An antibody that specifically binds to Trop2 or its antigen-binding fragment, its preparation method, and its application.

Abstract

This invention relates to an antibody that specifically binds to Trop2 or its antigen-binding fragment, as well as its preparation method and application, belonging to the field of biological immunotherapy technology. The antibody has good safety and therapeutic effect targeting Trop2, and can be used to prepare Trop2-targeting immune effector cells, providing a treatment or improvement approach for diseases related to Trop2 expression.

Description

Technical Field

[0001] This invention relates to the field of chimeric antigen receptor technology, specifically to an antibody that specifically binds to Trop2 or its antigen-binding fragment, as well as its preparation method and application. Background Technology

[0002] Traditional treatments for tumors, such as surgical resection, chemotherapy, and radiation therapy, damage normal tissues, have limitations, and limited effectiveness. In recent years, targeted therapies have emerged, designing therapeutic drugs at the cellular and molecular level that target known carcinogenic sites. Once in the body, these drugs specifically bind to and act on these carcinogenic sites, causing the tumor cells to die without harming surrounding normal tissue cells.

[0003] Trop2, short for Trophoblast Cell Surface Antigens 2, is a cell surface glycoprotein encoded by the TACSTD2 gene. Composed of a hydrophobic leader peptide, an extracellular domain, a transmembrane domain, and a cytoplasmic tail, Trop2 is a single-transmembrane glycoprotein measuring 35.7 kDa and acts as a calcium channel signal transducer. The N-terminus of the Trop2 protein is an extracellular domain (Trop2 EC), which is attached to the cell membrane via a single-direction transmembrane helix (TM) to the intracellular short tail (Trop2 IC). Its cytoplasmic tail contains a highly conserved phosphatidylinositol 4,5-bisphosphate (PIP2) binding sequence, indicating that PIP2 plays an important role in Trop2 signal transduction. In addition to the PIP2 binding motif, it also contains conserved tyrosine and serine phosphorylation sites. Trop2 is not expressed or is expressed at low levels in normal tissues, but it is overexpressed in a variety of malignant tumors such as breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer. It can promote the occurrence, invasion, metastasis and spread of tumors and plays a key role in the process of tumor growth. Therefore, Trop2 is considered a candidate target for tumor immunity.

[0004] Chimeric antigen receptor-T cells (CAR-T cells) are genetically modified T cells that can recognize specific target antigens in an MHC-unrestricted manner and continuously activate and expand. The 2012 International Society for Cell Therapy Annual Meeting indicated that biological immunotherapy has become the fourth treatment for tumors, alongside surgery, radiotherapy, and chemotherapy. It is a novel immunotherapy method targeting specific antigens on the surface of tumor cells. Numerous studies have shown that CAR-T cells can effectively recognize tumor antigens, elicit specific anti-tumor immune responses, and significantly improve patient survival.

[0005] Chimeric antigen receptors (CARs) are the core component of CAR-T therapy, endowing T cells with the ability to recognize tumor antigens in a HLA-independent manner. This allows CAR-modified T cells to recognize a wider range of targets compared to the natural T cell surface receptor (TCR). The basic design of a CAR includes a tumor-associated antigen (TAA) binding region (usually derived from the scFv segment of the antigen-binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region, and an intracellular signaling domain. The selection of the target antigen is a crucial determinant of the CAR's specificity, efficacy, and the safety of the genetically modified T cells themselves.

[0006] Single-chain antibodies are an important component of CARs. Traditionally, murine antibodies are used. However, due to the heterogeneity of murine antibodies, human anti-mouse antibody reaction (HAMA) can occur, which can lead to the rapid clearance of CAR-T cells in the circulatory system and loss of efficacy.

[0007] Currently, there are no reports of Trop2-binding molecules with good therapeutic efficacy and safety as described in this application, or of CAR-modified immune effector cells based on said Trop2-binding molecules. Summary of the Invention

[0008] This invention provides a Trop2 binding molecule comprising an anti-Trop2 antibody or an antigen-binding fragment thereof. The Trop2 binding molecule includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes a heavy chain complementarity-determining region (HCDR) as shown in any of SEQ ID NO:1-12, and the light chain variable region includes a light chain complementarity-determining region (LCDR) as shown in any of SEQ ID NO:13-24.

[0009] In one or more embodiments, the CDR sequence of the heavy chain variable region and the CDR sequence of the light chain variable region are selected from any one of the following:

[0010] (1) HCDR1 with sequence as shown in SEQ ID NO:1, HCDR2 with sequence as shown in SEQ ID NO:2, HCDR3 with sequence as shown in SEQ ID NO:3, LCDR1 with sequence as shown in SEQ ID NO:13, LCDR2 with sequence as shown in SEQ ID NO:14, and LCDR3 with sequence as shown in SEQ ID NO:15;

[0011] (2) HCDR1 with sequence as shown in SEQ ID NO:4, HCDR2 with sequence as shown in SEQ ID NO:5, HCDR3 with sequence as shown in SEQ ID NO:6, LCDR1 with sequence as shown in SEQ ID NO:16, LCDR2 with sequence as shown in SEQ ID NO:17, and LCDR3 with sequence as shown in SEQ ID NO:18;

[0012] (3) HCDR1 with sequence as shown in SEQ ID NO:7, HCDR2 with sequence as shown in SEQ ID NO:8, HCDR3 with sequence as shown in SEQ ID NO:9, LCDR1 with sequence as shown in SEQ ID NO:19, LCDR2 with sequence as shown in SEQ ID NO:20, and LCDR3 with sequence as shown in SEQ ID NO:21;

[0013] (4) HCDR1 with sequence as shown in SEQ ID NO:10, HCDR2 with sequence as shown in SEQ ID NO:11, HCDR3 with sequence as shown in SEQ ID NO:12, LCDR1 with sequence as shown in SEQ ID NO:22, LCDR2 with sequence as shown in SEQ ID NO:23, and LCDR3 with sequence as shown in SEQ ID NO:24.

[0014] In one or more embodiments, the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO:25-28; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO:29-32;

[0015] In one or more embodiments, the sequence of the heavy chain variable region and the light chain variable region is selected from any of the following:

[0016] (a) When the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:25, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:29;

[0017] (b) When the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:26, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:30;

[0018] (c) When the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:27, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:31;

[0019] (d) When the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:28, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:32.

[0020] In one or more embodiments, the antibody is at least one of a monoclonal antibody, a humanized antibody, a chimeric antibody, or a bispecific antibody; the antigen-binding fragment is at least one of Fab, F(ab'), F(ab')2, Fd, a single-chain antibody scFv, a disulfide-linked Fv(sdFv), or a single-domain antibody.

[0021] Another aspect of the present invention provides a chimeric antigen receptor comprising an optional signal peptide sequence, a Trop2 binding molecule as described in any embodiment herein, a hinge region, a transmembrane region, and an intracellular region.

[0022] In one or more embodiments, the intracellular region includes an intracellular co-stimulatory domain and / or an intracellular signaling domain.

[0023] In one or more embodiments, from the N-terminus to the C-terminus, the chimeric antigen receptor sequentially comprises a signal peptide, a Trop2 binding molecule as described in any of the embodiments herein, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signaling domain.

[0024] The present invention also provides a nucleic acid molecule having a sequence selected from any of the following:

[0025] (1) The coding sequence of the Trop2 binding molecule or chimeric antigen receptor described in any of the embodiments herein;

[0026] (2) and (1) are complementary sequences;

[0027] A 5-50bp fragment of any sequence from (3), (1), or (2).

[0028] In one or more embodiments, the fragment is a primer.

[0029] The present invention also provides a nucleic acid construct comprising the nucleic acid molecules described herein.

[0030] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or an integration vector.

[0031] The present invention also provides a host cell selected from:

[0032] (1) Expression and / or secretion of the Trop2 binding molecule or chimeric antigen receptor as described in any of the embodiments herein;

[0033] (2) Contains the nucleic acid molecules described herein; and / or

[0034] (3) Includes the nucleic acid constructs described herein.

[0035] In one or more embodiments, the host cell is an immune effector cell, preferably a T cell.

[0036] The present invention also provides a method for generating a Trop2 binding molecule according to any embodiment herein, comprising: culturing the host cells described herein under conditions suitable for generating a Trop2 binding molecule (e.g., an anti-Trop2 antibody or an antigen-binding fragment thereof, a monovalent or multivalent anti-Trop2 antibody, or a multispecific anti-Trop2 antibody), and optionally purifying the Trop2 binding molecule from the culture.

[0037] The present invention also provides a pharmaceutical composition comprising a Trop2 binding molecule, a nucleic acid molecule, a nucleic acid construct or a host cell as described in any embodiment herein, and a pharmaceutically acceptable excipient.

[0038] In one or more embodiments, the pharmaceutical composition is used to treat diseases or conditions related to Trop2 expression.

[0039] The present invention also provides the use of the Trop2 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct or host cell described in any embodiment herein in the preparation of activated immune cells (e.g. T cells).

[0040] The present invention also provides the use of the Trop2 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct or host cell described in any embodiment herein in the preparation of a medicament for the prevention or treatment of diseases or conditions associated with Trop2 expression.

[0041] In one or more embodiments, the disease or condition is selected from one or more of the following: breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, lung cancer, esophageal cancer, kidney cancer, bladder cancer, uterine cancer, and ovarian cancer.

[0042] The present invention also provides a method for treating or preventing diseases or conditions related to Trop2 expression, the method comprising administering to a patient in need a therapeutically effective amount of the Trop2 binding molecule or host cell as described in any embodiment of the present invention, or a pharmaceutical composition as described in any embodiment of the present invention.

[0043] The present invention also provides a kit for detecting Trop2, for example, to evaluate the efficacy of drug treatment or to diagnose cancer, said kit comprising a Trop2 binding molecule, nucleic acid molecule, nucleic acid construct or host cell as described in any embodiment herein.

[0044] In one or more embodiments, the kit further includes reagents for detecting the binding of Trop2 to the Trop2 binding molecule. For example, reagents for detecting the binding by an enzyme-linked immunosorbent assay (ELISA).

[0045] In one or more embodiments, the detection binding reagent is a detectable marker, such as biotin, that can be linked to a Trop2-binding molecule. The detectable marker is either linked to the Trop2-binding molecule or is present separately in the kit.

[0046] This invention also provides a non-diagnostic method for detecting the presence of Trop2 in a sample, the method comprising: incubating the sample with the Trop2 binding molecule as described in any embodiment of this invention, and detecting the binding of Trop2 to the Trop2 binding molecule, thereby determining the presence of Trop2 in the sample. The detection is performed using an enzyme-linked immunosorbent assay (ELISA).

[0047] The present invention also provides the use of the Trop2 binding molecule described in any embodiment herein in the preparation of kits for detecting Trop2 in samples, evaluating the efficacy of drug treatments, or diagnosing cancer.

[0048] The present invention has the following beneficial effects:

[0049] This invention provides a novel antibody that specifically recognizes Trop2 and CAR-modified cells containing the antibody. The antibody and cells have good therapeutic effects and safety in targeting Trop2, providing a therapeutic or ameliorative approach for diseases related to Trop2 expression. Attached Figure Description

[0050] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0051] Figure 1 This is an SDS-PAGE image of the recombinant human Trop2-avi-his antigen protein.

[0052] Figure 2 A schematic diagram of different clones of Trop2 CAR.

[0053] Figure 3 The CAR positivity rate of different clones of Trop2 CAR-T cells.

[0054] Figure 4 CD107a expression in different clones of Trop2 CAR-T cells.

[0055] Figure 5 INFγ secretion in different clones of Trop2 CAR-T cells at an effector-target ratio of 10:1.

[0056] Figure 6 INFγ secretion in different clones of Trop2 CAR-T cells at an effector-target ratio of 2:1.

[0057] Figure 7 IL-2 secretion in different clones of Trop2 CAR-T cells at an effector-target ratio of 10:1.

