CN115232213A

CN115232213A - Bispecific antibodies and uses thereof

Info

Publication number: CN115232213A
Application number: CN202210850692.3A
Authority: CN
Inventors: 曹国帅; 成赢; 李洋洋; 武玉伟
Original assignee: Hefei Tiangang Immune Drugs Co ltd
Current assignee: Hefei Tiangang Immune Drugs Co ltd
Priority date: 2022-07-19
Filing date: 2022-07-19
Publication date: 2022-10-25

Abstract

The invention provides a bispecific antibody and application thereof, wherein the antibody comprises: a first antigen binding region having binding activity for a CD3 molecule; and a second antigen-binding region having TROP2 molecule-binding activity. The bispecific antibody prepared by the invention can simultaneously target CD3 and TROP2, thereby mediating the killing of T cells on tumor cells and having stronger tumor inhibition capability.

Description

Bispecific antibodies and uses thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a bispecific antibody and application thereof, and more particularly relates to a recombinant antibody, a nucleic acid molecule, an expression vector, a recombinant cell, a composition, application of the recombinant antibody or an antigen binding fragment or the nucleic acid molecule or the expression vector or the recombinant cell or the composition in preparation of a medicament, the medicament, application of the recombinant antibody in preparation of a kit and the kit.

Background

The existing cancer treatment means comprise surgical resection, radiotherapy, chemotherapy, small molecule targeted therapy, antibody targeted therapy, macromolecule immunotherapy and other treatment methods, but the methods only play a limited role in part of cancer patients, and the cancer still has a problem of disturbing the life health of human beings.

TROP2 is a type I transmembrane protein, is called human trophoblast surface glycoprotein antigen 2, and has higher structural sequence similarity with epithelial adhesion molecule EPCAM. The research shows that TROP2 is highly expressed in various tumors, including pancreatic cancer, breast cancer, colon cancer, bladder cancer, oral squamous carcinoma, ovarian cancer and the like, and the high expression of TROP2 is closely related to the shortened survival period and poor prognosis of tumor patients, and the results suggest that TROP2 can be a potential tumor treatment target.

Until now, only one TROP 2-targeting drug has been approved, namely the TROP2 antibody-conjugated drug Trodelvy. Trodelvy consists of TROP2 monoclonal antibody and coupled irinotecan, and is essentially the delivery of chemotherapeutic drugs to tumors using antibody targeting.

In recent years, bispecific antibodies have become a focus of research in immunotherapy. The bispecific antibody is an artificial antibody containing two specific antigen binding sites, and can bridge between a target cell (tumor cell) and an effector cell (immune cell) to generate an effect function of targeted killing of tumors. Therefore, the development of a dual-antibody drug targeting CD3 and TROP2 is of great significance for the treatment and prevention of related cancers.

Disclosure of Invention

The present application is based on the discovery and recognition by the inventors of the following facts and problems:

TROP2 is a type I transmembrane protein that is expressed on the surface of myeloid cells, myeloid precursor cells (tumor cells) in acute myeloid patients, but not on the surface of normal stem cells, and thus, therapy targeting TROP2 is a potential therapy for myeloid cell leukemia.

The CD3 antibody single-chain variable fragment (ScFv) can be combined with T cells, the inventor designs a bispecific antibody targeting CD3 and TROP2, and obtains the dominant bispecific antibody CD3 xTROP 2 after a large amount of experimental screening, wherein the bispecific antibody can be simultaneously combined with TROP2 positive tumor cells and T cells, effectively mediates the killing of the T cells on the tumor cells, and has stronger anti-cancer capability.

Accordingly, in a first aspect of the invention, the invention provides a recombinant antibody. According to an embodiment of the invention, comprising: a first antigen binding region having binding activity for a CD3 molecule; and a second antigen-binding region having TROP2 molecule-binding activity. The recombinant antibody provided by the embodiment of the invention can be simultaneously combined with CD3 and TROP2, effectively mediates the killing effect of T cells on tumor cells, and has strong tumor inhibition capability.

In a second aspect of the invention, a nucleic acid molecule is presented. According to an embodiment of the invention, the nucleic acid molecule encodes the recombinant antibody of the first aspect. The recombinant antibody coded by the nucleic acid according to the embodiment of the invention can be simultaneously combined with CD3 and TROP2, effectively mediates the killing effect of T cells on tumor cells, and has stronger tumor inhibition capability.

In a third aspect of the invention, the invention features an expression vector. According to an embodiment of the invention, the nucleic acid molecule of the second aspect is carried. The expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein the control sequence is one or more control sequences that direct the expression of the nucleic acid molecule in a host. The expression vector provided by the embodiment of the invention can efficiently express the recombinant antibody in a suitable host cell, and the recombinant antibody can be simultaneously combined with CD3 and TROP2, effectively mediates the killing effect of T cells on tumor cells, and has stronger tumor inhibition capability.

In a fourth aspect of the invention, a method of making the recombinant antibody of the first aspect is provided. According to an embodiment of the invention, comprising: introducing the expression vector of the third aspect into a cell; culturing the cell under conditions suitable for protein expression and secretion so as to obtain the recombinant antibody. The recombinant antibody can be effectively obtained according to the method provided by the embodiment of the invention, can be combined with CD3 and TROP2 at the same time, effectively mediates the killing effect of T cells on tumor cells, and has stronger tumor inhibition capability.

In a fifth aspect of the invention, a recombinant cell is provided. According to an embodiment of the invention, the recombinant cell carries the nucleic acid of the second aspect, or the expression vector of the third aspect, or is capable of expressing the recombinant antibody of the first aspect. The recombinant cell is obtained by transfection or transformation of the expression vector. According to some embodiments of the present invention, the recombinant cell can express the recombinant antibody in a large amount and with high efficiency under appropriate conditions, and the recombinant antibody can be simultaneously combined with CD3 and TROP2, effectively mediate the killing effect of T cells on tumor cells, and has strong tumor inhibition ability.

In a sixth aspect of the invention, a composition is provided. According to an embodiment of the invention, comprising: the recombinant antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fifth aspect. As described above, the recombinant antibody of the embodiment of the present invention can effectively bind to CD3 and TROP2 protein molecules, and effectively mediate killing effects of T cells on tumor cells, and a composition, such as a food composition, a pharmaceutical composition, and the like, comprising the recombinant antibody or a nucleic acid molecule, an expression vector, or a recombinant cell capable of expressing the recombinant antibody also has a significant effect of treating or preventing tumors.

In a seventh aspect of the invention, the invention provides the use of a recombinant antibody according to the first aspect, a nucleic acid molecule according to the second aspect, an expression vector according to the third aspect, a recombinant cell according to the fifth aspect or a composition according to the sixth aspect for the manufacture of a medicament for the treatment or prevention of myeloid cell leukemia and a TROP 2-positive cancer. As described above, the recombinant antibody of the embodiment of the present invention can effectively bind to CD3 and TROP2 protein molecules, effectively mediate the killing effect of T cells on tumor cells, and drugs comprising a series of substances of the recombinant antibody also have significant effects on treating or preventing cancer.

In an eighth aspect of the invention, a medicament is presented. According to an embodiment of the invention, the medicament comprises: 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. As described above, the recombinant antibody of the embodiment 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, and effectively mediate the killing effect of T cells on tumor cells, so that a drug comprising the recombinant antibody or a series of substances capable of expressing the recombinant antibody also has a significant effect of treating or preventing cancer.

In a ninth aspect, the present invention proposes the use of a recombinant antibody, nucleic acid molecule, expression vector or recombinant cell as described above for the preparation of a kit for the detection of CD3 and/or TROP2. The recombinant antibody can bind to a CD3 and/or TROP2 protein, and under appropriate conditions, the nucleic acid molecule, the expression vector or the recombinant cell can express the recombinant antibody, and a kit can be prepared therefrom, so that the kit comprising the recombinant antibody or the nucleic acid molecule, the expression vector or the recombinant cell capable of expressing the recombinant antibody can be used for effectively detecting CD3 and/or TROP2. The kit can be used for scientific research, such as qualitative or quantitative detection of CD3 and/or TROP2 protein in a biological sample.

