Bispecific antibodies and their applications

Abstract

The present invention provides a bispecific antibody and its application, the recombinant antibody comprises a CDR sequence selected from at least one of SEQ ID NOs: 1-6 and B7H6 antibody variable region CDR sequences: SEQ ID NOs: 7-12, or an amino acid sequence having at least 95% identity thereto. The recombinant antibody prepared by the present invention can simultaneously target CD3 and B7H6, and has a significantly extended half-life and a stronger tumor suppression ability than single-targeting antibodies.

Description

[Technical Field]

[0001] The present invention belongs to the field of biopharmaceuticals and specifically relates to bispecific antibodies and their applications, and more specifically to recombinant antibodies, immune cells, nucleic acids, expression vectors, recombinant cells, compositions, the use of the above substances in drug preparation, and kits. [Background technology]

[0002] A bispecific antibody is an antibody that specifically binds to two antigen sites simultaneously. Bispecific antibodies for tumor therapy can be classified into three types based on their mechanism of action: effector cell redirection, immunomodulation, and targeting of double binding of tumor cell receptors. Here, antibodies for redirection function account for the majority, and the two tumor therapy antibodies currently on the market are also based on T cell redirection. Depending on the characteristics of the bispecific antibody, it can also be used in other therapeutic systems, such as for dual immunomodulation or targeting two molecules on the same cell membrane.

[0003] B7H6 is a type I transmembrane protein belonging to the B7 family, containing two immunoglobulin domains. B7H6 is also a ligand for the NK cell activation receptor NKp30. While B7H6 is expressed in many tumor cells, its mRNA has not been detected in normal human tissues or healthy peripheral blood mononuclear cells. In an inflammatory environment, some pro-inflammatory cytokines, such as IL-1β and TNF, can stimulate CD14+ and CD16+ monocytes and neutrophils to upregulate B7H6 expression. B7H6 has a broad tumor expression profile, including in leukemia, lymphoma, colorectal cancer, non-small cell lung cancer, breast cancer, ovarian cancer, gastric cancer, and liver cancer. B7H6 expression is also associated with metastasis in some cancers, but the role of B7H6 in most tumors is currently unknown. Although B7H6 is expressed in many tumors, there are currently few therapeutic options targeting this molecule. Researchers developed a bispecific antibody against B7H6, constructed in the BiTE form. They also determined that this form does not include the constant region of the natural antibody, resulting in a short half-life in the body and significantly limiting its use.

[0004] Among T-cell redirection-based bispecific antibodies, CD3 is a particularly important target. Unlike immune checkpoint inhibitor antibodies, CD3-related bispecific antibodies can mediate T-cell activation across TCRs and peptides (major histocompatibility complexes (pMHCs)), although the molecular mechanism of action at the synapse is very similar to that of conventional TCR-pMHC interactions. The activity of bispecific antibodies is influenced by CD3 affinity; high-affinity CD3 bispecific antibodies exhibit better killing effects in in vitro experiments but possess lethal cytokine release syndrome toxicity in vivo, which is very similar to chimeric antigen receptor (CAR) T-cell toxicity. Simultaneously, studies have shown that even very low-affinity CD3 antibody sequences can effectively stimulate T-cell activation after the construction of bispecific antibodies. When the affinity of the CD3 antibody is within an appropriate range (200 ± 78 nM) and the affinity of the tumor target-related antibody is high, the bispecific antibody encourages T cells to selectively localize to tumors rather than circulate peripherally, thus avoiding systemic activation. Therefore, the development of highly targeted bispecific antibodies has great value for tumor prevention and treatment. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] This application is based on the inventor's discoveries and knowledge regarding the following facts and problems. In this invention, the inventors have performed various combinations and screenings of CD3 and B7H6 antigen-binding fragments to design recombinant bispecific antibodies that specifically bind to CD3 and specifically bind to B7H6, and subsequently deliver the B7H6 molecule to tumor cells in a targeted manner. By binding to the B7H6 molecule on tumor cells, CD3 + These lymphocytes promote the formation of immune synapses with tumor cells, thereby activating B7H6 + The recombinant bispecific antibody kills tumor cells and has high CD3 and B7H6 binding activity, as well as high antitumor activity. [Means for solving the problem]

[0006] Therefore, in a first embodiment of the present invention, the present invention proposes a recombinant antibody. According to an embodiment of the present invention, the recombinant antibody comprises a CDR sequence selected from at least one of SEQ ID NO: 1 to 6 and B7H6 antibody variable region CDR sequences: SEQ ID NO: 7 to 12, or an amino acid sequence having at least 95% identity thereto. The recombinant antibody according to the embodiment of the present invention effectively binds to CD3 and B7H6, has potent in vivo and in vitro binding activity, has a long in vivo half-life, and has a significant antitumor effect.

[0007] RASQDIRNYLN(SEQ ID NO:1). YTSRLES (SEQ ID NO:2). QQGNTLPWT(SEQ ID NO:3). GYTMN (SEQ ID NO: 4). LINPYKGVSTYNQKFKD(SEQ ID NO:5). SGYYGDSDWYFDV (SEQ ID NO: 6). KASQSVDYDGDSYMN(SEQ ID NO:7). AASTLHS (SEQ ID NO: 8). QQSKEDPRT(SEQ ID NO:9). DYNMD (SEQ ID NO: 10). DINPNNGGTLYNQKFRG(SEQ ID NO:11). SEVFYGNYADY (SEQ ID NO: 12).

[0008] In a second aspect of the present invention, the present invention proposes a recombinant antibody. According to the examples of the present invention, the antibody has the amino acid sequences shown in SEQ ID NO: 49 and 63. The recombinant antibody according to the examples of the present invention effectively binds to CD3 and B7H6, has potent in vivo and in vitro binding activity, has a long in vivo half-life, and exhibits significant antitumor effects.

[0009] MDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGSGGGSEGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYGDSDWYFDVWGQGTLVTVSSEPKSCDKTHTCPPCPPAPEAAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:49). DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDTSISTAYMELSRLRSDDTAVYCARSEVGYNYADYWGQGTT VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK(SEQ ID NO:63)。

[0010] In a third aspect of the present invention, the present invention proposes a nucleic acid. According to an embodiment of the present invention, the nucleic acid 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 effectively binds to CD3 and B7H6, has potent in vivo and in vitro binding activity, has a long in vivo half-life, and has a significant antitumor effect.

[0011] In a fourth aspect of the present invention, the present invention proposes an expression vector. According to an embodiment of the present invention, the expression vector supports the nucleic acid molecule described in the third aspect. The expression vector may include a selectable control sequence, the control sequence being operably bound to the nucleic acid molecule, where the control sequence is one or more control sequences capable of directing the expression of the nucleic acid molecule in a host. The expression vector according to an embodiment of the present invention can efficiently and massively express the recombinant antibody in suitable host cells and can be effectively used for the specific treatment or prevention of tumors, particularly tumors expressing B7H6.