[0058] Figure 8 IL-2 secretion in different clones of Trop2 CAR-T cells at an effector-target ratio of 2:1.

[0059] Figure 9 This is the result of the killing experiment of target cells BxPC3-LUC-GFP by different clones of Trop2 CAR-T cells in Example 5.

[0060] Figure 10 This is the result of the killing experiment of different cloned Trop2 CAR-T cells on target cells U251-LUC-GFP in Example 5.

[0061] Figure 11 , 12 This is a dynamic diagram of the killing effect of different cloned Trop2 CAR-T cells on target cells in Example 6.

[0062] Figure 13 This is a statistical chart showing the 29H and 59H killing rates of different cloned Trop2 CAR-T cells against target cells in Example 6. Detailed Implementation

[0063] Through extensive and in-depth research and screening, the inventors discovered a class of anti-Trop2 antibodies and their antigen-binding fragments that can specifically recognize Trop2, bind to Trop2 with high affinity, and have good functional activity.

[0064] Specifically, the present invention first constructs a human Trop2 protein expression vector, expresses and purifies human Trop2 protein in eukaryotic cells, then constructs a human natural antibody phage display library, and pans to obtain anti-Trop2 antibodies with good targeting and safety, which can specifically bind to the extracellular domain of human Trop2.

[0065] The present invention also provides a chimeric antigen receptor (CAR) containing the anti-Trop2 antibody. Using a vector containing the coding sequence of this CAR to infect immune cells can yield immune effector cells with significant killing ability against tumor cells overexpressing Trop2. These immune effector cells can be used to treat or improve Trop2-related diseases, thus laying the foundation for the treatment of Trop2-positive tumors.

[0066] Antibody

[0067] In this article, "Trop2 binding molecules" are proteins that specifically bind to Trop2, including but not limited to antibodies, heavy chain antibodies, nanobodies, or their antigen-binding fragments.

[0068] In this document, the term "antibody" includes monoclonal antibodies (including full-length antibodies having the immunoglobulin Fc region), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), biantibodies and single-chain molecules, and antibody fragments, especially antigen-binding fragments, such as Fab, F(ab')2, Fd, and Fv. In this document, "antibody" and "immunoglobulin" are used interchangeably.

[0069] Traditional "antibodies" contain a basic four-chain antibody unit, a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has a variable domain (VH) at its N-terminus, followed by three (CH1, CH2, and CH3 for each α and γ chain) and four (CH1, CH2, CH3, and CH4 for μ and ε isoforms) constant domains (CH), and a hinge region located between the CH1 and CH2 domains. Each light chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its other end. Pairs of VH and VL together form an antigen-binding site. For the structure and properties of different classes of antibodies, see Basic and Clinical Immunology, 8th Edition, edited by Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw, Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6. Light chains from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on their constant domain amino acid sequences. Based on relatively minor differences in CH sequence and function, the γ and α types can be further subdivided into subclasses, such as those expressed in humans: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0070] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domains of the heavy and light chains are referred to as "VH" and "VL," respectively. These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and contain antigen-binding sites.

[0071] The term "variable" refers to the wide variation in certain segments within a variable domain within the antibody sequence. Variable domains mediate antigen binding and define the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the entire amino acid range spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (present in both light and heavy chain variable domains): HCDR1, HCDR2, and HCDR3 in the heavy chain variable domain, and LCDR1, LCDR2, and LCDR3 in the light chain variable domain. The more highly conserved portions of the variable domain are called backbone regions (FRs). The variable domains of both the natural heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a β-sheet conformation and are linked by three HVRs that form a loop and, in some cases, part of a β-sheet structure. The HVRs in each chain are held together very closely by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antibody's antigen-binding site. Typically, the structure of the variable region in the light chain is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the variable region in the heavy chain is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as antibody involvement in antibody-dependent cell-mediated cytotoxicity. There are several annotation schemes for antibody variable regions, including Chothia, Kabat, IMGT, and Contact. This article uses the IMGT annotation scheme as an example.

[0072] The “Fc region” (crystallizable fragment region), “Fc domain”, or simply “Fc” refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments from the CH2 and CH3 domains of the two antibody heavy chains; the Fc regions of IgM and IgE contain three heavy chain constant domains (CH domains 2–4) in each polypeptide chain. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the sequence segment from the amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, where the numbering is based on the EU index, as in Kabat. As used herein, the Fc region can be a native sequence Fc or a variant Fc.

[0073] An "antibody fragment" comprises a portion of a complete antibody, preferably the antigen-binding region and / or variable region of the complete antibody. The antibody fragment is preferably an antigen-binding fragment of the antibody. Examples of antibody fragments include Fab, Fab', F(ab'), F(ab')2, Fd, and Fv fragments; disulfide-linked Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragment whose half-life should be increased by chemical modification or by incorporation into liposomes. Antigen-binding fragments can be prepared using a variety of techniques, including but not limited to hydrolyzing and digesting complete antibody proteins, and by expression in host cells containing the antigen-binding fragment.

[0074] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a tightly bound, non-covalently linked heavy chain variable domain and a light chain variable domain. Six hypervariable rings (three rings each from the heavy and light chains) protrude from the folds of these two domains, contributing the amino acid residues for antigen binding and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only the three antigen-specific HVRs) can recognize and bind antigens, although with lower affinity than a complete binding site. "Single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing antibody VH and VL domains linked together into a single polypeptide chain. Preferably, the sFv polypeptide also includes a polypeptide linker between the VH and VL domains, allowing the sFv to form the desired antigen-binding structure.

[0075] In this document, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Compared to polyclonal antibody formulations (which typically consist of different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized through hybridoma culture, free from contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic that the antibody is obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies to be used according to the invention can be generated by a variety of techniques, including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and techniques for generating human or human-like antibodies from animals having partial or whole human immunoglobulin loci or genes encoding human immunoglobulin sequences, single-cell sequencing methods.

[0076] Monoclonal antibodies also include “chimeric” antibodies in this article, wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity.

[0077] The “humanized” form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that minimally contains sequences derived from non-human immunoglobulins. Therefore, a “humanized antibody” generally refers to a non-human antibody with a variable domain framework region that exchanges sequences found in human antibodies. Typically, in a humanized antibody, the entire antibody (except for the CDR) is encoded by human-derived polynucleotides or is identical to such an antibody (except for the CDR). The CDR (some or all of which are encoded by nucleic acids derived from non-human organisms) is transplanted into the β-sheet backbone of the variable region of the human antibody to produce an antibody whose specificity is determined by the transplanted CDR. Methods for producing such antibodies are well known in the art, for example, using mice with genetically engineered immune systems. In this invention, antibodies, single-chain antibodies, etc., all include humanized variants of the aforementioned antibodies.

[0078] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies generated by humans and / or produced using any of the techniques disclosed herein for generating human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.

[0079] In some embodiments, the present invention also provides antibodies or antigen-binding fragments thereof that bind to the same epitope on human Trop2 as the antigen-binding region of any anti-Trop2 antibody of the present invention, i.e., antibodies or antigen-binding fragments thereof capable of cross-competing with the antigen-binding region of any antibody of the present invention for binding to Trop2.

[0080] In this invention, the Trop2 binding molecule includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes the heavy chain complementarity determination region HCDR shown in any of SEQ ID NO:1-12, and the light chain variable region includes the light chain complementarity determination region LCDR shown in any of SEQ ID NO:13-24. Preferably, the CDR sequences of the heavy chain variable region and the light chain variable region of the Trop2 binding molecule of the present invention are selected from any one of the following: (a) HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, HCDR3 as shown in SEQ ID NO:3, and LCDR1 as shown in SEQ ID NO:13, LCDR2 as shown in SEQ ID NO:14, and LCDR3 as shown in SEQ ID NO:15; (b) HCDR1 as shown in SEQ ID NO:4, HCDR2 as shown in SEQ ID NO:5, HCDR3 as shown in SEQ ID NO:6, and LCDR1 as shown in SEQ ID NO:16, LCDR2 as shown in SEQ ID NO:17, and LCDR3 as shown in SEQ ID NO:18; (c) HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:8, HCDR3 as shown in SEQ ID NO:9, and LCDR1 as shown in SEQ ID NO:19, and LCDR3 as shown in SEQ ID NO:15. (d) LCDR2 as shown in NO:20, LCDR3 as shown in SEQ ID NO:21; (d) HCDR1 as shown in SEQ ID NO:10, HCDR2 as shown in SEQ ID NO:11, HCDR3 as shown in SEQ ID NO:12, LCDR1 as shown in SEQ ID NO:22, LCDR2 as shown in SEQ ID NO:23, and LCDR3 as shown in SEQ ID NO:24.

[0081] Preferably, the amino acid sequence of the heavy chain variable region of the Trop2 binding molecule of the present invention is shown in any one of SEQ ID NO:25-28; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO:29-32.

[0082] The Trop2 binding molecules described herein can be monovalent or multivalent antibodies, or multispecific antibodies, comprising one, two, or more of the anti-Trop2 antibodies or antigen-binding fragments described herein. Multispecificity can be against Trop2 and another antigen, or against two different epitopes of Trop2.

[0083] This invention also includes the antibody derivatives and analogs described herein. “Derivatives” and “analytes” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of this invention. The derivatives or analogs of this invention may be (i) polypeptides having substituents in one or more amino acid residues, or (ii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iii) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence, a secretory sequence, a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these derivatives and analogs are within the scope well known to those skilled in the art.

[0084] Without substantially affecting antibody activity, those skilled in the art can modify the antibody sequence of the present invention by one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) to obtain variants of the antibody or its functional fragment sequence. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids of similar or comparable properties generally do not alter protein function. For example, substitutions of amino acids with similar properties in the FR and / or Fc regions. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. For example, adding one or more amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. These are all considered to be included within the scope of protection of this invention.

[0085] The variants of the antibodies described herein include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibodies of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibodies of the present invention. In some embodiments, the sequences of the variants described herein may have at least 95%, 96%, 97%, 98%, or 99% homology with their source sequences. The sequence homology described herein can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The present invention also includes molecules having antibody heavy chain variable regions or light chain variable regions with CDRs, provided that their CDRs have at least 90% (preferably at least 95%, most preferably at least 98%) homology with the CDRs identified herein.

[0086] The antibodies of the present invention can be prepared using methods conventional in the art, such as hybridoma techniques. The single-chain antibodies of the present invention can be prepared using methods conventional in the art, such as phage display techniques well known in the art. Alternatively, the antibodies or single-chain antibodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with the sequence encoding the antibody of the present invention, and then the host cells can be cultured and the antibody purified. Transformation can be performed using any known method, including, for example, packaging polynucleotides in a virus (or viral vector) and transducing host cells with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Mammalian cell lines that can be used as hosts for expression are well known in the art, including but not limited to a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS) cells, human hepatocellular carcinoma cells (e.g., HepG2).

[0087] CAR

[0088] This invention also provides a chimeric antigen receptor (CAR) targeting Trop2. The CAR contains an optional signal peptide sequence, an antigen recognition region (i.e., the anti-Trop2 binding molecule described herein), a hinge region, a transmembrane region, and an intracellular region. The intracellular region includes one or more intracellular co-stimulatory domains and / or one or more intracellular signaling domains. The terms "hinge region," "transmembrane region," and "intracellular region" as used herein can all be selected from sequences of the hinge region, transmembrane region, and intracellular region in known CAR-T technologies.

[0089] The signal peptide, optionally selected for the CAR, can be chosen as needed. Generally, a signal peptide is a peptide sequence that directs the polypeptide to a desired site within the cell. The signal peptide directs the polypeptide to the cell's secretory pathway and allows the polypeptide to integrate and anchor to the lipid bilayer; the signal peptide can also be a membrane-localizing signal peptide. Exemplary signal peptides include CD8 signal peptide, CD28 signal peptide, CD4 signal peptide, or light chain signal peptide, the sequences of which are within the knowledge of those skilled in the art. The CD8 signal peptide suitable for use in this invention can be any of the various human CD8 signal peptide sequences commonly used in CARs in the art. In some embodiments, the amino acid sequence of the CD8 signal peptide comprises the sequence shown in SEQ ID NO:33.

