Bispecific antibodies and their applications

Abstract

The present invention provides a bispecific antibody and its application. The antibody comprises a first antigen-binding region that has CD3 binding activity and a second antigen-binding region that has TROP2 binding activity. The bispecific antibody prepared by the present invention can simultaneously target CD3 and TROP2, thereby mediating T cell killing of tumor cells and exhibiting strong tumor suppression capabilities.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to bispecific antibodies and applications thereof, and more specifically to recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, the use of the recombinant antibodies or antigen-binding fragments or nucleic acid molecules or expression vectors or recombinant cells or compositions in the preparation of drugs, drugs, the use of the recombinant antibodies in the preparation of kits, and kits. Background Art

[0002] Currently available cancer treatments include surgical resection, radiotherapy, chemotherapy, small molecule targeted therapy, antibody targeted therapy, and large molecule immunotherapy, but these methods only play limited roles in some cancer patients. Cancer remains a difficult problem that plagues human life and health.

[0003] TROP2, a type I transmembrane protein known as human trophoblast cell surface glycoprotein antigen 2, shares a high degree of structural sequence similarity with the epithelial adhesion molecule EPCAM. Studies have found that TROP2 is highly expressed in a variety of tumors, including pancreatic, breast, colon, bladder, oral squamous cell carcinoma, and ovarian cancer. Furthermore, high TROP2 expression is closely associated with shortened survival and poor prognosis in cancer patients. These findings suggest that TROP2 may be a potential target for cancer therapy.

[0004] To date, only one TROP2-targeted drug has been approved: the TROP2 antibody-drug conjugate Trodelvy. Composed of a TROP2 monoclonal antibody and conjugated irinotecan, Trodelvy essentially uses antibodies to target tumors and deliver chemotherapy drugs.

[0005] In recent years, bispecific antibodies have become a research hotspot in immunotherapy. Bispecific antibodies are artificial antibodies containing two specific antigen-binding sites. They can bridge the gap between target cells (tumor cells) and effector cells (immune cells), generating targeted tumor-killing effects. Therefore, the development of bispecific drugs targeting CD3 and TROP2 is of great significance for the treatment and prevention of related cancers. Summary of the Invention

[0006] This application is based on the inventor's discovery and understanding of the following facts and problems:

[0007] TROP2 is a type I transmembrane protein that is expressed on the surface of myeloid cells and myeloid progenitor cells (tumor cells) of acute myeloid leukemia patients, but not on the surface of normal stem cells. Therefore, therapy targeting TROP2 is a potential treatment for myeloid leukemia.

[0008] The single-chain variable fragment (ScFv) of the CD3 antibody can bind to T cells. The inventors designed a bispecific antibody targeting CD3 and TROP2. After a large number of experimental screenings, they obtained the advantageous bispecific antibody CD3×TROP2. This bispecific antibody can simultaneously bind to TROP2-positive tumor cells and T cells, effectively mediating T cell killing of tumor cells and having strong anti-cancer ability.

[0009] Therefore, in its first aspect, the present invention provides a recombinant antibody. According to an embodiment of the present invention, the antibody comprises: a first antigen-binding region having CD3 binding activity; and a second antigen-binding region having TROP2 binding activity. The recombinant antibody according to an embodiment of the present invention can simultaneously bind to CD3 and TROP2, effectively mediating T cell cytotoxicity against tumor cells and exhibiting strong tumor suppression capabilities.

[0010] In a second aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the recombinant antibody described in the first aspect. The recombinant antibody encoded by the nucleic acid according to the embodiment of the present invention can simultaneously bind to CD3 and TROP2, effectively mediating the killing effect of T cells on tumor cells, and exhibiting strong tumor suppression ability.

[0011] In the third aspect of the present invention, the present invention proposes an expression vector. According to an embodiment of the present invention, it carries the nucleic acid molecule described in the second aspect. The expression vector may include an optional control sequence, and the control sequence is operably connected to the nucleic acid molecule. Among them, the control sequence is one or more control sequences that can guide the expression of the nucleic acid molecule in the host. The expression vector proposed in the embodiment of the present invention can efficiently express the recombinant antibody in a suitable host cell, and the recombinant antibody can simultaneously bind to CD3 and TROP2, effectively mediating the killing effect of T cells on tumor cells, and has a strong tumor suppression ability.

[0012] In a fourth aspect, the present invention provides a method for preparing the recombinant antibody described in the first aspect. According to an embodiment of the present invention, the method comprises: introducing the expression vector described in the third aspect into cells; and culturing the cells under conditions suitable for protein expression and secretion to obtain the recombinant antibody. The method provided in an embodiment of the present invention can effectively obtain the recombinant antibody, which can simultaneously bind to CD3 and TROP2, effectively mediating the killing effect of T cells on tumor cells, and exhibiting strong tumor suppression ability.

[0013] In a fifth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the nucleic acid described in the second aspect, or the expression vector described in the third aspect, or is capable of expressing the recombinant antibody described in the first aspect. The recombinant cell is obtained by transfecting or transforming the expression vector. According to some specific embodiments of the present invention, the recombinant cell can efficiently and massively express the above-mentioned recombinant antibody under appropriate conditions. The recombinant antibody can simultaneously bind to CD3 and TROP2, effectively mediating the killing effect of T cells on tumor cells, and has a strong tumor suppression ability.

[0014] In its sixth aspect, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises: the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fifth aspect. As previously mentioned, the recombinant antibodies of the embodiments of the present invention can effectively bind to CD3 and TROP2 protein molecules, effectively mediating the killing effect of T cells on tumor cells. Compositions comprising the recombinant antibodies or nucleic acid molecules, expression vectors, or recombinant cells capable of expressing the recombinant antibodies, such as food compositions and pharmaceutical compositions, also have significant therapeutic or preventive effects on tumors.

[0015] In the seventh aspect of the present invention, the present invention provides the use of the recombinant antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, or the composition of the sixth aspect in the preparation of a medicament for treating or preventing myeloid leukemia and TROP2-positive cancer. As previously mentioned, the recombinant antibodies of the embodiments of the present invention can effectively bind to CD3 and TROP2 protein molecules, effectively mediating the killing effect of T cells on tumor cells. Medicaments containing a series of substances containing the recombinant antibodies also have significant effects in treating or preventing cancer.

[0016] In its eighth aspect, the present invention provides a drug. According to an embodiment of the present invention, the drug comprises: the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect. As previously mentioned, the recombinant antibodies of the embodiments of the present invention can effectively bind to CD3 on the surface of immune cells and TROP2 protein molecules on the surface of tumor cells, effectively mediating the killing effect of T cells on tumor cells. Therefore, drugs containing the recombinant antibodies or a series of substances capable of expressing the recombinant antibodies also have significant effects in treating or preventing cancer.

[0017] In its ninth aspect, the present invention provides the use of the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, or recombinant cells in the preparation of a kit for detecting CD3 and / or TROP2. The recombinant antibodies can bind to CD3 and / or TROP2 proteins. Under appropriate conditions, the nucleic acid molecules, expression vectors, or recombinant cells are capable of expressing the recombinant antibodies, and kits can be prepared using them. Therefore, a kit comprising the recombinant antibodies or nucleic acid molecules, expression vectors, or recombinant cells capable of expressing the recombinant antibodies can be used to effectively detect CD3 and / or TROP2. The kit can be used in scientific research, such as for the qualitative or quantitative detection of CD3 and / or TROP2 proteins in biological samples.

[0018] In the tenth aspect of the present invention, the present invention proposes a kit. According to an embodiment of the present invention, the kit comprises the recombinant antibody described above. The recombinant antibody provided according to the embodiment of the present invention can bind to CD3 and / or TROP2 protein. Under appropriate conditions, the nucleic acid molecule, expression vector or recombinant cell can express the recombinant antibody, and a kit can be prepared therefrom. Therefore, the kit comprising the recombinant antibody or the nucleic acid molecule, expression vector or recombinant cell capable of expressing the recombinant antibody can be used to effectively detect CD3 and / or TROP2. The kit can be used for scientific research, such as qualitatively or quantitatively detecting CD3 and / or TROP2 protein in biological samples, and can also be used to judge the status of an individual, such as after obtaining the TROP2 level of the individual, judging whether the TROP2 level is too high or too low than the normal level.