[0012] In a fifth aspect of the present invention, the present invention proposes recombinant cells. According to an embodiment of the present invention, the recombinant cells carry a nucleic acid molecule described in the third aspect, an expression vector described in the fourth aspect, or a recombinant antibody described in the first aspect. The recombinant cells are obtained by transfection or transformation with the expression vector. According to an embodiment of the present invention, the recombinant cells can efficiently and in large quantities express the recombinant antibody under appropriate conditions, and the recombinant cells can be effectively used for the specific treatment or prevention of tumors, particularly tumors expressing B7H6.

[0013] In a sixth aspect of the present invention, the present invention proposes a composition. According to the examples of the present invention, the composition comprises a recombinant antibody according to the first or second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, or a recombinant cell according to the fifth aspect. As described above, the recombinant antibody according to the examples of the present invention can effectively bind to CD3 or B7H6 protein molecules and can specifically recognize tumor cells having high expression of B7H6, thereby encouraging T cells to selectively localize to the tumor site rather than circulating peripherally, thus avoiding systemic activation. The drug containing the recombinant antibody also has a significant effect in treating or preventing tumors that express B7H6, with greater safety and fewer side effects.

[0014] In a seventh aspect of the present invention, the present invention proposes the use of a recombinant antibody according to the first or second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect, or a composition according to the sixth aspect in drug preparation. According to the examples of the present invention, the drug is used to treat or prevent tumors. As described above, the recombinant antibody according to the examples of the present invention can effectively bind to CD3 and B7H6 proteins and further specifically recognize tumor cells having high B7H6 expression. Drugs prepared using the recombinant antibody and the corresponding series of substances have a significant effect in treating or preventing tumors that similarly express B7H6, with greater safety and fewer side effects.

[0015] In the eighth aspect of the present invention, the present invention proposes a drug. According to an embodiment of the present invention, the drug includes the recombinant antibody described in the first aspect or the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect or the composition described in the sixth aspect, and the drug is used for the treatment and prevention of cancer. As described above, the recombinant antibody according to the embodiment of the present invention can effectively bind to CD3 and B7H6 proteins, and can further specifically recognize tumor cells with high expression of B7H6. The drug prepared using the recombinant antibody and a series of corresponding substances also has a significant effect on treating or preventing tumors expressing B7H6, and its safety is higher and the side effects are smaller.

[0016] In the ninth aspect of the present invention, the present invention proposes a kit. According to an embodiment of the present invention, the kit includes the recombinant antibody described in the first aspect or the second aspect. The antibody according to the embodiment of the present invention can effectively bind to CD3 and B7H6 protein molecules, and can further specifically recognize highly specific tumor cells expressing B7H6. Therefore, the recombinant antibody can be used to manufacture a kit for diagnosing or detecting at least one of rectal cancer, non-small cell lung cancer, breast cancer and liver cancer. The kit can be used for scientific research such as qualitative or quantitative detection of CD3 and / or B7H6 protein molecules in biological samples.

[0017] Additional aspects and advantages of the present invention will be shown in part from the following description, will become apparent in part from the following description, or will be understood through the practice of the present invention.

Brief Description of the Drawings

[0018] The above and / or additional aspects and advantages of the present invention will become apparent and easier to understand from the description of the embodiments with reference to the following drawings. [Figure 1] It is a structural schematic diagram of a recombinant bispecific antibody according to an embodiment of the present invention. [Figure 2A]This figure shows the results of the binding analysis between the CD3×B7H6 recombinant bispecific antibody according to an example of the present invention and the human B7H6 protein. [Figure 2B] This figure shows the results of the binding analysis between the CD3×B7H6 recombinant bispecific antibody and the human CD3 protein according to an example of the present invention. [Figure 2C] This is a graph showing the ELISA detection results of a CD3×B7H6 recombinant bispecific antibody and B7H6 and human CD3 biantigen according to an example of the present invention. [Figure 3A] This graph shows the analysis results of in vitro cytotoxicity experiments on HCT-15 cells using a CD3×B7H6 recombinant bispecific antibody and T cells according to an example of the present invention. [Figure 3B] This graph shows the analysis results of in vitro cytotoxicity experiments on LoVo cells using a CD3×B7H6 recombinant bispecific antibody and T cells according to an example of the present invention. [Figure 3C] This graph shows the results of in vitro cytotoxicity experiments on Hep G2 cells using a CD3×B7H6 recombinant bispecific antibody and T cells according to an example of the present invention. [Figure 3D] This graph shows the results of in vitro cytotoxicity experiments on SK-BR-3 cells using a CD3×B7H6 recombinant bispecific antibody and T cells according to an example of the present invention. [Figure 4A] This graph shows the analytical results of chemiluminescence values ​​detected after mixing T cells according to the embodiment of the present invention with Ho8910 cells overexpressing luciferase, adding a CD3×B7H6 recombinant bispecific antibody molecule at a gradient concentration, and reacting with a substrate after 24 hours. [Figure 4B] This graph shows the analysis results of chemiluminescence values ​​detected after mixing T cells according to an example of the present invention with K562 cells overexpressing luciferase, adding a CD3×B7H6 bispecific antibody molecule at a gradient concentration, and reacting with a substrate after 24 hours. [Figure 5A] This graph shows the results of the effect of the CD3×B7H6 recombinant bispecific antibody molecule on T cell proliferation when HCT-15 cells according to the embodiment of the present invention are present. [Figure 5B]This is a statistical analysis chart of the effect of the CD3×B7H6 recombinant bispecific antibody molecule on T cell proliferation in the presence of HCT-15 cells according to the embodiment of the present invention. [Figure 6A] This graph shows the effect of the CD3×B7H6 recombinant bispecific antibody molecule on the activation of CD4+ and CD8+ T cells when HCT-15 cells according to the embodiment of the present invention are present. [Figure 6B] This graph shows the effect of the CD3×B7H6 recombinant bispecific antibody molecule on the level of degranulation of CD4+ and CD8+ T cells when HCT-15 cells according to the embodiment of the present invention are present. [Figure 7A] This graph shows the effect of the CD3×B7H6 recombinant bispecific antibody molecule on IFN-γ secretion by T cells when HCT-15 cells according to the embodiment of the present invention are present. [Figure 7B] This graph shows the effect of the CD3×B7H6 recombinant bispecific antibody molecule on IL-2 secretion by T cells when HCT-15 cells according to the embodiment of the present invention are present. [Figure 7C] This graph shows the effect of the CD3×B7H6 recombinant bispecific antibody molecule on IL-10 secretion by T cells when HCT-15 cells according to the embodiment of the present invention are present. [Figure 7D] This graph shows the effect of the CD3×B7H6 recombinant bispecific antibody molecule on IL-17A secretion by T cells when HCT-15 cells according to the embodiment of the present invention are present. [Figure 8] This graph shows the results of the in vivo antitumor activity of the CD3×B7H6 recombinant bispecific antibody molecule on HCT-15 cells according to an example of the present invention. [Modes for carrying out the invention]

[0019] The embodiments of the present invention will be described in detail below, and examples of these embodiments are shown in the drawings. The embodiments described below with reference to the drawings are illustrative and are intended to be interpreted in accordance with the present invention, and should not be understood as limiting the present invention.