[0090] The hinge region of a chimeric antigen receptor is located between the extracellular antigen-binding region and the transmembrane region. The hinge region is an amino acid segment that typically exists between two domains of a protein and allows for protein flexibility and relative movement between the two domains. The hinge region can be a hinge region of a naturally occurring protein or a portion thereof. The hinge region of an antibody (such as IgG, IgA, IgM, IgE, or IgD antibodies) can also be used in the chimeric antigen receptor described herein. Non-naturally occurring peptides can also be used as the hinge region of the chimeric antigen receptor described herein. Exemplarily, the hinge region of a CAR is selected from the CD8α hinge region, the IgD hinge region, the IgG1 FcCH2CH3 hinge region, or the IgG4 Fc CH2CH3 hinge region, the sequences of which are within the knowledge of those skilled in the art. The CD8α hinge region suitable for use in this invention can be any of the various human CD8α hinge region sequences commonly used in CARs in the art. In some embodiments, the human CD8α hinge region comprises the sequence shown in SEQ ID NO:34.

[0091] The transmembrane region of a chimeric antigen receptor can form an α-helix, a complex of more than one α-helix, a β-barrel, or any other stable structure capable of translocating the cellular phospholipid bilayer. The transmembrane region can be of natural or synthetic origin. It can be selected from the transmembrane regions of the following proteins: CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or the α, β, or ζ chains of T-cell receptors. The human CD8α transmembrane region suitable for this invention can be any of the various human CD8α transmembrane region sequences commonly used in the art for CARs. In some embodiments, the amino acid sequence of the human CD8α transmembrane region comprises the sequence shown in SEQ ID NO:35.

[0092] Intracellular signaling regions (or intracellular signal transduction regions) are responsible for activating at least one normal effector function of immune effector cells expressing chimeric antigen receptors. For example, the effector function of T cells can be lytic activity or helper activity, including cytokine secretion. While the entire intracellular signal transduction region can generally be used, in many cases, using the whole chain is unnecessary. Regarding the use of truncated portions of intracellular signal transduction regions, such truncated portions can be used instead of the whole chain as long as they transduce effector function signals. Therefore, intracellular signal transduction regions include any truncated form of intracellular signal transduction regions sufficient to transduce effector function signals. The intracellular signaling domain of a CAR can be selected as needed, including but not limited to intracellular signaling domains derived from at least one of CD3ζ, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Preferably, the intracellular signaling region is derived from the human CD3ζ intracellular signaling region. Furthermore, the human CD3ζ intracellular signaling region has the amino acid sequence shown in SEQ ID NO:37.

[0093] In addition to stimulation by antigen-specific signals, many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate effector functions. The "co-stimulatory domain" can be the cytoplasmic portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to an associated binding chaperone on immune cells (such as T cells) that specifically binds to a co-stimulatory ligand, thereby enabling the immune cell to mediate a co-stimulatory response, such as, but not limited to, proliferation and survival. Suitable intracellular co-stimulatory domains can be selected as needed, including intracellular domains containing co-stimulatory signaling molecules, such as at least one of the intracellular domains derived from 4-1BB, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, and 41BBL. In some embodiments, the amino acid sequence of the 4-1BB co-stimulatory domain comprises the sequence shown in SEQ ID NO:36.

[0094] The aforementioned portions forming the chimeric antigen receptor of the present invention, such as the CD8 signal peptide, the anti-Trop2 single-chain antibody, the CD8α hinge region, the CD8α transmembrane region, the CD3ζ intracellular signal domain, and the 4-1BB co-stimulatory domain, can be directly linked to each other or linked via adapter sequences. The adapter sequence can be a known antibody-compatible adapter sequence, such as a G and S-containing adapter sequence. Typically, the adapter contains one or more repeating motifs. For example, the motif can be GGGS, GGGGS, SSSSG, GSGSA, and GGSGG. Preferably, the motifs are adjacent in the adapter sequence, with no inserted amino acid residues between the repeats. The adapter sequence can consist of 1, 2, 3, 4, or 5 repeating motifs. The length of the adapter can be 3 to 25 amino acid residues, for example, 3 to 15, 5 to 15, or 10 to 20 amino acid residues. In some embodiments, the adapter sequence is a polyglycine adapter sequence. The number of glycine residues in the linker sequence is not particularly limited, typically ranging from 2 to 20, for example, 2 to 15, 2 to 10, or 2 to 8. Besides glycine and serine, the linker may also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), and glutamine (Q). In some embodiments, the linker sequence is (GGGGS)n-linked, where n is an integer from 1 to 5.

[0095] In an exemplary embodiment, the CAR contains, from the N-terminus to the C-terminus, a CD8 signal peptide, the anti-Trop2 antibody described herein or its antigen-binding fragment, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signaling domain. In specific embodiments, exemplary CARs having the above structure are shown in any of SEQ ID NO:38-41.

[0096] It should be understood that in gene cloning, it is often necessary to design suitable restriction enzyme sites, which inevitably introduces one or more irrelevant residues at the end of the expressed amino acid sequence, without affecting the activity of the target sequence. To construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein, such as, but not limited to, suitable adaptor peptides, signal peptides, leader peptides, and terminal extensions. Therefore, the amino or carboxyl terminus of the CAR of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used herein. For example, the tags may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used for protein purification.

[0097] The antigen recognition region in the CAR of the present invention can be a variant of the aforementioned anti-Trop2 antibody or its functional fragment sequence. Furthermore, other parts of the CAR can also undergo sequence changes, resulting in a mutant with at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity with the CAR and retaining the biological activity of the CAR (e.g., activation of T cells). Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp.

[0098] The mutant also includes an amino acid sequence having one or more mutations (insertions, deletions, or substitutions) in the amino acid sequence of the CAR described in any embodiment, while still retaining the biological activity of the CAR. The number of mutations typically refers to 1-10, for example 1-8, 1-5, or 1-3. Substitution is preferably conserved. For example, in the art, conserved substitution with amino acids of similar or comparable properties generally does not alter the function of the protein or peptide. "Amino acids with similar or comparable properties" includes, for example, families of amino acid residues having similar side chains. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, replacing one or more sites in the polypeptide of the present invention with another amino acid residue from the same side chain class will not substantially affect its activity.

[0099] Nucleic acid

[0100] This invention also provides polynucleotides encoding the aforementioned antibodies or CARs. The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. This invention also includes degenerate variants of polynucleotide sequences encoding fusion proteins, i.e., nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences.

[0101] Therefore, the present invention also relates to polynucleotides that hybridize with the above-mentioned polynucleotide sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0102] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, a long fragment can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy or light chain can be fused with an expression tag (such as 6His) to form a fusion protein. The CAR sequence can also be obtained as described above. Alternatively, the sequences of the various parts of the CAR (signal peptide, antigen recognition region, hinge region, transmembrane region, or intracellular region) can be obtained as described above and then ligated to obtain the full-length CAR.

[0103] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms. Currently, DNA sequences encoding the proteins of this invention (or fragments thereof, or derivatives thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequences of this invention through chemical synthesis. The CAR portions can be sequentially cloned into a vector or integrated into a full-length CAR before cloning.

[0104] This invention also relates to nucleic acid constructs containing the polynucleotide sequences described herein, and one or more regulatory sequences operatively linked to these sequences. The polynucleotide sequences described herein can be manipulated in various ways to ensure the expression of the antibody or CAR. The nucleic acid constructs can be manipulated depending on the expression vector or requirements before insertion into a vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0105] The regulatory sequence can be a suitable promoter sequence. Promoter sequences are typically operatively linked to the coding sequence of the protein to be expressed. A promoter can be any nucleotide sequence that exhibits transcriptional activity in the chosen host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that host cell. An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the early promoter of simian virus 40 (SV40), mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ's sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the use of inducible promoters may also be considered. The use of inducible promoters provides a molecular switch that can turn on the expression of the polynucleotide sequence operatively linked to the inducible promoter during time-limited expression and turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0106] The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in a selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, the untranslated region of mRNA important for translation by the host cell. The leader sequence is operatively linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in a selected host cell can be used in this invention.

[0107] In some embodiments, the nucleic acid construct is a vector, such as a cloning vector, an expression vector, and an integration vector. Expression of the polynucleotide sequences of the present invention is typically achieved by operably linking the polynucleotide sequences of the present invention to an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Integration vectors contain components for integrating the target sequence into the cellular genome. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. These sequences (collectively referred to as “flanking sequences” in some embodiments) typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splicing sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylated sequence, a multi-connector region for inserting a nucleic acid encoding an antibody to be expressed, and optional marker elements.

[0108] Furthermore, the type of vector is not limited; for example, plasmids, phage particles, phage derivatives, animal viruses, and entrapments can be modified depending on the host cell to be introduced. Viral vector technology is well known in the art and has been described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

[0109] To assess the expression of CAR peptides or portions thereof, the vector introduced into cells may also contain one or both of an optional marker gene or reporter gene to facilitate the identification and selection of expressing cells from a population of cells seeking transfection or infection via a viral vector.

[0110] cell

[0111] The host cells suitable for introducing the nucleic acid constructs described herein can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, especially immune cells, preferably immune effector cells. Representative examples include: Escherichia coli, Streptomyces; Salmonella typhimurium bacterial cells; fungal cells such as yeast; Drosophila S2 or Sf9 insect cells; and animal cells such as CHO, COS7, and 293 cells.

[0112] "Immune effector cells" are immune cells capable of performing immune effector functions. In some embodiments, immune effector cells express at least FcγRIII and perform ADCC effector functions. Examples of immune effector cells mediating ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils. Preferably, immune effector cells are selected from at least one of: immune cells cultured and differentiated from pluripotent stem cells or embryonic stem cells, T lymphocytes, NK cells, peripheral blood mononuclear cells (PBMCs), and hematopoietic stem cells. More preferably, the immune effector cells are T lymphocytes (same as T cells). In some embodiments, T cells can be CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8-, or combinations thereof. In some embodiments, T cells produce IL-2, IFN, and / or TNF when expressing a chimeric antigen receptor and binding to target cells. In some implementations, CD8+ T cells lyse antigen-specific target cells when expressing chimeric antigen receptors and binding to target cells.

[0113] The T cells suitable for use in this invention can be of various types and origins. For example, T cells can be derived from PBMCs of patients with malignant solid tumors (e.g., pancreatic cancer). In some embodiments, after obtaining T cells, they can be activated by stimulation with an appropriate amount (e.g., 30–80 ng / ml, such as 50 ng / ml) of CD3 antibody, and then cultured in IL2 medium containing an appropriate amount (e.g., 30–80 IU / ml, such as 50 IU / ml) for later use.

[0114] Methods for introducing nucleic acids or vectors into mammalian cells are known in the art, and the vectors can be transferred into cells by physical, chemical, or biological methods. When the host is a prokaryote such as *Escherichia coli*, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, and liposome packaging can be used. In some embodiments, transduced or transfected immune effector cells proliferate in vitro after the introduction of nucleic acids or vectors.

[0115] The obtained transformants can be cultured using conventional methods to express the antibody or CAR encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0116] The peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0117] Uses and methods

[0118] By constructing a single-chain antibody library, the inventors screened single-chain antibodies that could bind to Trop2. Using these single-chain antibodies, the inventors constructed CAR and CAR-T cells. Cell-level experiments verified that the CAR-T cells have strong immune function, superior CD107a expression, IFN-γ and IL-2 secretion, and specific killing function against target cells, demonstrating significant in vivo efficacy.