[0019] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0021] Figure 1 Schematic diagram of the structure of a CD3×TROP2 recombinant bispecific antibody according to an embodiment of the present invention, wherein the connection position of the connecting peptide is not shown, and the first heavy chain constant region (left side) and the second heavy chain constant region (right side) of the recombinant bispecific antibody are connected by a knob-into-hole structure;

[0022] Figure 2 3 is a graph showing the detection results of the binding ability of the CD3×TROP2 recombinant bispecific antibody to the CD3E&D protein according to an embodiment of the present invention;

[0023] Figure 33 is a graph showing the results of the detection of the binding ability of the CD3×TROP2 recombinant bispecific antibody to Jurkat T cells according to an embodiment of the present invention;

[0024] Figure 4 3 is a graph showing the detection results of the binding ability of the CD3×TROP2 recombinant bispecific antibody to human peripheral blood CD8+ T cells according to an embodiment of the present invention;

[0025] Figure 5 3 is a graph showing the detection results of the binding ability of the CD3×TROP2 recombinant bispecific antibody to CHO-K1-TROP2 cells according to an embodiment of the present invention;

[0026] Figure 6 3 is a graph showing the detection results of the binding ability of the CD3×TROP2 recombinant bispecific antibody to human breast cancer MDA-MB-231 tumor cells according to an embodiment of the present invention;

[0027] Figure 7 This is a diagram showing the detection results of activating Jurkat-NFAT-lucia reporter cells using the CD3×TROP2 recombinant bispecific antibody according to an embodiment of the present invention;

[0028] Figure 8 This is a graph showing the detection results of the CD3×TROP2 recombinant bispecific antibody promoting PBMC to kill A375-TROP2 tumor cells according to an embodiment of the present invention. DETAILED DESCRIPTION

[0029] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0030] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0031] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0032] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0033] Herein, "bispecific antibody" refers to an antibody obtained by connecting peptide chains that can specifically recognize different protein molecules to the peptide chains of the heavy chain constant region and / or the light chain constant region, wherein the two chains of the heavy chain constant region are connected by a knobby hole structure.

[0034] The "knob into hole structure" herein refers to a knob-into-hole mutation formed in the CH3 region of the antibody heavy chain constant region to facilitate heavy chain engagement to form a heterodimer. For example, in the present application, this is achieved by mutating amino acids in the CH3 domain of the human IgG1 heavy chain constant region (T366S, L368A, Y407V, and Y349C mutations in one chain, i.e., the "hole"; and T366W and S354C mutations in the other chain, i.e., the "knob").

[0035] Herein, "operably linked" means that the exogenous gene is connected to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, can play their intended function of regulating the transcription and translation of the exogenous gene.

[0036] In its first aspect, the present invention provides a recombinant antibody comprising: a first antigen-binding region having CD3 binding activity; and a second antigen-binding region having TROP2 binding activity. The recombinant antibody according to an embodiment of the present invention can simultaneously bind to CD3 and TROP2, effectively mediating T cell cytotoxicity against tumor cells and exhibiting strong tumor suppression capabilities.

[0037] According to some specific embodiments of the present invention, the above-mentioned recombinant antibody may further include at least one of the following additional technical features:

[0038] According to some specific embodiments of the present invention, the first antigen-binding region comprises a CD3 single-chain antibody, the CD3 single-chain antibody comprises a CD3 antibody heavy chain variable region and a CD3 antibody light chain variable region, the C-terminus of the CD3 antibody heavy chain variable region is connected to the N-terminus of the CD3 antibody light chain variable region; or the C-terminus of the CD3 antibody light chain variable region is connected to the N-terminus of the CD3 antibody heavy chain variable region.

[0039] According to some specific embodiments of the present invention, the CD3 antibody heavy chain variable region includes: a heavy chain CDR shown in any one of SEQ ID NOs: 1-3.

[0040] According to some specific embodiments of the present invention, the CD3 antibody light chain variable region includes: a light chain CDR shown in any one of SEQ ID NOs: 4-6.

[0041] According to some specific embodiments of the present invention, the CD3 antibody heavy chain variable region comprises CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0042] GFTFNTYA (SEQ ID NO: 1).

[0043] IRSKYNNYAT (SEQ ID NO: 2).

[0044] VRHGNFGNSYVSWFAY (SEQ ID NO: 3).

[0045] According to some specific embodiments of the present invention, the CD3 antibody heavy chain variable region comprises CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively;

[0046] According to some specific embodiments of the present invention, the CD3 antibody light chain variable region comprises CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively;

[0047] TGAVTTSNY (SEQ ID NO: 4).

[0048] GTN (SEQ ID NO: 5).

[0049] ALWYSNLWV (SEQ ID NO: 6).

[0050] According to some specific embodiments of the present invention, the CD3 antibody heavy chain variable region comprises: the amino acid sequence shown in SEQ ID NO: 28.

[0051] EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO: 28).

[0052] According to some specific embodiments of the present invention, the CD3 antibody light chain variable region comprises: the amino acid sequence shown in SEQ ID NO:29.

[0053] ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSG SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 29).

[0054] According to some specific embodiments of the present invention, the CD3 single-chain antibody further comprises a connecting peptide 1, wherein the N-terminus of the connecting peptide 1 is connected to the C-terminus of the CD3 antibody heavy chain variable region, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the CD3 antibody light chain variable region; or the N-terminus of the connecting peptide 1 is connected to the C-terminus of the CD3 antibody light chain variable region, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the CD3 antibody heavy chain variable region.

[0055] According to some specific embodiments of the present invention, the connecting peptide 1 comprises the amino acid sequence shown in SEQ ID NO:16.

[0056] GGGGSGGGGSGGGGS (SEQ ID NO: 16).

[0057] According to some specific embodiments of the present invention, the CD3 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:13.

[0058] EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPG TPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 13).

[0059] According to some specific embodiments of the present invention, the first antigen binding region further comprises a first heavy chain constant region, wherein the C-terminus of the CD3 single-chain antibody is connected to the N-terminus of the first heavy chain constant region.

[0060] According to some specific embodiments of the present invention, the first heavy chain constant region includes a first hinge region and a first Fc peptide segment.

[0061] According to some specific embodiments of the present invention, the first Fc peptide segment includes a first CH2 region and a first CH3 region, and the C-terminus of the first CH2 region is connected to the N-terminus of the first CH3 region.

[0062] According to some specific embodiments of the present invention, the C-terminus of the first hinge region is connected to the N-terminus of the first Fc peptide segment.

[0063] According to some specific embodiments of the present invention, the first hinge region is a hinge region fragment of human, primate or mouse wild-type IgG1.

[0064] According to some specific embodiments of the present invention, the first CH2 region is a CH2 region fragment of human, primate or mouse wild-type IgG1.

[0065] According to some specific embodiments of the present invention, the first CH3 region has T366W and / or S354C mutations compared to the CH3 region fragment of human wild-type IgG1.

[0066] The amino acid sequence of the human wild-type IgG1 (L234A / L235A) antibody is:

[0067] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 31).

[0068] According to some specific embodiments of the present invention, the first antigen binding region further includes a connecting peptide 2, the N-terminus of the connecting peptide 2 is connected to the C-terminus of the CD3 single-chain antibody, and the C-terminus of the connecting peptide 2 is connected to the N-terminus of the first heavy chain constant region.

[0069] According to some specific embodiments of the present invention, the connecting peptide 2 includes the amino acid sequence shown in SEQ ID NO:17.

[0070] GGGGS (SEQ ID NO: 17).

[0071] According to some specific embodiments of the present invention, the first heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:18.

[0072] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18).

[0073] According to some specific embodiments of the present invention, the second antigen binding region includes a first peptide chain and a second peptide chain, and the first peptide chain includes: a TROP2 antibody heavy chain variable region.

[0074] According to some specific embodiments of the present invention, the TROP2 antibody heavy chain variable region includes: a heavy chain CDR shown in any one of SEQ ID NOs: 7-9.

[0075] According to some specific embodiments of the present invention, the CD3 antibody heavy chain variable region comprises CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.

[0076] GYTFTNYG (SEQ ID NO: 7).

[0077] INTYTGEP (SEQ ID NO: 8).

[0078] ARGGFGSSYWYFDV (SEQ ID NO: 9).