[0020] Furthermore, terms such as "first" and "second" are merely for the purpose of explaining the objective and cannot be considered to indicate or implicitly suggest relative importance, or to implicitly indicate a number that explicitly reveals a technical feature. Therefore, a feature defined as "first" or "second" may be explicitly or implicitly indicated to include at least one such feature, and in the description of this invention, unless otherwise specifically and clearly defined, the concept of "multiple" refers to at least two, for example, two or three.

[0021] Recombinant antibody In a first aspect of the present invention, the present invention proposes a recombinant antibody. According to some specific examples of the present invention, the recombinant antibody comprises a first antigen-binding domain that binds to CD3 and a second antigen-binding domain that specifically binds to B7H6. The recombinant antibody according to the examples of the present invention can effectively bind to CD3 on T cells and to B7H6 on tumor cells, has potent in vivo and in vitro binding activity, has a long in vivo half-life, and binds simultaneously to T cells and tumor cells, causing these two types of cells to form cellular synapses and exhibiting a significant antitumor effect.

[0022] According to some specific embodiments of the present invention, the recombinant antibody may further include at least one of the following additional technical features:

[0023] According to some specific embodiments of the present invention, the recombinant antibody comprises a CDR sequence selected from at least one of SEQ ID NO: 1-6 and B7H6 antibody variable region CDR sequences: SEQ ID NO: 7-12, or an amino acid sequence having at least 95% identity thereto. The recombinant antibody according to the embodiments of the present invention effectively binds to CD3 and B7H6, has potent in vivo and in vitro binding activity, has a long in vivo half-life, and exhibits significant antitumor effects.

[0024] RASQDIRNYLN(SEQ ID NO:1). YTSRLES (SEQ ID NO:2). QQGNTLPWT(SEQ ID NO:3). GYTMN (SEQ ID NO: 4). LINPYKGVSTYNQKFKD(SEQ ID NO:5). SGYYGDSDWYFDV (SEQ ID NO: 6). KASQSVDYDGDSYMN(SEQ ID NO:7). AASTLHS (SEQ ID NO: 8). QQSKEDPRT(SEQ ID NO:9). DYNMD (SEQ ID NO: 10). DINPNNGGTLYNQKFRG(SEQ ID NO:11). SEVFYGNYADY (SEQ ID NO: 12).

[0025] According to some specific embodiments of the present invention, a variable region of a CD3 antibody is included, wherein the variable region of the CD3 antibody has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the light chain variable region indicated by SEQ ID NO:13, the heavy chain variable region indicated by SEQ ID NO:14, or the amino acid sequences indicated by SEQ ID NO:13 and SEQ ID NO:14.

[0026] MDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK(SEQ ID NO:13). EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS(SEQ ID NO:14).

[0027] According to some specific embodiments of the present invention, the antibody comprises a variable region of a B7H6 antibody, the variable region of the B7H6 antibody having a light chain variable region indicated by SEQ ID NO:20 and a heavy chain variable region of an amino acid sequence indicated by SEQ ID NO:23, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identity with the amino acid sequences indicated by SEQ ID NO:20 and SEQ ID NO:23.

[0028] DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIK(SEQ ID NO:20). EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:23).

[0029] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the light chain variable region indicated by SEQ ID NO:16 and SEQ ID NO:22.

[0030] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the light chain variable region indicated by SEQ ID NO:16 and SEQ ID NO:23.

[0031] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the light chain variable region indicated by SEQ ID NO:16 and SEQ ID NO:24.

[0032] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the amino acid sequences shown in SEQ ID NO:16 and SEQ ID NO:25.

[0033] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the light chain variable region indicated by SEQ ID NO:17 and SEQ ID NO:22.

[0034] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the light chain variable region indicated by SEQ ID NO:17 and SEQ ID NO:23.

[0035] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the light chain variable region indicated by SEQ ID NO:17 and SEQ ID NO:24.

[0036] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has a light chain variable region indicated by SEQ ID NO:17 and a heavy chain variable region of the amino acid sequence indicated by SEQ ID NO:25, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identity with the amino acid sequences indicated by SEQ ID NO:17 and SEQ ID NO:25.

[0037] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the light chain variable region indicated by SEQ ID NO:19 and SEQ ID NO:22.

[0038] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has a light chain variable region indicated by SEQ ID NO:19 and a heavy chain variable region of the amino acid sequence indicated by SEQ ID NO:23, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identity with the amino acid sequences indicated by SEQ ID NO:19 and SEQ ID NO:23.

[0039] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has a light chain variable region indicated by SEQ ID NO:19 and a heavy chain variable region of the amino acid sequence indicated by SEQ ID NO:24, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identity with the amino acid sequences indicated by SEQ ID NO:19 and SEQ ID NO:24.

[0040] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has a light chain variable region indicated by SEQ ID NO:19 and a heavy chain variable region of the amino acid sequence indicated by SEQ ID NO:25, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identity with the amino acid sequences indicated by SEQ ID NO:19 and SEQ ID NO:25.

[0041] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the light chain variable region indicated by SEQ ID NO:20 and the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO:22, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the amino acid sequences indicated by SEQ ID NO:20 and SEQ ID NO:22.

[0042] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the light chain variable region indicated by SEQ ID NO:20 and the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO:24, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the amino acid sequences indicated by SEQ ID NO:20 and SEQ ID NO:24.

[0043] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the light chain variable region indicated by SEQ ID NO:20 and the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO:25, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the amino acid sequences indicated by SEQ ID NO:20 and SEQ ID NO:25.

[0044] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the light chain variable region indicated by SEQ ID NO:15 and SEQ ID NO:26.

[0045] According to some specific embodiments of the present invention, the variable region of the B7H6 antibody has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 99% identical to the light chain variable region indicated by SEQ ID NO:18 and SEQ ID NO:21.

[0046] DIVLTQSPVSLAVPLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGIPVRFSGSGSGTDFTLNIHPVEEEDAASYYCQQSKEDPRTFGGGTKLEIK(SEQ ID NO:15). DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGVPDRFSGSGSGTFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIK(SEQ ID NO:16). DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGIPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIK(SEQ ID NO:17). DIVLTQSPVSLAVPLGQRATISCKASQSVDYDADSYMNWYQQKPGQPPKLLIYAASTLHSGIPVRFSGSGSGTDFTLNIHPVEEEDAASYYCQQSKEDPRTFGGGTKLEIK(SEQ ID NO:18)。 DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIK(SEQ ID NO:19)。 EVLLQQSGPEVVKPGASVKITCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNNGGTLYNQKFRGRVTLIVDKSSSTAYMELRSLTSDDTAVYYCARSEVFYGNYADYWGQGTTLTVSS(SEQ ID NO:21)。 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWMGDINPNNGGTLYNQKFRGRVTMTVDTSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:22)。 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:24)。 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGKGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:25)。 EVLLQQSGPEVVKPGASVKITCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNNAGTLYNQKFRGRVTLIVDKSSSTAYMELRSLTSDDTAVYYCARSEVFYGNYADYWGQGTTLTVSS(SEQ ID NO:26).