[0119] All aspects of the antibodies, CARs, coding sequences, nucleic acid constructs, and cells described herein can be used to prepare drugs for the prevention or treatment of the various conditions and diseases described herein, which are diseases or conditions related to Trop2 expression, referring to diseases directly or indirectly caused by abnormal Trop2 expression, usually referring to diseases caused by Trop2 overexpression, such as cancer, including but not limited to: breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, lung cancer, and ovarian cancer.

[0120] This invention also includes a class of cell therapies comprising expressing the CAR described herein in immune cells (e.g., T cells) and administering a therapeutically effective amount of the cells to a recipient who requires them, the cells being capable of killing the recipient's tumor cells. Compared to antibody therapies, CAR-T cells can replicate in vivo, producing long-lasting durability that can lead to sustained tumor control. The anti-tumor immune response induced by CAR-T cells can be an active or passive immune response. Additionally, CAR-mediated immune responses can be part of an adoptive immunotherapy step, wherein CAR-T cells induce an immune response specific to the antigen-binding portion of the CAR.

[0121] The antibodies, nucleic acids, or CAR-modified cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as associated cytokines or cell populations. In this regard, the pharmaceutical compositions can be prepared by mixing an active pharmaceutical agent of desired purity with an optional pharmaceutically acceptable carrier in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dose and concentration used and may include at least one of buffers (e.g., neutral buffered saline, sulfate buffered saline), antioxidants, preservatives, isotonic agents, stabilizers, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), and surfactants. Furthermore, in order for the pharmaceutical compositions to be usable for in vivo administration, they must be sterile. The pharmaceutical compositions can be sterilized by filtration through a sterile filter membrane.

[0122] In some embodiments, the pharmaceutical composition may contain at least one additive selected from: a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, a growth inhibitor, and an active pharmaceutical agent required for the specific indication to be treated. The specific amount of the additive may be adjusted as needed. The pharmaceutical composition of the present invention may be administered in amounts described as “immunologically effective,” “antitumor effective,” “tumor-inhibitory effective,” or “therapeutic.” “Therapeutic” refers to a subject receiving the treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor volume, a decrease in the rate of cancer cell invasion into surrounding organs, or a decrease in the rate of tumor metastasis or tumor growth). When “immunologically effective,” “antitumor effective,” “tumor-inhibitory effective,” or “therapeutic” is indicated, the precise amount of the composition of the present invention to be administered may be determined by a physician, taking into account individual differences in the patient’s (subject’s) age, weight, tumor size, degree of infection or metastasis, and disease. Typically, a pharmaceutical composition including T cells as described herein may be administered in an amount of 10 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 6 The dosage is cells per kg of body weight. T-cell compositions can also be administered at these dosages multiple times. Cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a specific patient can be readily determined by a physician skilled in the medical field by monitoring the patient's disease signs and thus adjusting the treatment accordingly.

[0123] The composition can be administered in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The composition described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered by intravenous injection. The T-cell composition can be injected directly into the tumor, lymph node, or site of infection.

[0124] In some embodiments of the present invention, the CAR-T cells or compositions thereof of the present invention can be combined with other therapies known in the art. These therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressants. For example, treatment may be combined with radiotherapy or chemotherapy agents known in the art for treating mesothelin-mediated diseases.

[0125] In this article, "anti-tumor effect" refers to a biological effect that can be represented by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various cancer-related physiological symptoms.

[0126] The terms "patient," "subject," and "individual" are used interchangeably in this article to refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and their transgenic species.

[0127] Diagnostics, tests and kits

[0128] The binding molecules of this invention, due to their high affinity for Trop2, can be used for assays, such as binding assays, to detect and / or quantify Trop2 expressed in tissues or cells. Binding molecules, such as single-chain antibodies, can be used in studies further investigating the role of Trop2 in disease. The method for detecting Trop2 generally involves obtaining cell and / or tissue samples; detecting the level of Trop2 in the samples.

[0129] The Trop2 binding molecule of this invention can be used for diagnostic purposes to detect, diagnose, or monitor Trop2-related diseases and / or conditions. This invention provides methods for detecting the presence of Trop2 in samples using classic immunohistochemical methods known to those skilled in the art. Trop2 detection can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of Trop2 include ELISA, FACS, RIA, etc.

[0130] For diagnostic applications, binders such as single-chain antibodies are typically labeled with detectable labeling groups. Suitable labeling groups include (but are not limited to) the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinylated groups, or predetermined polypeptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pairs, binding sites for secondary antibodies, metal-binding domains, epitope tags), MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents. Various methods for labeling proteins are known in the art and can be used in carrying out this invention.

[0131] Another aspect of the invention provides a method for detecting the presence of a test molecule that competes with the antibody of the invention for binding to Trop2. An example of such determination would involve detecting the amount of free antibody in a solution containing a certain amount of Trop2, in the presence or absence of the test molecule. An increase in the amount of free antibody (i.e., antibody not bound to Trop2) would indicate that the test molecule is able to competitively bind to Trop2 with the antibody. In one embodiment, the antibody is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of the antibody.

[0132] This invention also provides a detection kit for detecting Trop2 levels, comprising an antibody that recognizes the Trop2 protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0133] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and materials used in the embodiments are conventional materials and methods in the art.

[0134] Example

[0135] Example 1: Construction and eukaryotic expression of recombinant human Trop2 protein expression vector

[0136] 1. Synthesis of gene sequences and construction of protein expression vectors

[0137] The human Trop2 protein sequence was downloaded from Uniport (https: / / www.uniprot.org / uniprot), optimized using an online codon optimization tool (http: / / www.jcat.de / #opennewwindow), and then sent to Sangon Biotech to synthesize the Trop2 extracellular gene sequence. Simultaneously, avi-tag and 6×his-tag nucleic acid sequences were added to the 3' end of this gene sequence, and the fusion gene sequence encodes the amino acid sequence shown in SEQ ID NO:46. The sequence of the fusion gene is shown in SEQ ID NO:47. Using molecular cloning, the splicing product was cloned into pTT5 using the TaKaRa seamless cloning kit to obtain the expression vector.

[0138] 2. Expression and purification of recombinant human Trop2 protein

[0139] Five days after transfecting 293T cells (ATCC) with the obtained expression vector, the culture supernatant was collected, and recombinant human Trop2 protein was purified using an AKTA Explorer 100 (GE) PCR machine. The Trop2 protein was stained with Coomassie Brilliant Blue after SDS-PAGE electrophoresis, revealing a size of approximately 36 kDa. The results are as follows: Figure 1 As shown.

[0140] Example 2: Preparation of anti-human Trop2 antibody

[0141] 1. Construction of scFv phage display library

[0142] A two-step method was used to construct a human natural antibody phage display library, which involved ligating the light chain variable region gene and the heavy chain variable region gene of the human natural antibody into the phage display vector in two steps.

[0143] 1) Gene amplification of variable regions in the κ and λ chains.

[0144] The human immunoglobulin κ chain variable region gene was amplified by PCR using human PBMC cDNA as a template, with forward primers (3z3-1-F) and reverse primers (Huvksc-R) for the κ chain variable region and by PCR using human PBMC cDNA as a template, with forward primers (3z3-1-F) and reverse primers (HuvLR-1) for the λ chain variable region. The PCR reaction conditions were as follows: 98℃ pre-denaturation for 45 seconds followed by temperature cycling: 98℃ denaturation for 15 seconds, 60℃ annealing for 20 seconds, 72℃ extension for 23 seconds, for 30 cycles, with a final extension at 72℃ for 5 minutes. After 1.5% agarose gel electrophoresis, the approximately 330 bp κ chain variable region gene fragment and λ chain variable region gene fragment were recovered using a gel extraction kit (Promega).

[0145] 2) Construction of κ-chain and λ-chain libraries

[0146] The κ-chain gene, λ-chain gene, and phage particle pcomb3sc were double-digested with NheI-HF and SalI-HF DNA endonucleases (NEB). The digested κ-chain and λ-chain genes were directly recovered using a gel extraction kit. The digested pcomb3sc vector was subjected to 1% agarose gel electrophoresis, and the 4100 bp vector fragment was recovered using a gel extraction kit (Promega). The κ-chain and λ-chain genes were ligated into the pcomb3sc vector using a T4 DNA ligase kit (Invitrogen). 1000 ng of pComb3sc, 200 ng each of κ and λ were used in multiple reaction tubes, 50 μL / tube, and ligation was incubated overnight at 16°C. A small amount of the ligation product was taken for agarose gel electrophoresis to assess ligation efficiency. The ligation product was desalted using a MECK MILLIPOREF microporous membrane. The desalted ligation products were electroporated into self-made TG1 electroporated competent cells to obtain κ-chain and λ-chain libraries. The κ-chain and λ-chain libraries were amplified overnight, and the κ-chain and λ-chain library plasmids were extracted the next day using a plasmid extraction kit (NucleoBond Xtra Maxi EF).

[0147] 3) Amplification of the VH gene in the heavy chain variable region

[0148] Human immunoglobulin heavy chain variable region (VH-MIX 1 / 7) and reverse primers (VH-MIX R), human immunoglobulin heavy chain variable region (VH-MIX 3) and reverse primers (VH-MIX R), and human immunoglobulin heavy chain variable region (VH-MIX 4) and reverse primers (VH-MIX R) were used. Human PBMC cDNA was used as a template for PCR amplification of the human immunoglobulin VH variable region gene. PCR reaction conditions were as follows: 98℃ pre-denaturation for 45 seconds followed by temperature cycling: 98℃ denaturation for 15 seconds, 60℃ annealing for 20 seconds, 72℃ extension for 23 seconds, for 30 cycles, with a final extension at 72℃ for 5 minutes. After 1% agarose gel electrophoresis, the approximately 330 bp VH gene fragment was recovered using a gel extraction kit (Promega).

[0149] 4) scFv library construction

[0150] The recovered VH fragment was mixed in equal proportions and digested with the aforementioned κ-chain and λ-chain library plasmids using SfiI DNA restriction enzyme at 50°C for 16 hours. The digested VH gene was directly recovered by column chromatography using a gel extraction kit. The digested κ-chain and λ-chain libraries were subjected to 1% agarose gel electrophoresis, and the approximately 4000 bp vector fragment was recovered using a gel extraction kit (Promega). The VH gene was ligated into the κ-chain and λ-chain vector libraries using a T4 DNA ligase kit (Invitrogen), i.e., pComb3sc-lambda / kappa (SfiI) 1000 ng, VH amplify mix (SfiI) 200 ng. Multiple reaction tubes were prepared, 50 μl / tube, and ligation was incubated overnight at 16°C. A small amount of the ligation product was taken for agarose gel electrophoresis to assess ligation efficiency. The ligation product was desalted using a MECK MILLIPOREF microporous membrane.

[0151] The ligation product was added to self-made TG1 electroporation competent cells, and then electroporation was performed using an electroporator. 50 μL of the bacterial culture was then serially diluted 10⁻⁶ times with PBS. 2 -10 5 10 μL of each serially diluted buffer was flow-lined onto an Amp / 2YT plate and incubated overnight at 37°C. The size of the phage antibody library was then counted and determined. The remaining electroporated bacteria were replenished with 2YT to a final volume of 500 mL, and then incubated overnight at 30°C and 220 rpm with 100 μg / mL ampicillin. The final scFv immunoglobulin library exceeded 3E10 cells. The electroporated antibody library was amplified overnight, and the bacterial cells were collected by centrifugation and stored at -80°C with 20% glycerol to a final concentration.

[0152] A portion of the frozen human natural antibody phage display library was inoculated into a 2YT culture set at an inoculation density of 0.1 OD. The bacterial culture was incubated at 37°C and 220 rpm for approximately 1.5 hours, until the bacterial density reached 0.6 OD. At this point, 20 times the number of M13KO7 phage cells were added and allowed to stand for 30 minutes for infection. The culture was then incubated overnight at 30°C and 220 rpm. The next day, the bacterial culture was centrifuged at 10,000 g, and the supernatant was collected. 1 / 4 volume of PEG / NaCl solution (20% PEG8000, 2.5 M NaCl) was added to the supernatant, mixed well, and incubated on ice for 1 hour. After the ice incubation, the culture was centrifuged at 8,000 g for 10 minutes, and the precipitate was collected. The precipitate was dissolved in 10% Glycerol / PBST to obtain the human natural antibody phage display library. The OD268 was measured, and the library was aliquoted into 1.5 mL centrifuge tubes (6 OD / tube) and stored at -80°C.