[0079] According to some specific embodiments of the present invention, the TROP2 antibody heavy chain variable region includes: the amino acid sequence shown in SEQ ID NO:14.

[0080] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTD DFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 14).

[0081] According to some specific embodiments of the present invention, the first peptide chain further includes a second heavy chain constant region, wherein the C-terminus of the TROP2 antibody heavy chain variable region is connected to the N-terminus of the second heavy chain constant region.

[0082] According to some specific embodiments of the present invention, the second heavy chain constant region includes: a CH1 region, a second hinge region and a second Fc peptide segment.

[0083] According to some specific embodiments of the present invention, the C-terminus of the CH1 region is connected to the N-terminus of the second hinge region, and the C-terminus of the second hinge region is connected to the N-terminus of the second Fc peptide segment.

[0084] According to some specific embodiments of the present invention, the second Fc peptide segment includes: a second CH2 and a second CH3 region, wherein the C-terminus of the second CH2 region is connected to the N-terminus of the second CH3 region.

[0085] According to some specific embodiments of the present invention, the CH1 region is the CH1 region of human, primate or mouse wild-type IgG1.

[0086] According to some specific embodiments of the present invention, the second hinge region is a hinge region fragment of human, primate or mouse wild-type IgG1.

[0087] According to some specific embodiments of the present invention, the second CH2 region is a CH2 region fragment of human, primate or mouse wild-type IgG1.

[0088] According to some specific embodiments of the present invention, the second CH3 region is a CH3 region fragment of human, primate or mouse wild-type IgG1.

[0089] According to some specific embodiments of the present invention, the second CH3 region has at least one of T366S, L368A, Y407V, and Y349C mutations compared to the CH3 region fragment of human wild-type IgG1.

[0090] According to some specific embodiments of the present invention, the second heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:19.

[0091] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 19).

[0092] According to some specific embodiments of the present invention, the second peptide chain includes: a TROP2 antibody light chain variable region.

[0093] According to some specific embodiments of the present invention, the TROP2 antibody light chain variable region includes: a heavy chain CDR shown in any one of SEQ ID NOs: 10-12.

[0094] According to some specific embodiments of the present invention, the TROP2 antibody light chain variable region includes CDR1, CDR2, and CDR3 sequences shown as SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

[0095] QDVSIA (SEQ ID NO: 10).

[0096] SAS (SEQ ID NO: 11).

[0097] QQHYITPLT (SEQ ID NO: 12).

[0098] According to some specific embodiments of the present invention, the TROP2 antibody light chain variable region includes: the amino acid sequence shown in SEQ ID NO:15, or the amino acid sequence shown in SEQ ID NO:15.

[0099] DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO: 15).

[0100] According to some specific embodiments of the present invention, the second peptide chain further includes a light chain constant region, wherein the C-terminus of the TROP2 antibody light chain variable region is connected to the N-terminus of the light chain constant region.

[0101] According to some specific embodiments of the present invention, the light chain constant region is a human, primate or mouse wild-type light chain constant region.

[0102] According to some specific embodiments of the present invention, the antibody light chain constant region is a human Kappa light chain constant region.

[0103] According to some specific embodiments of the present invention, the light chain constant region is the light chain constant region of human wild-type IgG1.

[0104] According to some specific embodiments of the present invention, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:30.

[0105] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 30).

[0106] According to some specific embodiments of the present invention, the first antigen binding region and the second antigen binding region are connected via a knob-into-hole structure.

[0107] According to some specific embodiments of the present invention, the knob-into-hole structure is formed by T366W and / or S354C mutations in the first CH3 region and at least one of T366S, L368A, Y407V, and Y349C mutations in the second CH3 region.

[0108] According to some specific embodiments of the present invention, the first peptide chain and the second peptide chain are connected via an inter-bond disulfide bond.

[0109] According to some specific embodiments of the present invention, the recombinant antibody comprises the amino acid sequence shown in SEQ ID NO: 20-22.

[0110] The first antigen-binding region comprises the following amino acid sequence:

[0111] EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPG TPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGSPPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 20).

[0112] The second antigen binding region - the first peptide chain has the following amino acid sequence:

[0113] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTD DFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 21).

[0114] The second antigen binding region - the second peptide chain has the following amino acid sequence:

[0115] DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC (SEQ ID NO: 22).

[0116] In a second aspect, the present invention provides a nucleic acid molecule encoding the recombinant antibody described in the first aspect. The recombinant antibody encoded by the nucleic acid molecule according to the embodiment of the present invention can simultaneously bind to CD3 and TROP2, effectively mediating the killing effect of T cells on tumor cells, and has strong tumor suppression ability.

[0117] According to some specific embodiments of the present invention, the nucleic acid has the nucleotide sequence shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25.

[0118] The nucleic acid molecule encoding the first antigen binding region includes the following nucleotide sequence:

[0119] gaagtgcaattgttggaaagtggaggtgggcttgtccaacctggcggttctctgaagttgagctgcgccgcatctggcttcaccttcaacacatatgccatg aactgggttcggcaggcaccaggaaaggggttggagtgggtcgcacggattaggtctaagtacaataattacgcaacttattacgccgactccgtgaaggacaggtttacaataagtcgtgacgattctaaaaatactgcctacttgcagatgaataatctcaaaacagaggacaccgcagtctactactgtgtgcgacacggaaactttgg taactcatacgtttcttggttcgcttattggggccaggggactttggtgaccgtttctagtggaggaggtggttctggaggtggagggtctggcggaggtggaagtgaattggtggtgactcaggaacctagcctcacagtgtctcctggcggaacagtcacattgacctgcagatcttccaccggcgccgtcaccacctccaattacgctaa ctgggtgcagcagaagcctggtcaggcacctaggggattgatcgggggcactaacaagagagctcctggaactccagccaggttcagtggttccttgctcggcgggaaggcagcacttactctgagcggagtccagcctgaggacgaggctgagtactactgtgccctgtggtacagcaatctgtgggtgtttggcgggggcacaaa actcaccgtgcttggaggtggcggatctccaaagtcttgcgacaagacacatacatgccctccatgtcctgcacctgaagctgccggtggcccaagtgttttcttgttcccccctaaacctaaagatacactgatgatttccaggacaccagaggtgacatgcgtagttgtggatgtatctcacgaggatccagaggtgaagttcaattggtacgtagacggcgtagaagtgcataacgccaagacaaagccacgggaggagcagtataattcaacctaccgcgtggtttctgtactgaccgtgctccatcaagatt ggctcaatggtaaggagtacaaatgcaaggtcagcaataaagcacttcccgcacccatcgagaaaacaatctccaaggcaaaaggacagccccgcgagcctcag gtttatactctccctccatgcagagaggagatgacaaagaaccaggtgtcactttggtgccttgtcaagggcttctatccatctgatattgccgttgagtggggagtcc aacgggcagcctgagaataattataagactactccccccgttcttgattccgacggcagcttctttctctacagcaagcttaccgtcgataagtcaagatggcaacag gggaatgtgttctcttgctccgtgatgcacgaagctttgcataaccattatacccaaaagagtctgtctctctcccccggaaaa (SEQ ID NO: 23).

[0120] The nucleic acid molecule encoding the second antigen binding region-first peptide chain includes the following nucleotide sequence:

[0121] caggtgcagttgcagcaatcaggctctgaactgaagaagcctggagccagtgtgaaggtcagctgcaaggcctccggctacacctttactaactatggtat gaactgggtgaaacaggcccccggtcagggcctgaagtggatgggctggattaatacatacaccggcgaacccacctacacagacgattttaaagggagattcgctttctccctggatacatcagtatcaacagcatatttgcaaatcagttccttgaaagctgatgatacagctgtttacttctgcgcaaggggtgggttcggaagttctta ctggtattttgacgtgtggggacagggttcccttgtgacagtgtcctccgcctctacaaaaggacctagcgttttccccctcgcaccctcctcaaagtctacctccggtgggactgccgcactgggctgtctcgtaaaggattattttcctgaacccgtgactgtgagctggaacagtggtgctttgacttccggagtacatacattccctgctgttc tccagagcagcggactttactctctgtccagcgttgtcaccgttcctagcagttctcttggcactcagacatacatctgcaatgtgaatcacaagcctagcaataccaaagtggataagaaagtcgagccaaagagttgcgacaagacccacacatgccccccctgcccagcccccgaggctgctgggggtccttccgtgttcctgttcccc ccaaagcccaaagacaccctgatgattagtagaactcctgaggtgacctgcgtcgtagtcgatgtgagccatgaagaccccgaagtgaagttcaattggtacgtggatggcgtcgaggtgcacaacgccaaaactaaaccccgggaagagcagtataactccacttatagggttgtgtccgtgctgactgtcctgcatcaggactggctgaatggcaaggagtacaagtgtaaggtatctaataaggccctgcccgcccctatcgagaaaacaatctctaaagcaaagggccaacccagagaaccccaggtctg cactctgcccccaagtcgggaagagatgactaagaaccaggtatccctcagctgcgccgttaagggcttctatccttccgacatcgcagtcgaatgggagtcaa acgggcagccagagaacaattacaagaccacccccacccgtacttgattccgatggaagttttttctggtctctaagcttaccgtggataagtctaggtggcagcagggcaacgtcttttcctgctccgtcatgcatgaggccctgcataatcactacacccagaagtccctgagtctgtcaccaggaaag(SEQ ID NO: 24).