[0047] According to some specific examples of the present invention, the invention further comprises linked peptides.

[0048] According to some specific embodiments of the present invention, the linked peptide has the amino acid sequence shown in SEQ ID NO:46. GGGGSGGGGSGGGGS (SEQ ID NO:46).

[0049] According to some specific embodiments of the present invention, the N-terminus of the linked peptide is linked to the C-terminus of the CD3 antibody light chain variable region, and the C-terminus of the linked peptide is linked to the N-terminus of the CD3 antibody heavy chain variable region.

[0050] According to some specific embodiments of the present invention, the N-terminus of the linked peptide is linked to the C-terminus of the B7H6 light chain variable region, and the C-terminus of the linked peptide is linked to the N-terminus of the B7H6 heavy chain variable region.

[0051] According to some specific embodiments of the present invention, the present invention further comprises a first Fc region and a second Fc region, wherein at least a portion of the first Fc region and the second Fc region is derived from at least one of a mouse-derived antibody, a human-derived antibody, a primate-derived antibody, or a variant thereof.

[0052] According to some specific embodiments of the present invention, at least a portion of the first Fc region and the second Fc region is derived from human IgG or a variant thereof.

[0053] According to some specific embodiments of the present invention, at least a portion of the first Fc region and the second Fc region is derived from human IgG1 or a variant thereof.

[0054] According to some specific embodiments of the present invention, the first Fc region has at least one of the S384C mutation and the T396W mutation compared to the wild-type IgG1 Fc region.

[0055] According to some specific embodiments of the present invention, the second Fc region has at least one of the Y380C mutation, T397S mutation, L399A mutation, and Y408V mutation compared to the wild-type IgG1 Fc region.

[0056] According to some specific embodiments of the present invention, the first antibody Fc region has an amino acid sequence indicated by SEQ ID NO:47, and the second antibody Fc region has an amino acid sequence indicated by SEQ ID NO:48.

[0057] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:47). EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:48).

[0058] In a second aspect of the present invention, the present invention proposes a recombinant antibody. According to the examples of the present invention, the antibody has the amino acid sequences indicated by SEQ ID NO: 49 and 63. The recombinant antibody according to the examples of the present invention can effectively bind to CD3 and B7H6, has potent in vivo and in vitro binding activity, has a long in vivo half-life, and has a significant antitumor effect.

[0059] MDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGG GGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTL VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:49). DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGGSGGGG SGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTT VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:63).

[0060] Preventive or therapeutic composition In a third aspect of the present invention, the present invention proposes a nucleic acid that encodes a recombinant antibody as described above. Recombinant antibodies encoded by nucleic acids according to some specific examples of the present invention can effectively bind to CD3 and B7H6, have potent in vivo and in vitro binding activity, have a long in vivo half-life, and have a significant antitumor effect.

[0061] According to some specific embodiments of the present invention, the nucleic acid has at least one of the nucleotide sequences shown in SEQ ID NO:69 and the nucleotide sequences shown in SEQ ID NO:70-87.

[0062] Nucleotide sequence encoding CD3 antibody

[0063] Nucleotide sequence encoding the B7H6-1 antibody

[0064] Nucleotide sequence encoding the B7H6-2 antibody

[0065] Nucleotide sequence encoding the B7H6-3 antibody

[0066] Nucleotide sequence encoding the B7H6-4 antibody

[0067] Nucleotide sequence encoding the B7H6-5 antibody

[0068] Nucleotide sequence encoding the B7H6-6 antibody

[0069] Nucleotide sequence encoding the B7H6-7 antibody

[0070] Nucleotide sequence encoding the B7H6-8 antibody

[0071] Nucleotide sequence encoding the B7H6-9 antibody

[0072] Nucleotide sequence encoding the B7H6-10 antibody

[0073] Nucleotide sequence encoding the B7H6-11 antibody

[0074] Nucleotide sequence encoding the B7H6-12 antibody

[0075] Nucleotide sequence encoding the B7H6-13 antibody

[0076] Nucleotide sequence encoding the B7H6-14 antibody

[0077] Nucleotide sequence encoding B7H6-15

[0078] Nucleotide sequence encoding the B7H6-16 antibody

[0079] Nucleotide sequence encoding the B7H6-17 antibody

[0080] Nucleotide sequence encoding the B7H6-18 antibody

[0081] It should be understood by those skilled in the art that the nucleic acids referred to in the specification and claims of this invention actually include either one or both of the complementary double strands. For convenience, although only one strand is often shown in this specification and claims, in practice, the other complementary strand is also shown. Furthermore, nucleic acid sequences in this application include either DNA or RNA form, and the disclosure of one means that the other is also disclosed.

[0082] In a fourth aspect of the present invention, the present invention proposes an expression vector that supports the nucleic acid molecule described above. The expression vector may include a selectable control sequence, which is operably bound to the nucleic acid molecule. The control sequence is one or more control sequences that can direct the expression of the nucleic acid molecule in a host. The expression vectors proposed in some specific embodiments of the present invention can efficiently express the recombinant antibody in suitable host cells and can be effectively used for the specific treatment or prevention of tumors, particularly tumors expressing B7H6.

[0083] In a fifth aspect of the present invention, the present invention proposes recombinant cells, the recombinant cells carrying a nucleic acid molecule as described in the third aspect, an expression vector as described in the fourth aspect, or a recombinant antibody as described in the first or second aspect. The recombinant cells are obtained by transfection or transformation with the expression vector. According to some specific examples of the present invention, the recombinant cells can efficiently express the recombinant antibody under suitable conditions, and the recombinant cells can be effectively used for the specific treatment or prevention of tumors, particularly tumors expressing B7H6.

[0084] Furthermore, the “suitable conditions” as described in this specification refer to conditions suitable for the expression of the recombinant antibody described in this application. As will be readily apparent to those skilled in the art, the conditions suitable for the expression of the recombinant antibody include, but are not limited to, a suitable transformation or transfection method, suitable transformation or transfection conditions, a healthy host cell state, a suitable host cell density, a suitable cell culture environment, and a suitable cell culture time. The “suitable conditions” are not particularly limited, and those skilled in the art will optimize the conditions for the expression of the recombinant antibody to the optimal extent according to the specific environment of their laboratory.

[0085] In a sixth aspect of the present invention, the present invention proposes a composition comprising a recombinant antibody according to the first or second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, or a recombinant cell according to the fifth aspect. As described above, the recombinant antibody according to the examples of the present invention effectively binds to CD3 or B7H6 protein molecules, further specifically recognizes tumor cells having high B7H6 expression, and encourages T cells to selectively localize to the tumor site rather than circulating peripherally, thereby avoiding systemic activation. The composition comprising the recombinant antibody and a corresponding series of substances, such as a food composition or a drug composition, similarly has a significant effect in treating or preventing tumors that express B7H6, with greater safety and fewer side effects.