[0153] 2. Selecting Trop2 scFv antibodies

[0154] 1) Recombinant human Trop2 protein coupled to streptavidin magnetic beads

[0155] Using a biotinylation kit (EasyBio) following the kit instructions, the avi-tag of recombinant human Trop2 protein was biotinylated to obtain biotinylated Trop2 protein. 10 μg of the biotinylated recombinant protein was added to 100 μL of streptavidin magnetic beads (DynaBeads 280) that had been washed three times with PBS. The mixture was placed on a rotary shaker at 20 rpm and coupled at room temperature for 1 hour, followed by washing three times with PBS.

[0156] 2) Blocking phage libraries and negative magnetic beads

[0157] Take one vial each of Kappa-scFv-lib and Lambda-scFv-lib, thaw them at room temperature, add 200 μL of 5% BSA / PBST to each, place them on a rotary shaker at 20 rpm, and incubate at room temperature for 1 hour. These phages are designated Input1. Simultaneously, take 100 μL of unconjugated protein DynaBeads 280, wash three times with PBS, add 1 mL of 1% BSA / PBS, and incubate under the same conditions for 1 hour.

[0158] 3) Blocking positive magnetic beads

[0159] Add 1 mL of 1% BSA / PBS to the above-conjugated Trop2 magnetic beads and rotate at 20 rpm for 1 hour at room temperature.

[0160] 4) Negative screening

[0161] To remove antibodies that interact with the magnetic beads, negative panning is necessary. A BSA-blocked phage library and unconjugated magnetic beads are mixed and incubated under the conditions described above for 1 hour. After incubation, the phage-magnetic bead mixture is placed on a magnetic rack. Once the beads have adhered to the walls, the supernatant is transferred to a new EP tube.

[0162] 5) Positive screening

[0163] The blocked Trop2 protein-conjugated magnetic beads were added to the negatively screened phage supernatant for positive screening. The mixture was rotated at 20 rpm for 1 hour at room temperature. After incubation, the beads were washed with 1 mL of PBST (0.1% Tween-20 in PBS), repeating the wash 10 times. After washing, 1 mL of 100 mM glycine (pH 2.0) was added, and the mixture was placed on a rotary shaker at 20 rpm for 10 minutes to elute. After elution, the EP tube was placed on a magnetic rack, and once the beads adhered to the walls, the eluent was transferred to a new EP tube. 0.2 mL of 1 M Tris-HCl solution (pH 8.0) was added to the eluent for neutralization. The neutralized eluent was added to 30 mL of TG1 bacterial culture with an OD600 of approximately 0.6 and allowed to stand for 30 minutes. Then, 20 times the number of M13KO7 phages were added and allowed to stand for another 30 minutes. Finally, 100 mL of 2YT medium and ampicillin and kanamycin at a final concentration of 100 μg / mL were added, and the mixture was incubated overnight at 30°C and 220 rpm. The next day, phages were harvested using the same method described above for harvesting phage libraries. The resulting phage was Input2.

[0164] 6) Repeated positive screening

[0165] The above selection method was repeated twice, that is, Input2 was subjected to another round of negative and positive selection to obtain Input3. The difference is that after the eluent obtained from Input3 was infected with TG1, M13KO7 was not added. Instead, 10 μl of bacterial culture was serially diluted with PBS, and 10 μl of the solution was used. 3 10 4 10 5 Spread 100 μl of bacterial culture from each of the three dilution gradients onto 2YT / Amp plates and incubate overnight at 30°C. Incubate the remaining bacterial culture overnight at 30°C and 220 rpm.

[0166] 7) ELISA screening for positive antibodies

[0167] Randomly pick TG1 monoclonal antibodies from the above plates using a toothpick and transfer them to 800 μL of a deep-well plate containing 10× self-induced 2 YT / Amp. Cover the deep-well plate with a breathable membrane and incubate at 37°C and 220 rpm for 3 hours, then incubate overnight at 30°C and 220 rpm. Coat each well of the ELISA plate with 100 ng of recombinant human Trop2 protein. The next day, take 50 μL of the culture medium from the deep-well plate to preserve the bacteria, and centrifuge the remaining medium at 4000 rpm for 10 minutes to remove the culture medium and retain the bacterial pellet. Add 100 μL of TES solution (20% sucrose, 0.1 mM EDTA, 50 mM Tris-HCl, pH 8.0) to each well, shake to resuspend the bacteria, incubate on ice for 30 minutes, then add 200 μL of ultrapure water and shake to mix for 30 minutes. After shaking, centrifuge at 4000 rpm for 10 minutes. The supernatant in the deep-well plate at this point is the periplasmic extract containing the antibody. Wash the ELISA plate three times with a plate washer, then add 200 μL of 1% BSA / PBS and block at 37°C for 1 hour. Remove the blocking solution from the ELISA plate, add 100 μL of the above-mentioned periplasmic extract, incubate at 37°C for 1 hour, wash three times with a plate washer, add 100 μL of Chicken anti-HA HRP (1% BSA / PBS), incubate at 37°C for 1 hour, wash three times with a plate washer, add 100 μL of TMB chromogenic solution, develop at 37°C for 10 minutes, and add 100 μL of stop solution. Read the OD450 value using a microplate reader. Perform Sanger sequencing on clones with OD450 values ​​three times higher than the background value to obtain the antibody gene sequence.

[0168] 8) Verify positive clones

[0169] Based on the sequencing results, clones with significant amino acid sequence differences in antibody CDR3 were selected, re-inoculated, and induced overnight. The ELISA method described above was then used to verify again whether the selected clones could bind to Trop2. Ultimately, four relatively good antibody sequences were obtained: L2C8, L2C9, L3G5, and K2F5.

[0170] The amino acid sequence of the heavy chain variable region of L2C8 is shown in SEQ ID NO:25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:29.

[0171] The amino acid sequence of the heavy chain variable region of L2C9 is shown in SEQ ID NO:26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:30.

[0172] The amino acid sequence of the heavy chain variable region of L3G5 is shown in SEQ ID NO:27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:31.

[0173] The amino acid sequence of the heavy chain variable region of K2F5 is shown in SEQ ID NO:28, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:32.

[0174] Example 3: Preparation of retroviral stock solution containing anti-human Trop2 chimeric antigen receptor element

[0175] 1. Preparation of chimeric antigen receptors targeting human Trop2 antigen

[0176] Genetic synthesis or cloning of single-chain antibodies (scFv) containing anti-human Trop2 antigen, chimeric antigen receptor sequences with hinge region, transmembrane region, and intracellular signaling segment, the structure of which is as follows: Figure 2 As shown. Based on the different loading of scFv and intracellular signals, the chimeric antigen receptors were named L2C8-BBz, L2C9-BBz, L3G5-BBz, and K2F5-BBz, respectively, with amino acid sequences shown in SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41, and nucleotide sequences shown in SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45, respectively.

[0177] Using the reverse transcription vector MSGV as the backbone, chimeric antigen receptor retroviral plasmids expressing L2C8, L2C9, L3G5, and K2F5 clones were constructed. Clones with correct sequencing were selected, inoculated into 200 ml of 2YT medium, and cultured overnight. Plasmid extraction was then performed according to the NucleoBond Xtra Maxi EF kit instructions.

[0178] 2. Retroviral packaging

[0179] The retrovirus was packaged using the cationic polymer PEI as follows: 36 μl of PEI and the retrovirus packaging plasmid (2 μg of main viral plasmid, 3.8 μg of Gag-pol, and 1.5 μg of vsvg) were diluted separately with 600 μl of serum-free DMEM; then, PEI / DMEM was added to the plasmid / DMEM mixture, vortexed to mix, and incubated at room temperature for 15 minutes; the plasmid-PEI complex was then added to pre-coated 293T cells. The medium was changed 16 h after transfection. The first viral supernatant was collected after 48 h, and the second viral supernatant was collected after 72 h. The supernatant was filtered through a 0.45 μm filter, aliquoted into 1.5 mL centrifuge tubes (1 mL / tube), and stored at -80°C for later use.

[0180] Example 4: Preparation of Trop2 CAR-T cells and determination of CAR positivity rate

[0181] 1. PBMC Isolation and Activation

[0182] After receiving a PBMC and verifying the patient's individual identification code, resuscitation was initiated. The cell density was adjusted to 1×10⁶ cells / mL using X-VIVO complete culture medium. 6 / mL. Gently pipette the PBMCs that have recovered overnight, filter through a 70μm cell sieve, and transfer to a 50ml centrifuge tube. Centrifuge at 1500rpm for 5min at room temperature, discarding the supernatant. Resuspend the cells in an appropriate amount of DPBS, mix 20μl with trypan blue at a 1:1 ratio, count the cells, calculate the viability and CD3+ cell count, then take the required volume of cells, centrifuge at 1500rpm for 5min at room temperature, discarding the supernatant for sorting. Calculate the amount of magnetic beads used according to a 1:1 ratio of CD3 / CD28 magnetic beads (LIFE) to CD3+ cells: Magnetic bead amount = [CD3+ cell count / 4 × 10⁻⁶] 5 **μl.** Cleaning the magnetic beads: Take a sterile flow cytometry tube, add 2ml of DPBS and magnetic beads, and let it stand on a magnetic rack for 1 min. Discard the supernatant. Remove the flow cytometry tube from the magnetic rack, resuspend the cells in an equal volume of DPBS or X-VIVO15, add the magnetic beads and cells to the cell suspension, mix, and incubate on a rotary mixer. Incubate at room temperature for 30 min. After incubation, gently transfer the cells to a sterile flow cytometry tube, and rinse the 15ml centrifuge tube with 1ml of DPBS, adding the rinse solution to the same flow cytometry tube. Transfer the sterile flow cytometry tube to a magnetic rack, let it stand for 1 min, and discard any unabsorbed liquid. Remove the sterile flow cytometry tube from the magnetic rack, resuspend the cells in 1ml of CAR-T medium, and rinse the tube wall twice with CAR-T medium. Collect all the CAR-T medium and transfer it to the same centrifuge tube. Adjust the cell density to 1×10⁻⁶ cells using CAR-T medium. 6 Add IL-2 to a final concentration of 200 IU / ml and incubate at 37°C in a 5% CO2 incubator for two days.

[0183] 2. Virus stock solution infection and culture

[0184] The activated T cells were adjusted to 5 × 10 5 Add 1 ml of T cells and 1 ml of viral stock solution to each well of a 24-well plate, along with 2 μl of polybrene. Centrifuge at 32°C, 2500 rpm for 1.5 h. Discard the supernatant and add 1 ml of T cell culture medium (containing 300 IU / ml IL-2) to each well. Incubate the plate at 37°C in a 5% CO2 incubator. 24 h post-infection, transfer to 6-well plates. Observe cell density daily and supplement with T cell culture medium containing 300 IU / ml IL-2 as needed to maintain T cell density at 1 × 10⁶ cells / ml. 6 Approximately 1 / ml, to promote cell proliferation.

[0185] 3. CAR positivity rate detection

[0186] The CAR positivity rate of retrovirally infected T lymphocytes was measured 72 hours after viral infection. NT values ​​of 1×10⁻⁶ cells were collected from the chimeric antigen receptor group containing L2C8, L2C9, L3G5, and K2F5 clones and the negative uninfected control group. 6 Cells were centrifuged to remove the culture medium, washed once with 500 μl PBS, and resuspended in 100 μl of PBS in a flow cytometry tube (BD). Biotin-labeled Trop2 antigen (1:200) was added and incubated at °C for 30 minutes. After washing once with PBS, secondary antibody PE-SA streptavidin (BioLegend) was added at a 1:100 ratio and incubated at °C in the dark for 30 minutes. After washing with 500 μl PBS, cells were resuspended in 200 μl PBS and analyzed by flow cytometry. The CAR-T positivity rate results are shown below. Figure 3 As shown.

