. After preparation, the target cells are spread in a 96-well cell culture plate, centrifuged at 250g for 5 minutes, and placed in a 37°C carbon dioxide incubator for 2 hours to promote target cell adhesion. 3) Collect the effector cell suspension, mix the T cells evenly, and count them using a hemocytometer. According to the amount of effector cells required for killing, take the effector cells, centrifuge at 1500rpm for 5 minutes, resuspend the effector cells separately with 1mL AIM-V killing medium (AIM-V + 4% FBS), and count them with trypan blue staining. 4) 1x10 target cells have been added to each well of a 96-well plate. 4 When the E:T (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 5 / well, prepare 1 volume of cells, use killing medium for 2-fold gradient dilution, and prepare effector cell suspensions with E:T=5:1 and E:T=2.5:1. 5) Target cells adhere to the wall for 2 hours, and after the effector cell suspension is prepared, plate it. It is necessary to set up effector cell blank group, target cell blank group, target cell maximum lysis group, and medium blank group (these groups must be set up to calculate the killing efficiency). After the plate is plated, seal the 96-well plate with sealing film and put it in a centrifuge, 250g, 3 up and 1 down for 5 minutes, remove the sealing film and put it in a 37℃ constant temperature incubator for incubation for 24 hours. 6) 45 minutes before the LDH killing test, add 10μL of lysis solution to each target cell maximum lysis well, and continue to incubate in a 37℃ constant temperature incubator. 7) After the substrate is taken out from -20℃ and the temperature drops to room temperature, gently shake it upside down to dissolve the substrate. 8) Take out the 96-well plate, seal it with sealing film, and place it in a centrifuge, 250G, 3 up and 1 down, centrifuge for 5 minutes. Take 50μL of supernatant from each well to a new 96-well plate, then add 50μL of substrate to each well and incubate in the dark for 10-15 minutes. Use an ELISA reader to detect the absorbance at 490nm. 9) Killing efficiency calculation formula: Cytotoxicity % = Experimental group killing / maximum killing x 100% = (experimental well - effector cell blank well - target cell blank well + culture medium background well) / (target cell maximum lysis well - culture medium volume correction well - target cell well + culture medium background well) x 100%.

[0132] Cytokine detection steps: After the effector T cells and target cells are co-incubated, the supernatant is collected in a 1.5mL EP tube for cytokine detection. a Sample processing: After the cell suspension is centrifuged at 1500rpm for 3min, the supernatant is collected in a new 1.5mL EP tube. b Standard preparation: Add 2mL standard diluent to dilute the standard lyophilized powder and let it stand at room temperature for 15min. Then dilute the standard by 2 times (a total of 10 gradients from the highest concentration to the blank dilution). c Add 50μL of standard or sample to a new EP tube, then add 50μL of magnetic beads and 50μL of detection antibody, mix evenly with a vortexer, and incubate at room temperature for 3h. d Add Wash Buffer to wash twice, discard the supernatant, add 200μL Wash Buffer to resuspend and detect cytokines IL-2 / IL-4 / IL-6 / IL-10 / IFN-γ / TNF on the machine.

[0133] The 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 anti-CD19 CAR-T cells, anti-Trop2 CAR-T cells had high specific killing on four solid tumor cell lines that highly expressed Trop2, pancreatic cancer cell line Bxpc3, bladder cancer cell line T24, non-small cell lung cancer cell lines PC-9 and H1975, and had obvious dose dependence. The higher the amount of CAR-T cells, the higher the killing level. However, there was no killing on Trop2 negative cell lines - Trop2 negative non-small cell lung cancer cell line A549, and myeloid leukemia cell line K562-CD19 that overexpressed CD19 ( Figure 7 ).

[0134] 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:

[0135] Compared with the anti-CD19 CAR-T cells in the control group, anti-Trop2 CAR-T cells released a large amount of IFN-r, TNF-α and IL-2 after being co-incubated with solid tumor cell lines that highly express Trop2 (pancreatic cancer cell Bxpc3 and triple-negative breast cancer cell MDA-MB-231) for 24 hours. However, the cytokines of the Trop2-negative cell line A549 were at a lower level ( Figure 8 ).

