WO2023273595A1

WO2023273595A1 - Antibody binding to trop2, bispecific antibody targeting trop2 and cd3, preparation methods therefor and uses thereof

Info

Publication number: WO2023273595A1
Application number: PCT/CN2022/090939
Authority: WO
Inventors: 袁清安; 白丽莉; 孟庆武; 李艺嘉; 赵立坤; 李延虎
Original assignee: 益科思特(北京)医药科技发展有限公司
Priority date: 2021-06-30
Filing date: 2022-05-05
Publication date: 2023-01-05
Also published as: CN115536747A

Abstract

The present invention relates to the technical field of genetic engineering antibodies, and specifically to an antibody binding to TROP2, a bispecific antibody targeting TROP2 and CD3, preparation methods therefor and the uses thereof. The antibody binding to TROP2 provided in the present invention has a good capacity for binding to TROP2 and a high affinity. The bispecific antibody binding to TROP2 and CD3 simultaneously has the excellent biological functions of an anti-TROP2 antibody and an anti-CD3 antibody, can build a bridge between tumor cells and immune effector cells, effectively activates the immune effector cells and a directed immune response, significantly enhances the effect of immune cells in killing tumor cells in addition to reducing the ADCC effect to the largest extent, is relatively safe, and has good application prospects in tumor immunotherapy.

Description

Antibody binding to TROP2, bispecific antibody targeting TROP2 and CD3, preparation method and application thereof

cross reference

This application claims the priority of the Chinese patent application No. 202110736045.5 filed on June 30, 2021, entitled "An antibody that binds TROP2 and a bispecific antibody targeting TROP2 and CD3, and its preparation method and application", The entire disclosure content thereof is incorporated herein by reference in its entirety.

technical field

The invention relates to the technical field of genetic engineering antibodies, in particular to an antibody binding to TROP2, a bispecific antibody targeting TROP2 and CD3, and a preparation method and application thereof.

Background technique

Antibody drugs are biomacromolecular drugs prepared by antibody engineering technology based on cell engineering technology and genetic engineering technology. They have the advantages of high specificity, uniform properties, and directional preparation for specific targets. Monoclonal antibodies are mainly used clinically in the following three aspects: tumor treatment, immune disease treatment and anti-infection treatment. Among them, tumor treatment is currently the most widely used field of monoclonal antibodies. Among the monoclonal antibody products that have entered clinical trials and are on the market, the number of products used for tumor treatment accounts for about 50%. Monoclonal antibody treatment of tumors is an immunotherapy that stimulates the immune system to kill target cells against specific targets of diseased cells. In order to enhance the effector function of antibodies, especially to improve the effect of killing tumor cells, people have tried various methods to modify antibody molecules.

There are several ways to obtain specific antibodies. The traditional hybridoma technology immunizes mice or rats, fuses the spleen cells with myeloma cells, then dilutes them (to one cell per well) and cultures them, and uses enzyme-linked immunosorbent assay (ELISA) to detect the difference between the supernatant of hybridoma cells and Immunizing the combination of antigens, and then screening monoclonal cell lines that can secrete antibodies that specifically bind to antigens. If this mouse-derived antibody is used in clinical drug development, it needs to be deimmunized by genetic engineering methods such as hybrid antibody technology (chimerization) and humanization technology (humanization). In order to eliminate the immunogenicity problem that must be solved in the future clinical practice from the initial stage, the transgenic mouse technology (transgenic mouse) replaces the variable region of the mouse antibody with the variable region of the human antibody, so that the variable region sequence of the monoclonal antibody obtained The main body is human, and then the constant region can be further replaced to obtain a fully human monoclonal antibody. Another technology is the phage display technology. Phage surface display technology is to construct engineered antibodies in test tubes, usually in the form of single-chain antibodies or Fabs. However, the gene fragments that make up engineered antibodies are derived from the gene pool of human immune cells, so the antibodies obtained are fully human. Antibodies obtained by multiple phage technologies have entered clinical research or been approved for the treatment of various diseases.

On the basis of monoclonal antibodies, in order to further improve drug efficacy and reduce side effects, new antibody drugs based on monoclonal antibodies include drug-conjugated antibodies (Antibody drug conjugate, ADC), bispecific antibodies (bispecific antibodies, The development of new protein and cell drugs such as bsAb) and CAR-T (chimeric antigen receptor T cells) is extremely rapid. Among them, the cell-bridging double antibody based on bispecificity has significantly improved the killing activity and specificity due to its ability to connect immune T cells and cancer target cells, and has a breakthrough clinical value. Much attention.

Bispecific antibodies can be obtained in a variety of ways, and their preparation methods mainly include: chemical coupling method, hybrid-hybridoma method and genetic engineering antibody preparation method. The chemical coupling method is to link two different monoclonal antibodies together by chemical coupling to prepare a bispecific monoclonal antibody, which is the earliest bispecific monoclonal antibody. The hybrid-hybridoma method is to produce bispecific monoclonal antibodies by means of cell hybridization or ternary hybridomas. These cell hybridomas or ternary hybridomas are fused by established hybridomas, or established hybridomas and hybridomas derived from mice The fusion of lymphocytes can only be used to produce murine bispecific antibodies, so its application is greatly limited. With the rapid development of molecular biology technology, various construction modes of genetically engineered humanized or fully human bispecific antibodies have emerged, mainly including bispecific microbodies, diabodies, and single-chain diabodies. , multivalent bispecific antibody four categories. At present, several genetically engineered bispecific antibody drugs have entered the clinical trial stage in the world, and have shown good application prospects.

Trophoblastic cell surface antigen 2 (TROP2) is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene, also known as tumor-associated calcium ion signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), gastrointestinal tumor-related Antigen (GA733-1), surface marker 1 (M1S1), the first cell surface glycoprotein identified in human placental trophoblast, was later found to be highly expressed in most human solid tumor cancer cells, and only limited or Limited expression in normal human tissues. TROP2 belongs to the GA733 protein family, and has a high structural sequence similarity with epithelial cell adhesion molecule (EpCAM, also known as TROP1, TACSTD1), with a homology of 49%. TROP2 consists of 323 amino acids, including 26 amino acids in the signal peptide, 248 amino acids in the extracellular region, 23 amino acids in the transmembrane region, and 26 amino acids in the cytoplasmic region. The schematic diagram of its structure is shown in Figure 1A. TROP2 mainly promotes the growth, proliferation and metastasis of tumor cells by regulating calcium ion signaling pathways, cell cycle protein expression and reducing fibronectin adhesion. TROP2 can also interact with β-catenin in the Wnt signaling cascade, thus acting on the transcription of nuclear oncogenes and cell proliferation. A large number of clinical studies and literature reports have shown that TROP2 is overexpressed in epithelial cancers such as breast cancer, pancreatic cancer, gallbladder cancer, colon cancer, gastric cancer, non-small cell lung cancer, prostate cancer, uterine cancer and oral squamous cell carcinoma, while it is normal in adults. There is little or no expression in tissues; overexpression of TROP2 in tumor tissues is closely related to poor prognosis of patients and metastasis of cancer cells, and affects the overall survival rate of patients at the same time. Therefore, TROP2 has become an attractive target in tumor molecular targeted therapy.

TROP2 is a transmembrane protein whose extracellular domain is widely distributed on a variety of tumor cells, making it a natural candidate for targeted therapy. The limited tissue expression of TROP2 reduces the toxicity of treatment, which is also the advantage of targeting TROP2 therapy. Various forms of drugs, such as antibodies targeting TROP2, antibody conjugates, and drug combinations, are under development. The utility of anti-TROP2 antibodies conjugated to other chemotherapeutic agents has been demonstrated in various preclinical studies. The antibody drug conjugate (ADC) IMMU-132 for the treatment of TROP2-overexpressing epithelial malignancies has been approved by the FDA (April 2020). The new antibody-conjugated drug Sacituzumab govitecan (IMMU-132) is based on TROP2 as the target, and the humanized antibody hRS7 is used as the targeting carrier to couple with the active metabolite SN38 of irinotecan, which can be used to treat a variety of epithelial malignant tumors Such as breast cancer (triple negative breast cancer), ovarian cancer, small cell lung cancer, etc. In addition, other humanized anti-TROP2 IgG-SN-38 conjugates, such as anti-TROP2hRS7-CL2A-SN-38 antibody drug conjugates, have been shown to be effective in various tumor cell lines (Calu-3, Capan-1, BxPC -3 and COLO-205) have significant specific anticancer effects in xenograft models. In summary, it is of great significance to develop a specific anti-TROP2 antibody, especially a fully humanized anti-TROP2 antibody with good TROP2 antigen binding.

The CD3 molecule on the surface of T cells consists of four subunits δ, ε, γ, and ζ, with molecular masses of 18.9kDa, 23.1kDa, 20.5kDa, and 18.7kDa, and lengths of 171, 207, 182, and 164, respectively. Amino acid residues, which together form 6 peptide chains, are often tightly combined with T cell receptor (TCR) to form a TCR-CD3 complex containing 8 peptide chains. The schematic diagram of its structure is shown in Figure 1B. This complex has the functions of T cell activation, signal transduction and stable TCR structure. CD3 cytoplasm contains immunoreceptor tyrosine-based activation motif (immunoreceptor tyrosine-based activation motif, ITAM), TCR recognizes and binds the antigenic peptide presented by MHC (major histo-compatibility complex) molecules, resulting in the activation of CD3 ITAM The tyrosine residues in the conserved sequence are phosphorylated by the tyrosine protein kinase p56lck in T cells, and then recruit other tyrosine protein kinases (such as ZAP-70) containing the SH2 (Scr homology 2) domain. The phosphorylation of ITAM and the combination with ZAP-70 is one of the important biochemical reactions in the early stage of T cell activation signal transduction process. Therefore, the function of the CD3 molecule is to transduce the activation signal generated by the recognition of the antigen by the TCR. Based on the function of CD3, it is of great significance to develop a bispecific antibody that can be used for immunotherapy and simultaneously binds TROP2 and CD3.