[0122] The gene encoding the second antigen-binding region - the first peptide chain has the nucleotide sequence shown below:

[0123] gacatccagctgacccagtctcctagttccttgtctgcatcagtgggagaccgggtaagcatcacatgcaaagcctcacaggatgttagtattgct gttgcctggtatcagcaaaagcctggtaaggcacctaaactgttgatttatagtgctagctaccgttacaccggggtgcctgatcgatttagcgggagtgg aagtggtaccgattttactctgaccatctcttccctccagcccgaggatttcgccgtgtattattgtcaacagcattacattacacctctgacattcggtgc agggacaaaggtggagatcaaacgaaccgtggccgctccaagtgtgtttatctttcccccctccgacgaacagctgaagagcgggaccgcttctgtgg tgtgtttgctgaacaatttctaccctcgagaggccaaggtgcagtggaaggtagataatgctctgcagagcgggaattctcaggagtctgtcacagagc aggatagcaaggacagcacctactctttgagctccactctcaccctgtccaaggccgactatgagaaacacaaagtatatgcctgtgaggtgacacatc agggtctctccagtcccgtgaccaaatctttcaataggggggagtgc (SEQ ID NO: 25).

[0124] It should be noted that, for the nucleic acids mentioned in the specification and claims of the present invention, those skilled in the art should understand that they actually include any one or both of the complementary double strands. For convenience, in this specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually also disclosed. In addition, the nucleic acid sequences in this application include DNA or RNA forms, and the disclosure of one of them means that the other is also disclosed.

[0125] In the third aspect of the present invention, the present invention proposes an expression vector carrying the nucleic acid molecule described in the second aspect. The expression vector may include an optional control sequence, which is operably connected to the nucleic acid molecule. Among them, the control sequence is one or more control sequences that can guide the expression of the nucleic acid molecule in the host. The expression vector proposed in the embodiment of the present invention can efficiently express the recombinant antibody in a suitable host cell, and the recombinant antibody can simultaneously bind to CD3 and TROP2, effectively mediating the precise and efficient killing of tumor cells by T cells, and has a strong tumor suppression ability.

[0126] In the fourth aspect of the present invention, the present invention proposes a method for preparing the recombinant antibody described in the first aspect, comprising: introducing the expression vector described in the third aspect into cells; culturing the cells under conditions suitable for protein expression and secretion to obtain the recombinant antibody. The method proposed in some specific embodiments of the present invention can effectively obtain the recombinant antibody, which can simultaneously bind to CD3 and TROP2, effectively mediate the killing effect of T cells on tumor cells, and has strong tumor suppression ability. According to some specific embodiments of the present invention, the cells are not particularly limited, and either prokaryotic cells or eukaryotic cells can be used. When the cells are eukaryotic cells, such as mammalian cells, the expression efficiency of the recombinant antibody is higher.

[0127] According to some specific embodiments of the present invention, the cell is a eukaryotic cell.

[0128] According to some embodiments of the present invention, the eukaryotic cell is a mammalian cell. According to some embodiments of the present invention, when the cell is a eukaryotic cell, such as a mammalian cell, the expression efficiency of the recombinant antibody is higher.

[0129] According to some specific embodiments of the present invention, the eukaryotic cells do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0130] In a fifth aspect, the present invention provides a recombinant cell, which carries the nucleic acid molecule described in the second aspect, or the expression vector described in the third aspect, or expresses the recombinant antibody described in the first aspect. The recombinant cell is obtained by transfecting or transforming the expression vector. According to some specific embodiments of the present invention, the recombinant cell can efficiently and massively express the recombinant antibody under appropriate conditions. The recombinant antibody can simultaneously bind to CD3 and TROP2, effectively mediating the killing effect of T cells on tumor cells, and has strong tumor suppression ability.

[0131] It should be noted that the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells may be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Trichoderma, insect cells such as S. frugiperda, plant cells such as tobacco, or mammalian cells such as BHK cells, CHO cells, COS cells, or myeloma cells. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0132] It should be noted that the "suitable conditions" described in this specification refer to conditions suitable for the expression of the recombinant antibodies described herein. It will be readily understood by those skilled in the art that conditions suitable for the expression of recombinant antibodies include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the recombinant antibodies according to the specific environment of the laboratory.

[0133] In its sixth aspect, the present invention provides a composition comprising: the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fifth aspect. As previously mentioned, the recombinant antibodies of the embodiments of the present invention can effectively bind to CD3 and TROP2 protein molecules and promote T cells to accurately and efficiently kill tumor cells. Compositions containing such recombinant antibodies, such as food compositions and pharmaceutical compositions, also have significant therapeutic or preventive effects on tumors.

[0134] It should be noted that the composition includes combinations separated in time and / or space, as long as they can work together to achieve the purpose of the present invention. For example, the components contained in the composition can be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0135] In the seventh aspect of the present invention, the present invention provides the use of the recombinant antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, or the composition of the sixth aspect in the preparation of a medicament for treating or preventing myeloid leukemia and TROP2-positive cancer. As previously mentioned, the recombinant antibodies of the embodiments of the present invention can effectively bind to CD3 and TROP2 protein molecules, prompting T cells to accurately and efficiently kill tumor cells. Therefore, a series of drugs containing the recombinant antibodies also have significant effects in treating or preventing myeloid leukemia and TROP2-positive cancer.

[0136] According to some specific embodiments of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

[0137] According to some specific embodiments of the present invention, the TROP2-positive cancer includes at least one of the following: pancreatic cancer, breast cancer, colon cancer, bladder cancer, oral squamous cell carcinoma and ovarian cancer.

[0138] In an eighth aspect, the present invention provides a drug comprising: the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect. As previously mentioned, the recombinant antibodies of the embodiments of the present invention can effectively bind to CD3 and TROP2 protein molecules, prompting T cells to accurately and efficiently kill tumor cells. Therefore, a drug comprising a series of substances containing the recombinant antibodies also has significant effects in treating or preventing cancer.

[0139] According to some specific embodiments of the present invention, the above-mentioned medicine may further include at least one of the following additional technical features:

[0140] According to some specific embodiments of the present invention, the medicament is used to treat or prevent myeloid leukemia and TROP2-positive cancer.

[0141] According to some specific embodiments of the present invention, the TROP2-positive cancer includes at least one of the following: pancreatic cancer, breast cancer, colon cancer, bladder cancer, oral squamous cell carcinoma and ovarian cancer.

[0142] According to some specific embodiments of the present invention, the present invention comprises a pharmaceutically acceptable carrier and an effective amount of the antibody active ingredient.

[0143] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0144] As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents.

[0145] The medicament of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to) saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be compatible with the mode of administration. The dosage form of the medicament of the present invention is an injection, oral preparation (tablets, capsules, oral liquid), transdermal preparation, or sustained-release preparation. For example, it can be prepared using conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The medicament is preferably manufactured under sterile conditions.

[0146] The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, and the like. For example, depending on the exigencies of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0147] The pharmaceutically acceptable carriers of the present invention include, but are not limited to, water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be compatible with the mode of administration, as is well known to those skilled in the art.