[0136] The above in vitro results prove that anti-Trop2 CAR-T cells can be stimulated and activated by Trop2-positive target cells in vitro, and produce a series of cytokines related to immune activation. At the same time, they can also undergo specific proliferation and achieve a good killing effect on target cells. So next, in order to explore whether anti-Trop2 CAR-T cells can also play a similar tumor-killing function in vivo. We conducted in vivo experiments in mice for verification. We constructed a human pancreatic cancer mouse model to explore the anti-tumor effector function of anti-Trop2 CAR-T cells in vivo.

[0137] Example 6 In vivo animal experiment of anti-Trop2 CAR-T cells

[0138] 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;

[0139] Animal preparation: 6-week-old female NCG mice weighing 20-25 g (NOD / ShiLtJGpt-Prkdc em26Il2rg em26 / Gpt, from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., strain type KnoCL out, strain number T001475, background NOD / ShiltJGpt) were ordered one week before inoculation. Animal husbandry and experimental operations were strictly carried out in accordance with SPF standards. Animal experiments were approved by the Ethics Committee of East China Normal University and strictly followed the guidelines of the Animal Research Committee. The mice were kept in a specified-pathogen-free (SPF) environment, at a room temperature of 26°C, with sterile feed and sterile water. The mice were allowed to adapt to the environment for 7 days before starting related experiments.

[0140] 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 bottle, digest for 6-8 minutes, add 6ml of RPMI1640 complete medium to each bottle to terminate digestion, 1500rpm, 9-9, centrifuge for 5min, discard the supernatant, then resuspend and mix with RPMI1640 basal medium, 1500rpm, 9-9, centrifuge for 5min, discard the supernatant, and repeat washing once. 2) Inoculation: After rinsing twice, count with trypan blue staining, determine cell viability (more than 95%), and adjust the cell density to 5.0×10 with basal medium. 7 / ml, use 75% alcohol to disinfect the skin under the armpit of the NCG mouse forelimb, mix the cell suspension, inject 100ul of the cell suspension subcutaneously with an insulin needle, then slowly pull out the needle and continue to feed at the 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 once 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, 9 up and 9 down, centrifuge for 5min, discard the supernatant, rinse twice with AIMV basal medium, count with trypan blue staining, determine 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: mouse tumor volume reaches 100mm 3The CAR-T cell infusion regimen was 1×10 7 Cell volume / time, a total of 4 times of reinfusion, CAR-T reinfusion every 4 days ( Fig.10 ). Reinfusion operation: Use 75% alcohol to disinfect the tail of NCG mice, mix the cell suspension, and use an insulin needle to inject 100ul of CAR-T cell suspension through the tail vein of the mouse, then slowly pull out the needle, disinfect the wound and stop bleeding for 20s, and continue to feed at the SPF level.

[0141] The in vivo experimental method of anti-Trop2 CAR-T cells in the 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, with 6 mice in each group. The experimental mice were treated with 4 CAR-T cell infusions (1×10 7 / only / times), with a 3-day interval in between.

[0142] The in vivo animal experiment method of anti-Trop2 CAR-T cells of 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, with 5 mice in each group. The experimental mice were treated with 4 CAR-T cell infusions (1×10 7 / only / times), with a 3-day interval in between.

[0143] During the treatment process, we continuously observed and recorded the tumor size, 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 could significantly inhibit tumor growth compared with the anti-CD19 CAR-T cell group and the untreated group. Fig. 9 Anti-Trop2 CAR-T cells specifically kill MDA-MB-231 triple-negative breast cancer cells in vivo and significantly inhibit tumor growth. Among them, a is the tumor volume curve, and b is the mouse weight change curve. Compared with the control group and CD19 CART treatment group mice, 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. Fig.10Anti-Trop2 CAR-T cells specifically kill H1975 lung cancer cells in vivo and significantly inhibit tumor growth. Among them, a is the tumor volume curve, and b is the mouse weight change curve. Compared with the control group and CD19 CART treatment group mice, 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.

[0144] 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 ( Fig. 9 and 10 ).

[0145] The in vivo results of 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.