[0187] Example 5: Functional analysis of anti-human Trop2 CAR-T cells

[0188] 1. Analysis of CD107a expression in anti-human Trop2 CAR-T cells

[0189] CAR-T cells containing different antibody clones were mixed with target cells (Trop2-positive human pancreatic cancer cell line BxPC3) and control target cells (human glioma cells U251) at a 1:1 effector-target ratio (both effector cells and target cells were 3 × 10⁻⁶). 5 (A total of CAR-T cells) were mixed and incubated at 37°C in a 5% CO2 incubator for 4 hours. The proportion of CD107a in CAR-T cells was then detected by flow cytometry. This evaluated the degranulation response of CAR-T cells after stimulation by target cells. The flow cytometry results of CD107a expression are shown below. Figure 4 As shown.

[0190] 2. Detection of cytokine secretion capacity of anti-human Trop2 CAR-T cells

[0191] CAR-T cells containing different antibody clones were mixed with target cells (Trop2-positive cell line BxPC3) and control target cells (Trop2-negative cell line U251) at effector-target ratios of 10:1 and 2:1, respectively (target cells were 3 × 10⁻⁶ cells per cell line). 4 After incubation for 24 hours, the supernatant was collected, and the secretion of IFN-γ and IL-2 was detected using ELISA (enzyme-linked immunosorbent assay). IFN-γ and IL-2 were detected using the Erbitsin Human IFN-gamma ELISA Kit and Human IL-2 ELISA Kit, and the experimental procedures were performed according to the product instructions. The results of IFN-γ secretion detection are as follows: Figure 5, Figure 6 As shown in the figure. The results of IL-2 secretion detection are as follows. Figure 7 , Figure 8 As shown.

[0192] 3. Anti-human Trop2 CAR-T cell toxicity assay

[0193] The CAR-T cell cytotoxicity assay evaluates the in vitro function of CAR-T cells by detecting their cytotoxic effect on target cells. Different effector-to-target ratios (3 × 10⁻⁶) were used. 4 Using a baseline of 10 target cells (with effector-to-target ratios of 10:1 and 2:1), T cells were co-cultured with Trop2-positive target cells (Bxpc3-LUC-GFP) stably expressing firefly luciferase, and control target cells (U251-LUC-GFP). A positive control consisting only of target cells was also included. After overnight incubation at 37°C, 100 μl of luciferase reaction substrate was added to the culture system, and fluorescence values ​​were detected. The killing efficiency was calculated using the following formula: Killing efficiency = (Fluorescence value of positive control well - Fluorescence value of experimental well) / (Fluorescence value of positive control well) × 100%. Experimental groupings and analysis results are as follows: Figure 9 , Figure 10 As shown.

[0194] Example 6: Comparison of CAR-T cell cytotoxicity based on different anti-human Trop2 single-chain antibodies

[0195] During the research and development process, the applicant obtained multiple antibodies. In addition to the four antibodies L2C8, L2C9, L3G5, and K2F5 mentioned in this paper, other antibodies screened included L1A8, L2G1, K2G1, and L1F4. CAR plasmids were constructed according to the methods in Examples 3 and 4 of this paper, virus was coated onto them, and T cells were infected to obtain eight CAR-T cells based on different scFvs. These CAR-T cells differ only in the scFv sequence of the CAR. For example, the scFv amino acid sequence of L1F4-BBz is SEQ ID NO:48 (heavy chain CDR1, CDR2, and CDR3 are shown as SEQ ID NO:48, sections 26-33, 51-60, and 99-110aa, respectively; light chain CDR1, CDR2, and CDR3 are shown as SEQ ID NO:48, sections 178-185, 203-205, and 242-252aa, respectively).

[0196] Then, the Agilent xCELLigence RTCA SP killer sorter was used to perform in vitro killing experiments on BxPC3 cells at different effective-to-target ratios (3:1, 1:1 and 1:3).

[0197] (1) Based on the number of samples, first add 50 μl of the culture medium corresponding to the target cells to the 96-well resistance plate and then measure the baseline.

[0198] (2) Target cell plating: Take out the resistance plate and place it in a clean bench. First, add 100 μl (1×10⁻⁶) of the target cell plate. 4 (Number) BxpC3 target cells, let stand at room temperature for half an hour, allow the cells to settle naturally to the bottom of the plate, and then load them onto the machine.

[0199] (3) T cell plating: The next day, when the target cell cell index increased to approximately 1, the procedure was paused. The resistance plate was removed, and 100 μl of CAR-T cells were added at effector-to-target ratios of 3:1, 1:1, and 1:3, along with control empty T cells. The killing dynamics of each clone are shown in the figure below. Figure 11 , 12 As shown (green curves represent target cells in culture medium, red curves represent target cells in T cells, and the bottom curve represents target cells in CAR-T cells), the killing kinetics plot shows that L2C8, L2C9, L3G5, and K2F5 have significant inhibitory effects on target cells and kill them rapidly. L1A8, L2G1, K2G1, and L1F4 do not inhibit target cells as effectively as L2C8, L2C9, L3G5, and K2F5. The killing rates at 29 hours and 59 hours were calculated based on the killing kinetics plot. Figure 13 It can be seen that L2C8, L2C9, L3G5, and K2F5 have better lethality than other clones at different effective-to-target ratios.