[0086] In an eighth aspect of the present invention, the present invention proposes a drug comprising a recombinant antibody according to the first or second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect, or a composition according to the sixth aspect, wherein the drug is used for the treatment and prevention of cancer. As described above, the recombinant antibody according to the examples of the present invention effectively binds to CD3 or B7H6 protein molecules and further specifically recognizes tumor cells having high B7H6 expression, promoting the selective localization of T cells to the tumor site rather than circulating peripherally, thus avoiding systemic activation. Drugs containing the recombinant antibody system also have a significant effect in treating or preventing tumors that express B7H6, with greater safety and fewer side effects.

[0087] According to some specific embodiments of the present invention, the cancer includes at least one of rectal cancer, non-small cell lung cancer, breast cancer, and liver cancer.

[0088] The drugs according to some specific embodiments of the present invention comprise a pharmaceutically acceptable vector and an effective amount of the antibody active component.

[0089] As used herein, the terms “effective dose” or “effective dosage” refer to the amount that produces function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0090] As used herein, a “pharmaceutically acceptable” ingredient is a substance that is applicable to humans and / or mammals but without excessive adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., has a reasonable benefit / risk ratio. The term “pharmaceutically acceptable vector” refers to a vector used to administer therapeutic agents, including a variety of excipients and diluents.

[0091] The drug of the present invention comprises a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable vector. Such vectors include (but are not limited to) saline, buffer, glucose, water, glycerin, ethanol, and combinations thereof. The usual drug formulation should match the method of administration, and the dosage forms of the drug of the present invention are injection, oral formulation (tablets, capsules, oral solutions), transdermal formulation, and sustained-release formulation. For example, it is manufactured by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The drug is preferably manufactured under sterile conditions.

[0092] The effective dose of the active ingredient described in this invention varies depending on the method of administration and the severity of the disease being treated. The selection of a preferred effective dose can be determined by a person skilled in the art through various factors (e.g., clinical trials). These factors include, but are not limited to, pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, and half-life, the severity of the disease being treated in the patient, the patient's weight, the patient's immune status, and the route of administration. For example, depending on the pressing needs of the treatment situation, different doses may be administered multiple times a day, or the dose may be reduced proportionally.

[0093] The pharmaceutically acceptable vectors described in this invention include (but are not limited to) water, saline solution, liposomes, lipids, proteins, protein-antibody complexes, peptides, cellulose, nanogels, or combinations thereof. The choice of vector should match the administration method, which are well known to those skilled in the art.

[0094] use In a seventh aspect of the present invention, the present invention proposes the use of a recombinant antibody according to the first or second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect, or a composition according to the sixth aspect in drug preparation, wherein the drug is used to treat or prevent tumors. As described above, the recombinant antibodies provided in some specific examples of the present invention can effectively bind to CD3 and B7H6 proteins, and furthermore, can specifically recognize highly specific tumor cells that express B7H6, and drugs prepared using the recombinant antibodies and the corresponding series of substances similarly have a significant effect in treating or preventing tumors that express B7H6, with greater safety and fewer side effects.

[0095] According to some specific embodiments of the present invention, the above use may further include at least one of the following additional technical features:

[0096] According to some specific embodiments of the present invention, the tumor includes at least one of rectal cancer, non-small cell lung cancer, breast cancer, and liver cancer.

[0097] kit In a ninth aspect of the present invention, the present invention proposes a kit comprising a recombinant antibody as described in the first or second aspect. The recombinant antibody provided in some specific examples of the present invention can bind to the B7H6 protein, and for this reason, a kit comprising the recombinant antibody can be used to effectively diagnose or detect tumors that highly express the B7H6 protein.

[0098] According to some specific embodiments of the present invention, the kit is used to diagnose or detect at least one of rectal cancer, non-small cell lung cancer, breast cancer, and liver cancer. The recombinant antibodies provided in some specific embodiments of the present invention can effectively bind to CD3 and B7H6 protein molecules and can specifically recognize highly specific tumor cells expressing B7H6. For this reason, the recombinant antibodies can be used to manufacture a kit for diagnosing or detecting at least one of rectal cancer, non-small cell lung cancer, breast cancer, and liver cancer, and the kit can be used in scientific studies such as qualitative or quantitative detection of CD3 and / or B7H6 protein molecules in biological samples.

[0099] Treatment and methods for the disease In a tenth aspect of the present invention, the present invention proposes the use of recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, or drugs as described above in the treatment or prevention of cancer. As described above, if the recombinant antibody can specifically recognize the CD3 and / or B7H6 protein, has an appropriate CD3 antibody affinity, and the tumor target-associated antibody has high affinity, the bispecific antibody selectively localizes T cells to the tumor site rather than the peripheral circulation, effectively treating and preventing cancer while avoiding systemic activation. For this reason, recombinant antibodies according to the examples of the present invention, compositions of the recombinant antibody, drugs, and a series of substances that can be expressed under appropriate conditions to obtain the recombinant antibody, such as the nucleic acid molecules, expression vectors, and recombinant cells, can all be used to treat or prevent cancer.

[0100] According to some specific embodiments of the present invention, the cancer includes at least one of rectal cancer, non-small cell lung cancer, breast cancer, and liver cancer.

[0101] In an eleventh aspect of the present invention, the present invention proposes a method for treating or preventing cancer. According to some specific embodiments of the present invention, the method includes administering to a subject at least one of the following: 1) a recombinant antibody as described above, 2) a nucleic acid molecule as described above, 3) an expression vector as described above, 4) recombinant cells as described above, or 5) a composition as described above. As described above, if the recombinant antibody can specifically recognize the CD3 and / or B7H6 protein, the CD3 antibody affinity is within an appropriate range, and the tumor target-associated antibody has high affinity, the bispecific antibody selectively localizes T cells to the tumor site rather than the peripheral circulation, effectively treating and preventing cancer while avoiding systemic activation. For this reason, the method according to the embodiments of the present invention can effectively treat and prevent cancer and avoid systemic activation of T cells.

[0102] According to some specific embodiments of the present invention, the cancer includes at least one of rectal cancer, non-small cell lung cancer, breast cancer, and liver cancer.

[0103] The following describes the examples in detail. Unless specific techniques or conditions are indicated in the examples, the procedures should be carried out in accordance with the techniques or conditions described in the literature of the relevant art, or in accordance with the product specifications. Unless the manufacturer is indicated for the reagents or equipment used, they are conventional products available commercially.