Contents of the invention

The first object of the present invention is to provide a TROP2-binding antibody and its active fragment.

The second object of the present invention is to provide bispecific antibodies and active fragments thereof that bind to TROP2 and CD3.

The third object of the present invention is to provide nucleic acids encoding TROP2 antibodies and bispecific antibodies binding to TROP2 and CD3.

The fourth object of the present invention is to provide the use of the above-mentioned TROP2 antibody, bispecific antibody binding to TROP2 and CD3 or an active fragment thereof.

Specifically, the present invention provides the following technical solutions:

First, the present invention provides a TROP2 antibody, which includes a heavy chain variable region and a light chain variable region, and the CDR1, CDR2, and CDR3 of the heavy chain variable region have amino acids shown in SEQ ID NO.1-3 respectively sequence, or it respectively has the amino acid sequence shown in SEQ ID NO.1-3 as a reference sequence, and contains one or more combinations of the following mutations:

(1) The first S of the amino acid sequence shown in SEQ ID NO.1 is mutated to N;

(2) The fourth P of the amino acid sequence shown in SEQ ID NO.2 is mutated to R, K or G;

(3) The first P in the amino acid sequence shown in SEQ ID NO.3 is mutated to H and A;

(4) The second N of the amino acid sequence shown in SEQ ID NO.3 is mutated to E;

The CDR1, CDR2 and CDR3 of the light chain variable region have the amino acid sequences shown in SEQ ID NO.4-6 respectively, or they respectively have the amino acid sequences shown in SEQ ID NO.4-6 as the reference sequence, An amino acid sequence comprising a combination of one or more of the following mutations:

(1) The fifth G of the amino acid sequence shown in SEQ ID NO.4 is mutated to N or E;

(2) The 7th S mutation of the amino acid sequence shown in SEQ ID NO.4 is R or K

(3) The first A of the amino acid sequence shown in SEQ ID NO.5 is mutated to R;

(4) The third S of the amino acid sequence shown in SEQ ID NO.5 is mutated to G, H or R;

(5) The fourth S of the amino acid sequence shown in SEQ ID NO.5 is mutated into K.

The present invention screens the anti-TROP2 genetically engineered single-chain antibody from the fully synthetic single-chain human antibody library, obtains the variable region sequence of the antibody, constructs a mutation library through a point mutation kit, and obtains clones with high affinity. The DNA was mixed to assemble a single-chain antibody combinatorial library in a recombinant way, and after screening, a high-affinity TROP2 antibody that binds to human TROP2 was obtained.

The amino acid sequence pattern of the antibody variable region provided by the present invention is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In this application, the division of regions of FR and CDR is based on the Kabat nomenclature system. Here, FR1-4 represent four framework regions, and CDR1-3 represent three hypervariable regions. FR1-4 can be isolated from the constant region sequence (such as the most commonly used amino acids of human immunoglobulin light and heavy chain class, subclass or subfamily), or can be isolated from a single human antibody framework region or from different Combination of framework region genes.

Further, the present invention provides a TROP2 antibody, which includes a heavy chain variable region and a light chain variable region, and the CDR1 of the heavy chain variable region has the amino acid sequence shown in any one of SEQ ID NO.1, 7, CDR2 has the amino acid sequence shown in any one of SEQ ID NO.2,8,9, and CDR3 has the amino acid sequence shown in any one of SEQ ID NO.3,10;

The CDR1 of the light chain variable region has the amino acid sequence shown in any one of SEQ ID NO.4, 11, 12, the CDR2 has the amino acid sequence shown in any one of SEQ ID NO.5, 13, 14, and the CDR3 has the amino acid sequence shown in SEQ ID NO. The amino acid sequence shown in any one of 6 and 15.

After further screening, the present invention provides the following TROP2 antibodies with higher TROP2 binding affinity, and the CDR of the heavy chain variable region is any of the following:

(1) CDR1 has the amino acid sequence shown in SEQ ID NO.1, CDR2 has the amino acid sequence shown in SEQ ID NO.2, and CDR3 has the amino acid sequence shown in SEQ ID NO.3;

(2) CDR1 has the amino acid sequence shown in SEQ ID NO.7, CDR2 has the amino acid sequence shown in SEQ ID NO.9, and CDR3 has the amino acid sequence shown in SEQ ID NO.3;

(3) CDR1 has the amino acid sequence shown in SEQ ID NO.7, CDR2 has the amino acid sequence shown in SEQ ID NO.8, and CDR3 has the amino acid sequence shown in SEQ ID NO.3;

(4) CDR1 has the amino acid sequence shown in SEQ ID NO.7, CDR2 has the amino acid sequence shown in SEQ ID NO.8, and CDR3 has the amino acid sequence shown in SEQ ID NO.10;

The CDRs of the light chain variable region are any of the following:

(1) CDR1 has the amino acid sequence shown in SEQ ID NO.4, CDR2 has the amino acid sequence shown in SEQ ID NO.5, and CDR3 has the amino acid sequence shown in SEQ ID NO.6;

(2) CDR1 has the amino acid sequence shown in SEQ ID NO.4, CDR2 has the amino acid sequence shown in SEQ ID NO.13, and CDR3 has the amino acid sequence shown in SEQ ID NO.15;

(3) CDR1 has the amino acid sequence shown in SEQ ID NO.11, CDR2 has the amino acid sequence shown in SEQ ID NO.13, and CDR3 has the amino acid sequence shown in SEQ ID NO.15;

(4) CDR1 has the amino acid sequence shown in SEQ ID NO.12, CDR2 has the amino acid sequence shown in SEQ ID NO.13, and CDR3 has the amino acid sequence shown in SEQ ID NO.15;

(5) CDR1 has the amino acid sequence shown in SEQ ID NO.12, CDR2 has the amino acid sequence shown in SEQ ID NO.14, and CDR3 has the amino acid sequence shown in SEQ ID NO.15;

(6) CDR1 has the amino acid sequence shown in SEQ ID NO.4, CDR2 has the amino acid sequence shown in SEQ ID NO.14, and CDR3 has the amino acid sequence shown in SEQ ID NO.15;

Among the above-mentioned antibodies, the following antibodies have higher binding affinity to TROP2, and the CDRs of the heavy chain variable region and the CDRs of the light chain variable region are any of the following:

(1) CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.1, CDR2 has the amino acid sequence shown in SEQ ID NO.2, and CDR3 has the amino acid sequence shown in SEQ ID NO.3; light chain CDR1 of the variable region has the amino acid sequence shown in SEQ ID NO.4, CDR2 has the amino acid sequence shown in SEQ ID NO.5, and CDR3 has the amino acid sequence shown in SEQ ID NO.6;

(2) CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.7, CDR2 has the amino acid sequence shown in SEQ ID NO.8, and CDR3 has the amino acid sequence shown in SEQ ID NO.3; light chain CDR1 of the variable region has the amino acid sequence shown in SEQ ID NO.4, CDR2 has the amino acid sequence shown in SEQ ID NO.13, and CDR3 has the amino acid sequence shown in SEQ ID NO.15;

(3) CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.7, CDR2 has the amino acid sequence shown in SEQ ID NO.8, and CDR3 has the amino acid sequence shown in SEQ ID NO.3; light chain CDR1 of the variable region has the amino acid sequence shown in SEQ ID NO.12, CDR2 has the amino acid sequence shown in SEQ ID NO.13, and CDR3 has the amino acid sequence shown in SEQ ID NO.15;

(4) CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.7, CDR2 has the amino acid sequence shown in SEQ ID NO.8, and CDR3 has the amino acid sequence shown in SEQ ID NO.3; light chain CDR1 of the variable region has the amino acid sequence shown in SEQ ID NO.12, CDR2 has the amino acid sequence shown in SEQ ID NO.14, and CDR3 has the amino acid sequence shown in SEQ ID NO.15.

On the basis of the CDR regions of the above-mentioned heavy chain and light chain variable regions, the present invention provides the sequences of the heavy chain and light chain variable regions of these TROP2 antibodies:

The heavy chain variable region has the amino acid sequence shown in any one of SEQ ID NO.16, 18-20, and the light chain variable region has the amino acid sequence shown in any one of SEQ ID NO.17, 21-25;

Alternatively, compared with the aforementioned sequence, the heavy chain variable region and the light chain variable region have at least one of the following two requirements: a) binding to the same antigenic epitope; b) sequence identity greater than 70%, 80%, 85% %, 90%, 97%, 98% or 99% of the amino acid sequence.

Further, the antibody with the following variable region sequence shows more excellent TROP2 binding affinity: the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.16, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.17 The amino acid sequence shown;

Alternatively, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.19, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.21;

Alternatively, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.19, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.23;

Alternatively, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.19, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.24.

Optionally, the Fc fragment of the heavy chain of the above-mentioned TROP2 antibody is the Fc fragment of a human or humanized antibody, and the human or humanized antibody is IgG1, IgG2, IgA, IgE, IgM, IgG4 or IgD.