[0148] In its ninth aspect, the present invention provides the use of the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, or recombinant cells in the preparation of a kit for detecting CD3 and / or TROP2. The recombinant antibodies can bind to CD3 and / or TROP2 proteins. Under appropriate conditions, the nucleic acid molecules, expression vectors, or recombinant cells are capable of expressing the recombinant antibodies, and kits can be prepared using them. Therefore, a kit comprising the recombinant antibodies or nucleic acid molecules, expression vectors, or recombinant cells capable of expressing the recombinant antibodies can be used to effectively detect CD3 and / or TROP2. The kit can be used in scientific research, such as for the qualitative or quantitative detection of CD3 and / or TROP2 proteins in biological samples.

[0149] In the tenth aspect of the present invention, the present invention proposes a kit, which comprises the recombinant antibody, nucleic acid molecule, expression vector or recombinant cell described above. The recombinant antibody provided according to the embodiment of the present invention can bind to CD3 and / or TROP2 protein. Under appropriate conditions, the nucleic acid molecule, expression vector or recombinant cell can express the recombinant antibody, and a kit can be prepared therefrom. Therefore, the kit comprising the recombinant antibody or the nucleic acid molecule, expression vector or recombinant cell capable of expressing the recombinant antibody can be used to effectively detect CD3 and / or TROP2. The kit can be used for scientific research, such as qualitatively or quantitatively detecting CD3 and / or TROP2 protein in biological samples, and can also be used to judge the status of an individual, such as after obtaining the TROP2 level of the individual, judging whether the TROP2 level is too high or too low.

[0150] According to some specific embodiments of the present invention, the kit is used to detect CD3 and / or TROP2.

[0151] The following examples are described in detail. Where specific techniques or conditions are not specified in the examples, the experiments were performed according to those described in the literature in the art or according to the product instructions. Reagents or instruments used, for which the manufacturer is not specified, are commercially available conventional products.

[0152] Example 1 Preparation of CD3×TROP2 Bispecific Antibody

[0153] In this example, bispecific antibodies were produced. The specific experimental procedures were as follows: ExpiCHO cells (purchased from Thermo Fisher) were cultured using ExpiCHO Expression Medium (purchased from Thermo Fisher, A2910001) and the cell concentration was adjusted to 6×10 6 / mL to obtain an ExpiCHO cell solution. A pTT5 vector (synthesized by Suzhou Jinweizhi Co., Ltd.) containing the three-chain encoding genes for the first and second antigen-binding regions (shown in SEQ ID NOs: 23, 24, and 25, respectively) was added to 2 mL of OptiSFM medium (Thermo Fisher, 12309019) to obtain Solution A. The first antigen-binding region includes a nucleotide sequence encoding a CD3 single-chain antibody (SEQ ID NO: 13), connecting peptide 1 (SEQ ID NO: 16), connecting peptide 2 (SEQ ID NO: 17), and the first heavy chain constant region (SEQ ID NO: 18). The second antigen-binding region-first peptide chain encoding gene includes a nucleotide sequence encoding a TROP2 heavy chain variable region (SEQ ID NO: 14) and a second heavy chain constant region (SEQ ID NO: 19). 160 μL of ExpiFectamine CHO transfection reagent (Thermo Fisher, A29130) was added to 2 mL of OptiSFM medium to obtain Solution B. Solution A and Solution B were then mixed to obtain a transfection mixture, which was then added to 50 mL of ExpiCHO cell solution within 5 minutes. After culturing for one day at 37°C and 5% CO₂, 8 mL of feed and a 300 μL Enhancer (Thermo Fisher, A29130) were added. The cells were then transferred to 32°C and 5% CO₂ for 9 days of culture, and the culture supernatant was harvested, with 8 mL of feed added on day 5. The bispecific antibody CD3×TROP2 was affinity purified from the culture supernatant using a Protein A purification column (GE) to obtain the antibody CD3×TROP2. The antibody CD3×TROP2 has the amino acid sequences set forth in SEQ ID NO:20 (first antigen-binding region), SEQ ID NO:21 (second antigen-binding region - first peptide chain), and SEQ ID NO:22 (second antigen-binding region - second peptide chain).

[0154] The gene encoding the first antigen-binding region includes the nucleotide sequence shown below:

[0155] gaagtgcaattgttggaaagtggaggtgggcttgtccaacctggcggttctctgaagttgagctgcgccgcatctggcttcaccttcaacacatatgccatg aactgggttcggcaggcaccaggaaaggggttggagtgggtcgcacggattaggtctaagtacaataattacgcaacttattacgccgactccgtgaaggacaggtttacaataagtcgtgacgattctaaaaatactgcctacttgcagatgaataatctcaaaacagaggacaccgcagtctactactgtgtgcgacacggaaactttgg taactcatacgtttcttggttcgcttattggggccaggggactttggtgaccgtttctagtggaggaggtggttctggaggtggagggtctggcggaggtggaagtgaattggtggtgactcaggaacctagcctcacagtgtctcctggcggaacagtcacattgacctgcagatcttccaccggcgccgtcaccacctccaattacgctaa ctgggtgcagcagaagcctggtcaggcacctaggggattgatcgggggcactaacaagagagctcctggaactccagccaggttcagtggttccttgctcggcgggaaggcagcacttactctgagcggagtccagcctgaggacgaggctgagtactactgtgccctgtggtacagcaatctgtgggtgtttggcgggggcacaaa actcaccgtgcttggaggtggcggatctccaaagtcttgcgacaagacacatacatgccctccatgtcctgcacctgaagctgccggtggcccaagtgttttcttgttcccccctaaacctaaagatacactgatgatttccaggacaccagaggtgacatgcgtagttgtggatgtatctcacgaggatccagaggtgaagttcaattggtacgtagacggcgtagaagtgcataacgccaagacaaagccacgggaggagcagtataattcaacctaccgcgtggtttctgtactgaccgtgctccatcaagatt ggctcaatggtaaggagtacaaatgcaaggtcagcaataaagcacttcccgcacccatcgagaaaacaatctccaaggcaaaaggacagccccgcgagcctcag gtttatactctccctccatgcagagaggagatgacaaagaaccaggtgtcactttggtgccttgtcaagggcttctatccatctgatattgccgttgagtggggagtcc aacgggcagcctgagaataattataagactactccccccgttcttgattccgacggcagcttctttctctacagcaagcttaccgtcgataagtcaagatggcaacag gggaatgtgttctcttgctccgtgatgcacgaagctttgcataaccattatacccaaaagagtctgtctctctcccccggaaaa (SEQ ID NO: 23).

[0156] The second antigen binding region-first peptide chain encoding gene includes the nucleotide sequence shown below:

[0157] caggtgcagttgcagcaatcaggctctgaactgaagaagcctggagccagtgtgaaggtcagctgcaaggcctccggctacacctttactaactatggtat gaactgggtgaaacaggcccccggtcagggcctgaagtggatgggctggattaatacatacaccggcgaacccacctacacagacgattttaaagggagattcgctttctccctggatacatcagtatcaacagcatatttgcaaatcagttccttgaaagctgatgatacagctgtttacttctgcgcaaggggtgggttcggaagttctta ctggtattttgacgtgtggggacagggttcccttgtgacagtgtcctccgcctctacaaaaggacctagcgttttccccctcgcaccctcctcaaagtctacctccggtgggactgccgcactgggctgtctcgtaaaggattattttcctgaacccgtgactgtgagctggaacagtggtgctttgacttccggagtacatacattccctgctgttc tccagagcagcggactttactctctgtccagcgttgtcaccgttcctagcagttctcttggcactcagacatacatctgcaatgtgaatcacaagcctagcaataccaaagtggataagaaagtcgagccaaagagttgcgacaagacccacacatgccccccctgcccagcccccgaggctgctgggggtccttccgtgttcctgttcccc ccaaagcccaaagacaccctgatgattagtagaactcctgaggtgacctgcgtcgtagtcgatgtgagccatgaagaccccgaagtgaagttcaattggtacgtggatggcgtcgaggtgcacaacgccaaaactaaaccccgggaagagcagtataactccacttatagggttgtgtccgtgctgactgtcctgcatcaggactggctgaatggcaaggagtacaagtgtaaggtatctaataaggccctgcccgcccctatcgagaaaacaatctctaaagcaaagggccaacccagagaaccccaggtct gcactctgcccccaagtcgggaagagatgactaagaaccaggtatccctcagctgcgccgttaagggcttctatccttccgacatcgcagtcgaatgggagtcaaacgggcagccagagaacaattacaagaccaccccacccgtacttgattccgatggaagtttttttctggtctctaagcttaccgtggataagtctaggtggcagcag ggcaacgtcttttcctgctccgtcatgcatgaggccctgcataatcactacacccagaagtccctgagtctgtcaccaggaaag(SEQ ID NO:24).