In a tenth aspect of the invention, a kit is provided. According to an embodiment of the invention, the kit comprises the recombinant antibody as described above. The recombinant antibody provided according to the embodiments of the present invention can bind to CD3 and/or TROP2 protein, and under suitable conditions, the nucleic acid molecule, the expression vector or the recombinant cell can express the recombinant antibody, and a kit can be prepared therefrom, so that the kit comprising the recombinant antibody or the nucleic acid molecule, the expression vector or the recombinant cell capable of expressing the recombinant antibody can be used for effectively detecting CD3 and/or TROP2. The kit can be used for scientific research, such as qualitative or quantitative detection of CD3 and/or TROP2 protein in a biological sample, and can also be used for judging the individual state, such as judging whether the TROP2 level of the individual is higher or lower than the normal level after obtaining the TROP2 level of the individual.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a schematic structural diagram of a CD3 XTROP 2 recombinant bispecific antibody according to an embodiment of the present invention, in which the linking position of the linking peptide is not given, and the first heavy chain constant region (left side) and the second heavy chain constant region (right side) of the recombinant bispecific antibody are linked by a knob-endo-hole structure;

FIG. 2 is a graph showing the results of the detection of the binding ability of a CD3 XTROP 2 recombinant bispecific antibody to a CD3E & D protein according to the embodiment of the present invention;

FIG. 3 is a graph showing the results of measuring the binding ability of a CD3 XTROP 2 recombinant bispecific antibody to Jurkat T cells according to the embodiment of the present invention;

FIG. 4 is a graph showing the results of measuring the binding ability of the CD3 XTROP 2 recombinant bispecific antibody to human peripheral blood CD8+ T cells according to the embodiment of the present invention;

FIG. 5 is a graph showing the results of measuring the binding ability of the CD3 XTROP 2 recombinant bispecific antibody to CHO-K1-TROP2 cells according to the example of the present invention;

FIG. 6 is a graph showing the results of the measurement of the binding ability of the CD3 XTROP 2 recombinant bispecific antibody to human breast cancer MDA-MB-231 tumor cells according to the embodiment of the present invention;

FIG. 7 is a graph showing the results of detection of activation of Jurkat-NFAT-lucia reporter cells by CD3 XTROP 2 recombinant bispecific antibody according to the embodiment of the present invention;

FIG. 8 is a graph showing the results of the assay of CD3 XTROP 2 recombinant bispecific antibody promoting PBMC killing of A375-TROP2 tumor cells according to the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. 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, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

Herein, "bispecific antibody" refers to a polypeptide obtained by linking a peptide chain specifically recognizing different protein molecules to a heavy chain constant region and/or a light chain constant region, respectively, wherein both chains of the heavy chain constant region are linked by a knob-into-hole structure.

The "Knob-in-hole structure" herein is a button (Knob) knot (hole) mutation formed in the CH3 region of the heavy chain constant region of an antibody to facilitate heavy chain engagement and heterodimer formation, and is achieved, for example, by mutating amino acids in the CH3 region of the heavy chain constant region of human IgG1 (T366S, L368A, Y407V, Y349C mutations in one chain, i.e., "hole", and T366W, S354C mutations in the other chain, i.e., "Knob") in the present application.

"operably linked" herein refers to the attachment of a foreign gene to a vector such that control elements within the vector, such as transcriptional and translational control sequences and the like, are capable of performing their intended function of regulating the transcription and translation of the foreign gene.

In a first aspect of the invention, the invention provides a recombinant antibody comprising: a first antigen binding region having binding activity for a CD3 molecule; and a second antigen-binding region having TROP2 molecule-binding activity. The recombinant antibody provided by the embodiment of the invention can be combined with CD3 and TROP2 at the same time, effectively mediates the killing effect of T cells on tumor cells, and has strong tumor inhibition capability.

According to some embodiments of the invention, the recombinant antibody may further comprise at least one of the following additional technical features:

according to some embodiments of the 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 linked to the N-terminus of the CD3 antibody light chain variable region; or the C end of the CD3 antibody light chain variable region is connected with the N end of the CD3 antibody heavy chain variable region.

According to some embodiments of the invention, the CD3 antibody heavy chain variable region comprises: the heavy chain CDR set forth in any one of SEQ ID NO 1-3.

According to some embodiments of the invention, the CD3 antibody light chain variable region comprises: a light chain CDR set forth in any one of SEQ ID NOs 4-6.

According to some embodiments of the invention, the CD3 antibody heavy chain variable region comprises a heavy chain variable region as set forth in SEQ ID NOs: 1. the amino acid sequence of SEQ ID NO: 2. SEQ ID NO:3, CDR1, CDR2 and CDR3 sequences shown in the specification.

GFTFNTYA(SEQ ID NO：1)。

IRSKYNNYAT(SEQ ID NO：2)。

VRHGNFGNSYVSWFAY(SEQ ID NO：3)。

According to some embodiments of the invention, the CD3 antibody heavy chain variable region comprises a heavy chain variable region as set forth in SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO:3, CDR1, CDR2, CDR3 sequences shown;

according to some embodiments of the invention, the CD3 antibody light chain variable region comprises a heavy chain variable region as set forth in SEQ ID NO: 4. the amino acid sequence of SEQ ID NO: 5. SEQ ID NO:6, CDR1, CDR2, CDR3 sequences shown in;

TGAVTTSNY(SEQ ID NO：4)。

GTN(SEQ ID NO：5)。

ALWYSNLWV(SEQ ID NO：6)。

according to some embodiments of the invention, the CD3 antibody heavy chain variable region comprises: 28, SEQ ID NO.

EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS(SEQ ID NO：28)。

According to some embodiments of the invention, the CD3 antibody light chain variable region comprises: 29, SEQ ID NO.

ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSG SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL(SEQ ID NO：29)。

According to some embodiments of the invention, the CD3 single-chain antibody further comprises a linker peptide 1, wherein the N-terminus of the linker peptide 1 is linked to the C-terminus of the heavy chain variable region of the CD3 antibody, and the C-terminus of the linker peptide 1 is linked to the N-terminus of the light chain variable region of the CD3 antibody; or the N end of the connecting peptide 1 is connected with the C end of the variable region of the light chain of the CD3 antibody, and the C end of the connecting peptide 1 is connected with the N end of the variable region of the heavy chain of the CD3 antibody.

According to some embodiments of the invention, the linker peptide 1 comprises SEQ ID NO: 16.

GGGGSGGGGSGGGGS(SEQ ID NO：16)。

According to some embodiments of the invention, the CD3 single chain antibody comprises the amino acid sequence shown in SEQ ID NO 13.

EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSG GGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPG TPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL(SEQ ID NO：13)。

According to some embodiments of the 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 linked to the N-terminus of the first heavy chain constant region.

According to some embodiments of the invention, the first heavy chain constant region comprises a first hinge region and a first Fc peptide fragment.

According to some embodiments of the invention, the first Fc peptide fragment comprises a first CH2 region and a first CH3 region, the C-terminus of the first CH2 region is linked to the N-terminus of the first CH3 region.

According to some embodiments of the invention, the C-terminus of the first hinge region is linked to the N-terminus of the first Fc peptide fragment.

According to some embodiments of the invention, the first hinge region is a hinge region fragment of a wild-type IgG1 of human, primate or murine origin.

According to some embodiments of the invention, the first CH2 region is a CH2 region fragment of a wild-type IgG1 of human, primate or murine origin.

According to some embodiments of the invention, the first CH3 region has a T366W and/or S354C mutation compared to a CH3 region fragment of a human wild-type IgG 1.

The amino acid sequence of the human wild type IgG1 (L234A/L235A) antibody is as follows:

PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:31)。

according to some embodiments of the invention, the first antigen binding region further comprises a linker peptide 2, the N-terminus of the linker peptide 2 is linked to the C-terminus of the CD3 single chain antibody, and the C-terminus of the linker peptide 2 is linked to the N-terminus of the first heavy chain constant region.