Preferably, the heavy chain of the TROP2 antibody has the amino acid sequence shown in any one of SEQ ID NO.27, 29, and the light chain has the amino acid sequence shown in any one of SEQ ID NO.28, 30; or, the heavy chain, light chain Compared with the aforementioned sequence, the chain has at least one of the following two: a) binding to the same antigenic epitope; b) sequence identity greater than 70%, 80%, 85%, 90%, 97%, 98% or 99% amino acid sequence.

The present invention provides a TROP2 antibody having the following heavy chain and light chain full-length sequences: the heavy chain has the amino acid sequence shown in SEQ ID NO.27, and the light chain has the amino acid sequence shown in SEQ ID NO.28; or, the heavy chain has the amino acid sequence shown in SEQ ID NO.28; The amino acid sequence shown in SEQ ID NO.29, the light chain has the amino acid sequence shown in SEQ ID NO.30.

The human anti-human TROP2 antibody provided by the invention binds to human TROP2 with an affinity of 4.35nM-7.5nM. The antibody binds to cells expressing TROP2, and the cells can be human epithelial malignant tumors (gastric cancer, cervical cancer, breast cancer, lung cancer, prostate cancer, colon cancer, esophageal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, intrauterine mucosa Serous papillary carcinoma and other tumors) cells.

The present invention also provides single-chain antibodies, Fab antibodies, miniature antibodies, chimeric antibodies, full antibody immunoglobulin IgG1, IgG2, IgA, IgE, IgM, IgG4 or IgD, bispecific antibodies, Multispecific Antibodies.

Further, the present invention provides a bispecific antibody that binds to TROP2 and CD3, which includes: a first domain that binds to trophoblast cell surface antigen 2, and a second domain that binds to the T cell surface antigen CD3, wherein the first The domains include a heavy chain variable region and a light chain variable region, which are the heavy chain variable region and light chain variable region of the TROP2 antibody described above.

Preferably, the first structural domain of the bispecific antibody is two complete light chain-heavy chain pairs connected by disulfide bonds, and the amino acid sequences of the heavy chain and the light chain are the heavy chain of the above-mentioned TROP2 antibody. Amino acid sequences of chain and light chain.

For the second structural domain that binds to the CD3 antigen, the heavy chain variable region preferably has the amino acid sequence shown in SEQ ID NO.31, and the light chain variable region preferably has the amino acid sequence shown in SEQ ID NO.32;

Preferably, the light chain variable region and the heavy chain variable region of the second structural domain are connected into a single-chain antibody through a connecting peptide, and the single-chain antibody has the amino acid sequence shown in SEQ ID NO.33.

The bispecific antibody of the present invention is preferably designed with the following structure: the first domain contains two complete light chain-heavy chain pairs, and the second domain contains two single-chain antibodies, which are connected by any of the following methods Symmetrical structure:

(1) The C-terminals of the two single-chain antibodies of the second domain are respectively connected to the N-terminals of the two heavy chains of the first domain through a connecting peptide;

(2) The N-terminals of the two single-chain antibodies of the second domain are respectively connected to the C-terminals of the two heavy chains of the first domain through a connecting peptide;

The present invention finds that for the sequences of the above-mentioned first domain and the second domain, the bispecific antibody with the above-mentioned symmetrical structure can better retain the first domain and the second domain than bispecific antibodies with other structures. The specific antigen-binding ability of the primary antibody of the structural domain, and the excellent biological function of binding TROP2 and CD3, have obvious advantages in production technology and medicinal properties. The present invention has developed a bispecific antibody binding to TROP2 and CD3 having the above-mentioned antibody molecular structure. The bispecific antibody has a specific targeting effect and can efficiently stimulate a oriented immune response to kill tumor cells.

Preferably, the amino acid sequence of the connecting peptide used to connect the first domain and the second domain is (GGGGX)n, wherein X is Gly or Ser, and n is a natural number of 1-4.

As a preferred embodiment of the present invention, the amino acid sequence of the connecting peptide is shown in SEQ ID NO.34.

As an example of a bispecific antibody with the above structure, on the basis of the above-mentioned TROP2 antibody and CD3 antibody, the present invention constructs a bispecific antibody that simultaneously binds to TROP2 and CD3, and its structure and sequence are as follows:

The heavy chain of the first structural domain has the amino acid sequence shown in SEQ ID NO.35 or 36 after connecting the second structural domain with the connecting peptide, and the light chain has the amino acid sequence shown in SEQ ID NO.28 or 30 .

Preferably, the heavy chain of the first structural domain has the amino acid sequence shown in SEQ ID NO.35 after connecting the second structural domain with the connecting peptide, and the light chain has the amino acid sequence shown in SEQ ID NO.28, Alternatively, the heavy chain of the first structural domain and the second structural domain have the amino acid sequence shown in SEQ ID NO.36 after connecting the second structural domain, and the light chain has the amino acid sequence shown in SEQ ID NO.30.

The sequences shown in SEQ ID NO.1-36 disclosed and claimed above include "conservative sequence modifications", that is, nucleotide and amino acid sequences that do not significantly affect and change the binding characteristics of the antibody or the antibody containing the amino acid sequence grooming. Such conservative sequence modifications include nucleotide or amino acid substitutions, additions or deletions. Modifications can be introduced into SEQ ID NO.1-36 by standard techniques in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include amino acid residues being replaced by amino acid residues with similar side chains or other amino acid residue substitution. In the art, families of amino acid residues having similar side chains have been defined. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), and uncharged polar side chains. Chain amino acids (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, valine amino acid, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (such as threonine, valine, isoleucine) and Amino acids with aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to replace a non-essential amino acid residue in a human anti-TROP2 antibody with another amino acid residue from the same side chain family.

Therefore, the antibody having the amino acid sequence disclosed above and/or the antibody comprising the amino acid sequence disclosed above includes antibodies substantially encoded by similar sequences modified by conservative sequences, or antibodies containing similar sequences modified by conservative sequences, both should be regarded as the scope of the present invention.

The present invention also provides nucleic acid molecules encoding the TROP2 antibody and nucleic acid molecules encoding the bispecific antibody binding to TROP2 and CD3.

Considering the degeneracy of codons, the gene encoding the antibody of the present invention can be modified in its coding region without changing the amino acid sequence to obtain the gene encoding the same antibody. Those skilled in the art can artificially synthesize and modify the gene according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

The present invention also provides biological materials containing the nucleic acid molecules, and the biological materials include recombinant DNA, expression cassettes, vectors, host cells, engineering bacteria or cell lines.

Wherein, the vectors include but are not limited to cloning vectors and expression vectors, and may be vectors such as plasmid vectors, viral vectors, and transposons.

The host cell or cell line may be a cell or cell line derived from microorganisms or animals.

The present invention also provides a method for preparing the TROP2 antibody or the bispecific antibody binding to TROP2 and CD3, which includes: introducing the nucleic acid encoding the antibody into a host cell to obtain a host stably expressing the bispecific antibody Cells; host cells are cultured, and the antibody is obtained through separation and purification.

When preparing the TROP2 antibody or the bispecific antibody that binds to TROP2 and CD3, those skilled in the art can select conventional host cells, expression vectors, methods for introducing expression vectors into host cells, and antibody isolation as required. Purification method.

Based on the functions of the TROP2 antibody and the bispecific antibody binding to TROP2 and CD3 provided by the present invention, the present invention provides a TROP2 antibody, a bispecific antibody binding to TROP2 and CD3, a nucleic acid molecule encoding the antibody, and a nucleic acid molecule containing the nucleic acid molecule. Any of the following applications of biomaterials:

(1) Application in the preparation of medicines for diagnosing, preventing or treating diseases related to TROP2 expression;

(2) Application in the preparation of drugs for diagnosing, preventing or treating diseases targeting TROP2;

(3) Application in the preparation of drugs for killing cells expressing TROP2;

(4) Application in the preparation of detection reagents for TROP2 and/or CD3;

(5) Application in the preparation of related reagents suitable for CAR-T therapy;

(6) Application in preparation of immunotoxin or labeled antibody.

The above-mentioned diseases related to TROP2 expression are preferably tumors with high expression/overexpression of TROP2, especially epithelial malignant tumors expressing TROP2 (including but not limited to gastric cancer, cervical cancer, breast cancer, lung cancer, prostate cancer, colon cancer, esophageal cancer, etc.) Cancer, pancreatic cancer, head and neck cancer, ovarian cancer, intrauterine mucosal serous papillary cancer and other tumors).

Preferably, the drug is an antineoplastic drug.

The above-mentioned labeled antibody may be a radioisotope-labeled antibody.

The bispecific antibodies provided by the invention are useful in therapy and diagnosis per se. Antibodies can be labeled, cross-linked or conjugated and fused with other proteins or polypeptide molecules to form complexes (such as cytotoxic substances, radioactive toxins and/or chemical molecules, etc.) for diagnosis and treatment.

The present invention provides a multispecific antibody, a fusion protein, an immunotoxin, a drug or a detection reagent comprising the TROP2 antibody or the bispecific antibody binding to TROP2 and CD3.

The immunotoxins described above comprise TROP2 antibodies or bispecific antibodies linked to cytotoxic agents in various formats.

The various linking forms are antibody labeling, in vitro cross-linking or molecular coupling. The cytotoxic agents include chemical molecules, radioactive isotopes, polypeptides, toxins and other substances that can kill or induce cell death.