[0146] All publications and patent applications cited in this specification are incorporated herein by reference, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In addition, any theory, mechanism, proof or discovery described herein is intended to further enhance the 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 illustrative rather than restrictive. Sequence Listing <110> Shanghai Ucardi Biopharmaceutical Technology Co., Ltd. <120> Antibodies or Trop2 antigen-binding fragments binding to Trop2 and anti-Trop2 CARs and applications thereof <130> WHYY-NP-21-101030 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 118 <212> PRT <213> Homo sapiens <400> 1 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Asp Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Gly Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gln Ser Ser Ser Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 2 <211> 107 <212> PRT <213> Humans (Homo sapiens) <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Pro Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Val Lys 100 105 <210> 3 <211> 8 <212> PRT <213> Humans (Homo sapiens) <400> 3 Gly Phe Thr Phe Ser Ser Tyr Trp 1 5 <210> 4 <211> 8 <212> PRT <213> Humans (Homo sapiens) <400> 4 Ile See See See See See Tyr Ile 1 5 <210> 5 <211> 11 <212> PRT <213> Humans (Homo sapiens) <400> 5 Ala Arg Glu Gln Ser Ser Ser Gly Phe Asp Tyr 1 5 10 <210> 6 <211> 6 <212> PRT <213> Humans (Homo sapiens) <400> 6 Gln Ser Ile Ser Ser Trp 1 5 <210> 7 <211> 3 <212> PRT <213> Humans (Homo sapiens) <400> 7 Light Ala Ser 1 <210> 8 <211> 9 <212> PRT <213> Humans (Homo sapiens) <400> 8 Gln Gln Tyr Asn Ser Tyr Pro Leu Thr 1 5 <210> 9 <211> 5 <212> PRT <213> Humans (Homo sapiens) <400> 9 Watch Tyr Trp Met Watch 1 5 <210> 10 <211> 17 <212> PRT <213> Humans (Homo sapiens) <400> 10 Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 11 <211> 9 <212> PRT <213> Humans (Homo sapiens) <400> 11 Glu Gln Ser Ser Ser Gly Phe Asp Tyr 1 5 <210> 12 <211> 11 <212> PRT <213> Humans (Homo Sapiens) <400> 12 Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala 1 5 10 <210> 13 <211> 7 <212> PRT <213> Humans (Homo sapiens) <400> 13 Lys Ala Ser Ser Leu Glu Ser 1 5 <210> 14 <211> 9 <212> PRT <213> Humans (Homo sapiens) <400> 14 Gln Gln Tyr Asn Ser Tyr Pro Leu Thr 1 5 <210> 15 <211> 7 <212> PRT <213> Humans (Homo sapiens) <400> 15 Gly Phe Thr Phe Ser Ser Tyr 1 5 <210> 16 <211> 6 <212> PRT <213> Humans (Homo sapiens) <400> 16 See See See See See Bull 1 5 <210> 17 <211> 118 <212> PRT <213> Homo sapiens <400> 17 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu 115 <210> 18 <211> 105 <212> PRT <213> Homo sapiens <400> 18 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 1 5 10 15 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 20 25 30 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 35 40 45 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 50 55 60 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 65 70 75 80 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 85 90 95 Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 <210> 19 <211> 107 <212> PRT <213> Homo sapiens <400> 19 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 20 <211> 107 <212> PRT <213> Homo sapiens <400> 20 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 21 <211> 354 <212> DNA <213> Artificial sequence <400> 21 gaggtgcagc tggtgcagtc tgggggaggc ttggtccagc ccggggggc cctgagactc 60 tcctgtgaag actctggatt caccttcagt agctattgga tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatcc attagtagta gtagtagtta catatactac 180 gcagactcag tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240 ctacagatga acagcctgag aggcgaggac acggctgttt attactgtgc gagggaacag 300 tctagcagtg gctttgacta ctggggccag ggaaccctgg tcaccgtctc ctca 354 <210> 22 <211> 321 <212> DNA <213> Artificial sequence <400> 22 60. gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc atcacgtgcc gggccagtca gagtattagt agctggttgg cctggtatca gcagaaacca gggaaagccc ctaaactcct gatctataag gcgtctagtt tagaaagtgg ggtcccacca aggttcagcg gcagtggatc tggggcagaa ttcactctca ccatcagcag cctgcagcct 240 gatgattttg caacttatta ctgccaacag father atccgctcac tttcggcgga gggaccaagg tggaggtcaa a <210> 23 <211> 8251 <212> DNA <213> Individual sequence(Artificial sequence) <400> 23 agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta 180 father gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acggggttatg gcccttgcgt 300 gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg 360 aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt 420 tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg 480 tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct 540 ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt 600 ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg 660 gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc 720 tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg 780 gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc 840 aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag 900 ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag 960 gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt 1020 ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca 1080 cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gttttttct tccatttcag gtgtcgtgag gaagatctct agaagctggg taccttgtgc ccggggcgcca ccatggagtt tgggctgagc tggctttttc 1260. ttgtcgcgat tcttaagggt gtccagtgcg agacgactcc tcgacgagga agcttgacat 1320 ccagatgacc cagagccctt ccagcctgag cgctagcgtg ggagacaggg tgaccatcac ctgcagggct agccaggatg tgagcaccgc cgtggcctgg tatcagcaga agcctggcaa ggcccccaag ctgctgatct acagcgccag cttcctgtac agcggagtgc ccagcagatt cagcggcagc ggagcggca ccgatttcac cctgaccatc agcagcctgc agcccgagga 1560. cttcgctacc tactactgcc agcagtacct gtaccacccc gccacctttg gccagggcac 1620 caaggtggag atcaagcgta cggtggctgc accatctgtc ttcatcttcc cgccatctga 1680. tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa tcgggtaact cccaggagag 1800 tgtcacagag caggacagca aggacagcac ctacagcctc agcagcaccc tgacgctgag 1860 caaagcagac tacgagaaac acaaagtcta cgcctgcgaa gtcacccatc agggcctgag 1920 ctcgcccgtc acaaagagct tcaacagggg agagtgtatc caaacggagg agcgtctccg 1980 cttcgaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg 2040 gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt 2100 ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag 2160 gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct 2220 acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa gttgagccca 2280 aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc ctggggggac 2340 cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg 2400 aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt 2460 acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca 2520 gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg 2580 agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca 2640 aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggatgagc 2700 tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg 2760 ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc 2820 tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc 2880 agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc 2940 agaagagcct ctccctgtct ccgggtaaat gagcggccgc tcgaggccgg caaggccgga 3000 tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa 3060 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg 3120 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt 3180 gtgggaggtt ttttaaagca agtaaacct ctacaaatgt ggtatggctg attatgatcc 3240 ggctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggat 3300 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca 3360 gcgggtgttg gcgggtgtcg gggcgcagcc atgaggtcga ctctagagga tcgatccccg 3420 ccccggacga actaaacctg actacgacat ctctgcccct tcttcgcggg gcagtgcatg 3480 taatcccttc agttggttgg tacaacttgc caactgggcc ctgttccaca tgtgacacgg 3540 gggggggacca aacacaaagg ggttctctga ctgtagttga catccttata aatggatgtg 3600 cacatttgcc aacactgagt ggctttcatc ctggagcaga ctttgcagtc tgtggactgc 3660 aacacaacat tgcctttatg tgtaactctt ggctgaagct cttacaccaa tgctggggga 3720 catgtacctc ccaggggccc aggagaacta cgggaggcta caccaacgtc aatcagaggg 3780 gcctgtgtag ctaccgataa gcggacctc aagagggcat tagcaatagt gtttataagg 3840 cccccttgtt aaccctaaac gggtagcata tgcttccccgg gtagtagtat atactatcca 3900 gactaaccct aattcaatag catatgttac ccaacgggaa gcatatgcta tcgaattagg 3960 gttagtaaaa gggtcctaag gaacagcgat atctcccacc ccatgagctg tcacggtttt 4020 atttacatgg ggtcaggatt ccacgagggt agtgaaccat tttagtcaca agggcagtgg 4080 ctgaagatca aggagcgggc agtgaactct cctgaatctt cgcctgcttc ttcattctcc 4140 ttcgtttagc taatagaata actgctgagt tgtgaacagt aaggtgtatg tgaggtgctc 4200 gaaaacaagg tttcaggtga cgcccccaga ataaaatttg gacggggggt tcagtggtgg 4260 cattgtgcta tgacaccaat ataaccctca caaacccctt gggcaataaa tactagtgta 4320 ggaatgaaac attctgaata tctttaacaa tagaaatcca tggggtgggg acaagccgta 4380 aagactggat gtccatctca cacgaattta tggctatggg caacacataa tcctagtgca 4440 atatgatact ggggttatta agatgtgtcc caggcaggga ccaagacagg tgaaccatgt 4500 tgttacactc tatttgtaac aaggggaaag agagtggacg ccgacagcag cggactccac 4560 tggttgtctc taacaccccc gaaaattaaa cggggctcca cgccaatggg gcccataaac 4620 aaagacaagt ggccactcttt ttttttgaaa ttgtggagtg ggggcacgcg tcagccccca 4680 cacgccgccc tgcggttttg gactgtaaaa taagggtgta ataacttggc tgattgtaac 4740 cccgctaacc actgcggtca aaccacttgc ccacaaaacc actaatggca ccccggggaa 4800 tacctgcata agtaggtggg cgggccaaga taggggcgcg attgctgcga tctggaggac 4860 aaattacaca cacttgcgcc tgagcgccaa gcacagggtt gttggtcctc atattcacga 4920 ggtcgctgag agcacggtgg gctaatgttg ccatgggtag catatactac ccaaatatct 4980 ggatagcata tgctatccta atctatatct gggtagcata ggctatccta atctatatct 5040 gggtagcata tgctatccta atctatatct gggtagtata tgctatccta atttatatct 5100 gggtagcata ggctatccta atctatatct gggtagcata tgctatccta atctatatct 5160 gggtagtata tgctatccta atctgtatcc gggtagcata tgctatccta atagagatta 5220 gggtagtata tgctatccta attatctct gggtagcata tactacccaa atatctggat 5280 agcatatgct atcctaatct atatctgggt agcatatgct atcctaatct atatctgggt 5340 5400. 