According to some embodiments of the invention, the linker peptide 2 comprises SEQ ID NO: 17.

GGGGS(SEQ ID NO：17)。

According to some embodiments of the invention, the first heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO 18.

PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：18)。

According to some embodiments of the invention, the second antigen binding region comprises a first peptide chain and a second peptide chain, the first peptide chain comprising: TROP2 antibody heavy chain variable region.

According to some embodiments of the invention, the TROP2 antibody heavy chain variable region comprises: the heavy chain CDR set forth in any one of SEQ ID NO 7-9.

According to some embodiments of the invention, the CD3 antibody heavy chain variable region comprises a heavy chain variable region as set forth in SEQ ID NOs: 7. SEQ ID NO: 8. The amino acid sequence of SEQ ID NO:9, CDR1, CDR2, CDR3 sequences shown.

GYTFTNYG(SEQ ID NO：7)。

INTYTGEP(SEQ ID NO：8)。

ARGGFGSSYWYFDV(SEQ ID NO：9)。

According to some embodiments of the invention, the TROP2 antibody heavy chain variable region comprises: 14, SEQ ID NO.

QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTD DFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS(SEQ ID NO： 14)。

According to some embodiments of the invention, 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 linked to the N-terminus of the second heavy chain constant region.

According to some specific embodiments of the invention, the second heavy chain constant region comprises: a CH1 region, a second hinge region, and a second Fc peptide fragment.

According to some embodiments of the invention, the C-terminus of the CH1 region is linked to the N-terminus of the second hinge region, and the C-terminus of the second hinge region is linked to the N-terminus of the second Fc peptide fragment.

According to some specific embodiments of the invention, the second Fc peptide fragment comprises: a second CH2 and a second CH3 region, wherein the C terminal of the second CH2 region is connected with the N terminal of the second CH3 region.

According to some embodiments of the invention, the CH1 region is a CH1 region of a wild-type IgG1 of human, primate or murine origin.

According to some embodiments of the invention, the second hinge region is a hinge region fragment of a wild-type IgG1 of human, primate or murine origin.

According to some embodiments of the invention, the second CH2 region is a CH2 region fragment of a wild-type IgG1 of human, primate or murine origin.

According to some embodiments of the invention, the second CH3 region is a CH3 region fragment of a wild-type IgG1 of human, primate or murine origin.

According to some embodiments of the invention, the second CH3 region has at least one of the T366S, L368A, Y407V, Y349C mutations compared to a CH3 region fragment of human wild type IgG 1.

According to some embodiments of the invention, the second heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO 19.

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：19)。

According to some embodiments of the invention, the second peptide chain comprises: TROP2 antibody light chain variable region.

According to some embodiments of the invention, the TROP2 antibody light chain variable region comprises: the heavy chain CDR set forth in any one of SEQ ID NO 10-12.

According to some embodiments of the invention, the TROP2 antibody light chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 10. SEQ ID NO: 11. the amino acid sequence of SEQ ID NO:12, CDR1, CDR2, CDR3 sequences shown.

QDVSIA(SEQ ID NO：10)。

SAS(SEQ ID NO：11)。

QQHYITPLT(SEQ ID NO：12)。

According to some embodiments of the invention, the TROP2 antibody light chain variable region comprises: 15 or an amino acid sequence shown as SEQ ID NO 15.

DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGS GTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK(SEQ ID NO：15)。

According to some embodiments of the invention, the second peptide chain further comprises a light chain constant region, wherein the C-terminus of the TROP2 antibody light chain variable region is linked to the N-terminus of the light chain constant region.

According to some embodiments of the invention, the light chain constant region is a wild-type light chain constant region of human, primate or murine origin.

According to some embodiments of the invention, the antibody light chain constant region is a human Kappa light chain constant region.

According to some embodiments of the invention, the light chain constant region is a light chain constant region of a human wild-type IgG 1.

According to some embodiments of the invention, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO 30.

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：30)。

According to some embodiments of the invention, the first antigen-binding region and the second antigen-binding region are linked by a knob-into-hole structure.

According to some embodiments of the invention the knob-into-hole structure is formed by a mutation of T366W and/or S354C of the first CH3 region and a mutation of at least one of T366S, L368A, Y407V, Y349C of the second CH3 region.

According to some embodiments of the invention, the first peptide chain and the second peptide chain are linked by an inter-bond disulfide bond.

According to some embodiments of the invention, the recombinant antibody comprises the amino acid sequence shown in SEQ ID NO 20-22.

The first antigen binding region comprises the amino acid sequence:

EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSG GGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPG TPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVS LWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK(SEQ ID NO：20)。

the second antigen binding region-the first peptide chain has the amino acid sequence shown below:

QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTD DFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：21)。

second antigen binding region-second peptide chain has the amino acid sequence shown below:

DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGS GTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC(SEQ ID NO：22)。

in a second aspect of the invention, the invention provides a nucleic acid molecule encoding the recombinant antibody of the first aspect. The recombinant antibody coded by the nucleic acid molecule according to the embodiment of the invention can be simultaneously combined with CD3 and TROP2, effectively mediates the killing effect of T cells on tumor cells, and has stronger tumor inhibition capability.

According to some embodiments of the invention, the nucleic acid has the sequence of SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO: 25.

The nucleic acid molecule encoding the first antigen-binding region comprises the nucleotide sequence:

gaagtgcaattgttggaaagtggaggtgggcttgtccaacctggcggttctctgaagttgagctgcgccgcatctggcttcaccttcaacacatatgccatg aactgggttcggcaggcaccaggaaaggggttggagtgggtcgcacggattaggtctaagtacaataattacgcaacttattacgccgactccgtgaaggacag gtttacaataagtcgtgacgattctaaaaatactgcctacttgcagatgaataatctcaaaacagaggacaccgcagtctactactgtgtgcgacacggaaactttgg taactcatacgtttcttggttcgcttattggggccaggggactttggtgaccgtttctagtggaggaggtggttctggaggtggagggtctggcggaggtggaagtg aattggtggtgactcaggaacctagcctcacagtgtctcctggcggaacagtcacattgacctgcagatcttccaccggcgccgtcaccacctccaattacgctaa ctgggtgcagcagaagcctggtcaggcacctaggggattgatcgggggcactaacaagagagctcctggaactccagccaggttcagtggttccttgctcggc gggaaggcagcacttactctgagcggagtccagcctgaggacgaggctgagtactactgtgccctgtggtacagcaatctgtgggtgtttggcgggggcacaaa actcaccgtgcttggaggtggcggatctccaaagtcttgcgacaagacacatacatgccctccatgtcctgcacctgaagctgccggtggcccaagtgttttcttgtt cccccctaaacctaaagatacactgatgatttccaggacaccagaggtgacatgcgtagttgtggatgtatctcacgaggatccagaggtgaagttcaattggtac gtagacggcgtagaagtgcataacgccaagacaaagccacgggaggagcagtataattcaacctaccgcgtggtttctgtactgaccgtgctccatcaagattgg ctcaatggtaaggagtacaaatgcaaggtcagcaataaagcacttcccgcacccatcgagaaaacaatctccaaggcaaaaggacagccccgcgagcctcag gtttatactctccctccatgcagagaggagatgacaaagaaccaggtgtcactttggtgccttgtcaagggcttctatccatctgatattgccgttgagtgggagtcca acgggcagcctgagaataattataagactactccccccgttcttgattccgacggcagcttctttctctacagcaagcttaccgtcgataagtcaagatggcaacag gggaatgtgttctcttgctccgtgatgcacgaagctttgcataaccattatacccaaaagagtctgtctctctcccccggaaaa(SEQ ID NO：23)。

the nucleic acid molecule encoding the second antigen binding region-the first peptide chain comprises the following nucleotide sequence:

caggtgcagttgcagcaatcaggctctgaactgaagaagcctggagccagtgtgaaggtcagctgcaaggcctccggctacacctttactaactatggtat gaactgggtgaaacaggcccccggtcagggcctgaagtggatgggctggattaatacatacaccggcgaacccacctacacagacgattttaaagggagattc gctttctccctggatacatcagtatcaacagcatatttgcaaatcagttccttgaaagctgatgatacagctgtttacttctgcgcaaggggtgggttcggaagttctta ctggtattttgacgtgtggggacagggttcccttgtgacagtgtcctccgcctctacaaaaggacctagcgttttccccctcgcaccctcctcaaagtctacctccggt gggactgccgcactgggctgtctcgtaaaggattattttcctgaacccgtgactgtgagctggaacagtggtgctttgacttccggagtacatacattccctgctgttc tccagagcagcggactttactctctgtccagcgttgtcaccgttcctagcagttctcttggcactcagacatacatctgcaatgtgaatcacaagcctagcaatacca aagtggataagaaagtcgagccaaagagttgcgacaagacccacacatgccccccctgcccagcccccgaggctgctgggggtccttccgtgttcctgttcccc ccaaagcccaaagacaccctgatgattagtagaactcctgaggtgacctgcgtcgtagtcgatgtgagccatgaagaccccgaagtgaagttcaattggtacgtg gatggcgtcgaggtgcacaacgccaaaactaaaccccgggaagagcagtataactccacttatagggttgtgtccgtgctgactgtcctgcatcaggactggctg aatggcaaggagtacaagtgtaaggtatctaataaggccctgcccgcccctatcgagaaaacaatctctaaagcaaagggccaacccagagaaccccaggtct gcactctgcccccaagtcgggaagagatgactaagaaccaggtatccctcagctgcgccgttaagggcttctatccttccgacatcgcagtcgaatgggagtcaa acgggcagccagagaacaattacaagaccaccccacccgtacttgattccgatggaagtttttttctggtctctaagcttaccgtggataagtctaggtggcagcag ggcaacgtcttttcctgctccgtcatgcatgaggccctgcataatcactacacccagaagtccctgagtctgtcaccaggaaag(SEQ ID NO：24)。

the gene encoding the second antigen-binding region-the first peptide chain has a nucleotide sequence shown below:

gacatccagctgacccagtctcctagttccttgtctgcatcagtgggagaccgggtaagcatcacatgcaaagcctcacaggatgttagtattgct gttgcctggtatcagcaaaagcctggtaaggcacctaaactgttgatttatagtgctagctaccgttacaccggggtgcctgatcgatttagcgggagtg gaagtggtaccgattttactctgaccatctcttccctccagcccgaggatttcgccgtgtattattgtcaacagcattacattacacctctgacattcggtgc agggacaaaggtggagatcaaacgaaccgtggccgctccaagtgtgtttatctttcccccctccgacgaacagctgaagagcgggaccgcttctgtgg tgtgtttgctgaacaatttctaccctcgagaggccaaggtgcagtggaaggtagataatgctctgcagagcgggaattctcaggagtctgtcacagagc aggatagcaaggacagcacctactctttgagctccactctcaccctgtccaaggccgactatgagaaacacaaagtatatgcctgtgaggtgacacatc agggtctctccagtcccgtgaccaaatctttcaataggggggagtgc(SEQ ID NO:25)。

it is to be noted that, for nucleic acids mentioned in the present specification and claims, a person skilled in the art will understand that any one or two of the complementary double strands are actually included. For convenience, in the specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequences in the present application include DNA forms or RNA forms, one of which is disclosed, meaning that the other is also disclosed

In a third aspect, the present invention provides an expression vector carrying the nucleic acid molecule of the second aspect. The expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein the control sequence is one or more control sequences that direct the expression of the nucleic acid molecule in a host. The expression vector provided by the embodiment of the invention can efficiently express the recombinant antibody in a suitable host cell, and the recombinant antibody can be simultaneously combined with CD3 and TROP2, effectively mediates the accurate and efficient killing of T cells on tumor cells, and has stronger tumor inhibition capacity.

In a fourth aspect of the invention, there is provided a method of producing the recombinant antibody of the first aspect, comprising: introducing the expression vector of the third aspect into a cell; culturing the cell under conditions suitable for protein expression and secretion so as to obtain the recombinant antibody. The method provided by some specific embodiments of the present invention can effectively obtain the recombinant antibody, and the recombinant antibody can simultaneously bind to CD3 and TROP2, effectively mediate killing effect of T cells on tumor cells, and has strong tumor inhibition ability. According to some embodiments of the present invention, the cell is not particularly limited, and prokaryotic cells or eukaryotic cells can be used, and the recombinant antibody is expressed efficiently when the cell is eukaryotic cells, such as mammalian cells.

According to some embodiments of the invention, the cell is a eukaryotic cell.

According to some embodiments of the invention, the eukaryotic cell is a mammalian cell. According to some embodiments of the invention, the recombinant antibody is expressed more efficiently when the cell is a eukaryotic cell, such as a mammalian cell.

According to some embodiments of the invention, the eukaryotic cell does not comprise an animal germ cell, a fertilized egg, or an embryonic stem cell.

In a fifth aspect, the present invention provides a recombinant cell carrying the nucleic acid molecule of the second aspect, or the expression vector of the third aspect, or expressing the recombinant antibody of the first aspect. The recombinant cell is obtained by transfection or transformation of the expression vector. According to some embodiments of the present invention, the recombinant cell can efficiently express a large amount of the recombinant antibody under appropriate conditions, and the recombinant antibody can be simultaneously combined with CD3 and TROP2, effectively mediate the killing effect of T cells on tumor cells, and has strong tumor inhibition ability.

It is to be noted that the recombinant cell of the present invention is not particularly limited, and may be a prokaryotic cell, a eukaryotic cell or a phage. The prokaryotic cell can be escherichia coli, bacillus subtilis, streptomyces or proteus mirabilis and the like. The eukaryotic cell can be a fungus such as pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces and trichoderma, an insect cell such as a meadow armyworm, a plant cell such as tobacco, and a mammalian cell such as a BHK cell, a CHO cell, a COS cell and a myeloma cell. In some embodiments, the recombinant cells of the 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.

The term "suitable conditions" as used herein refers to conditions suitable for the expression of the recombinant antibody as described herein. It will be readily understood by those skilled in the art that suitable conditions for recombinant antibody expression include, but are not limited to, suitable transformation or transfection means, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the conditions for the expression of the recombinant antibody optimally according to the specific circumstances of the laboratory.

In a sixth aspect of the invention, the invention provides a composition comprising: the recombinant antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fifth aspect. As described above, the recombinant antibody of the embodiments of the present invention can effectively bind to CD3 and TROP2 protein molecules and promote T cells to kill tumor cells with high efficiency, and compositions, such as food compositions, pharmaceutical compositions, and the like, comprising the recombinant antibody also have significant effects of treating or preventing tumors.

It is to be noted that the compositions include temporally and/or spatially separated combinations as long as they can work together to achieve the objects of the present invention. For example, the ingredients contained in the composition may be administered to the subject in bulk, or separately. When the ingredients contained in the composition are administered separately to a subject, the individual ingredients may be administered to the subject simultaneously or sequentially.

In a seventh aspect of the invention, the invention provides the use of a recombinant antibody according to the first aspect, a nucleic acid molecule according to the second aspect, an expression vector according to the third aspect, a recombinant cell according to the fifth aspect or a composition according to the sixth aspect for the manufacture of a medicament for the treatment or prevention of myeloid cell leukemia and a TROP 2-positive cancer. As described above, the recombinant antibody of the embodiment of the present invention can effectively bind to CD3 and TROP2 protein molecules, so as to promote T cells to kill tumor cells accurately and efficiently, and therefore, drugs comprising a series of substances of the recombinant antibody also have significant effects on treating or preventing myeloid leukemia and TROP 2-positive cancer.

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

according to some specific embodiments of the invention, the TROP 2-positive cancer comprises at least one of: pancreatic cancer, breast cancer, colon cancer, bladder cancer, oral squamous carcinoma, and ovarian cancer.

In an eighth aspect of the invention, there is provided a medicament comprising: 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. As described above, the recombinant antibody of the embodiment of the present invention can effectively bind to CD3 and TROP2 protein molecules, and promote T cells to kill tumor cells accurately and efficiently, so that drugs containing a series of substances of the recombinant antibody also have significant effects on treating or preventing cancer.