[0200] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. sequence list <110> Shanghai Hengrunda Biotechnology Co., Ltd. <120> An antibody that specifically binds to Trop2 or its antigen-binding fragment, its preparation method, and its application. <130> / <160> 48 <170> SIPOSequenceListing 1.0 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <400> 1 Gly Phe Ser Val Arg Asn Asn Tyr 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial sequence <400> 2 Val Phe Pro Gly Gly Ser Ser 1 5 <210> 3 <211> 15 <212> PRT <213> Artificial sequence <400> 3 Ala Arg Asp Ser Gly Ser Pro Leu Trp Arg Gly His Phe Gln Tyr 1 5 10 15 <210> 4 <211> 8 <212> PRT <213> Artificial sequence <400> 4 Gly Phe Thr Phe Ser Asn Tyr Gly 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial sequence <400> 5 Ile Trp Ser Asp Gly Thr Tyr Thr 1 5 <210> 6 <211> 15 <212> PRT <213> Artificial sequence <400> 6 Ala Lys Ser Leu Thr Pro Leu Gly Gly Ser Phe Arg Ile Gly Asp 1 5 10 15 <210> 7 <211> 8 <212> PRT <213> Artificial sequence <400> 7 Gly Gly Ser Ile Ser Ser His Tyr 1 5 <210> 8 <211> 7 <212> PRT <213> Artificial sequence <400> 8 Ile His Ser Ser Gly Ile Thr 1 5 <210> 9 <211> 13 <212> PRT <213> Artificial sequence <400> 9 Ala Arg Gly Leu Arg Leu Thr Asp Asp Ala Phe Asp Ile 1 5 10 <210> 10 <211> 8 <212> PRT <213> Artificial sequence <400> 10 Gly Asp Asn Phe Ser Thr Asn Trp 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial sequence <400> 11 Ile Tyr Pro Gly Asp Ser Asp Ser 1 5 <210> 12 <211> 12 <212> PRT <213> Artificial sequence <400> 12 Ala Arg Tyr Gln Gly Ser Thr Thr Pro Phe Asp Tyr 1 5 10 <210> 13 <211> 9 <212> PRT <213> Artificial sequence <400> 13 Ser Ala Asn Ile Gly Ala Gly Tyr Asp 1 5 <210> 14 <211> 3 <212> PRT <213> Artificial sequence <400> 14 Asp Asn Asn 1 <210> 15 <211> 11 <212> PRT <213> Artificial sequence <400> 15 Gly Thr Trp Asp Thr Ser Leu Ser Ala Trp Val 1 5 10 <210> 16 <211> 9 <212> PRT <213> Artificial sequence <400> 16 Ser Ser Asn Ile Gly Ala Gly Tyr Asp 1 5 <210> 17 <211> 3 <212> PRT <213> Artificial sequence <400> 17 Asp Asn Thr 1 <210> 18 <211> 11 <212> PRT <213> Artificial sequence <400> 18 Gln Ser Tyr Asp Ser Arg Leu Asn Ala Leu Val 1 5 10 <210> 19 <211> 9 <212> PRT <213> Artificial sequence <400> 19 Ser Ser Asp Val Gly Gly Tyr Asn Tyr 1 5 <210> 20 <211> 3 <212> PRT <213> Artificial sequence <400> 20 Asp Val Ser 1 <210> twenty one <211> 10 <212> PRT <213> Artificial sequence <400> twenty one Ser Ser Tyr Thr Ser Ser Asn Thr Leu Ala 1 5 10 <210> twenty two <211> 6 <212> PRT <213> Artificial sequence <400> twenty two Gln Ser Ile Asn Ser Asn 1 5 <210> 23 <211> 3 <212> PRT <213> Artificial sequence <400> 23 Ala Ala Ser 1 <210> 24 <211> 9 <212> PRT <213> Artificial sequence <400> 24 Gln Gln Ser Tyr Ser Thr Pro Leu Thr 1 5 <210> 25 <211> 121 <212> PRT <213> Artificial sequence <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Gln Arg Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Ser Val Arg Asn Asn 20 25 30 Ala Ile Val Phe Pro Gly Gly Ser Ser Tyr His Ala Asp Ser Val Lys 35 40 45 Ala Ile Val Phe Pro Gly Gly Ser Ser Tyr His Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Leu Ser Lys Asn Ser Val Tyr Leu 65 70 75 80 Glu Met Asn Ser Leu Arg Glu Asp Asp Thr Gly Val Tyr Phe Cys Ala 85 90 95 Arg Asp Ser Gly Ser Pro Leu Trp Arg Gly His Phe Gln Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 26 <211> 122 <212> PRT <213> Artifical sequence <400> 26 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Met 1 5 10 15 Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Ile Ile Trp Ser Asp Gly Thr Tyr Thr Tyr Tyr Gly Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Thr Leu His 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Leu Thr Pro Leu Gly Gly Ser Phe Arg Ile Gly Asp Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 27 <211> 119 <212> PRT <213> Artificial sequence <400> 27 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Ile Cys Thr Val Ser Gly Gly Ser Ile Ser Ser His 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile His Ser Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Met 50 55 60 Gly Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Pro Leu 65 70 75 80 Lys Val Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Leu Arg Leu Thr Asp Asp Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115 <210> 28 <211> 119 <212> PRT <213> Artificial sequence <400> 28 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Thr Ile Ser Cys Lys Ser Ser Gly Asp Asn Phe Ser Thr Asn 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Ser Arg Tyr Ser Pro Ser Phe 50 55 60 Glu Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gln Gly Ser Thr Thr Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 29 <211> 111 <212> PRT <213> Artificial sequence <400> 29 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ala Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe 50 55 60 Ser Gly Ser Asn Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu 65 70 75 80 Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Thr Ser 85 90 95 Leu Ser Ala Trp Val Phe Gly Gly Gly Thr Arg Leu Thr Val Leu 100 105 110 <210> 30 <211> 111 <212> PRT <213> Artificial sequence <400> 30 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Asp Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Arg 85 90 95 Leu Asn Ala Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 31 <211> 110 <212> PRT <213> Artificial sequence <400> 31 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile His Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Phe Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Asn Thr Leu Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 32 <211> 107 <212> PRT <213> Artificial sequence <400> 32 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 33 <211> 21 <212> PRT <213> Artificial sequence <400> 33 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> 34 <211> 45 <212> PRT <213> Artificial sequence <400> 34 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 35 40 45 <210> 35 <211> 24 <212> PRT <213> Artificial sequence <400> 35 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 36 <211> 48 <212> PRT <213> Artifical sequence <400> 36 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 1 5 10 15 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 20 25 30 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg 35 40 45 <210> 37 <211> 111 <212> PRT <213> Artifical sequence <400> 37 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 1 5 10 15 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 20 25 30 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 35 40 45 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 50 55 60 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 65 70 75 80 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 85 90 95 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 38 <211> 512 <212> PRT <213> Artifical sequence <400> 38 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 Glu Val Gln Leu Val Glu Ser Gly Gly Ala Leu 20 25 30 Val Gln Arg Gly Gly Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe 35 40 45 Ser Val Arg Asn Asn Tyr Ile Thr Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Ala Ile Val Phe Pro Gly Gly Ser Ser Tyr His 65 70 75 80 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Leu Ser Lys 85 90 95 Asn Ser Val Tyr Leu Glu Met Asn Ser Leu Arg Glu Asp Asp Thr Gly 100 105 110 Val Tyr Phe Cys Ala Arg Asp Ser Gly Ser Pro Leu Trp Arg Gly His 115 120 125 Phe Gln Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gln 130 135 140 Ala Gly Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 145 150 155 160 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ala Ser Ala Gln Ser Val 165 170 175 Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln Arg Val Thr 180 185 190 Ile Ser Cys Thr Gly Ser Ser Ala Asn Ile Gly Ala Gly Tyr Asp Val 195 200 205 His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr 210 215 220 Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 225 230 235 240 Asn Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln Thr Gly 245 250 255 Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Thr Ser Leu Ser Ala 260 265 270 Trp Val Phe Gly Gly Gly Thr Arg Leu Thr Val Leu Thr Thr Thr Pro 275 280 285 Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 290 295 300 Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 305 310 315 320 Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu 325 330 335 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 340 345 350 Cys Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 355 360 365 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 370 375 380 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 385 390 395 400 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 405 410 415 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 420 425 430 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 435 440 445 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 450 455 460 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 465 470 475 480 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 485 490 495 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505 510 <210> 39 <211> 513 <212> PRT <213> Artificial sequence <400> 39 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 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val 20 25 30 Val Gln Pro Gly Met Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Ser Asn Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Gly Ile Ile Trp Ser Asp Gly Thr Tyr Thr Tyr 65 70 75 80 Tyr Gly Asp Ser Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95 Glu Asn Thr Leu His Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Lys Ser Leu Thr Pro Leu Gly Gly Ser Phe 115 120 125 Arg Ile Gly Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 130 135 140 Gln Ala Gly Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ala Ser Ala Gln Ser 165 170 175 Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln Arg Val 180 185 190 Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp 195 200 205 Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile 210 215 220 Tyr Asp Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly 225 230 235 240 Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln Ala 245 250 255 Asp Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Arg Leu Asn 260 265 270 Ala Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Thr Thr Thr 275 280 285 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 290 295 300 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 305 310 315 320 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 325 330 335 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 340 345 350 Tyr Cys Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr 355 360 365 Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu 370 375 380 Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu 385 390 395 400 Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 405 410 415 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 420 425 430 Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 435 440 445 Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 450 455 460 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 465 470 475 480 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 485 490 495 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 500 505 510 Arg <210> 40 <211> 509 <212> PRT <213> Artificial Sequence <400> 40 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 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu 20 25 30 Leu Lys Pro Ser Glu Thr Leu Ser Leu Ile Cys Thr Val Ser Gly Gly 35 40 45 Ser Ile Ser Ser His Tyr Trp Ser Trp Ile Arg Gln Thr Pro Gly Lys 50 55 60 Gly Leu Glu Trp Ile Gly Trp Ile His Ser Ser Gly Ile Thr Asn Tyr 65 70 75 80 Asn Pro Ser Leu Met Gly Arg Val Thr Met Ser Val Asp Thr Ser Lys 85 90 95 Asn Gln Phe Pro Leu Lys Val Asn Ser Val Thr Ala Ala Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Gly Leu Arg Leu Thr Asp Asp Ala Phe Asp 115 120 125 Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gln Ala Gly 130 135 140 Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Gly Gly Gly Gly Ser Gly Gly Gly Ala Ser Ala Gln Ser Ala Leu Thr 165 170 175 Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser 180 185 190 Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser Trp 195 200 205 Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile His Asp Val 210 215 220 Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Phe Gly Ser Lys Ser 225 230 235 240 Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu 245 250 255 Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Asn Thr Leu Ala Phe 260 265 270 Gly Gly Gly Thr Lys Leu Thr Val Leu Thr Thr Thr Pro Ala Pro Arg 275 280 285 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 290 295 300 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 305 310 315 320 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 325 330 335 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Phe 340 345 350 Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 355 360 365 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 370 375 380 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 385 390 395 400 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 405 410 415 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 420 425 430 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 435 440 445 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 450 455 460 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 465 470 475 480 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 485 490 495 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505 <210> 41 <211> 506 <212> PRT <213> Artificial sequence <400> 41 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 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30 Lys Lys Pro Gly Glu Ser Leu Thr Ile Ser Cys Lys Ser Ser Gly Asp 35 40 45 Asn Phe Ser Thr Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys 50 55 60 Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Ser Arg 65 70 75 80 Tyr Ser Pro Ser Phe Glu Gly Gln Val Thr Ile Ser Ala Asp Lys Ser 85 90 95 Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr 100 105 110 Ala Met Tyr Tyr Cys Ala Arg Tyr Gln Gly Ser Thr Thr Pro Phe Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gln Ala Gly 130 135 140 Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Gly Gly Gly Gly Ser Gly Gly Gly Ala Ser Ala Asp Ile Gln Met Thr 165 170 175 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 180 185 190 Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser Asn Leu Asn Trp Tyr Gln 195 200 205 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser 210 215 220 Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 225 230 235 240 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 245 250 255 Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly 260 265 270 Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr 275 280 285 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 290 295 300 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 305 310 315 320 Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val 325 330 335 Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Phe Ser Val Val 340 345 350 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 355 360 365 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 370 375 380 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 385 390 395 400 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 405 410 415 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 420 425 430 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 435 440 445 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 450 455 460 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 465 470 475 480 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 485 490 495 Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505 <210> 42 <211> 1536 <212> DNA <213> Artificial sequence <400> 42 atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60 cctgaagtgc agctggtgga gtctggcgga gccttggtcc agcggggggg gtccctgaga 120 ctctcctgtt cagcctctgg attcagcgtc aggaacaact acataacgtg ggtccgccag 180 gctccaggga agggactgga gtgggtcgca atcgtttttc ccggtggtag ttcataccac 240 gcagattctg tgaagggccg attcaccatc tccagagacc tttctaagaa ttcggtgtat 300 cttgaaatga acagcctgcg agaggacgac acgggtgtat atttctgtgc gcgcgatagt 360 gggagtcccc tttggcgagg ccactttcaa tactggggcc aggggaccac ggtcaccgtc 420 tcctcaggcc aggccggccc gggaggtggt ggctctggag gtggtggctc tggaggtggc 480 ggttctggag gtggcggttc tggaggtggc gctagcgctc agtctgtgtt gacgcagccg 540 ccctcagtgt ctggggcccc agggcagagg gtcaccatct cctgcactgg gagcagcgcc 600 aacatcgggg caggttatga tgtacactgg taccagcagc ttccaggaac agcccccaaa 660 ctcctcattt atgacaataa taagcgaccc tcagggattc ctgaccgatt ctctggctcc 720 aactctggca cgtcggccac cctgggcatc accggactcc agactggaga cgaggccgat 780 tattactgcg gaacatggga taccagcctg agtgcttggg tgttcggcgg agggaccagg 840 ttgaccgtcc taactacaac tccagcaccc agacccccta cacctgctcc aactatcgca 900 agtcagcccc tgtcactgcg ccctgaagcc tgtcgccctg ctgccggggg agctgtgcat 960 actcggggac tggactttgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 1020 ggggtccttc tcctgtcact ggttatcacc ctttactgca ggttcagtgt cgtgaagaga 1080 ggccggaaga agctgctgta catcttcaag cagcctttca tgaggcccgt gcagactacc 1140 caggaggaag atggatgcag ctgtagattc cctgaagagg aggaaggagg ctgtgagctg 1200 agagtgaagt tctcccgaag cgcagatgcc ccagcctatc agcagggaca gaatcagctg 1260 tacaacgagc tgaacctggg aagacgggag gaatacgatg tgctggacaa aaggcggggc 1320 agagatcctg agatgggcgg caaaccaaga cggaagaacc cccaggaagg tctgtataat 1380 gagctgcaga aagacaagat ggctgaggcc tactcagaaa tcgggatgaa gggcgaaaga 1440 aggagagga aaggccacga cggactgtac caggggctga gtacagcaac aaaagacacc tatgacgctc tgcacatgca ggctctgcca from <210> 43 <211> 1539 <212> DNA <213> Artificial sequence (Artificial sequence) <400> 43 atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg cctgaggtgc agctgttgga gtctggggga ggcgtggtcc agcctgggat gtccctgaga 120 gtctcctgtg cagcgtctgg attcaccttc agtaactatg gcatgcactg ggtccgccag 180 gctccaggca agggactgga gtgggtgggga attatatggt ctgatggaac ttatacatat 240 300. ccggagact ccgtgcaggg ccgattcacc atctccagag acaactccga gaacaccctg catctgcaaa tgaatagcct gagagccgag gacacggctg tgtattactg tgcgaaatcc ctgacccccc ttggggggag cttccgtatt ggcgactggg gccagggac cctggtcacc 420 gtctcctcag gccaggccgg cccggggaggt ggtggctctg gaggtggtgg ctctggaggt 480 ggcggttctg gaggtggcgg ttctggaggt ggcgctagcg ctcagtctgt gttgacgcag 540 ccgccctcag tgtctggggc cccagggcag agggtcacca tctcctgcac tgggagcagc 600 tccaacatcg gggcaggtta tgatgtacac tggtaccagc agcttccagg gacagccccc 660 aaactcctca tctatgataa caccaatcgg ccctcagggg tccctgaccg attctctggc 720 tccaagtctg gcacctcagc ctccctggcc atcactgggc tccaggctga cgatgagggt 780 gattattact gccagtccta cgacagcaga ctgaatgctt tggtattcgg cggagggacc 840 aagctgaccg tcctaactac aactccagca cccagacccc ctacacctgc tccaactatc gcaagtcagc ccctgtcact gcgccctgaa gcctgtcgcc ctgctgccgg gggagctgtg 960 catactcggg gactggactt tgcctgtgat atctacatct gggcgccctt ggccgggact 1020 tgtggggtcc ttctcctgtc actggttatc accctttact gcaggttcag tgtcgtgaag 1080 agggccgga agaagctgct gtacatcttc aagcagcctt tcatgaggcc cgtgcagact acccaggagg aagatggatg cagctgtaga ttccctgag aggaggagg aggctgtgag ctgagagtga agttctcccg aagcgcagat gccccagcct atcagcaggg acagaatcag ctgtacaacg agctgaacct gggaagacgg gaggaatacg atgtgctgga caaaaggcgg ggcagagatc ctgagatggg cggcaaacca agacggaga acccccagga aggtctgtat aatgagctgc agaaagacaa gatggctgag gcctactcag aatcgggat gaagggcgaa agaaggagag gaaaaggcca cgacggactg taccaggggc tgagtacagc aacaaaagac acctatgacg ctctgcacat gcaggctctg ccaccaga 1539 <210> 44 <211> 1527 <212> DNA <213> Artificial sequence (Artificial sequence) <400> 44 atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg cctcagctgc agctgcagga gtcgggccca ggactgctga agccttcgga gaccctgtcc 120 ctcatttgca ctgtctctgg tggctccatc agtagtcact actggagttg gatccggcag accccaggga agggactgga gtggattggg tggatccatt ccagtggggat caccaactac aacccctccc tcatgggtcg agtcaccatg tcagtggaca cgtccaagaa ccagttcccc 300. ctgaaggtga actctgtgac cgctgcggac acggccgtct attactgtgc gagaggtctg agactaactg atgatgcttt tgatatctgg ggccaaggga caatggtcac cgtctcctca 420 ggccaggccg gcccgggagg tggtggctct ggaggtggtg gctctggagg tggcggttct 480 ggaggtggcg gttctggagg tggcgctagc gctcagtctg ccctgactca gcctgcctcc 540 gtgtctgggt ctcctggaca gtcgatcacc atctcctgca ctggaaccag cagtgacgtt 600 ggtggttata actatgtctc ctggtaccaa caacacccag gcaaagcccc caaactcatg 660 attcatgatg tcagtaatcg gccctcaggg gtttctaatc gcttctttgg ctccaagtct 720 ggcaacacgg cctccctgac catctctggg ctccaggctg aggacgaggc tgattattac 780 tgcagctcat atacaagcag caacactctc gcgttcggcg gagggaccaa gctgaccgtc 840 ctaactacaa ctccagcacc cagaccccct acacctgctc caactatcgc aagtcagccc 900 ctgtcactgc gccctgaagc ctgtcgccct gctgccgggg gagctgtgca tactcgggga 960 ctggactttg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg tggggtcctt 1020 ctcctgtcac tggttatcac cctttactgc aggttcagtg tcgtgaagag aggccggaag 1080 aagctgctgt acatcttcaa gcagcctttc atgaggcccg tgcagactac ccaggaggaa 1140 gatggatgca gctgtagatt ccctgaagag gaggaaggag gctgtgagct gagagtgaag 1200 ttctcccgaa gcgcagatgc cccagcctat cagcagggac agaatcagct gtacaacgag 1260 ctgaacctgg gaagacggga ggaatacgat gtgctggaca aaaggcgggg cagagatcct 1320 gagatgggcg gcaaaccaag acggaagaac ccccaggaag gtctgtataa tgagctgcag 1380 aaagacaaga tggctgaggc ctactcagaa atcgggatga agggcgaaag aaggagagga 1440 aaaggccacg acggactgta ccaggggctg agtacagcaa caaaagacac ctatgacgct 1500 ctgcacatgc aggctctgcc accaaga 1527 <210> 45 <211> 1518 <212> DNA <213> Artifical sequence <400> 45 atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60 cctcaggtcc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgacg 120 atctcctgta agagttctgg agacaacttt agcacgaact ggatcggctg ggtgcgccag 180 atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgatagcaga 240 tacagtccgt ccttcgaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 300 tacctgcagt ggagcagcct gaaggcctcg gacaccgcca tgtattactg tgcgagatat 360 caagggtcta ccaccccctt tgactactgg ggccagggaa ccacggtcac cgtctcctca 420 ggccaggccg gcccgggagg tggtggctct ggaggtggtg gctctggagg tggcggttct 480 ggaggtggcg gttctggagg tggcgctagc gctgacatcc agatgaccca gtctccatcc 540 tccctgtctg catctgtagg agacagagtc accatcactt gccgggcaag tcagagcatt 600 aacagtaatt taaattggta tcagcagaaa ccagggaaag cccctaagct cctgatctat 660 gctgcatcca gtttgcaaag tggggtccca tcaaggttca gtggcagtgg atctgggaca 720 gatttcactc tcaccatcag cagtctgcaa cctgaagatt ttgcaactta ctactgtcaa 780 cagagttaca gtaccccgct cactttcggc ggagggacca aggtggagat caaaactaca 840 actccagcac ccagaccccc tacacctgct ccaactatcg caagtcagcc cctgtcactg 900 cgccctgaag cctgtcgccc tgctgccggg ggagctgtgc atactcgggg actggacttt 960 gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 1020 ctggttatca ccctttactg caggttcagt gtcgtgaaga gaggccggaa gaagctgctg 1080 tacatcttca agcagccttt catgaggccc gtgcagacta cccaggagga agatggatgc 1140 agctgtagat tccctgaaga ggaggaagga ggctgtgagc tgagagtgaa gttctcccga 1200 agcgcagatg ccccagccta tcagcaggga cagaatcagc tgtacaacga gctgaacctg 1260 ggaagacggg aggaatacga tgtgctggac aaaaggcggg gcagagatcc tgagatgggc 1320 ggcaaaccaa gacggaagaa cccccaggaa ggtctgtata atgagctgca gaaagacaag 1380 atggctgagg cctactcaga aatcgggatg aagggcgaaa gaaggagagg aaaaggccac 1440 gacggactgt accaggggct gagtacagca acaaaagaca cctatgacgc tctgcacatg 1500 caggctctgc caccaaga 1518 <210> 46 <211> 271 <212> PRT <213> Artificial sequence <400> 46 His Thr Ala Ala Gln Asp Asn Cys Thr Cys Pro Thr Asn Lys Met Thr 1 5 10 15 Val Cys Ser Pro Asp Gly Pro Gly Gly Arg Cys Gln Cys Arg Ala Leu 20 25 30 Gly Ser Gly Met Ala Val Asp Cys Ser Thr Leu Thr Ser Lys Cys Leu 35 40 45 Leu Leu Lys Ala Arg Met Ser Ala Pro Lys Asn Ala Arg Thr Leu Val 50 55 60 Arg Pro Ser Glu His Ala Leu Val Asp Asn Asp Gly Leu Tyr Asp Pro 65 70 75 80 Asp Cys Asp Pro Glu Gly Arg Phe Lys Ala Arg Gln Cys Asn Gln Thr 85 90 95 Ser Val Cys Trp Cys Val Asn Ser Val Gly Val Arg Arg Thr Asp Lys 100 105 110 Gly Asp Leu Ser Leu Arg Cys Asp Glu Leu Val Arg Thr His His Ile 115 120 125 Leu Ile Asp Leu Arg His Arg Pro Thr Ala Gly Ala Phe Asn His Ser 130 135 140 Asp Leu Asp Ala Glu Leu Arg Arg Leu Phe Arg Glu Arg Tyr Arg Leu 145 150 155 160 His Pro Lys Phe Val Ala Ala Val His Tyr Glu Gln Pro Thr Ile Gln 165 170 175 Ile Glu Leu Arg Gln Asn Thr Ser Gln Lys Ala Ala Gly Asp Val Asp 180 185 190 Ile Gly Asp Ala Ala Tyr Tyr Phe Glu Arg Asp Ile Lys Gly Glu Ser 195 200 205 Leu Phe Gln Gly Arg Gly Gly Leu Asp Leu Arg Val Arg Gly Glu Pro 210 215 220 Leu Gln Val Glu Arg Thr Leu Ile Tyr Tyr Leu Asp Glu Ile Pro Pro 225 230 235 240 Lys Phe Ser Met Lys Arg Leu Thr Ala Ser Gly Leu Asn Asp Ile Phe 245 250 255 Glu Ala Gln Lys Ile Glu Trp His Glu His His His His His His 260 265 270 <210> 47 <211> 813 <212> DNA <213> Artificial sequence <400> 47 cacaccgccg cccaggacaa ctgcacctgc cccaccaaca agatgaccgt gtgcagcccc 60 gacggccccg gcggccgctg ccagtgccgc gccctgggca gcggcatggc cgtggactgc 120 agcaccctga ccagcaagtg cctgctgctg aaggcccgca tgagcgcccc caagaacgcc 180 cgcaccctgg tgcgccccag cgagcacgcc ctggtggaca acgacggcct gtacgacccc 240 gactgcgacc ccgagggccg cttcaaggcc cgccagtgca accagaccag cgtgtgctgg 300 tgcgtgaaca gcgtgggcgt gcgccgcacc gacaagggcg acctgagcct gcgctgcgac 360 gagctggtgc gcacccacca catcctgatc gacctgcgcc accgccccac cgccggcgcc 420 ttcaaccaca gcgacctgga cgccgagctg cgccgcctgt tccgcgagcg ctaccgcctg 480 caccccaagt tcgtggccgc cgtgcactac gagcagccca ccatccagat cgagctgcgc 540 cagaacacca gccagaaggc cgccggcgac gtggacatcg gcgacgccgc ctactacttc 600 gagcgcgaca tcaagggcga gagcctgttc cagggccgcg gcggcctgga cctgcgcgtg 660 cgcggcgagc ccctgcaggt ggagcgcacc ctgatctact acctggacga gatccccccc 720 aagttcagca tgaagcgcct gaccgctagc ggtctgaacg acatcttcga ggctcagaaa 780 atcgaatggc acgaacatca tcaccatcac cat 813 <210> 48 <211> 265 <212> PRT <213> Artificial sequence <400> 48 Gln Met Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Asp Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr Thr Glu Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg Gly Gly Trp Gly Ser Asn Trp Phe Asp Pro Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gln Ala Gly Pro Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Ala Ser Ala Gln Ser Val Val Thr Gln Pro Pro 145 150 155 160 Ser Leu Ser Ala Ala Pro Gly Gln Ala Val Thr Ile Ser Cys Ser Gly 165 170 175 Ser Asn Ser Asn Ile Gly Gly Asn Tyr Val Ser Trp Tyr Leu His Leu 180 185 190 Pro Gly Thr Ala Pro Lys Leu Leu Ile His Asp Asp Asn Glu Arg Pro 195 200 205 Ser Gly Ile Pro Asp Arg Phe Ser Ala Ser Lys Ser Gly Thr Ser Ala 210 215 220 Thr Leu Gly Ile Thr Gly Leu Gln Thr Gly Asp Glu Ala Asp Tyr Phe 225 230 235 240 Cys Gly Ala Trp Asp Arg Thr Leu Ser Ala Trp Val Phe Gly Gly Gly 245 250 255 Thr Lys Gly Asp Arg Pro Ser Gln Ala 260 265