The above-mentioned fusion protein comprises the complex of the above-mentioned TROP2 antibody or bispecific antibody provided by the present invention and other proteins or polypeptide molecules with certain functions.

Specifically, the fusion protein can be a recombinant expression vector constructed by linking antibody genes with immunotoxin or cytokine genes, and the recombinant fusion protein molecule can be obtained through mammalian cells or other expression systems.

The above-mentioned drugs and detection reagents may also contain other active ingredients or adjuvants allowed in the field of pharmacy and detection reagents (such as: antibody components and pharmacologically acceptable delivery molecules or solutions. Among them, the therapeutic group Divided into sterile, can be freeze-dried at low temperature).

The anti-TROP2 antibody and bispecific antibody provided by the present invention can inhibit one or more biological activities induced by TROP2. These antibodies function by depleting TROP2 on the cell surface after binding to TROP2 and internalizing the complex. All interfering functions possessed by TROP2 antagonists are equally considered objects of the present invention.

The beneficial effects of the present invention are:

The present invention uses genetic engineering and phage surface display library technology to screen specific anti-TROP2 antibodies from the single-chain antibody library of natural full human sequences. The TROP2 antibodies provided by the present invention have good ability to bind TROP2. ELISA detection and flow cytometry detection can specifically bind to TROP2 on the surface of MDA-MB-231, MDA-MB-468, MCF7 and other cells, indicating that the target specificity is good, and the binding affinity to human TROP2 is 4.35nM-7.5 nM, has a good prospect for therapeutic application.

On this basis, the present invention uses genetic engineering and antibody engineering methods to construct a bispecific antibody that binds to TROP2 and CD3 comprising a single-chain antibody and a complete monoclonal antibody structure, and the bispecific antibody fusion protein retains a complete monoclonal antibody structure, and has a highly stable symmetrical structure, which better retains the biological functions of anti-CD3 single-chain antibody and anti-TROP2 monoclonal antibody, and realizes a bispecific antibody molecule with excellent anti-TROP2 and anti-CD3 The biological function of monoclonal antibodies can build a bridge between tumor cells and immune effector cells, effectively activate immune effector cells and guide immune responses, significantly enhance the efficacy of immune cells in killing tumor cells, and at the same time minimize ADCC effect, with higher security. In addition, due to the completely symmetrical structure of the bispecific antibody provided by the present invention, it will not produce protein isomers of other structures when expressed in the host, thereby greatly reducing the difficulty of extraction and purification processes, and has the advantages of simple preparation, The advantage of high yield has broad application prospects in tumor immunotherapy.

The present invention provides human TROP2-specific antibody candidate molecules for the development of anti-tumor antibody drugs targeting TROP2 targets, CAR-T reagent development, prevention and treatment. The bispecific antibody of the present invention can simultaneously bind immune cells and tumor cells, guide T immune response, specifically and effectively kill tumor cells, and can be developed as an antibody drug for epithelial malignant tumors.

Description of drawings

Figure 1 is a schematic diagram of the protein structure and TCR structure of TROP2 in the background technology of the present invention, wherein, A is the structure of the TROP2 antigen, source: Goldenberg DM, Stein R, Sharkey RM.The emergence of trophoblast cell-surface antigen 2 (TROP-2 )as a novel cancer target.Oncotarget.2018 Jun 22;9(48):28989-29006.doi:10.18632/oncotarget.25615.PMID:29989029;PMCID:PMC6034748;B is the T cell receptor (TCR) complex structure And the composition of CD3. Source: https://www.researchgate.net/publication/299549376.

Figure 2 is the flow cytometry (FACS) analysis of the combination of TROP2 and the lead clone 1F7 in Example 2 of the present invention, wherein,

A: negative control; B: positive control; C: candidate clone IF7.

Fig. 3 is the analysis of the affinity of 1F7 and its affinity matured mutant F7A to human and cynomolgus TROP2 by plasmon resonance (SPR) technology in Example 3 of the present invention, where the ordinate is the binding reaction unit (RU).

Figure 4 is a schematic diagram of the structure of the anti-TROP2×CD3 bispecific antibody constructed based on the FIST platform in Example 4 of the present invention, wherein, A: the double antibody constructed with N-terminal fusion (nFIST): the anti-CD3 single chain antibody is fused to the anti-CD3 The N-terminal of the VH of the TROP2 antibody; B: the double antibody constructed by C-terminal fusion (cFIST): the anti-CD3 single-chain antibody is fused to the C-terminal of the Fc of the anti-TROP2 antibody.

Fig. 5 is the SPR analysis of the affinity of the single-chain antibody K3 to CD3 in Example 4 of the present invention.

Figure 6 is the SDS-PAGE electrophoresis of bispecific antibodies F7AK3 and 1F7K3 in Example 5 of the present invention, wherein A and C are reducing SDS-PAGE electrophoresis; B and D are non-reducing SDS-PAGE electrophoresis; A and B are 1F7 SDS-PAGE electrophoresis results of K3 bispecific antibody; C and D are SDS-PAGE electrophoresis results of F7AK3 bispecific antibody; lane M represents protein molecular weight standard, and lane 1 is target protein.

Fig. 7 is the HPLC-SEC purity analysis peak shape of the bispecific antibodies F7AK3 and K3F7A purified in Example 5 of the present invention, wherein, A is the bispecific antibody F7AK3; B is the bispecific antibody 1F7 K3.

Fig. 8 is the combination of F7AK3 and TROP2 analyzed by flow cytometry and the combination of F7AK3 and T cells in Example 6 of the present invention, wherein, A: flow cytometry analysis of the combination of F7AK3 to TROP2 cell lines; B-G: gradient analysis of F7AK3 to TROP2 The binding capacity of each cell line;

Figure 9 shows the bridging effect of F7AK3 on TROP2 positive cells and T cells in Example 6 of the present invention.

Figure 10 is the detection of the killing ability of T cells on human breast cancer cells (MCF, MDA-MB-231, MDA-MB-468, HCC1395) mediated by the binding of F7AK3 double antibody to TROP2 antigen in Example 7 of the present invention, wherein, A, B, C, and D are MCF, MDA-MB-231, MDA-MB-468, and HCC1395 cells, respectively.

detailed description

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Example 1 Screening of anti-human TROP2 antibody from natural human antibody phage surface display library

Antibody library technology is a method of cloning all antibody variable region genes of a certain animal (including humans) into plasmids or phages. After the latter infects E. coli, antibody fragments are expressed on the surface of phage particles, or on the periplasm of E. coli, cytoplasm in the pulp. Then use the target antigen to screen the clones carrying specific antibody genes from the antibody library, so as to obtain the corresponding specific antibody technology. Various antibodies needed in basic research and clinical development have been screened from the antibody library, such as tumor-associated membrane protein antigens, autoantigens related to autoimmune diseases, and antibodies against viral antigens of viral diseases. These show the great application potential of antibody library technology in basic research and antibody drug development. In particular, obtaining fully human monoclonal antibodies from human antibody libraries overcomes the difficulty of obtaining human monoclonal antibodies using mouse hybridoma technology; The immunogenicity of the antibody is much lower than that of animal-derived antibodies, so it is safer.

The natural antibody repertoire of non-immune people uses healthy people as the source of antibody genes, and these people are called donors. Due to the use of all natural human antibody sequences, the antibodies obtained have very low immunogenicity to the human immune system. Using molecular biology and DNA manipulation techniques, use an RNA extraction kit (such as QIAGEN's RNeasy Mini Kit, catalog number 74104) to extract total RNA from the donor's peripheral blood mononuclear cells (PBMC). Take equal amounts from one RNA sample and combine them, and use a reverse transcription kit (such as ThermoFisher Scientific's cDNA synthesis kit (SuperScript ^TM IV Reverse Transcriptase) to synthesize the light and heavy chain gene cDNA. Using these antibody cDNAs as templates, in In one round of amplification, carry out PCR amplification under the cooperation of each subgroup-specific upstream primer and constant region primer, and obtain all member genes of each subgroup.In the second round of PCR, the heavy chain with the bridge can be The variable region (VH) 3'-end primer set and the light chain variable region (VL) 5'-end primer set with a bridge were respectively combined with the other end primer (containing a specific restriction endonuclease site ) coordination, so that the obtained VH and VL can take the head link to form a single-chain antibody (scFv) gene. Utilize the specific enzyme cleavage sites of these scFv genes at the 5'-end and 3'-end, these scFv The gene is cloned into a lysogenic phagemid (phagemid, such as M13 phagemid) vector to construct an antibody library. The size of the library reaches 5 billion colony-forming units (cfu).