According to some embodiments of the present invention, the above-mentioned medicament may further comprise at least one of the following additional technical features:

according to some embodiments of the invention, the medicament is for treating or preventing myeloid cell leukemia and TROP2 positive cancer.

According to some embodiments of the invention, a pharmaceutically acceptable carrier and an effective amount of the antibody active ingredient are included.

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in, and is acceptable to, a human and/or an animal.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The medicament of the invention contains a safe and effective amount of the active ingredient of the invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The medicament preparation is matched with the administration mode generally, and the medicament of the invention is in the dosage forms of injection, oral preparation (tablet, capsule, oral liquid), transdermal agent and sustained release agent. For example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The medicament is preferably manufactured under sterile conditions.

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, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as urgently required by the condition being treated.

The pharmaceutically acceptable carrier of the present invention includes (but is not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptidic substances, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched with the mode of administration, which is well known to those skilled in the art.

In a ninth aspect of the invention, the invention provides the use of a recombinant antibody, nucleic acid molecule, expression vector or recombinant cell as described above in the preparation of a kit for the detection of CD3 and/or TROP2. The recombinant antibody can bind to the CD3 and/or TROP2 protein, and under appropriate conditions, the nucleic acid molecule, the expression vector or the recombinant cell can express the recombinant antibody, and a kit can be prepared therefrom, so that the kit comprising the recombinant antibody or the nucleic acid molecule, the expression vector or the recombinant cell capable of expressing the recombinant antibody can be used for effectively detecting the CD3 and/or TROP2. The kit can be used for scientific research, such as qualitative or quantitative detection of CD3 and/or TROP2 protein in a biological sample.

In a tenth aspect of the invention, the invention provides a kit comprising a recombinant antibody, nucleic acid molecule, expression vector or recombinant cell as described above. The recombinant antibody provided according to the embodiments of the present invention can bind to CD3 and/or TROP2 protein, and the nucleic acid molecule, the expression vector or the recombinant cell can express the recombinant antibody under suitable conditions, and a kit can be prepared therefrom, so that the kit comprising the recombinant antibody or the nucleic acid molecule, the expression vector or the recombinant cell capable of expressing the recombinant antibody can be used for effectively detecting CD3 and/or TROP2. The kit can be used in scientific research, such as qualitative or quantitative detection of CD3 and/or TROP2 protein in a biological sample, and can also be used for judging the state of an individual, such as judging whether the TROP2 level of the individual is higher or lower than a normal level after obtaining the TROP2 level of the individual.

According to some embodiments of the invention, the kit is for detecting CD3 and/or TROP2.

The embodiments will be described in detail below. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 preparation of CD3 XTROP 2 bispecific antibody

This example carries out the production of bispecific antibodies, the specific experimental procedure is as follows: expicCHO cells (purchased from Thermo Fisher) were cultured in ExpicCHO Expression Medium Medium (purchased from Thermo Fisher, A2910001) and adjusted to 6X 10 cell concentration ⁶ Per mL, expicHO cell solution was obtained. pTT5 vector (synthesized by King Kong, suzhou) containing three strands of encoding genes (SEQ ID NOS: 23, 24 and 25, respectively) having a first antigen-binding region and a second antigen-binding region was added to 2mL of OptiSFM medium (Thermo Fisher, 12309019) to obtain solution A. Wherein the first antigen binding region comprises a nucleotide sequence encoding a CD3 single chain antibody (SEQ ID NO: 13), a linker peptide 1 (SEQ ID NO: 16), a linker peptide 2 (SEQ ID NO: 17) and a first heavy chain constant region (SEQ ID NO: 18), and the second antigen binding region-first peptide chain encoding gene comprises 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 μ L Expifeactinamine CHO transfection reagent (Thermofisiher, A29130) was added to 2mL OptiSFM medium to obtain solution B. Then, solution a and solution B were mixed to obtain a transfection mixture, and the transfection mixture was added to 50mL of expichho cell solution in its entirety within 5 minutes. At 37 ℃ C, 5% CO ₂ After culturing under the conditions for 1 day, 8mL of Feed and 300. Mu. Of L hancer (Thermo Fisher, A29130) were added, and the mixture was subjected to transformation at 32 ℃ and 5% CO ₂ Culture supernatants were harvested after 9 days of culture under conditions, with 8mL of Feed added on day 5. Bispecific antibody was affinity purified from the culture supernatant using Protein a purification column (GE) to obtain antibody CD3 × TROP2. The antibody CD3 XTROP 2 has the amino acid sequence as shown 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).

The first antigen binding region encoding gene comprises a nucleotide sequence as shown below:

gaagtgcaattgttggaaagtggaggtgggcttgtccaacctggcggttctctgaagttgagctgcgccgcatctggcttcaccttcaacacatatgccatg aactgggttcggcaggcaccaggaaaggggttggagtgggtcgcacggattaggtctaagtacaataattacgcaacttattacgccgactccgtgaaggacag gtttacaataagtcgtgacgattctaaaaatactgcctacttgcagatgaataatctcaaaacagaggacaccgcagtctactactgtgtgcgacacggaaactttgg taactcatacgtttcttggttcgcttattggggccaggggactttggtgaccgtttctagtggaggaggtggttctggaggtggagggtctggcggaggtggaagtg aattggtggtgactcaggaacctagcctcacagtgtctcctggcggaacagtcacattgacctgcagatcttccaccggcgccgtcaccacctccaattacgctaa ctgggtgcagcagaagcctggtcaggcacctaggggattgatcgggggcactaacaagagagctcctggaactccagccaggttcagtggttccttgctcggc gggaaggcagcacttactctgagcggagtccagcctgaggacgaggctgagtactactgtgccctgtggtacagcaatctgtgggtgtttggcgggggcacaaa actcaccgtgcttggaggtggcggatctccaaagtcttgcgacaagacacatacatgccctccatgtcctgcacctgaagctgccggtggcccaagtgttttcttgtt cccccctaaacctaaagatacactgatgatttccaggacaccagaggtgacatgcgtagttgtggatgtatctcacgaggatccagaggtgaagttcaattggtac gtagacggcgtagaagtgcataacgccaagacaaagccacgggaggagcagtataattcaacctaccgcgtggtttctgtactgaccgtgctccatcaagattgg ctcaatggtaaggagtacaaatgcaaggtcagcaataaagcacttcccgcacccatcgagaaaacaatctccaaggcaaaaggacagccccgcgagcctcag gtttatactctccctccatgcagagaggagatgacaaagaaccaggtgtcactttggtgccttgtcaagggcttctatccatctgatattgccgttgagtgggagtcca acgggcagcctgagaataattataagactactccccccgttcttgattccgacggcagcttctttctctacagcaagcttaccgtcgataagtcaagatggcaacag gggaatgtgttctcttgctccgtgatgcacgaagctttgcataaccattatacccaaaagagtctgtctctctcccccggaaaa(SEQ ID NO:23)。

the second antigen binding region-first peptide chain encoding gene comprises the nucleotide sequence shown as follows:

caggtgcagttgcagcaatcaggctctgaactgaagaagcctggagccagtgtgaaggtcagctgcaaggcctccggctacacctttactaactatggtat gaactgggtgaaacaggcccccggtcagggcctgaagtggatgggctggattaatacatacaccggcgaacccacctacacagacgattttaaagggagattc gctttctccctggatacatcagtatcaacagcatatttgcaaatcagttccttgaaagctgatgatacagctgtttacttctgcgcaaggggtgggttcggaagttctta ctggtattttgacgtgtggggacagggttcccttgtgacagtgtcctccgcctctacaaaaggacctagcgttttccccctcgcaccctcctcaaagtctacctccggt gggactgccgcactgggctgtctcgtaaaggattattttcctgaacccgtgactgtgagctggaacagtggtgctttgacttccggagtacatacattccctgctgttc tccagagcagcggactttactctctgtccagcgttgtcaccgttcctagcagttctcttggcactcagacatacatctgcaatgtgaatcacaagcctagcaatacca aagtggataagaaagtcgagccaaagagttgcgacaagacccacacatgccccccctgcccagcccccgaggctgctgggggtccttccgtgttcctgttcccc ccaaagcccaaagacaccctgatgattagtagaactcctgaggtgacctgcgtcgtagtcgatgtgagccatgaagaccccgaagtgaagttcaattggtacgtg gatggcgtcgaggtgcacaacgccaaaactaaaccccgggaagagcagtataactccacttatagggttgtgtccgtgctgactgtcctgcatcaggactggctg aatggcaaggagtacaagtgtaaggtatctaataaggccctgcccgcccctatcgagaaaacaatctctaaagcaaagggccaacccagagaaccccaggtct gcactctgcccccaagtcgggaagagatgactaagaaccaggtatccctcagctgcgccgttaagggcttctatccttccgacatcgcagtcgaatgggagtcaa acgggcagccagagaacaattacaagaccaccccacccgtacttgattccgatggaagtttttttctggtctctaagcttaccgtggataagtctaggtggcagcag ggcaacgtcttttcctgctccgtcatgcatgaggccctgcataatcactacacccagaagtccctgagtctgtcaccaggaaag(SEQ ID NO:24)。

the second antigen binding region-second peptide chain encoding gene comprises the nucleotide sequence shown as follows:

example 2 identification of the binding Capacity of bispecific antibodies to CD3E & D

An ELISA assay was used to determine the binding properties of the CD3 XTROP 2 antibody obtained in example 1. And coating the CD3E & D protein into a 96-well plate, and judging the binding property of the antibody and the CD3E & D by using the strength of a signal after the antibody is added.

CD3E & D protein (from Acro) was diluted to 2. Mu.g/mL with PBS buffer, added to a 96-well plate in a volume of 100. Mu.L/well, and allowed to stand overnight at 4 ℃. The 96-well plate was aspirated off PBS buffer, and after washing the plate 6 times with PBST (0.1% Tween 20 in PBS, pH7.2), 200. Mu.L/well PBS/10% BSA was added, and blocking was performed by incubation at 37 ℃ for 2h. After removing the blocking solution and washing the plate 6 times with PBST, the test CD3 XTROP 2 antibody was diluted to an appropriate concentration using 100. Mu.L/well of PBST/0.05% BSA and then incubated at 37 ℃ for 1h. The reaction system was removed and after washing the plate 6 times with PBST, HRP (horseradish peroxidase) -labeled rabbit anti-human IgG secondary antibody (Boshide, BA 1070) was diluted with PBST/0.05% BSA at 100. Mu.L/well and incubated at 37 ℃ for 1h. After the completion of incubation, the plate was washed 6 times with PBST, 80. Mu.L/well of TMB (tetramethylbenzidine) was added, the plate was incubated at room temperature for 3min, and the reaction was terminated by adding 80. Mu.L/well of 4M sulfuric acid. The absorbance was read at 450mm using a microplate reader. The results of the specific experiments are shown in fig. 2, indicating that the CD3 × TROP2 antibodies of the invention are capable of binding CD3E & D.

Example 3 characterization of the binding Capacity of bispecific antibodies to Jurkat T cells

This example uses flow cytometry to examine the binding properties of the CD3 × TROP2 bispecific antibody described in example 1 and uses the intensity of the signal after bispecific antibody addition to determine the binding properties of bispecific antibody and Jurkat T. The specific experimental procedures were as follows:

jurkat T cells were diluted 1X 10 with PBS ⁶ (ii)/mL, added to a 1.5mL EP tube in a volume of 90. Mu.L/tube, to which 10. Mu.L/tube rat serum was added, and blocked at 4 ℃ for 30min; after blocking, a series of concentration gradients of 0.1, 1, 10, 30, 100, 300. Mu.g/mL) of CD3 XTROP 2 bispecific antibody and hIgG (control IgG1, biolegend, QA16A 12) 10. Mu.L/tube were added, incubated at 4 ℃ for 30min, 1mL PBS was added to the EP tube, centrifuged at 4 ℃ and 100g for 5min, the supernatant was discarded, the pellet was washed once with PBS, the supernatant was discarded after centrifugation, the cells were resuspended with 100. Mu.L/tube PBS, and 1. Mu.L/tube of Alexa-647-labeled rat anti-human Fc antibody two was added theretoAnti (Biolegend, M1310G 05) and incubated at 4 ℃ for 30min in the absence of light. Washed twice with PBS, centrifuged and the supernatant discarded. The cells were resuspended in 200. Mu.L/tube PBS and assayed by flow cytometry, and the results are shown in FIG. 3, which illustrates that bispecific antibodies of the invention are capable of binding to Jurkat T cells.

Example 4 bispecific antibody and human peripheral blood CD8 ⁺ Characterization of binding Capacity of T cells

This example uses flow cytometry to examine the binding properties of the CD3 XTROP 2 bispecific antibody described in example 1 and to determine the strength of the signal after bispecific antibody addition for use in the determination of bispecific antibody and human peripheral blood CD8 ⁺ T binding properties. The specific experimental procedures were as follows:

human peripheral blood mononuclear cells (Chimaphila sp.) were diluted 5X 10 with PBS ⁶ (ii)/mL, added to a 1.5mL EP tube in a volume of 90. Mu.L/tube, to which 10. Mu.L/tube rat serum was added, and blocked at 4 ℃ for 30min; after blocking, a series of concentration gradients (0.1, 1, 10, 30, 100, 300. Mu.g/mL) of CD3 XTROP 2 bispecific antibody and hIgG (control IgG1, biolegend, QA16A 12) 10. Mu.L/tube were added, incubated at 4 ℃ for 30min, 1mL PBS was added to the EP tube, centrifuged at 4 ℃ and 100G for 5min, the supernatant was discarded, the precipitate was washed once with PBS, the supernatant was discarded after centrifugation, the cells were resuspended in 100. Mu.L/tube PBS, 1. Mu.L/tube of Alexa-647 labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G 05) and 1. Mu.L/tube of FITC labeled mouse anti-human CD8 antibody (Invitrogen, OKT 8) were added thereto, and incubated at 4 ℃ for 30min in the dark. Washed twice with PBS, centrifuged and discarded the supernatant. The cells were resuspended in 200. Mu.L/tube PBS and assayed by flow cytometry, and the results are shown in FIG. 4, which illustrates that the bispecific antibody of the present invention is capable of binding to human peripheral blood T cells.

Example 5 characterization of the binding Capacity of bispecific antibodies to CHO-K1-TROP2 cells

This example uses flow cytometry to examine the binding properties of the bispecific antibody described in example 1 and uses the intensity of the signal after the addition of the bispecific antibody to determine the binding properties of the bispecific antibody and CHO-K1-TROP2 cells. The specific experimental procedures were as follows:

HEK293Tcells were as per 5X 10 ⁵ Cells/well were plated in six-well plates and incubated overnight in DMEM medium without double antibody. The medium was discarded before transfection and 1mL of fresh DMEM medium without double antibody was added. The coding sequence of TROP2 protein (SEQ ID NO: 26), pMD2G and psPAX2 vector (total 3. Mu.g) were inserted between EcoRI and BamHI at the cleavage site of pLVX-EF1a-IRES-puro vector in a ratio of 2 ⁴ To a 6-well plate of CHO-K1 cells, polybrene (Sigma) was added at a final concentration of 4. Mu.g/mL, and the cells were cultured for 12 hours. The supernatant was then discarded and fresh complete DMEM medium was added. The obtained cells were CHO-K1-TROP2 cells.

MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAV DCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSV GVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPT IQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSM KRLTAGLIAVIVVVVVALVAGMAVLVITNRRKSGKYKKVEIKELGELRKEPSL(SEQ ID NO:27)。

CHO-K1-TROP2 cells were diluted 1X 10 with PBS ⁶ To 1.5mL of EP tube was added 90. Mu.L/tube, 10. Mu.L/tube of rat serum was added, and the mixture was blocked at 4 ℃ for 30min. Respectively adding a series of concentration gradients (0.1, 1, 10, 30, 100,300. Mu.g/mL) of CD3 XTROP 2 bispecific antibody, human IgG (control IgG1, biolegend, QA16A 12) 10. Mu.L/tube, and incubated at 4 ℃ for 30min. After the incubation, 1mL of PBS was added to the EP tube, centrifuged at 100g at 4 ℃ for 5min, the supernatant was discarded, and the precipitate was washed with PBS. After centrifugation, the supernatant was discarded, the cells were resuspended in 100. Mu.L/tube of PBS, and after the resuspension, a 1. Mu.L/tube Alexa-647-labeled secondary rat anti-human Fc antibody (Biolegend, M1310G 05) was added thereto, and the mixture was incubated at 4 ℃ for 30min in the absence of light. Washed twice with PBS, centrifuged and discarded the supernatant. The cells were resuspended in 200. Mu.L/tube PBS and assayed by flow cytometry, and the results are shown in FIG. 5, which further shows that the bispecific antibody CD3 XTROP 2 of the present invention is able to bind to CHO-K1-TROP2 cells.

Example 6 characterization of the binding Capacity of bispecific antibodies to human Breast cancer MDA-MB-231

This example uses flow cytometry to examine the binding properties of the bispecific antibody described in example 1 and to determine the binding properties of the bispecific antibody and MDA-MB-231 tumor cells based on the intensity of the signal after the addition of the bispecific antibody. The specific experimental procedures were as follows:

MDA-MB-231 cells were diluted 1X 10 with PBS ⁶ PermL, in a volume of 90. Mu.L/tube in a 1.5mL EP tube, 10. Mu.L/tube rat serum was added thereto, and the tube was sealed at 4 ℃ for 30min. A series of concentration gradients (0.1, 1, 10, 30, 100, 300. Mu.g/mL) of CD3 XTROP 2 bispecific antibody and human IgG (control IgG1, biolegend, QA16A 12) 10. Mu.L/tube were added and incubated at 4 ℃ for 30min. After the incubation, 1mL of PBS was added to the EP tube, centrifuged at 100g at 4 ℃ for 5min, the supernatant was discarded, and the precipitate was washed with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100. Mu.L/tube of PBS, and after resuspension, a 1. Mu.L/tube of Alexa-647-labeled secondary rat anti-human Fc antibody (Biolegend, M1310G 05) was added thereto, and incubated at 4 ℃ for 30min in the absence of light. Washed twice with PBS, centrifuged and discarded the supernatant. The cells were resuspended in 200. Mu.L/tube PBS and assayed by flow cytometry, and the results of the particular experiment are shown in FIG. 6, which further shows that the bispecific antibody CD3 XTROP 2 of the present invention is able to bind to human breast cancer MDA-MB-231 cells.

Example 7: identification of bispecific antibodies promoting activation of Jurkat-NFAT-lucia reporter cells

This example identifies the ability of the bispecific antibody described in example 1 to cross-link TROP2 on the target cell surface and CD3 on the effector cell surface, promoting T cell activation, using Jurkat-NFAT-lucia reporter methodology, and the relative intensity of the chemiluminescent signal (RLU) is used to determine the ability of the bispecific antibody to cross-link the target cell and the T cell, and thereby activate the T cell:

(1) CHO-K1-TROP2 cells were diluted 1X 10 with complete RPMI-1640 medium ⁵ PermL, into a 96-well plate in a volume of 100. Mu.L/well.

(2) CD3 XTROP 2 bispecific antibody was diluted to 500. Mu.g/mL, 100. Mu.g/mL, 20. Mu.g/mL, 4. Mu.g/mL, 0.8. Mu.g/mL, 160ng/mL, 32ng/mL, 6.4ng/mL using complete RPMI-1640 medium, and added to a 96-well plate in a volume of 20. Mu.L/well.

(3) Jurkat-NFAT-lucia cells were diluted to 1.25X 10 with complete RPMI-1640 medium ⁵ mL, added to 96-well plates in a volume of 80. Mu.L/well.

(4) The reaction system obtained in step (3) was subjected to 5% CO at 37 ℃ C ₂ Culturing in an incubator for 24h.

(5) 50 μ L of culture supernatant was aspirated and added to a 96-well plate, and then luciferase substrate was added to the plate in a volume of 50 μ L/well.

(6) Chemiluminescence was detected using a multifunctional microplate reader.

The specific experimental results are shown in fig. 7, and further show that the bispecific antibody CD3 × TROP2 of the present invention can bridge target cells and T cells, promoting T cell activation.

Example 8 bispecific antibodies promote PBMC killing of tumor cells

In this example, the effect of the bispecific antibody obtained in example 1 on killing of A375-TROP2 cells by PBMC was examined by constructing a reaction system of the tumor cells + PBMC + bispecific antibody at different concentrations, as follows:

HEK293T cells according to 5X 10 ⁵ Cells/well were plated in six-well plates and incubated overnight in DMEM medium without double antibody. Medium was discarded before transfection and 1mL of fresh D without diabody was addedMEM medium. The coding sequence of TROP2 protein (SEQ ID NO: 26), pMD2G, and psPAX2 vector (total 3. Mu.g) were inserted between EcoRI and BamHI at the cleavage site of pLVX-EF1a-IRES-puro vector in a ratio of 2 ⁴ A375 cells were plated in 6-well plates and incubated for 12h with polybrene (Sigma) at a final concentration of 4. Mu.g/mL. The supernatant was then discarded and fresh complete DMEM medium was added. The obtained cells are A375-TROP2 cells.

(1) Adding complete RPMI-1640 culture medium into a 16-well RTCA plate according to the volume of 50 mu L/well, and performing on-machine calibration;

(2) A375-TROP2 cells were diluted to 2X 10 with complete RPMI-1640 medium ⁵ mL, added to the RTCA plate obtained in step (1) in a volume of 50. Mu.L/well, respectively, and then CO 5% ₂ Detecting the cell coefficient for 24h by using an xCELLigence RTCA TP device under the condition;

(3) The CD3 XTROP 2 bispecific antibody and the CD27 XTROP 2 bispecific antibody (produced in this laboratory) were diluted to a series of concentration gradients (0.32, 1.6, 8, 40, 200, 1000 ng/mL) with complete RPMI-1640 medium and added to the RTCA plate obtained in step (2) in a volume of 20. Mu.L/well;

(4) PBMC (Cyanin Biopsis) were diluted to 1.25X 10 with complete RPMI-1640 medium ⁶ Adding the solution to the RTCA plate obtained in the step (3) in a volume of 80 mu L/hole;

(5) The reaction system obtained in step (4) was subjected to 5% CO at 37 ℃ C ₂ The cell coefficient was measured using an xCelLigence RTCA TP instrument for 48h.

The specific experimental results are shown in fig. 8, which further shows that the bispecific antibody of the present invention can promote PBMC to kill TROP 2-expressing positive tumor cells.

The experimental results show that the bispecific antibody obtained by the invention can be combined with T cells and tumor cells, bridge the T cells and the tumor cells, and promote the activation of the T cells and the tumor cells.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A recombinant antibody, comprising:

a first antigen binding region having binding activity for a CD3 molecule; and

a second antigen-binding region having TROP2 molecule binding activity.

2. The recombinant antibody according to claim 1, wherein 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, and the C-terminus of the CD3 antibody heavy chain variable region is linked to the N-terminus of the CD3 antibody light chain variable region; or the C end of the CD3 antibody light chain variable region is connected with the N end of the CD3 antibody heavy chain variable region;

optionally, the CD3 antibody heavy chain variable region comprises: the heavy chain CDR set forth in any one of SEQ ID NO 1-3;

optionally, the CD3 antibody light chain variable region comprises: a light chain CDR set forth in any one of SEQ ID NOs 4-6;

optionally, the CD3 antibody heavy chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 1. the amino acid sequence of SEQ ID NO: 2. the amino acid sequence of SEQ ID NO:3, CDR1, CDR2, CDR3 sequences;

optionally, the CD3 antibody light chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 4. the amino acid sequence of SEQ ID NO: 5. SEQ ID NO:6, CDR1, CDR2, CDR3 sequences shown in;

optionally, the CD3 antibody heavy chain variable region comprises: 28 in SEQ ID NO;

optionally, the CD3 antibody light chain variable region comprises: 29, SEQ ID NO.