[0104] Example 1: Design and construction of a CD3×B7H6 bispecific antibody molecule

[0105] In this embodiment, by screening multiple configurations, the inventors determined a bispecific antibody configured as scFv-Fc, which is named CD3×B7H6. The antibody contains two monovalent units, one of which is in the anti-CD3 ScFv-Fc form and the other is in the anti-B7H6 ScFv-Fc form. A single linked peptide connects the light chain variable region and the heavy chain variable region of the CD3 antibody, and a similar linked peptide connects the light chain variable region and the heavy chain variable region of the B7H6 antibody. The linked peptide has the amino acid sequence shown in SEQ ID NO:46. The CD3 antibody has the light chain variable region shown in SEQ ID NO:13 and the heavy chain variable region shown in SEQ ID NO:14. The B7H6 antibody has the light chain variable region shown in SEQ ID NO:15-20 and the heavy chain variable region shown in SEQ ID NO:21-26. Here, the inventors screened different combinations of B7H6 antibody variable regions and CD3 antibody variable regions to construct 18 CD3×B7H6 recombinant antibody molecules. By altering the Fc of the monovalent unit through amino acid mutation, the amino acids in the Fc region of anti-CD3 changed to cysteine ​​(S384C) at position 384 and tryptophan (T396W) at position 396, while the amino acids in the Fc region of anti-B7H6 changed to cysteine ​​(Y380C) at position 380, serine (T397S) at position 397, alanine (L399A) at position 399 and valine (Y408V) at position 408. Although homodimer formation is not easy, heterodimer formation is easy, and this heterodimer is a bispecific antibody CD3×B7H6. The specific structure is shown in Figure 1, and the 18 CD3×B7H6 recombinant antibody molecules are SEQ It has a CD3 single-chain antibody indicated by ID NO:27, a B7H6 single-chain antibody indicated by SEQ ID NO:28-45, a first antibody Fc region indicated by SEQ ID NO:47, and a second antibody Fc region indicated by SEQ ID NO:48, both of which are linked by a knob-into-hole structure after the above mutation.

[0106] Using general molecular biological techniques, nucleic acid sequences encoding CD3 and B7H6(1-18) monovalent unit polypeptide chains were cloned into the expression vector pTT5 (Yuho Seibutsu, catalog number VT2202), respectively. The specific nucleotide sequences are at least one of the nucleotide sequences shown in SEQ ID NO:69 and SEQ ID NO:70-87. Simultaneously, in order to efficiently express and secrete the signal peptide into the culture medium in CHO cells, a lead peptide of a mouse-derived antibody kappa chain was selected as the secretion signal peptide and inserted into the expression vector. The signal peptide is located at the N-terminus of the antibody variable region, and its amino acid sequence is METDTLLLWVLLLWVPGSTG (SEQ ID NO:68). The nucleotide sequence encoding the signal peptide is shown in SEQ ID NO:88.

[0107] ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCGGCTCCACCGGC (SEQ ID NO:88).

[0108] CD3 single chain antibody MDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSE VQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS(SEQ ID NO:27).

[0109] B7H6-1 single chain antibody DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWMGDINPNNGGTLYNQKFRGRVTMTVDTSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:28).

[0110] B7H6-2 single-chain antibody DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:29).

[0111] B7H6-3 single-chain antibody DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:30).

[0112] B7H6-4 single-chain antibody DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGKGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:31).

[0113] B7H6-5 single-chain antibody DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGIPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWMGDINPNNGGTLYNQKFRGRVTMTVDTSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:32).

[0114] B7H6-6 single-chain antibody DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGIPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:33).

[0115] B7H6-7 single-chain antibody DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGIPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:34).

[0116] B7H6-8 single-chain antibody DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGIPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGKGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:35).

[0117] B7H6-9 single-chain antibody DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWMGDINPNNGGTLYNQKFRGRVTMTVDTSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:36).

[0118] B7H6-10 single-chain antibody DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:37).

[0119] B7H6-11 single-chain antibody DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:38).

[0120] B7H6-12 single-chain antibody DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGKGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:39).

[0121] B7H-13 single-chain antibody DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWMGDINPNNGGTLYNQKFRGRVTMTVDTSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:40).

[0122] B7H6-14 single-chain antibody DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:41).

[0123] B7H6-15 single-chain antibody DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:42).

[0124] B7H6-16 single-chain antibody DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGG SEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGKGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSS(SEQ ID NO:43).

[0125] B7H6-17 single chain antibody DIVLTQSPVSLAVPLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGIPVRFSGSGSGTDFTLNIHPVEEEDAASYYCQQSKEDPRTFGGGTKLEIKGGGGSGGGGSGGGG SEVLLQQSGPEVVKPGASVKITCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNNAGTLYNQKFRGRVTLIVDKSSSTAYMELRSLTSDDTAVYYCARSEVFYGNYADYWGQGTTLTVSS(SEQ ID NO:44).

[0126] B7H6-18 single chain antibody DIVLTQSPVSLAVPLGQRATISCKASQSVDYDADSYMNWYQQKPGQPPKLLIYAASTLHSGIPVRFSGSGSGTDFTLNIHPVEEEDAASYYCQQSKEDPRTFGGGTKLEIKGGGGSGGGGSGGGG SEVLLQQSGPEVVKPGASVKITCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNNGGTLYNQKFRGRVTLIVDKSSSTAYMELRSLTSDDTAVYYCARSEVFYGNYADYWGQGTTLTVSS(SEQ ID NO:45).

[0127] Amino acid sequence of the first Fc region EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:47).

[0128] Amino acid sequence of the second Fc region EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:48).

[0129] Example 2: Expression and purification of CD3×B7H6 bispecific antibody

[0130] 2.1 Expression of CD3×B7H6 bispecific antibody molecule ExpiCHO-S cells (Gibco, catalog number A29127) were transfected instantaneously with a pTT5 vector carrying CD3×B7H6 chain coding genes to prepare bispecific molecules that bind to CD3 and B7H6. Here, each of the 18 CD3×B7H6 chain coding genes has an anti-CD3 ScFv-Fc, indicated by SEQ ID NO:49, and an anti-B7H6 ScFv-Fc, indicated by SEQ ID NO:50-67. The specific experimental procedure is as follows: On the day before transfection, ExpiCHO-S cells were raised to a cell density of (3-4)×10⁶ 6The solution was adjusted to 1 / mL and cultured overnight at 37°C with 8% CO2 and 120 rpm. On the day of transfection, the cells were 7 × 10⁶. 6 -1 × 10 7 Once the cells have grown to 6 × 10¹ / mL and the viability exceeds 95%, prepare for transfection and infuse the cells in fresh, preheated ExpiCHO medium (Gibco, catalog number A2910002) in 6 × 10¹⁶ units. 6 Diluted to 1 / mL, the plasmid containing the CD3 polypeptide chain and the B7H6 polypeptide chain was transfected into ExpiCHO-S cells in a 1:1 mass ratio using ExpiFectamine CHO transfection reagent (Gibco, catalog number A29129). The cells were then cultured at 37°C with 8% CO2 and 120 rpm. 18-22 hours after transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were homogeneously mixed and immediately added to the transfected cells. The cells were then homogeneously mixed and cultured at 32°C with 5% CO2 and 120 rpm. On day 5 after transfection, 8 mL of ExpiCHO Feed was re-added to the cells, homogeneously mixed, and culture continued. Cell number and viability changes were observed daily. Wait until cell viability fell below 80%, or after 10-14 days of culture, cells were obtained by centrifugation. The supernatant was purified or frozen at -80°C for reserve use.