Biopanning refers to the process of screening an antibody library with a specific target to obtain a specific monoclonal antibody. In order to obtain human antibodies specific for human TROP2 antigen, panning was performed by liquid phase screening. The general steps of the liquid phase panning method are as follows: firstly, the commercialized TROP2 antigen (TROP2-Fc fusion protein, Acrobiosystems, cat. molecular. An appropriate amount of biotin-labeled TROP2 antigen was bound to streptavidin-coupled magnetic beads (eg, Dynabeads ^™ M-280 Streptavidin, ThermoFisher Scientific, Cat# 11205D). Thaw a human antibody library containing 10 billion phage particles expressing different antibodies. Both magnetic beads and antibody libraries were blocked with 4% non-fat dry milk solution (4% MPBS) to eliminate non-specific interaction sites. Since there may be streptavidin that has not been bound by avidin on the magnetic beads, and because the TROP2 antigen molecule is a fusion protein composed of human Fc, it is necessary to add a sufficient amount (50-100 times) to the thawed antibody library at the same time. streptavidin and human antibody Fc in excess of the amount of target protein used) to remove antibody clones that bind them. Add the blocked antibody library solution together with the magnetic beads (total volume <1ml) into a 1.5ml Eppendorf tube, and incubate for 2 hours at room temperature with rotation and mixing to allow TROP2 to bind to the specific phage antibody. After the incubation, place the Eppendorf tube on the magnetic stand for 1 minute to separate the magnetic beads and the solution. Use a pipette gun to completely remove the solution part as much as possible, replace with a new tip (tip), add 1ml of washing solution PBST (add Tween 20 with a final concentration of 0.05% to the phosphate buffer solution (PBS) solution) to the Eppendorf tube, keep away from the Use a pipette to gently suspend the magnetic beads on the magnetic stand, and place them back on the magnetic stand to separate the magnetic beads and the solution. The solution was removed and this was repeated three times. Then change the washing solution to PBS and wash the magnetic beads 3 times. After washing, non-specific and low-affinity phage antibodies are mostly removed, and specific phage antibodies remain on the magnetic beads. Add eluent (10mM Glycine, pH2.0) to the magnetic beads, resuspend the magnetic beads, let stand at room temperature for 10 minutes, separate the magnetic beads and the solution with a magnetic stand, draw the solution into a clean Eppendorf tube, add 1/ 10 1M Tris solution (pH8.0) to neutralize the solution, which is the eluate, which contains thousands of phage antibodies with different binding affinity to the TROP2 antigen, thus completing the first round of panning.

In order to further harvest specific antibody clones with relatively high affinity, more rounds of panning are required. To this end, the phage antibody solution eluted from the first round of panning was used to infect Escherichia coli (such as TG1 strain) in the logarithmic phase that can be infected by M13 phage to obtain an infection solution. Take a small amount of infection solution and perform a series of 10-fold serial dilutions (usually diluted to one millionth of the stock solution, and take the last three gradients for coating) to determine the titer of the first round of output eluate (output) ( titer), the titer is also called the first round of maximum diversity, usually the output titer after the first round of panning is below 10E6 cfu. Spread all the remaining infection solution on the bacterial culture plate containing the corresponding antibiotics and culture overnight to obtain colonies; scrape off the colony layer and resuspend in liquid medium, take enough resuspension containing the diversity of the first round of output to In a shaking flask containing sufficient liquid medium (2YT-CG, 2YT medium is added Carbenicillin and glucose, the final concentration is 100 μg/ml and 2% respectively), dilute the resuspension to below 0.1OD600 and start culturing until Logarithmic phase, that is, OD ₆₀₀ reaches about 0.5. In order to make these antibodies obtained in the first round of panning reappear on the surface of phage particles, take 10ml of bacterial liquid, add helper phage M13K07 to make the multiplicity of infection 20:1, and keep it at 37°C for 30 minutes (this stage is called for phage rescue). Centrifuge, resuspend the cells with 50ml expression medium (2YT-AK, add Carbenicillin and Kanamycin to 2YT medium, the final concentrations are 100μg/ml and 30μg/ml respectively), place at 30°C, 200 rpm, and culture overnight . The next day, the culture supernatant was harvested by centrifugation, 1/5 volume of PEG8000/NaCl (PEG-8000 20%, NaCl 2.5M) was added, mixed well, and incubated on ice for 1 hour. High-speed centrifugation (11500×g) for 30 minutes to harvest phage antibody particles. Resuspend the pellet with 1ml PBS solution and centrifuge again at high speed to remove bacterial debris. The supernatant is the amplification solution after the first round of panning, and each antibody clone contained in it has been amplified more than ten thousand times. This amplification solution can be used for the second round of panning experiments. The operation of the second round of panning is exactly the same as that of the first round except that it is increased to 6 times (6/6) in PBST/PBS. In the third round, the number of washes can be further increased to 10/10. Multiple rounds of panning usually effectively enrich specific clones. Although the diversity is significantly reduced, their affinity is relatively high, which is convenient for subsequent monoclonal screening.

In order to obtain specific monoclonal antibodies, a monoclonal phage enzyme-linked immunosorbent assay (Monophage ELISA) is required. For this purpose, single colonies separated well in the second and/or third round of serial dilution were individually inoculated into 96-well culture plates containing 2YT-AG (93 colonies per plate, leaving three wells as a negative control), cultured overnight, this is the master plate. Inoculate the bacterial solution in each well of the master plate into a new culture plate and grow to the logarithmic phase, perform the above-mentioned phage rescue, and express the antibody of each clone on the surface of the phage. Coat TROP2 antigen (1 μg/ml) on common 96-well enzyme-linked plate. Each individually expressed monoclonal phage antibody bacterial solution was added to the TROP2 plate, followed by an appropriate secondary antibody (mouse anti-M13 monoclonal antibody) and horseradish peroxidase (HRP)-coupled tertiary antibody (rabbit anti-mouse polyclonal antibody) Antibody), add HRP substrate for color development, and read the absorbance value (450nM). The method for judging TROP2 positive clones is: the signal is more than 3 times higher than that of negative control wells. After analysis, clones corresponding to multiple wells were positive for the TROP2 antigen, and these clones were collectively referred to as hits.

Inoculate these hit bacteria liquids into 3ml 2YT-CG from the corresponding wells of the motherboard, place at 37°C, 200 rpm, and culture overnight. The next day, the phagemid DNA was extracted, and the sequence of the single-chain antibody region containing each hit was determined with specific primers. The DNA sequences of the coding regions were translated into amino acid sequences, and multiple sequence comparisons (CLUSTALW, website link https://www.genome.jp/tools-bin/clustalw) were performed on them to determine the clone specificity. After analysis, these hits belonged to two different clones in sequence, one of which was named 1F7, thus obtaining the variable region sequence of the fully human antibody against human TROP2 antigen. The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of 1F7 are shown in SEQ ID NO.1-3 respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO.4- 6, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.17. The amino acid sequence of the single-chain antibody composed of the light chain and heavy chain variable regions of 1F7 is shown in SEQ ID NO.26.

Example 2 Antibody binding and functional verification

In order to verify whether the obtained TROP2 antibody clone binds to the purified TROP2 antigen and TROP2 expressed on the cell membrane surface, the gene of the 1F7 single-chain antibody was cloned into the eukaryotic expression vector pFH (manufactured by Ekest) to obtain the plasmid pFH-1F7. In this vector, the scFv gene is fused with the Fc gene of human IgG4 to express the protein in the form of scFv-Fc, which can be affinity purified with Potein-A or labeled with (HRP or fluorescein) anti-human Fc antibody detection.

After obtaining the scFv-Fc protein of 1F7, the binding of 1F7 to the TROP2-expressing CHO cell line (manufactured by EKSTER) was detected by flow cytometry, and it was confirmed that 1F7 specifically binds to the TROP2 antigen on the surface of the cell membrane (see Fig. 2). The angle percentage (positive percentage) in the figure represents the binding strength of the antibody to the cell surface antigen. The positive percentage of clone 1F7 was 89.33%, thus verifying the ability of the antibody to bind to the human cell surface antigen TROP2.

Example 3 Antibody affinity maturation

In vitro affinity maturation is a rapid directed molecular evolution operation: mutations are introduced at the DNA level and screened at the protein binding level. For the 1F7 antibody, in order to avoid introducing mutations that may enhance immunogenicity or significantly change the structure of the antibody, the mutation sites are limited to the sequence of the CDR region, and each position is subjected to saturation mutations, and each mutation is expressed separately. Tested separately.

The operation process of affinity maturation is as follows: firstly, the light and heavy chain variable region genes of 1F7 are respectively cloned into the pUFL vector (pUFL is a plasmid capable of expressing antibody Fab in Escherichia coli, prepared by Ecosite), and the Fab form of 1F7. Design a full set of single-point random mutation primers in the CDR region, and perform PCR mutations on each CDR position with the point mutation kit (QuikChange Lightning Multi Site-Directed Mutagenesis Kit, Agilent catalog number 210515), respectively, and transform BL21 (DE3) competent bacteria, Plates of monoclonal colonies are prepared separately and these mutants are collectively referred to as single point mutation libraries. At the same time, the Fab vector of the parental 1F7 was also transformed. Pick >30 colonies to prepare DNA for sequence analysis of the mutation region to evaluate the mutation efficiency. When the mutation efficiency is appropriate (greater than 80%), pick 92 colonies according to the mutation of each CDR position to a 96-well culture plate containing the corresponding culture medium (containing 100 μg/ml Carbenicillin+0.1% glucose in 2YT), and pick in addition Take 3 parental colonies and leave 3 wells as blank controls. The plate was placed at 37°C, cultured at 300 rpm for 6 hours, added IPTG to a final concentration of 1mM, transferred to 30°C, 300 rpm, cultured overnight, and Fab fragments were expressed and secreted into the medium.