[0158] The second antigen-binding region-second peptide chain-encoding gene comprises the nucleotide sequence shown below:

[0159] gacatccagctgacccagtctcctagttccttgtctgcatcagtgggagaccgggtaagcatcacatgcaaagcctcacaggatgttagtattgct gttgcctggtatcagcaaaagcctggtaaggcacctaaactgttgatttatagtgctagctaccgttacaccggggtgcctgatcgatttagcgggagtgg aagtggtaccgattttactctgaccatctcttccctccagcccgaggatttcgccgtgtattattgtcaacagcattacattacacctctgacattcggtgc agggacaaaggtggagatcaaacgaaccgtggccgctccaagtgtgtttatctttcccccctccgacgaacagctgaagagcgggaccgcttctgtgg tgtgtttgctgaacaatttctaccctcgagaggccaaggtgcagtggaaggtagataatgctctgcagagcgggaattctcaggagtctgtcacagagc aggatagcaaggacagcacctactctttgagctccactctcaccctgtccaaggccgactatgagaaacacaaagtatatgcctgtgaggtgacacatcagggtctctccagtcccgtgaccaaatctttcaataggggggagtgc (SEQ ID NO: 25).

[0160] Example 2 Identification of the Binding Ability of Bispecific Antibodies to CD3E & D

[0161] ELISA experiments were used to detect the binding properties of the CD3×TROP2 antibody obtained in Example 1. CD3E&D protein was coated into a 96-well plate, and the strength of the signal after the addition of the antibody was used to determine the binding properties of the antibody and CD3E&D.

[0162] CD3 E&D protein (purchased from Acro) was diluted to 2 μg / mL in PBS buffer and added to a 96-well plate at a volume of 100 μL / well. The plate was incubated at 4°C overnight. The PBS buffer was aspirated from the 96-well plate, and the plate was washed six times with PBST (pH 7.2 PBS containing 0.1% Tween 20). The plate was then blocked by adding 200 μL / well of PBS / 10% BSA and incubating at 37°C for 2 hours. The blocking buffer was removed, and the plate was washed six times with PBST. The CD3×TROP2 antibody to be tested was diluted to the appropriate concentration in 100 μL / well of PBST / 0.05% BSA, and then incubated at 37°C for 1 hour. Remove the reaction system, wash the plate 6 times with PBST, and dilute HRP (horseradish peroxidase) labeled rabbit anti-human IgG secondary antibody (Boster, BA1070) with 100 μL / well PBST / 0.05% BSA, and incubate at 37°C for 1 hour. After incubation, wash the plate 6 times with PBST, add 80 μL / well TMB (tetramethylbenzidine), incubate at room temperature for 3 minutes, and add 80 μL / well 4M sulfuric acid to terminate the reaction. Read the absorbance value at 450mm with a microplate reader. The specific experimental results are as follows. Figure 2 As shown, it was demonstrated that the CD3×TROP2 antibody of the present invention can bind to CD3E&D.

[0163] Example 3 Identification of the Binding Ability of Bispecific Antibodies to Jurkat T Cells

[0164] This example uses flow cytometry to detect the binding properties of the CD3×TROP2 bispecific antibody described in Example 1. The strength of the signal after the addition of the bispecific antibody is used to determine the binding properties of the bispecific antibody to Jurkat T cells. The specific experimental procedures are as follows:

[0165] Dilute Jurkat T cells to 1 × 10 6 / mL, 90 μL / tube were added to 1.5 mL EP tubes, and 10 μL / tube of rat serum was added. The cells were blocked at 4°C for 30 min. After blocking, a series of concentration gradients (0.1, 1, 10, 30, 100, and 300 μg / mL) of CD3×TROP2 bispecific antibody and hIgG (control IgG1, Biolegend, QA16A12) were added at 10 μL / tube. The cells were incubated at 4°C for 30 min. 1 mL of PBS was added to the EP tubes, and the cells were centrifuged at 4°C at 100 g for 5 min. The supernatant was discarded, and the pellet was washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100 μL / tube of PBS. 1 μL / tube of Alexa-647-labeled rat anti-human Fc secondary antibody (Biolegend, M1310G05) was added. The cells were incubated at 4°C for 30 min in the dark. The cells were washed twice with PBS, and the supernatant was discarded after centrifugation. Resuspend the cells with 200 μL / tube PBS and detect them by flow cytometry. The specific experimental results are as follows Figure 3 As shown, it is shown that the bispecific antibody of the present invention can bind to Jurkat T cells.

[0166] Example 4 Bispecific Antibody and Human Peripheral Blood CD8 + Identification of T cell binding capacity

[0167] In this example, flow cytometry was used to detect the binding characteristics of the CD3×TROP2 bispecific antibody described in Example 1, and the strength of the signal after the addition of the bispecific antibody was used to determine the binding characteristics of the bispecific antibody and human peripheral blood CD8 + The binding characteristics of T. The specific experimental procedures are as follows:

[0168] Human peripheral blood mononuclear cells (Sai Li Biotechnology) were diluted to 5×10 6 / mL, added to a volume of 90 μL / tube in a 1.5 mL EP tube, and 10 μL / tube of rat serum was added thereto, and the cells were blocked at 4°C for 30 min. After blocking, a series of concentration gradients (0.1, 1, 10, 30, 100, and 300 μg / mL) of CD3×TROP2 bispecific antibody and hIgG (control IgG1, Biolegend, QA16A12) were added at 10 μL / tube, and incubated at 4°C for 30 min. Then, 1 mL of PBS was added to the EP tube, and the tube was centrifuged at 4°C and 100 g for 5 min. The supernatant was discarded, and the pellet was washed again with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100 μL / tube of PBS. 1 μL / tube of Alexa-647-labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G05) and 1 μL / tube of FITC-labeled mouse anti-human CD8 antibody (Invitrogen, OKT8), incubate at 4℃ in the dark for 30min. Wash twice with PBS, centrifuge and discard the supernatant. Resuspend the cells with 200μL / tube PBS and detect by flow cytometry. The specific experimental results are shown in Figure 2. Figure 4 As shown, it is shown that the bispecific antibody of the present invention can bind to human peripheral blood T cells.

[0169] Example 5 Identification of the Binding Ability of Bispecific Antibodies to CHO-K1-TROP2 Cells

[0170] This example uses flow cytometry to detect the binding properties of the bispecific antibody described in Example 1, and the strength of the signal after the addition of the bispecific antibody is used to determine the binding properties of the bispecific antibody to CHO-K1-TROP2 cells. The specific experimental procedures are as follows:

[0171] HEK293T cells were grown at 5×10 5Plate cells / well in a six-well plate and culture overnight in DMEM medium without the antibody. Before transfection, discard the medium and add 1 mL of fresh DMEM medium without the antibody. pLVX-EF1a-TROP2-IRES-puro (the coding sequence of the TROP2 protein (SEQ ID NO: 26) was inserted between the restriction sites EcoRI and BamHI of the pLVX-EF1a-IRES-puro vector), pMD2G, and psPAX2 vector (3 μg in total) were added to 200 μL of serum-free DMEM medium at a ratio of 2:1:1, and then 12 μg of polyetherimide (PEI, Polysciences Co., Ltd.) was added. The obtained TROP2 protein has the amino acid sequence shown in SEQ ID NO: 27; after mixing, the mixture was allowed to stand for 16 minutes, and then all the liquid was added to the above-mentioned six-well plate containing HEK293T cells. After culturing for 6 hours, the culture medium was discarded and fresh complete DMEM medium was added for culturing. 48 hours after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter (Millipore) to obtain the viral supernatant. All the viral supernatant was added to a 1×10 4 Polybrene (Sigma) was added to a 6-well plate containing CHO-K1 cells at a final concentration of 4 μg / mL and cultured for 12 hours. The supernatant was then discarded and fresh complete DMEM medium was added. The resulting cells are CHO-K1-TROP2 cells.