Claims

1. An anti-Trop2 antibody or its antigen-binding fragment, characterized in that, The anti-Trop2 antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region; The heavy chain variable region includes HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3; The light chain variable region includes LCDR1 with the sequence shown in SEQ ID NO:13, LCDR2 with the sequence shown in SEQ ID NO:14, and LCDR3 with the sequence shown in SEQ ID NO:15; Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:29; The antibody is a monoclonal antibody; The antigen-binding fragment is Fab, F(ab'), F(ab')2, single-chain antibody scFv, or Fv linked by a disulfide bond.

2. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor consists of a signal peptide, an antigen-binding domain, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signal domain, from the N-terminus to the C-terminus. The antigen-binding domain is the antigen-binding fragment of the anti-Trop2 antibody according to claim 1, and the antigen-binding fragment is a single-chain antibody scFv.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-Trop2 antibody of claim 1 or its antigen-binding fragment, or encodes the chimeric antigen receptor of claim 2.

4. A nucleic acid construct, characterized in that, The nucleic acid construct comprises the nucleic acid molecule of claim 3; The nucleic acid construct is a cloning vector, expression vector, or integration vector.

5. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 3 or the nucleic acid construct of claim 4, and expresses the anti-Trop2 antibody of claim 1 or its antigen-binding fragment, or expresses the chimeric antigen receptor of claim 2.

6. A method for generating the anti-Trop2 antibody of claim 1 or its antigen-binding fragment, characterized in that, include: The host cells of claim 5 are cultured under conditions suitable for producing the anti-Trop2 antibody or its antigen-binding fragment, and the anti-Trop2 antibody or its antigen-binding fragment is purified from the culture.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the host cell as described in claim 5 and pharmaceutically acceptable excipients.

8. A kit for detecting Trop2, characterized in that, The kit comprises the anti-Trop2 antibody or its antigen-binding fragment as described in claim 1, and reagents for detecting the binding of Trop2 to the anti-Trop2 antibody or its antigen-binding fragment.

9. Use of the anti-Trop2 antibody of claim 1 or its antigen-binding fragment in the preparation of a kit for detecting Trop2 in a sample.

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