3. The recombinant antibody according to claim 2, wherein the CD3 single-chain antibody further comprises a linker peptide 1, wherein the N-terminus of the linker peptide 1 is linked to the C-terminus of the heavy chain variable region of the CD3 antibody, and the C-terminus of the linker peptide 1 is linked to the N-terminus of the light chain variable region of the CD3 antibody; or the N end of the connecting peptide 1 is connected with the C end of the variable region of the light chain of the CD3 antibody, and the C end of the connecting peptide 1 is connected with the N end of the variable region of the heavy chain of the CD3 antibody;

optionally, the linker peptide 1 comprises SEQ ID NO: 16;

optionally, the CD3 single chain antibody comprises an amino acid sequence shown in SEQ ID NO. 13.

4. The recombinant antibody of claim 2, wherein said first antigen binding region further comprises a first heavy chain constant region, wherein the C-terminus of said CD3 single chain antibody is linked to the N-terminus of said first heavy chain constant region;

optionally, the first heavy chain constant region comprises a first hinge region and a first Fc peptide segment;

optionally, the first Fc peptide fragment comprises a first CH2 region and a first CH3 region, the C-terminus of the first CH2 region is linked to the N-terminus of the first CH3 region;

optionally, the C-terminus of the first hinge region is linked to the N-terminus of the first Fc peptide fragment;

optionally, the first hinge region is a hinge region fragment of a wild-type IgG1 of human, primate or murine origin;

optionally, the first CH2 region is a CH2 region fragment of a wild-type IgG1 of human, primate or murine origin;

optionally, the first CH3 region has a T366W and/or S354C mutation compared to a CH3 region fragment of a human wild type IgG 1;

optionally, the first antigen binding region further comprises a linker peptide 2, the N-terminus of the linker peptide 2 is linked to the C-terminus of the CD3 single chain antibody, and the C-terminus of the linker peptide 2 is linked to the N-terminus of the first heavy chain constant region;

optionally, the linker peptide 2 comprises SEQ ID NO: 17;

optionally, the first heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO 18.

5. The recombinant antibody according to claim 1, wherein the second antigen binding region comprises a first peptide chain and a second peptide chain, the first peptide chain comprising: TROP2 antibody heavy chain variable region;

optionally, the TROP2 antibody heavy chain variable region comprises: the heavy chain CDR set forth in any one of SEQ ID NO 7-9;

optionally, the CD3 antibody heavy chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 7. the amino acid sequence of SEQ ID NO: 8. SEQ ID NO:9, CDR1, CDR2, CDR3 sequences;

optionally, the TROP2 antibody heavy chain variable region comprises: 14, SEQ ID NO;

optionally, 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 linked to the N-terminus of the second heavy chain constant region;

optionally, the second heavy chain constant region comprises: a CH1 region, a second hinge region, and a second Fc peptide fragment;

optionally, the C-terminus of the CH1 region is linked to the N-terminus of the second hinge region, which is linked to the N-terminus of the second Fc peptide fragment;

optionally, the second Fc peptide fragment comprises: a second CH2 region and a second CH3 region, wherein the C end of the second CH2 region is connected with the N end of the second CH3 region;

optionally, the CH1 region is a CH1 region of a wild-type IgG1 of human, primate or murine origin;

optionally, the second hinge region is a hinge region fragment of a wild-type IgG1 of human, primate or murine origin;

optionally, the second CH2 region is a CH2 region fragment of a wild-type IgG1 of human, primate or murine origin;

optionally, the second CH3 region is a CH3 region fragment of a wild-type IgG1 of human, primate or murine origin;

optionally, the second CH3 region has at least one of T366S, L368A, Y407V, Y349C mutations compared to a CH3 region fragment of human wild type IgG 1;

optionally, the second heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO 19.

6. The recombinant antibody according to claim 1, wherein said second peptide chain comprises: TROP2 antibody light chain variable region;

optionally, the TROP2 antibody light chain variable region comprises: the heavy chain CDR set forth in any one of SEQ ID NO 10-12;

optionally, the TROP2 antibody light chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 10. the amino acid sequence of SEQ ID NO: 11. SEQ ID NO:12, CDR1, CDR2, CDR3 sequences;

optionally, the TROP2 antibody light chain variable region comprises: 15, SEQ ID NO;

optionally, the second peptide chain further comprises a light chain constant region, wherein the C-terminus of the TROP2 antibody light chain variable region is linked to the N-terminus of the light chain constant region;

optionally, the light chain constant region is a wild-type light chain constant region of human, primate, or murine origin;

optionally, the antibody light chain constant region is a human Kappa light chain constant region;

optionally, the light chain constant region is a light chain constant region of a human wild-type IgG 1;

optionally, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO 30.

7. The recombinant antibody according to any one of claims 1 to 6, wherein the first antigen-binding region and the second antigen-binding region are linked by a knob-into-hole structure;

optionally, said knob-into-hole structure is formed by a mutation of T366W and/or S354C of said first CH3 region and a mutation of at least one of T366S, L368A, Y407V, Y349C of said second CH3 region;

optionally, the first peptide chain and the second peptide chain are linked by an inter-bond disulfide bond;

optionally, the recombinant antibody comprises an amino acid sequence shown in SEQ ID NO 20-22.

8. A nucleic acid molecule encoding the recombinant antibody of any one of claims 1 to 7.

9. The nucleic acid of claim 8, wherein the nucleic acid molecule has the sequence of SEQ ID NO: 23-25.

10. An expression vector carrying the nucleic acid molecule of claim 8 or 9.

11. A method of producing the recombinant antibody of any one of claims 1-7, comprising:

introducing the expression vector of claim 10 into a cell;

culturing said cells under conditions suitable for protein expression and secretion so as to obtain said recombinant antibody;

optionally, the cell is a eukaryotic cell.

12. A recombinant cell carrying the nucleic acid molecule of claim 8 or 9, or the expression vector of claim 10, or capable of expressing the recombinant antibody of any one of claims 1-7.

13. A composition, comprising:

the recombinant antibody according to any one of claims 1 to 7, the nucleic acid molecule according to claim 8 or 9, the expression vector according to claim 10 or the recombinant cell according to claim 12.

14. Use of the recombinant antibody of any one of claims 1-7, the nucleic acid molecule of claim 8 or 9, the expression vector of claim 10, the recombinant cell of claim 12, or the composition of claim 13 in the manufacture of a medicament for treating or preventing myeloid cell leukemia and TROP 2-positive cancer.

15. The use according to claim 14, wherein the TROP 2-positive cancer comprises at least one of: pancreatic cancer, breast cancer, colon cancer, bladder cancer, oral squamous carcinoma, and ovarian cancer.

16. A medicament, comprising: the recombinant antibody of any one of claims 1-7, the nucleic acid molecule of claim 8 or 9, the expression vector of claim 10, the recombinant cell of claim 12, or the composition of claim 13.

17. The medicament of claim 16, further comprising a pharmaceutically acceptable excipient;

optionally, the medicament is for the treatment or prevention of myeloid cell leukemia and TROP2 positive cancer;

optionally, the TROP 2-positive cancer comprises at least one of: pancreatic cancer, breast cancer, colon cancer, bladder cancer, oral squamous carcinoma, and ovarian cancer.

18. Use of the recombinant antibody of any one of claims 1-7, the nucleic acid molecule of claim 8 or 9, the expression vector of claim 10, or the recombinant cell of claim 12 in the preparation of a kit for the detection of CD3 and/or TROP2.

19. A kit comprising the recombinant antibody of any one of claims 1-7, the nucleic acid molecule of claim 8 or 9, the expression vector of claim 10, or the recombinant cell of claim 12.

20. The kit of claim 19, wherein the kit is for detecting CD3 and/or TROP2.