[0172]

[0173] MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAV DCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSV GVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSM KRLTAGLIAVIVVVVVALVAGMAVLVITNRRKSGKYKKVEIKELGELRKEPSL(SEQ ID NO:27).

[0174] CHO-K1-TROP2 cells were diluted to 1×10 6 / mL, 90 μL / tube were added to 1.5 mL EP tubes. 10 μL / tube of rat serum was added and the cells were blocked at 4°C for 30 min. A series of concentration gradients (0.1, 1, 10, 30, 100, and 300 μg / mL) of CD3×TROP2 bispecific antibody and human IgG (control IgG1, Biolegend, QA16A12) were added at 10 μL / tube and incubated at 4°C for 30 min. After incubation, 1 mL of PBS was added to the EP tubes, and the cells were centrifuged at 100g for 5 min at 4°C. The supernatant was discarded and the pellet was washed once with PBS. After centrifugation, the supernatant was discarded and the cells were resuspended in 100 μL / tube of PBS. After resuspension, 1 μL / tube of Alexa-647-labeled rat anti-human Fc secondary antibody (Biolegend, M1310G05) was added and incubated at 4°C for 30 min in the dark. Wash twice with PBS, centrifuge and discard the supernatant. Resuspend the cells with 200 μL / tube PBS and detect them by flow cytometry. The specific experimental results are as follows Figure 5 As shown, it is further demonstrated that the bispecific antibody CD3×TROP2 of the present invention can bind to CHO-K1-TROP2 cells.

[0175] Example 6 Identification of the Binding Ability of Bispecific Antibodies to Human Breast Cancer MDA-MB-231

[0176] This example uses flow cytometry to detect the binding properties of the bispecific antibody described in Example 1, and the strength of the signal after the addition of the bispecific antibody is used to determine the binding properties of the bispecific antibody to MDA-MB-231 tumor cells. The specific experimental procedures are as follows:

[0177] Dilute MDA-MB-231 cells to 1 × 10 6 / mL, 90 μL / tube were added to 1.5 mL EP tubes. 10 μL / tube of rat serum was added and the cells were blocked at 4°C for 30 min. A series of concentration gradients (0.1, 1, 10, 30, 100, and 300 μg / mL) of CD3×TROP2 bispecific antibody and human IgG (control IgG1, Biolegend, QA16A12) were added at 10 μL / tube and incubated at 4°C for 30 min. After incubation, 1 mL of PBS was added to the EP tubes, and the cells were centrifuged at 100g for 5 min at 4°C. The supernatant was discarded and the pellet was washed once with PBS. After centrifugation, the supernatant was discarded and the cells were resuspended in 100 μL / tube of PBS. After resuspension, 1 μL / tube of Alexa-647-labeled rat anti-human Fc secondary antibody (Biolegend, M1310G05) was added and incubated at 4°C for 30 min in the dark. Wash twice with PBS, centrifuge and discard the supernatant. Resuspend the cells with 200 μL / tube PBS and detect them by flow cytometry. The specific experimental results are as follows Figure 6 As shown, it is further demonstrated that the bispecific antibody CD3×TROP2 of the present invention can bind to human breast cancer MDA-MB-231 cells.

[0178] Example 7: Identification of Bispecific Antibodies Promoting Activation of Jurkat-NFAT-Lucia Reporter Cells

[0179] This example uses the Jurkat-NFAT-Lucia reporter system to identify the ability of the bispecific antibody described in Example 1 to cross-link TROP2 on the surface of target cells and CD3 on the surface of effector cells to promote T cell activation. The relative chemiluminescence signal (RLU) is used to determine the ability of the bispecific antibody to bridge target cells and T cells, thereby activating T cells:

[0180] (1) CHO-K1-TROP2 cells were diluted to 1×10 5 / mL, added to a 96-well plate, with an addition volume of 100 μL / well.

[0181] (2) The CD3×TROP2 bispecific antibody was diluted to 500 μg / mL, 100 μg / mL, 20 μg / mL, 4 μg / mL, 0.8 μg / mL, 160 ng / mL, 32 ng / mL, and 6.4 ng / mL in complete RPMI-1640 medium, and added to a 96-well plate at a volume of 20 μL / well.

[0182] (3) Jurkat-NFAT-lucia cells were diluted to 1.25×10 5 / mL, added to a 96-well plate, with an addition volume of 80 μL / well.

[0183] (4) The reaction system obtained in step (3) was cultured in a 37°C, 5% CO2 incubator for 24 h.

[0184] (5) 50 μL of culture supernatant was aspirated and added to a 96-well plate. Luciferase substrate was then added to the plate at a volume of 50 μL / well.

[0185] (6) Detect chemiluminescence using a multifunctional enzyme reader.

[0186] Specific experimental results such as Figure 7 As shown, it is further demonstrated that the bispecific antibody CD3×TROP2 of the present invention can bridge target cells and T cells and promote T cell activation.

[0187] Example 8 Bispecific Antibodies Promote PBMCs to Kill Tumor Cells

[0188] This example tests the effect of the bispecific antibody obtained in Example 1 on the killing of A375-TROP2 cells by PBMC. The test is performed by constructing a reaction system of tumor cells + PBMC + bispecific antibodies at different concentrations. The specific experimental procedures are as follows:

[0189] HEK293T cells were grown at 5×10 5 Plate cells / well in a six-well plate and culture overnight in DMEM medium without the antibody. Before transfection, discard the medium and add 1 mL of fresh DMEM medium without the antibody. pLVX-EF1a-TROP2-IRES-puro (the coding sequence of the TROP2 protein (SEQ ID NO: 26) was inserted between the restriction sites EcoRI and BamHI of the pLVX-EF1a-IRES-puro vector), pMD2G, and psPAX2 vector (3 μg in total) were added to 200 μL of serum-free DMEM medium at a ratio of 2:1:1, and then 12 μg of polyetherimide (PEI, Polysciences Co., Ltd.) was added. The obtained TROP2 protein has the amino acid sequence shown in SEQ ID NO: 27; after mixing, the mixture was allowed to stand for 16 minutes, and then all the liquid was added to the above-mentioned six-well plate containing HEK293T cells. After culturing for 6 hours, the culture medium was discarded and fresh complete DMEM medium was added for culturing. 48 hours after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter (Millipore) to obtain the viral supernatant. All the viral supernatant was added to a 1×10 4 A375 cells were cultured in a 6-well plate with polybrene (Sigma) at a final concentration of 4 μg / mL and incubated for 12 hours. The supernatant was then discarded and fresh complete DMEM medium was added. The resulting cells are A375-TROP2 cells.

[0190] (1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate the plate.

[0191] (2) A375-TROP2 cells were diluted to 2×10 5 / mL, and added to the RTCA plate obtained in step (1) at a volume of 50 μL / well, and then the cell coefficient was detected using the xCELLigence RTCA TP device at 37°C and 5% CO2 for 24 hours;

[0192] (3) Dilute CD3×TROP2 bispecific antibody and CD27×TROP2 bispecific antibody (produced in our laboratory) into a series of concentration gradients (0.32, 1.6, 8, 40, 200, 1000 ng / mL) using complete RPMI-1640 medium and add them to the RTCA plate obtained in step (2) at a volume of 20 μL / well;

[0193] (4) PBMC (Sai Li Biotechnology) were diluted to 1.25×10 6 pcs / mL, added to the RTCA plate obtained in step (3), with an addition volume of 80 μL / well;

[0194] (5) The reaction system obtained in step (4) was incubated at 37°C and 5% CO2 for 48 hours using an xCELLigence RTCA TP instrument to detect the cell coefficient.

[0195] Specific experimental results such as Figure 8 As shown, it is further shown that the bispecific antibody of the present invention can promote PBMC to kill TROP2-expressing positive tumor cells.

[0196] It can be seen from the above experimental results that the bispecific antibody obtained by the present invention can bind to T cells and tumor cells, bridge T cells and tumor cells, and promote T cell activation and tumor cell killing.