Through preliminary experiments, the conditions of the enzyme-linked reaction were optimized so that the A450 absorption value of the 1F7 parental antibody Fab to the antigen TROP2 was slightly higher than the background signal by about 3 times. Coat TROP2 antigen at 0.25 μg/ml on the enzyme-linked plate one day in advance, add the secreted and expressed mutant Fab to the wells the next day, use HRP-coupled anti-human Fab as the secondary antibody, develop color with the substrate, and read A450 to obtain Clones corresponding to wells whose signals were 1.5 times higher than the highest value of parental wells were called hits. Collect all the hits in the light variable region, re-induce expression and enzyme-linked experiments to verify that they do have higher affinity than the parental antibody. Sequence analysis of the mutant region of the plasmid of the repeat-positive clone is performed to obtain the mutation information of the CDR amino acid. This process is called primary screening. In this way, all the mutation information in the CDR region that is beneficial to the improvement of affinity is obtained: the mutation sites that provide affinity in the heavy chain variable region CDR are shown in Table 1, and the mutation sites that provide affinity in the light chain variable region CDR are shown in Table 1. Table 2.

Table 1 The beneficial mutations in the CDR of the heavy chain variable region of 1F7 for improving the affinity

CDR区位置CDR region location	亲本氨基酸parent amino acid	有益的突变氨基酸Beneficial mutant amino acids
H31H31	SS	NN
H53H53	NN	R，K，GR, K, G
H99H99	GG	H，AH,A
H100H100	DD.	EE.

Table 2 The beneficial mutations in the CDR of the light chain variable region of 1F7 for improving the affinity

CDR区位置CDR region location	亲本氨基酸parent amino acid	有益的突变氨基酸Beneficial mutant amino acids
L28L28	GG	N,EN,E
L30L30	SS	R,KR,K
L50L50	AA	RR
L52L52	SS	G,H,RG,H,R
L53L53	SS	KK

After obtaining all the single mutation information in the entire CDR region that is beneficial to the improvement of affinity from the primary library through enzyme-linked assay screening, redesign new multi-point mutation primers to include the main beneficial mutations, and construct mutations again with the point mutation kit library, this library is called a combinatorial library. In order to screen all the combinations of mutations in the variable regions of the light and heavy chains as much as possible, combinatorial libraries containing multiple mutations in the variable regions of the light and heavy chains were respectively constructed and screened. From the light chain variable region combinatorial library of parental heavy chain variable region, the top 10 clones with the highest affinity were screened, and their light chain variable region amino acid sequences contained 5 kinds of mutant sequences, such as SEQ ID NO.21-25 As shown, CDR1 of its light chain variable region has the amino acid sequence shown in any one of SEQ ID NO.4, 11, 12, CDR2 has the amino acid sequence shown in any one of SEQ ID NO.2, 13, 14, and CDR3 has the amino acid sequence shown in any one of SEQ ID NO.2, 13, 14 The amino acid sequence shown in any one of SEQ ID NO.3, 15.

From the heavy chain variable region combinatorial library of the parental light chain variable region, the amino acid sequences of the 10 clones with the highest affinity were screened, and the amino acid sequences of their heavy chain variable region contained three mutant sequences, as shown in SEQ ID As shown in NO.18-20, the CDR1 of the heavy chain variable region has the amino acid sequence shown in any one of SEQ ID NO.1 and 7, and the CDR2 has the amino acid sequence shown in any one of SEQ ID NO.2, 8, or 9 , CDR3 has the amino acid sequence shown in any one of SEQ ID NO.3,10.

At the same time, mix the positive all heavy chain variable region mixed clones and all positive light chain variable region clones obtained from the above screening, prepare their DNA mixed plasmids respectively, and assemble them into the final Fab antibody combination by enzyme digestion and linking recombination library. This library was screened, the top 10 positions with the highest signal were picked, and their DNA sequences were analyzed to obtain 3 different Fab clones, which had the same heavy chain variable region, and their amino acid sequences were shown in SEQ ID NO.19; Their light chain variable region amino acid sequences are shown in SEQ ID NO.21-24.

Using flow cytometry (FACS) to detect the TROP2 binding ability of the above screened TROP2 antibody and different cell lines, the results show that, compared to the parental antibody 1F7; Shows significantly improved apparent affinity in flow cytometric analysis. ELISA detection showed that the intrinsic affinity of the parental antibody 1F7 was about 7.5nM, while that of the mutant F7A was 4.35nM.

In order to test whether the affinity of the obtained mutants was improved, one of the mutant F7A and the 1F7 parental antibody in the form of IgG were prepared, expressed and purified, respectively. The amino acid sequence of CDR1 of the heavy chain variable region of 1F7 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.3; the light chain can be The amino acid sequence of CDR1 in the variable region is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of CDR3 is shown in SEQ ID NO.6; the amino acid sequence of the heavy chain variable region As shown in SEQ ID NO.16, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.17; the full-length sequence of the light chain is shown in SEQ ID NO.30, and the full-length sequence of the heavy chain is shown in SEQ ID NO. 29. Wherein the amino acid sequence of CDR1 of the heavy chain variable region of F7A is shown in SEQ ID NO.7, the amino acid sequence of CDR2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR3 is shown in SEQ ID NO.3; The amino acid sequence of CDR1 in the variable region is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is shown in SEQ ID NO.13, the amino acid sequence of CDR3 is shown in SEQ ID NO.15, and the amino acid sequence of the heavy chain variable region The sequence is shown in SEQ ID NO.19, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.21; the full-length sequence of the light chain is shown in SEQ ID NO.28, and the full-length sequence of the heavy chain is shown in SEQ ID NO .27 shown. The expressed and purified antibody products were detected by surface plasmon resonance (SPR), and their binding curves to human and cynomolgus monkey TROP2 proteins are shown in FIG. 3 . The affinity analysis data are shown in Table 3. The results showed that the binding of mutant F7A to human TROP2 was 1.74 times higher than that of 1F7; the affinity to monkey TROP2 was 3.18 times higher. The cross ratio between human and monkey was reduced from 5.38 times to 2.94 times (Table 3).

Table 3 SPR analysis of the affinity of 1F7 and F7A to human and monkey TROP2

抗体Antibody	抗原antigen	ka(1/Ms)ka(1/Ms)	kd(1/s)kd(1/s)	亲和力KD(M)Affinity KD(M)
1F71F7	Human TROP-2Human TROP-2	1.18E+051.18E+05	8.92E-048.92E-04	7.57E-097.57E-09
1F71F7	Rhesus TROP-2Rhesus TROP-2	2.31E+042.31E+04	9.42E-049.42E-04	4.07E-084.07E-08
F7AF7A	Human TROP-2Human TROP-2	1.92E+051.92E+05	8.34E-048.34E-04	4.35E-094.35E-09
F7AF7A	Rhesus TROP-2Rhesus TROP-2	1.28E+051.28E+05	1.65E-031.65E-03	1.28E-081.28E-08

Example 4 Design of TROP2×CD3 bispecific antibody

In this example, a bispecific antibody was designed using the tumor cell surface antigen TROP2 and the immune cell surface antigen CD3 as targets.

Combining protein structure design software and a large number of manual experimental screening, the present invention screened and determined bispecific antibodies with symmetrical structures including single-chain antibody units and monoclonal antibody units in a variety of bispecific antibody structures that bind TROP2 and CD3 structure. The present invention refers to this technical platform as FIST (fusion of IgG and scFv technology). For example, the anti-TROP2 monoclonal antibody unit can be used as an IgG antibody, including two complete light chain-heavy chain pairs (that is, containing complete Fab and Fc domains, and the heavy chain and light chain are connected by disulfide bonds), anti-TROP2 CD3 can be used as a single chain antibody unit, including 2 single chain antibodies (ScFv). The single-chain antibody and the monoclonal antibody are connected by a connecting peptide. For the connection method of the single-chain antibody and the monoclonal antibody, the following connection method can be designed to obtain a bispecific antibody with a symmetrical structure. The schematic diagram of its structure is shown in Figure 4 shown.

nFIST was obtained by linking the C-terminal of the single-chain antibody to the N-terminal of the heavy chain variable region (VH) of the monoclonal antibody (Figure 4A). The single-chain antibody can also be fused to the C-terminus of IgG-Fc via a linker peptide to obtain cFIST (Figure 4B). The variable region sequence of the anti-CD3 single-chain antibody UCHT1 was obtained from the literature (Beverley, P.C. & Callard, R.E. Distinctive functional characteristics of human "T" lymphocytes defined by E rosetting or a monoclonal anti-T cell antibody (1981) Eur.J.Immunol. 11,329-334) and transformed by humanization (Shalaby et.al., Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene. (1992) J Exp Med. Jan 1; 175 (1): 217 -25). The single-chain antibody thus constructed is named K3 (SEQ ID NO.33), which contains two cysteines (Cys) inserted in the variable region of the heavy chain and the variable region of the light chain respectively, which are formed after folding A pair of interchain disulfide bonds, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.32. . Using the surface plasmon resonance technique, the binding force of the single-chain antibody form K3 to human CD3 was detected, and the results are shown in FIG. 5 .

Example 5 Preparation of TROP2×CD3 bispecific antibody

According to the bispecific antibody coding gene designed in the form of cFIST in Example 4 and cloned into the expression vector pG4HK, two bispecific antibodies F7AK3 and 1F7K3 were obtained, and their heavy chain amino acid sequences were shown in SEQ ID NO.35, 36, respectively. Shown; Light chain amino acid sequence shown in SEQ ID NO.28,30 respectively. The expression plasmids corresponding to 1F7K3 and F7AK3 were stably transfected into CHO-K1 respectively, and the double antibody protein was expressed.