[0131] 2.2 Purification of CD3×B7H6 bispecific antibody molecules 2.1 The supernatant obtained from the experiment was filtered through a 0.22 μm filter membrane. Antibodies containing the Fc structural domain were captured from the expression supernatant using a Mabselect prism A affinity chromatography column (GE Corporation, catalog number 17549854). The chromatography column was equilibrated with pH 7.2 phosphate buffer, and the supernatant was passed through the affinity chromatography column. Elution was performed with elution buffer (100 mM citrate, pH 2.7). Finally, the solution was concentrated and replaced with PBS buffer. The purified antibody was identified by SDS-PAGE as having a purity of 95% or higher.

[0132] CD3 ScFv-Fc amino acid sequence MDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGG GGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTL VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:49).

[0133] B7H6-1ScFv-Fc amino acid sequence DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWMGDINPNNGGTLYNQKFRGRVTMTVDTSISTAYMELSRLRSDDTAVYCARSEVGYNYADYWGQGTT VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK(SEQ ID NO:50)。

[0134] B7H6-2 ScFv-Fc amino acid sequence DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDTSISTAYMELSRLRSDDTAVYCARSEVGYNYADYWGQGTT VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK(SEQ ID NO:51)。

[0135] B7H6-3 ScFv-Fc amino acid sequence DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYCARSEVGYNYADYWGQGTT VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK(SEQ ID NO:52)。

[0136] B7H6-4 ScFv-Fc amino acid sequence DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGKGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYCARSEVGYNYADYWGQGTT VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK(SEQ ID NO:53)。

[0137] B7H6-5 ScFv-Fc amino acid sequence DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGIPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWMGDINPNNGGTLYNQKFRGRVTMTVDTSISTAYMELSRLRSDTAVYCARSEVGYNYADYWGQGTT VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK(SEQ ID NO:54)。

[0138] B7H6-6 ScFv-Fc amino acid sequence DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGIPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDTSISTAYMELSRLRSDDTAVYCARSEVGYNYADYWGQGTT VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK(SEQ ID NO:55)。

[0139] B7H6-7 ScFv-Fc amino acid sequence DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGIPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGSGGGSEGGGSESEVQLVQSGAEVKKPGASVKVSCKASGYTFDTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSSEPKSCDKTHTCPPCPPAPEAAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:56).

[0140] B7H6-8 ScFv-Fc amino acid sequence DIVLTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGIPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGSGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFDTYNMDWVRQAPGKGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSSEPKSCDKTHTCPPCPPAPEAAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:57).

[0141] B7H6-9 ScFv-Fc amino acid sequence DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWMGDINPNNGGTLYNQKFRGRVTMTVDTSISTAYMELSRLRSDDTAVYCARSEVGYNYADYWGQGTT VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK(SEQ ID NO:58)。

[0142] B7H6-10 ScFv-Fc amino acid sequence DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARSEVGYNYADYWGQGTT VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK(SEQ ID NO:59)。

[0143] B7H6-11 ScFv-Fc amino acid sequence DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYCARSEVGYNYADYWGQGTT VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK(SEQ ID NO:60)。

[0144] B7H6-12 ScFv-Fc amino acid sequence DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGKGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYCARSEVGYNYADYWGQGTT VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK(SEQ ID NO:61)。

[0145] B7H6-13 ScFv-Fc amino acid sequence DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWMGDINPNNGGTLYNQKFRGRVTMTVDTSISTAYMELSRLRSDDTAVYCARSEVGYNYADYWGQGTT VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK(SEQ ID NO:62)。

[0146] B7H6-14 ScFv-Fc amino acid sequence DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDTSISTAYMELSRLRSDDTAVYCARSEVGYNYADYWGQGTT VTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK(SEQ ID NO:63)。

[0147] B7H6-15 ScFv-Fc amino acid sequence DIVLTQTPLSLSVTPGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGSGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFDTDYNMDWVRQAPGQGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSSEPKSCDKTHTCPPCPPAPEAAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:64).

[0148] B7H6-16 ScFv-Fc amino acid sequence DIVLTQTPLSLVTGQPASISCKASQSVDYDGDSYMNWYLQKPGQPPQLLIYAASTLHSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSKEDPRTFGQGTKLEIKGGGGSGGGSGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFDTYNMDWVRQAPGKGLEWIGDINPNNGGTLYNQKFRGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARSEVFYGNYADYWGQGTTVTVSSEPKSCDKTHTCPPCPPAPEAAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:65).

[0149] B7H6-17 ScFv-Fc amino acid sequence DIVLTQSPVSLAVPLGQRATICSKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASTLHSGIPVRFSGSGSGTDFTLNIHPVEEDAASYCQQSKEDPRTFGGGTKLEIKGGGGSGGGGSGGGSEVLLQQSGPEVVKPGASVKITCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNNAGTLYNQKFRVTLIVDKSSSTAYMELRSLTSDTAVYCARSEVYGYNYADYWGQGTT LTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPG(SEQ ID NO:66)。

[0150] B7H6-18 ScFv-Fc amino acid sequence DIVLTQSPVSLAVPLGQRATISCKASQSVDYDADSYMNWYQQKPGQPPKLLIYAASTLHSGIPVRFSGSGSGTDFTLNIHPVEEEDAASYYCQQSKEDPRTFGGGTKLEIKGGGGGSGGGG SGGGGSEVLLQQSGPEVVKPGASVKITCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNNGGTLYNQKFRGRVTLIVDKSSSTAYMELRSLTSDDTAVYYCARSEVFYGNYADYWGQGTT LTVSSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:67).

[0151] Example 3: Detection of binding activity between CD3×B7H6 bispecific antibody and antigen (ELISA)

[0152] This example detects the binding activity of the CD3×B7H6 bispecific antibody to a single antigen (B7H6 antigen or CD3 antigen), and the binding activity of the CD3×B7H6 bispecific antibody to a dual antigen (B7H6 antigen and CD3 antigen) simultaneously. The specific experimental procedure is as follows.

[0153] 3.1 Measurement of binding activity of CD3×B7H6 bispecific antibody to single antigen Human B7H6 (ACRO biosystems, catalog number B76-H52H8) and human CD3 antigen (ACRO biosystems, catalog number CDD-H52W1) were each diluted to 2 μg / mL in coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6), and 100 μL was added per well to an enzyme-conjugated plate and incubated overnight at 4°C. The plates were then washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) was added to the plate and incubated at room temperature for 2 hours. The plates were then washed three times with PBST. The corresponding bispecific antibody was added to each well and incubated at room temperature for 1 hour. The plates were then washed three times with PBST. HRP-rabbit anti-human IgG secondary antibody (boster, catalog no. BA1070), diluted in 100 μL of blocking buffer, was added to each well and incubated at room temperature for 1 hour. The wells were washed three times with PBST, TMB was added to each well, and the mixture was allowed to react in the dark at room temperature for 2–5 minutes. The reaction in each well was stopped with 2 M sulfuric acid, and the OD450 value was finally read using a microplate reader.