[0197] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0198] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A recombinant antibody, characterized in that include: a first antigen-binding region having CD3 molecule-binding activity; and A second antigen-binding region, wherein the second antigen-binding region has TROP2 molecule binding activity, wherein: The first antigen-binding region comprises a CD3 single-chain antibody, the CD3 single-chain antibody comprising a CD3 antibody heavy chain variable region and a CD3 antibody light chain variable region, the C-terminus of the CD3 antibody heavy chain variable region being connected to the N-terminus of the CD3 antibody light chain variable region; or the C-terminus of the CD3 antibody light chain variable region being connected to the N-terminus of the CD3 antibody heavy chain variable region; the CD3 antibody heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the CD3 antibody light chain variable region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; The first antigen-binding region further comprises a first heavy chain constant region, wherein the C-terminus of the CD3 single-chain antibody is connected to the N-terminus of the first heavy chain constant region; the first heavy chain constant region comprises a first hinge region and a first Fc peptide segment; the first Fc peptide segment comprises a first CH2 region and a first CH3 region, wherein the C-terminus of the first CH2 region is connected to the N-terminus of the first CH3 region; the C-terminus of the first hinge region is connected to the N-terminus of the first Fc peptide segment; the first hinge region is a hinge region fragment of human, primate or murine wild-type IgG1; the first CH2 region is a CH2 region fragment of human, primate or murine wild-type IgG1, and the first heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 18; The second antigen binding region includes a first peptide chain and a second peptide chain, wherein the first peptide chain includes: a TROP2 antibody heavy chain variable region; the TROP2 antibody heavy chain variable region includes SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 9 shows the CDR1, CDR2, and CDR3 sequences; the first peptide chain further comprises a second heavy chain constant region, wherein the C-terminus of the TROP2 antibody heavy chain variable region is connected to the N-terminus of the second heavy chain constant region; the second heavy chain constant region comprises: a CH1 region, a second hinge region, and a second Fc peptide segment; the C-terminus of the CH1 region is connected to the N-terminus of the second hinge region, and the C-terminus of the second hinge region is connected to the N-terminus of the second Fc peptide segment; the second Fc peptide segment comprises: a second CH2 and a second CH3 region, wherein the C-terminus of the second CH2 region is connected to the N-terminus of the second CH3 region; the CH1 region is the CH1 region of human, primate, or mouse wild-type IgG1; the second hinge region is a hinge region fragment of human, primate, or mouse wild-type IgG1; the second CH2 region is a CH2 region fragment of human, primate, or mouse wild-type IgG1; the second CH3 region is a CH3 region fragment of human, primate, or mouse wild-type IgG1, and the second heavy chain constant region comprises SEQ ID The amino acid sequence shown in NO:19; The second peptide chain comprises: a TROP2 antibody light chain variable region; the TROP2 antibody light chain variable region comprises CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; the second peptide chain further comprises a light chain constant region, wherein the C-terminus of the TROP2 antibody light chain variable region is connected to the N-terminus of the light chain constant region; the light chain constant region is a wild-type light chain constant region of human, primate, or mouse origin, and comprises the amino acid sequence shown in SEQ ID NO: 30; The first antigen binding region and the second antigen binding region are connected through a knob-into-hole structure, and the first peptide chain and the second peptide chain are connected through an inter-bond disulfide bond.

2. The recombinant antibody according to claim 1, characterized in that The CD3 antibody heavy chain variable region includes: the amino acid sequence shown in SEQ ID NO:

28.

3. The recombinant antibody according to claim 1, characterized in that The CD3 antibody light chain variable region includes: the amino acid sequence shown in SEQ ID NO:

29.

4. The recombinant antibody according to claim 1, characterized in that The CD3 single-chain antibody further includes a connecting peptide 1, wherein the N-terminus of the connecting peptide 1 is connected to the C-terminus of the CD3 antibody heavy chain variable region, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the CD3 antibody light chain variable region; or the N-terminus of the connecting peptide 1 is connected to the C-terminus of the CD3 antibody light chain variable region, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the CD3 antibody heavy chain variable region.

5. The recombinant antibody according to claim 4, characterized in that The connecting peptide 1 includes the amino acid sequence shown in SEQ ID NO:

16.

6. The recombinant antibody according to claim 1, characterized in that The CD3 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:

13.

7. The recombinant antibody according to claim 1, characterized in that The first CH3 region has T366W and / or S354C mutations compared to the CH3 region fragment of human wild-type IgG1.

8. The recombinant antibody according to claim 1, characterized in that The first antigen binding region further includes a connecting peptide 2, the N-terminus of the connecting peptide 2 is connected to the C-terminus of the CD3 single-chain antibody, and the C-terminus of the connecting peptide 2 is connected to the N-terminus of the first heavy chain constant region.

9. The recombinant antibody according to claim 8, characterized in that The connecting peptide 2 includes the amino acid sequence shown in SEQ ID NO:

17.

10. The recombinant antibody according to claim 1, characterized in that The TROP2 antibody heavy chain variable region includes: the amino acid sequence shown in SEQ ID NO:

14.

11. The recombinant antibody according to claim 1, characterized in that Compared with the CH3 region fragment of human wild-type IgG1, the second CH3 region has at least one of T366S, L368A, Y407V, and Y349C mutations.

12. The recombinant antibody according to claim 1, characterized in that The TROP2 antibody light chain variable region includes: the amino acid sequence shown in SEQ ID NO:

15.

13. The recombinant antibody according to claim 1, characterized in that The antibody light chain constant region is a human Kappa light chain constant region.

14. The recombinant antibody according to claim 1, characterized in that The light chain constant region is a light chain constant region of human wild-type IgG1.

15. The recombinant antibody according to claim 1, characterized in that The knob-into-hole structure is formed by mutations T366W and / or S354C in the first CH3 region and at least one of mutations T366S, L368A, Y407V, and Y349C in the second CH3 region.

16. The recombinant antibody according to claim 1, characterized in that The recombinant antibody comprises the amino acid sequence shown in SEQ ID NO: 20-22.

17. A nucleic acid molecule, characterized in that The nucleic acid encodes the recombinant antibody according to any one of claims 1 to 16.

18. The nucleic acid according to claim 17, characterized in that The nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 23-25.

19. An expression vector, characterized in that Carrying the nucleic acid molecule according to claim 17 or 18.

20. A method for preparing the recombinant antibody according to any one of claims 1 to 16, characterized in that: include: introducing the expression vector according to claim 19 into a cell; The cells are cultured under conditions suitable for protein expression and secretion to obtain the recombinant antibody.

21. The method according to claim 20, characterized in that The cells are eukaryotic cells.

22. A recombinant cell, characterized in that The recombinant cell carries the nucleic acid molecule of claim 17 or 18, or the expression vector of claim 19, or is capable of expressing the recombinant antibody of any one of claims 1 to 16.

23. A composition, characterized in that include: The recombinant antibody according to any one of claims 1 to 16, the nucleic acid molecule according to claim 17 or 18, the expression vector according to claim 19, or the recombinant cell according to claim 22.

24. Use of the recombinant antibody of any one of claims 1-16, the nucleic acid molecule of claim 17 or 18, the expression vector of claim 19, the recombinant cell of claim 22, or the composition of claim 23 in the preparation of a medicament for treating or preventing TROP2-positive cancer, wherein the TROP2-positive cancer includes at least one of the following: pancreatic cancer, breast cancer, colon cancer, bladder cancer, oral squamous cell carcinoma, and ovarian cancer.

25. A drug, characterized in that include: The recombinant antibody of any one of claims 1 to 16, the nucleic acid molecule of claim 17 or 18, the expression vector of claim 19, the recombinant cell of claim 22, or the composition of claim 23.

26. The drug according to claim 25, characterized in that The drug further includes pharmaceutically acceptable excipients.

27. The drug according to claim 25, characterized in that The drug is used for treating or preventing TROP2-positive cancer, which includes at least one of the following: pancreatic cancer, breast cancer, colon cancer, bladder cancer, oral squamous cell carcinoma and ovarian cancer.

28. Use of the recombinant antibody according to any one of claims 1 to 16, the nucleic acid molecule according to claim 17 or 18, the expression vector according to claim 19, or the recombinant cell according to claim 22 in preparing a kit for detecting CD3 and / or TROP2.

29. A kit, characterized in that The kit comprises the recombinant antibody according to any one of claims 1 to 16, the nucleic acid molecule according to claim 17 or 18, the expression vector according to claim 19, or the recombinant cell according to claim 22.

30. The kit according to claim 29, characterized in that The kit is used for detecting CD3 and / or TROP2.

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