5400. 5400. 5400. 5400. 5400. 5400. 5400. 5400. 5400. 5400. 5400 5460. 5460. 5460. 5460. 5460. 5460. 5460. 5460. 5460. 5460 agcatatgct atcctaatct atatctgggt agcatatgct atcctaatct gtatccgggt agcatatgct atcctcatgc atatacagtc agcatgat acccagtagt agagtgggag tgctatcctt tgcatatgcc gccacctccc aagggggcgt gattttcgc tgcttgtcct 5640. tttcctgctg cttatcgatg aaacatgaga attcttgaag acgaaagggc ctcgtgatac gcctattttt attack gtcatgata taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 5820. tcaaatatgt atccgctcat gagacaata ccctgataaa tgcttcaata atccgctcaat aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 5940 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 6120 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 6180 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 6240 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttactcttctg 6300 aaacgatcg gaggaccgaa ggagctaacc gcttttttgc aacacatggg ggatcatgta 6360 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 6420 6480 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 6540 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 6600 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 6660 gttatctaca cgacggggag tcaggcaact atggatgaac gaatagaca gatcgctgag 6720 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt 6780 tagattgatt taaaacttca ttttaattt aaaaggatct aggtgaagat cctttttgat 6840 aatctcatga ccaaaatccc ttaacgtgag tttcgttcc actgagcgtc agaccccgta 6900 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 6960 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 7020 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag 7080 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 7140 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 7200 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 7260 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 7320 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 7380 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 7440 gggttcgcc acctctgact tgagcgtcga ttttgtgat gctcgtcagg ggggcggagc 7500 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 7560 gctcacatga agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa 7620 cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc 7680 tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccccgagat gcgccgcgtg 7740 cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc 7800 acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg 7860 tgccgccctg cttcatcccc gtggcccgtt gctcgcgttt gctggcggtg tcactggccc 7920 cgtgggttag ggacggggtc ccccatgggg aatggtttat ggttcgtggg ggtattatt 7980 ttgggcgttg cgtggggtca ggtccacgac tggactgagc agacagaccc atggtttttg 8040 gatggcctgg gcatggaccg catgtactgg cgcgacacga acaccgggcg tctgtggctg 8100 ccaaaacaccc ccgaccccca aaaaccaccg cgcggatttc tggcgtgcca agctagtcga 8160 ccaattctca tgtttgacag cttatcatcg cagatccggg caacgttgtt gccattgctg 8220 caggcgcaga actggtaggt atggaagatc t 8251 <210> 24 <211> 7930 <212> DNA <213> Artificial sequence <400> 24 agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga 60 gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa 120 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta 180 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca 240 ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt 300 gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg 360 aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt 420 tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg 480 tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct 540 ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt 600 ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg 660 gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc 720 tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg 780 gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc 840 aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag 900 ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag 960 gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt 1020 ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca 1080 cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa 1140 gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gaagatctct 1200 agaagctggg taccttgtgc ccgggcgcca ccatggacat gcgcgtgccc gcccagctgc 1260 tgggcctgct gctgctgtgg ttccccggct cgcgatgcga gacgactcct cgacgaggaa 1320 gcttatgcct ccaccaaggg acccagcgtg ttcccctg ccccctgttc cagatccacc 1380 tccgaaagca cagccgctct cggctgcctg gtcaggatt acttccctga gcccgtgaca 1440 gtctcctgga atagcggcgc tctgacctcc ggcgtgcata ccttccctgc tgtgctgcaa 1500 tcctccggac tgtacagcct gagcagcgtg gtcaccgtgc cttcctccag cctgggaacc 1560 aaaacctaca