In order to obtain high-purity bispecific antibodies, follow the steps below for purification. (1) Feed liquid pretreatment: the supernatant of the fermentation culture was centrifuged at 2000 rpm for 10 min, and then filtered through a 0.22 μM filter membrane. (2) Affinity chromatography: Use Mabselect SuRe affinity chromatography column (purchased from GE Company, Cat. No. 18-5438-02) to capture the antibody in the pretreated fermentation broth, and equilibrate buffer (10mM PB, 0.1M NaCl, pH 7.0) to fully equilibrate the chromatography column, pass through the affinity chromatography column, and elute with elution buffer (0.1M citric acid, pH 3.0). (3) Cation exchange chromatography: The sample prepared by affinity chromatography was further purified by molecular sieve exchange chromatography, buffer (50 mM PBS, 0.2 Man Na ₂ SO ₄ , pH 6.7).

1F7K3 and F7AK3 were detected by SDS-PAGE and HPLC-SEC. The results of SDS-PAGE are shown in FIG. 6 , and the results of HPLC-SEC detection are shown in FIG. 7 . The test results showed that the bispecific antibodies 1F7K3 and F7AK3 were successfully prepared through expression and purification, and the monomer purity of the purified bispecific antibodies was above 95%.

Example 6 Detection of binding activity of bispecific antibody F7AK3 to TROP2 cells and T cells by flow cytometry

In order to verify the binding of F7AK3 to TROP2 cell line and T cells, a bispecific antibody of F7A and single-chain antibody K3 was constructed in the form of cFIST.

Take breast cancer cell lines MCF, MDA-MB-231, MDA-MB-468, HCC1395, CD3 ⁺ T cells, resuspend in PBS at 1×10 ⁶ cells/reaction tube, add 1ml binding buffer (containing 0.5% w/v BSA+2mM EDTA in PBS), rinse the cells once, centrifuge (350×g, 4° C., 5 min), and then resuspend the cells with 200 μl binding buffer. Add bispecific antibody and F7AK3 to 5 μg/ml, and incubate on ice for 45 minutes. Cells were washed once as above and resuspended in 100 μl of binding buffer. 5 μl of fluorescently labeled secondary antibody (PE anti-human IgG Fc Antibody, Biolegend, 409304) was added to the sample tube. Cells were rinsed once as above. After the cells were resuspended in 200 μl of PBS, they were tested on a machine (Beckman). The flow cytometric analysis charts for each cell line are shown in Figure 8A. The binding gradient analysis is shown in BE of FIG. 8 , and the binding to CD3 of human T cells is shown in FIG. 8F . The EC50 analysis data are shown in Table 4 and G of Figure 8 . The results showed that F7AK3 can bind to TNBC cell lines with different TROP2 expression levels in a concentration-dependent manner, and can also effectively bind to CD3 of human T cells, with a difference of 2-10 times in EC50 (Table 4). At the same time, as shown in Figure 9, F7AK3 can cross-link T cells and TNBC cancer cells (such as MDA-MB-468) to form cell clusters (synapse).

Table 4 The binding of F7AK3 to TROP2 ⁺ cell lines and CD3 ⁺ T cells detected by flow cytometry

the	MCFMCF	MDA-MB-231MDA-MB-231	MDA-MB-468MDA-MB-468	HCC1395HCC1395	CD3 ⁺T细胞 CD3 ⁺ T cells
EC50(ng/ml)EC50(ng/ml)	204.8204.8	89.0689.06	66.7766.77	53.2453.24	568.3568.3

Example 7 Detection of in vitro cell killing efficiency mediated by bispecific antibodies

In this example, MCF, MDA-MB-231, MDA-MB-468, HCC1395, and murine cell 4T1 were used as target cells, and PBMC were used as immune effector cells to detect the killing effect of the target cells mediated by the bispecific antibody F7AK3 . The specific experimental steps are as follows:

1) Target cell preparation: culture target cells, blow evenly, count, centrifuge at 1000 rpm for 5 min, and wash once with PBS. After the target cells were centrifuged and washed, the density was adjusted to 0.2×10 ⁶ /ml with GT-T551 medium, and 50 μl was added to each well, so the number of cells in each well was 10,000.

2) PBMC preparation: use PBMC as effector cells. Take out the PBMC frozen in the liquid nitrogen tank (refer to cell freezing and recovery), thaw, add to a 15ml centrifuge tube containing PBS or GT-T551 medium, centrifuge at 1000rpm for 5min, and wash twice with PBS or GT-T551 medium. Count the number of cells, activity and density, adjust the density of viable cells to 2×10 ⁶ /ml, add 50 μl to each well, then there are 100,000 cells in each well.

3) Antibody dilution: The bispecific F7AK3 was diluted with GT-T551 medium, and the initial antibody concentration was adjusted to 10 nM. Dilute in a ratio of 1:5. Add 100 μl of the diluted antibody to the prepared cells above, mix well, put the 96-well plate back into the incubator, and detect the killing effect after 24 hours.

4) Detection: distinguish dead and living cells by PMA staining, and then calculate the killing efficiency by cell counting.

5) Data processing: the formula for calculating the killing ratio of target cells is as follows:

Target cell killing ratio (percentage)=100×(reading value of target cell only-reading value of detection well)/reading value of detection well only.

The antibody concentration corresponding to the killing ratio of target cells in all the detection wells was converted into log10, and a curve was drawn using this as the abscissa and the killing ratio as the vertical axis. The killing concentration-gradient curves of F7AK3 on sensitive cell lines MCF, MDA-MB-231, MDA-MB-468, and HCC1395 are shown in Figure 10; the killing percentages of different cells are shown in Table 5. F7AK3 exhibits different killing abilities to different TROP2 ⁺ cells.

Table 5 Bispecific antibody-mediated target cell killing effect

the	MCFMCF	MDA-MB-231MDA-MB-231	MDA-MB-468MDA-MB-468	HCC1395HCC1395
杀伤效率(％)Killing efficiency (%)	2525	5050	7575	8282

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Industrial Applicability

The invention relates to the technical field of genetic engineering antibodies, in particular to an antibody binding to TROP2, a bispecific antibody targeting TROP2 and CD3, and a preparation method and application thereof. The TROP2 antibody provided by the present invention has a good ability to bind TROP2 and has high affinity; the bispecific antibody that binds TROP2 and CD3 has excellent biological functions of anti-TROP2 and anti-CD3 antibodies, and can bind between tumor cells and immune effector cells Build a bridge between them, effectively activate immune effector cells and oriented immune responses, significantly enhance the efficacy of immune cells in killing tumor cells, and at the same time minimize the ADCC effect. Good application prospects.

Claims

A TROP2 antibody, characterized in that it includes a heavy chain variable region and a light chain variable region, and the CDR1, CDR2, and CDR3 of the heavy chain variable region have the amino acid sequences shown in SEQ ID NO.1-3 respectively, Alternatively, they respectively have amino acid sequences that take the amino acid sequence shown in SEQ ID NO.1-3 as a reference sequence and contain one or a combination of the following mutations:

(1) The first S of the amino acid sequence shown in SEQ ID NO.1 is mutated to N;

(2) The fourth P of the amino acid sequence shown in SEQ ID NO.2 is mutated to R, K or G;

(3) The first P in the amino acid sequence shown in SEQ ID NO.3 is mutated to H and A;

(4) The second N of the amino acid sequence shown in SEQ ID NO.3 is mutated to E;

The CDR1, CDR2 and CDR3 of the light chain variable region have the amino acid sequences shown in SEQ ID NO.4-6 respectively, or they respectively have the amino acid sequences shown in SEQ ID NO.4-6 as the reference sequence, An amino acid sequence comprising a combination of one or more of the following mutations:

(1) The fifth G of the amino acid sequence shown in SEQ ID NO.4 is mutated to N or E;

(2) The seventh S of the amino acid sequence shown in SEQ ID NO.4 is mutated to R or K;

(3) The first A of the amino acid sequence shown in SEQ ID NO.5 is mutated to R;

(4) The third S of the amino acid sequence shown in SEQ ID NO.5 is mutated to G, H or R;

(5) The fourth S of the amino acid sequence shown in SEQ ID NO.5 is mutated into K.
A TROP2 antibody, characterized in that it comprises a heavy chain variable region and a light chain variable region, the CDR1 of the heavy chain variable region has the amino acid sequence shown in any one of SEQ ID NO.1 and 7, and the CDR2 has the amino acid sequence shown in SEQ ID NO.1, 7. The amino acid sequence shown in any one of ID NO.2, 8, 9, CDR3 has the amino acid sequence shown in any one of SEQ ID NO.3, 10;

The CDR1 of the light chain variable region has the amino acid sequence shown in any one of SEQ ID NO.4, 11, 12, the CDR2 has the amino acid sequence shown in any one of SEQ ID NO.5, 13, 14, and the CDR3 has the amino acid sequence shown in SEQ ID NO. 6. The amino acid sequence shown in any one of 15;

Preferably, the CDRs of the heavy chain variable region are any of the following:

(1) CDR1 has the amino acid sequence shown in SEQ ID NO.1, CDR2 has the amino acid sequence shown in SEQ ID NO.2, and CDR3 has the amino acid sequence shown in SEQ ID NO.3;

(2) CDR1 has the amino acid sequence shown in SEQ ID NO.7, CDR2 has the amino acid sequence shown in SEQ ID NO.9, and CDR3 has the amino acid sequence shown in SEQ ID NO.3;

(3) CDR1 has the amino acid sequence shown in SEQ ID NO.7, CDR2 has the amino acid sequence shown in SEQ ID NO.8, and CDR3 has the amino acid sequence shown in SEQ ID NO.3;