catgcaacgt ggaccacaag cccagcaca ccaagtgga caagagggtg 1620 gagtccaagt acggaccccc ttgtcctcc tgccctgctc ctgaagccgc tggaggacct 1680 agcgtgttcc tgttccccc caagcccaag gaccctca tgatctccag gaccccgag 1740 gtgacctgtg tcgtggtgga cgtgagccaa gaggaccccg aggtgcagtt caactggtac 1800 gtggatggcg tcgaggtcca taacgccaag accaagccta gggaggagca gttcacagc 1860 acctacagag tggtgagcgt cctgaccgtg ctccaccaag actggctgaa cggcaggaa 1920 tacaagtgca aggtctccaa cagggactc ccttcctcca tcgagaagac catcagcaag 1980 gccaagggcc agcccagaga acccaagctc tacacactgc cccccagcca agaggaatg 2040 accaagaacc aggtgagcct gacctgcctg gtgaaaggct tctaccccag cgacattgct 2100 gtcgaatggg agagcaacgg ccaacccgag aacaactaca agaccacccc ccctgtgctc 2160 gacagcgacg gctccttctt cctctacagc aggctgacag tggacaagtc caggtggcaa 2220 gagggcaatg tcttcagctg tagcgtcatg cacgaggccc tccacaacca ctacacccag 2280 aagagcctgt ccctctccct gggctgacca aacggaggag cgtctcccgt acggtggctg 2340 caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga actgcctctg 2400 ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg aaggtggata 2460 acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc aggacagca 2520 cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct 2580 acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc ttcaacaggg 2640 gagagtgttg agcggccgct cgaggccggc aaggccggat ccagacatga taagatacat 2700 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat 2760 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa 2820 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa 2880 gtaaaacctc tacaaatgtg gtatggctga ttatgatccg gctgcctcgc gcgtttcggt 2940 gatgacggtg aaaacctctg acacatgcag ctcccggata cggtcacagc ttgtctgtaa 3000 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg 3060 ggcgcagcca tgaggtcgac tctagaggat cgatccccgc cccggacgaa ctaaacctga 3120 ctacgacatc tctgcccctt cttcgcgggg cagtgcatgt aatcccttca gttggttggt 3180 acaacttgcc aactgggccc tgttccacat gtgacacggg gggggaccaa acacaaaggg 3240 gttctctgac tgtagttgac atccttataa atggatgtgc acatttgcca acactgagtg 3300 gctttcatcc tggagcagac tttgcagtct gtggactgca acacaacatt gcctttatgt 3360 gtaactcttg gctgaagctc ttacaccaat gctgggggac atgtacctcc caggggccca 3420 ggaagactac gggaggctac accaacgtca atcagagggg cctgtgtagc taccgataag 3480 cggaccctca agagggcatt agcaatagtg tttataaggc ccccttgtta accctaaacg 3540 ggtagcatat gcttcccggg tagtagtata tactatccag actaacccta attcaatagc 3600 atatgttacc caacgggaag catatgctat cgaattaggg ttagtaaaag ggtcctaagg 3660 aacagcgata tctcccaccc catgagctgt cacggtttta tttacatggg gtcaggattc 3720 cacgagggta gtgaaccatt ttagtcacaa gggcagtggc tgaagatcaa ggagcgggca 3780 gtgaactctc ctgaatcttc gcctgcttct tcattctcct tcgtttagct aatagaataa 3840 ctgctgagtt gtgaacagta aggtgtatgt gaggtgctcg aaaacaaggt ttcaggtgac 3900 gcccccagaa taaaatttgg acggggggtt cagtggtggc attgtgctat gacaccaata 3960 taaccctcac aaaccccttg ggcaataaat actagtgtag gaatgaaaca ttctgaatat 4020 ctttaacaat agaaatccat ggggtgggga caagccgtaa agactggatg tccatctcac 4080 acgaatttat ggctatgggc aacacataat cctagtgcaa tatgatactg gggttattaa 4140 gatgtgtccc aggcagggac caagacaggt gaaccatgtt gttacactct atttgtaaca 4200 aggggaaaga gagtggacgc cgacagcagc ggactccact ggttgtctct aacacccccg 4260 aaaattaaac ggggctccac gccaatgggg cccataaaca aagacaagtg gccactcttt 4320 tttttgaaat tgtggagtgg gggcacgcgt cagcccccac acgccgccct gcggttttgg 4380 actgtaaaat aagggtgtaa taacttggct gattgtaacc ccgctaacca ctgcggtcaa 4440 accacttgcc cacaaaacca ctaatggcac cccgggggaat acctgcataa gtaggtgggc 4500 gggccaagat aggggcgcga ttgctgcgat ctggagaca aattacacac acttgcgcct 4560 gagcgccaag cacagggttg ttggtcctca tattcacgag gtcgctgaga gcacggtggg 4620 ctaatgttgc catgggtagc atatactacc caaatatctg gatagcatat gctatcctaa 4680 tctatatctg ggtagcatag gctatcctaa tctatatctg ggtagcatat gctatcctaa 4740 tctatatctg ggtagtatat gctatcctaa tttatatctg ggtagcatag gctatcctaa 4800 tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagtatat gctatcctaa 4860 tctgtatccg ggtagcatat gctatcctaa tagagattag ggtagtatat gctatcctaa 4920 tttatatctg ggtagcatat actacccaaa tatctggata gcatatgcta tcctaatcta 4980 tatctgggta catatgcta tcctaatcta tatctgggta catataggcta tcctaatcta 5040 tatctgggta gcatatgcta tcctaatcta tatctgggta gtatatgcta tcctaattta 5100 tatctgggta ccatagcta tcctaatcta tatctgggta ccatagcta tcctaatcta 5160 tatctgggta gtatatgcta tcctaatctg tatccgggta gcatatgcta tcctcatgca 5220 tatacagtca catatgata cccagtagta gagtgggagt gctatccttt catatgccg 5280 ccacctccca aggggcgtg aattttcgct gcttgtcctt ttcctgctgc ttatcgatga 5340 taagctgtca aacatgagaa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta 5400 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcgggggaaat 5460 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 5520 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 5580 catttccgtg tcgcccttt tccctttttt gcggcatttt gccttcctgt ttttgctcac 5640 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 5700 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 5760 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc 5820 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 5880 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 5940 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 6000 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 6060 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg 6120 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 6180 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 6240 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 6300 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 6360 caggcaacta tggatgacg aatagacag atcgctgaga taggtgcctc actgattag cattggtac tgtcagacca agtttactca fathercttt agttgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct tacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 6660 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 6720 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 6960. 7020. 7020. 7020. 7020. 7020. 7020. 100% agaaaggcgg acaggtatcc ggtaagcggc agggtcgga caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 7140 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 7200 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgaa gctgtccctg 7260 atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc 7320 ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa 7380 gacgtagccc agcgcgtcgg ccccgagatg cgccgcgtgc ggctgctgga gatggcggac 7440 gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga 7500 ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccctgc ttcatccccg 7560 tggcccgttg ctcgcgtttg ctggcggtgt cactggcccc gtgggttagg gacggggtcc 7620 cccatgggga atggtttatg gttcgtgggg gttattattt tgggcgttgc gtggggtcag 7680 gtccacgact ggactgagca gacagaccca tggtttttgg atggcctggg catggaccgc 7740 atgtactggc gcgacacgaa caccgggcgt ctgtggctgc caaacacccc cgacccccaa 7800 aaaccaccgc gcggatttct ggcgtgccaa gctagtcgac caattctcat gtttgacagc 7860 ttatcatcgc agatccgggc aacgttgttg ccattgctgc aggcgcagaa ctggtaggta 7920 tggaagatct 7930 <210> 25 <211> 21 <212> PRT <213> Artificial sequence <400> 25 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 26 <211> 240 <212> PRT <213> Homo sapiens <400> 26 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Pro Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Val Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln 115 120 125 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 130 135 140 Glu Asp Ser Gly Phe Thr Phe Ser Ser Tyr Trp Met Ser Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Ser Ser 165 170 175 Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Gly Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gln Ser Ser 210 215 220 Ser Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 225 230 235 240 <210> 27 <211> 47 <212> PRT <213> Artificial sequence <400> 27 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 35 40 45 <210> 28 <211> 22 <212> PRT <213> Artificial sequence <400> 28 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 1 5 10 15 Val Ile Thr Leu Tyr Cys 20 <210> 29 <211> 42 <212> PRT <213> Artificial sequence <400> 29 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 30 <211> 112 <212> PRT <213> Artificial sequence <400> 30 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 31 <211> 63 <212> DNA <213> Individual sequence(Artificial sequence) <400> 31 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg ccg 63 <210> 32 <211> 720 <212> DNA <213> Individual sequence(Artificial sequence) <400> 32 60. gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc atcacgtgcc gggccagtca gagtattagt agctggttgg cctggtatca gcagaaacca gggaaagccc ctaaactcct gatctaag gcgtctagtt tagaaagtgg ggtcccacca aggttcagcg gcagtggatc tggggcagaa ttcactctca ccatcagcag cctgcagcct 240 gatgattttg caacttatta ctgccaacag father atccgctcac tttcggcgga gggaccaagg tggaggtcaa aggtggcggt ggctcggggcg gtggtgggtc gggtggcggc 360 ggatctgagg tgcagctggt gcagtctggg ggaggcttgg tccagcccgg ggggtccctg 420 agactctcct gtgaagactc tggattcacc ttcagtagct attggatgag ctgggtccgc 480 caggctccag ggaaggggct ggagtgggtc tcatccatta gtagtagtag tagttacata 540 actcagcg actcagtga gggccgattc accatctcca gagacaacgc caagaactca ctgtatctac agatgaacag cctgagaggc gaggacacgg ctgtttatta ctgtgcgagg gcagtcta gcagtggctt tgactactgg ggccaggga ccctggtcac cgtctcctca <210> 33 <211> 141 <212> DNA <213> Individual sequence(Artificial sequence) <400> 33 60. accacgacgc cagcgccgcg accaccaca ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcta c 141 <210> 34 <211> 66 <212> DNA <213> Individual sequence(Artificial sequence) <400> 34 atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt tactgc 66 <210> 35 <211> 126 <212> DNA <213> Artificial sequence <400> 35 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120 gaactg 126 <210> 36 <211> 336 <212> DNA <213> Artificial sequence <400> 36 agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgc 336