(4) CDR1 has the amino acid sequence shown in SEQ ID NO.7, CDR2 has the amino acid sequence shown in SEQ ID NO.8, and CDR3 has the amino acid sequence shown in SEQ ID NO.10;

The CDRs of the light chain variable region are any of the following:

(1) CDR1 has the amino acid sequence shown in SEQ ID NO.4, CDR2 has the amino acid sequence shown in SEQ ID NO.5, and CDR3 has the amino acid sequence shown in SEQ ID NO.6;

(2) CDR1 has the amino acid sequence shown in SEQ ID NO.4, CDR2 has the amino acid sequence shown in SEQ ID NO.13, and CDR3 has the amino acid sequence shown in SEQ ID NO.15;

(3) CDR1 has the amino acid sequence shown in SEQ ID NO.11, CDR2 has the amino acid sequence shown in SEQ ID NO.13, and CDR3 has the amino acid sequence shown in SEQ ID NO.15;

(4) CDR1 has the amino acid sequence shown in SEQ ID NO.12, CDR2 has the amino acid sequence shown in SEQ ID NO.13, and CDR3 has the amino acid sequence shown in SEQ ID NO.15;

(5) CDR1 has the amino acid sequence shown in SEQ ID NO.12, CDR2 has the amino acid sequence shown in SEQ ID NO.14, and CDR3 has the amino acid sequence shown in SEQ ID NO.15;

(6) CDR1 has the amino acid sequence shown in SEQ ID NO.4, CDR2 has the amino acid sequence shown in SEQ ID NO.14, and CDR3 has the amino acid sequence shown in SEQ ID NO.15;

More preferably, the CDR of the heavy chain variable region and the CDR of the light chain variable region are any of the following:

(1) CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.1, CDR2 has the amino acid sequence shown in SEQ ID NO.2, and CDR3 has the amino acid sequence shown in SEQ ID NO.3; light chain CDR1 of the variable region has the amino acid sequence shown in SEQ ID NO.4, CDR2 has the amino acid sequence shown in SEQ ID NO.5, and CDR3 has the amino acid sequence shown in SEQ ID NO.6;

(2) CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.7, CDR2 has the amino acid sequence shown in SEQ ID NO.8, and CDR3 has the amino acid sequence shown in SEQ ID NO.3; light chain CDR1 of the variable region has the amino acid sequence shown in SEQ ID NO.4, CDR2 has the amino acid sequence shown in SEQ ID NO.13, and CDR3 has the amino acid sequence shown in SEQ ID NO.15;

(3) CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.7, CDR2 has the amino acid sequence shown in SEQ ID NO.8, and CDR3 has the amino acid sequence shown in SEQ ID NO.3; light chain CDR1 of the variable region has the amino acid sequence shown in SEQ ID NO.12, CDR2 has the amino acid sequence shown in SEQ ID NO.13, and CDR3 has the amino acid sequence shown in SEQ ID NO.15;

(4) CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.7, CDR2 has the amino acid sequence shown in SEQ ID NO.8, and CDR3 has the amino acid sequence shown in SEQ ID NO.3; light chain CDR1 of the variable region has the amino acid sequence shown in SEQ ID NO.12, CDR2 has the amino acid sequence shown in SEQ ID NO.14, and CDR3 has the amino acid sequence shown in SEQ ID NO.15.
The TROP2 antibody according to claim 2, wherein the heavy chain variable region has the amino acid sequence shown in any one of SEQ ID NO.16, 18-20, and the light chain variable region has SEQ ID NO.17 , the amino acid sequence shown in any one of 21-25;

Alternatively, compared with the aforementioned sequence, the heavy chain variable region and the light chain variable region have at least one of the following two requirements: a) binding to the same antigenic epitope; b) sequence identity greater than 70%, 80%, 85% %, 90%, 97%, 98% or 99% of the amino acid sequence;

Preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.16, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.17;

Alternatively, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.19, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.21;

Alternatively, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.19, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.23;

Alternatively, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.19, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.24.
The TROP2 antibody according to any one of claims 1 to 3, wherein the Fc fragment of the heavy chain is the Fc fragment of a human or humanized antibody, and the human or humanized antibody is IgG1, IgG2, IgA , IgE, IgM, IgG4 or IgD.
The TROP2 antibody according to any one of claims 1 to 4, wherein the heavy chain of the antibody has the amino acid sequence shown in any one of SEQ ID NO.27, 29, and the light chain has the amino acid sequence shown in SEQ ID NO.28, 30 any one of the amino acid sequences shown;

Alternatively, compared with the aforementioned sequence, the heavy chain and light chain have at least one of the following two requirements: a) binding to the same antigenic epitope; b) sequence identity greater than 70%, 80%, 85%, 90%, 97% %, 98% or 99% of the amino acid sequence;

Preferably, the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO.27, and the light chain has the amino acid sequence shown in SEQ ID NO.28; or, the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO.29 The amino acid sequence shown, the light chain has the amino acid sequence shown in SEQ ID NO.30.
Single chain antibody, Fab antibody, miniature antibody, chimeric antibody, full antibody immunoglobulin IgG1, IgG2, IgA, IgE, IgM, IgG4 or IgD containing the TROP2 antibody described in any one of claims 1 to 5, bispecific Antibodies, multispecific antibodies.
A bispecific antibody binding to TROP2 and CD3, characterized in that it comprises:

The first domain, which binds trophoblast surface antigen 2,

and, the second domain, which binds the T cell surface antigen CD3,

The first structural domain comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region are the heavy chain of the TROP2 antibody according to any one of claims 1 to 3 Variable region and light chain variable region.
The bispecific antibody binding TROP2 and CD3 according to claim 7, characterized in that the first domain is two complete light chain-heavy chain pairs connected by disulfide bonds, the heavy chain and light chain The amino acid sequence of the chain is the amino acid sequence of the heavy chain and the light chain of the TROP2 antibody as claimed in claim 5.
The bispecific antibody binding to TROP2 and CD3 according to claim 7 or 8, wherein the heavy chain variable region of the second domain has the amino acid sequence shown in SEQ ID NO.31, and the light chain can be The variable region has the amino acid sequence shown in SEQ ID NO.32;

Preferably, the light chain variable region and the heavy chain variable region of the second structural domain are connected into a single-chain antibody through a connecting peptide, and the single-chain antibody has the amino acid sequence shown in SEQ ID NO.33.
The bispecific antibody that binds to TROP2 and CD3 according to any one of claims 7 to 9, wherein the second domain contains two single-chain antibodies, and the bispecific antibody is obtained through any of the following methods: A symmetrical structure connected in several ways:

(1) The C-terminals of the two single-chain antibodies of the second domain are respectively connected to the N-terminals of the two heavy chains of the first domain through a connecting peptide;

(2) The N-terminals of the two single-chain antibodies of the second domain are respectively connected to the C-terminals of the two heavy chains of the first domain through a connecting peptide;

Preferably, the amino acid sequence of the connecting peptide is (GGGGX)n, wherein X is Gly or Ser, and n is a natural number of 1-4;

More preferably, the amino acid sequence of the connecting peptide is shown in SEQ ID NO.34.
The bispecific antibody binding to TROP2 and CD3 according to any one of claims 7 to 10, characterized in that the heavy chain of the first domain and the second domain have SEQ ID after being connected via a connecting peptide The amino acid sequence shown in NO.35 or 36, the light chain has the amino acid sequence shown in SEQ ID NO.28 or 30;

Preferably, the heavy chain of the first structural domain has the amino acid sequence shown in SEQ ID NO.35 after connecting the second structural domain with the connecting peptide, and the light chain has the amino acid sequence shown in SEQ ID NO.28, Alternatively, the heavy chain of the first structural domain and the second structural domain have the amino acid sequence shown in SEQ ID NO.36 after connecting the second structural domain, and the light chain has the amino acid sequence shown in SEQ ID NO.30.
A nucleic acid molecule, characterized in that it encodes the TROP2 antibody according to any one of claims 1-5, or it encodes the bispecific antibody binding to TROP2 and CD3 according to any one of claims 7-11.
The biological material containing the nucleic acid molecule of claim 12, said biological material comprising recombinant DNA, expression cassette, vector, host cell, engineering bacteria or cell line.
The preparation method of the TROP2 antibody according to any one of claims 1 to 5 or the bispecific antibody binding to TROP2 and CD3 according to any one of claims 7 to 11, characterized in that it comprises: encoding the antibody The nucleic acid is introduced into the host cell to obtain the host cell stably expressing the bispecific antibody; the host cell is cultured, and the antibody is obtained through separation and purification.
The TROP2 antibody of any one of claims 1 to 5 or the bispecific antibody binding to TROP2 and CD3 of any one of claims 7 to 11 or the nucleic acid molecule of claim 12 or the nucleic acid molecule of claim 13 Any of the following applications of biomaterials:

(1) Application in the preparation of medicines for diagnosing, preventing or treating diseases related to TROP2 expression;

(2) Application in the preparation of drugs for diagnosing, preventing or treating diseases targeting TROP2;

(3) Application in the preparation of drugs for killing cells expressing TROP2;

(4) Application in the preparation of detection reagents for TROP2 and/or CD3;

(5) Application in the preparation of related reagents suitable for CAR-T therapy;

(6) Application in preparation of immunotoxin or labeled antibody.
A multispecific antibody, fusion protein, immunotoxin, drug or detection comprising the TROP2 antibody according to any one of claims 1 to 5 or the bispecific antibody binding to TROP2 and CD3 according to any one of claims 7 to 11 reagent.