Claims

1. An antibody or a Trop2 antigen-binding fragment that binds to Trop2, characterized in that: Include: a1) in accordance with the IMGT numbering system: a heavy chain variable region with 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 with VL CDR1 of SEQ ID NO.6, VL CDR2 of SEQ ID NO.7 and VLCDR3 of SEQ ID NO.8; or a2) a heavy chain variable region having VH CDR1 of SEQ ID NO.9, VH CDR2 of SEQ ID NO.10 and VHCDR3 of SEQ ID NO.11; a light chain variable region having VL CDR1 of SEQ ID NO.12, VL CDR2 of SEQ ID NO.13 and VL CDR3 of SEQ ID NO.14 in accordance with the Kabat numbering system; or a3) following the Chothia numbering system: a heavy chain variable region with VH CDR1 of SEQ ID NO.15, VH CDR2 of SEQ ID NO.16 and VH CDR3 of SEQ ID NO.11; a light chain variable region with VL CDR1 of SEQ ID NO.12, VL CDR2 of SEQ ID NO.13 and VL CDR3 of SEQ ID NO.14; The antibody binding to Trop2 is 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) a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; or b7) The Fv fragment consists 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 with an amino acid sequence as shown in SEQ ID NO.1 and a light chain variable region with an amino acid sequence as shown in 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 a nucleotide sequence of SEQ ID NO.21 encoding a heavy chain variable region and a nucleotide sequence of SEQ ID NO.22 encoding a 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 CD8leader-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; wherein, The tumor can express Trop2; the tumor is one or more of pancreatic cancer, gastric cancer, colorectal cancer, breast cancer, prostate cancer, lung cancer, oral squamous cell carcinoma and bladder cancer.

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