[0154] The specific experimental results are shown in Figure 2A. Figure 2A shows the binding status of CD3×B7H6 bispecific antibody molecules, which consist of different B7H6 and CD3 sequences, to the B7H6 antigen. All of these bispecific antibody molecules can bind to the B7H6 antigen, and the binding activity of CD3×B7H6-14 is the strongest. Therefore, subsequent experiments will mainly focus on CD3×B7H6-14, and Figure 2B shows that the CD3×B7H6-14 bispecific antibody molecule can bind to the CD3 antigen.

[0155] 3.2 Measurement of binding activity of CD3×B7H6 bispecific antibody and biantigen Human B7H6 (ACRO biosystems, catalog number B76-H82Wb) was diluted to 2 μg / mL in coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6), and 100 μL was added per well to an enzyme-conjugated plate and incubated overnight at 4°C. The plates were then washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) was added to the plate and incubated at room temperature for 2 hours. The plates were then washed three times with PBST. The corresponding bispecific antibody was added to each well and incubated at room temperature for 1 hour. The plates were then washed three times with PBST. Human CD3 antigen (ACRO biosystems, catalog number CDD-H52W1), diluted in 100 μL of blocking buffer, was added to each well and incubated at room temperature for 1 hour. The plates were washed three times with PBST. 100 μL of HRP-anti-His tagged secondary antibody (Genscript, catalog number A00612), diluted in blocking buffer, was added to each well and incubated at room temperature for 1 hour. The plates were washed three times with PBST, TMB was added to each well, and the reaction was allowed to proceed at room temperature in the dark for 2–5 minutes. Each well was stopped with 2 M sulfuric acid, and the OD450 value was finally read using a microplate reader. The specific results, as shown in Figure 2C, show that the OD450 reading gradually increased with increasing concentration of the CD3×B7H6-14 bispecific antibody molecule, indicating that this bispecific antibody molecule can simultaneously bind to human CD3 and B7H6.

[0156] Example 4: In vitro detection of death of bispecific antibody molecules

[0157] 4.1 Isolation of human peripheral blood mononuclear cells (PBMCs) Anticoagulated human peripheral blood was added to an equal volume of sterile PBS. A 50 mL sterile centrifuge tube was taken, and 25 mL of diluted peripheral blood was gently added to 15 mL of lymphopropyl alcohol (GE Healthcare, catalog number 17144003). The interface was kept clearly still throughout the process, and centrifugation was performed at 21°C at 400 g for 30 min, accelerating and decelerating the centrifuge. After centrifugation, the inside of the tube was divided into three layers. The middle layer was the lymphocyte layer. The middle white film layer was aspirated into a new 50 mL centrifuge tube, which was then filled with PBS to 50 mL and centrifuged at 500 g for 10 min. The supernatant was discarded, 1 mL of PBS was added and gently blown uniformly, the tube was filled with PBS to 40 mL and centrifuged at 250 g for 10 min.

[0158] After centrifugation, discard the supernatant, resuspend in 1 mL of MACS buffer (0.5% BSA, 2.5 mM EDTA-PBS), dilute, and count.

[0159] 4.2 Isolation of purified T cells Human T cells were isolated from peripheral blood using magnetic bead sorting (Miltenyi, catalog number 130096535). After collecting the PBMCs, the supernatant was completely discarded, and 10 7 After resuspending the cells with 40 μL of MACS buffer for each individual cell, an additional 10 μL of T Cell Biotin-Antibody Cocktail was added, mixed uniformly, and incubated at 4°C for 10 minutes. 7 For each individual cell, add an additional 30 μL of MACS buffer and 20 μL of T Cell MicroBead Cocktail, mix thoroughly, and incubate at 4°C for 15 min. Place the LS separation column in a magnetic field, wet the separation column with 3 mL of MACS buffer, add the cell suspension to the separation column, wash the centrifuge tube with 1 mL of MACS buffer, add the separation column, and collect the effluent. Wash the separation column with 3 mL of MACS buffer and collect the effluent. After mixing thoroughly, count the cells. Centrifuge 300 g of cell suspension at 4°C for 10 min, then resuspend in culture medium.

[0160] 4.3 Detection of in vitro killing of adherent cultured tumor cells Digest the tumor cells in adherent culture and then count them, and adjust the cell density to 2×10 5 / mL. Select the RTCA instrument (Agilent), instrument type DP, select the experimental mode, and input cell information and drug information. Enter the schedule setting experimental steps, add 50 μL of fresh medium (89% RPMI 1640 medium + 10% fetal bovine serum + 1% penicillin) to the plate, then place it in the instrument and close it, and click the first step to start. After Done, take out the plate, add 100 μL of cell suspension, let it stand at room temperature for 15 - 30 min to prevent edge effects, place it in the instrument, and click Start. After growing to the logarithmic phase, pause temporarily, add 50 μL of purified T cells (1×10 6 / mL), add the bispecific antibody at gradient concentrations, click Start, and analyze after a certain period of time.

[0161] As shown in the figure, the specific experimental results show that as the concentration of the CD3×B7H6 - 14 bispecific antibody increases, the killing efficiency of T cells against HCT - 15 cells (Figure 3A), LoVo cells (Figure 3B), Hep G2 cells (Figure 3C), and SK - BR - 3 cells (Figure 3D) gradually increases, indicating that CD3×B7H6 - 14 can promote the killing of B7H6+ tumor cells by T cells.

[0162] Example 5 Detection experiment of in vitro killing of floating cells by luciferase reaction method Collect the tumor cells overexpressing luciferase and then adjust the cell density to 2×10 5 / mL. As described in Examples 4.1 and 4.2, separate, purify, and collect T cells and adjust the cell density to 5×10 5Adjust to / mL. Transfer 50 μL each of tumor cells and purified T cells to a 96-well flat plate, add bispecific antibodies at a gradient concentration, and mix homogeneously. Incubate at 37°C in 5% CO2 for 4 hours, then add 100 μL (15 mg / mL) of fluorescein potassium salt solution (Goldbio, catalog no. LUCK-1G) per well, mix rapidly and homogeneously, and detect chemiluminescence values ​​with a microplate reader.

[0163] As shown in the figure, the specific experimental results indicate that as the concentration of the CD3×B7H6-14 bispecific antibody increases, the chemiluminescence values ​​of Ho8910 and K562 cells gradually decrease, resulting in fewer surviving tumor cells. This demonstrates that CD3×B7H6-14 can significantly promote the death of Ho8910 cells (Figure 4A) and K562 cells (Figure 4B) by T cells.

[0164] Example 6: Detecting the proliferation status of T cells in the presence of B7H6-positive tumor cells. To measure T cell proliferation, this example was set up as a 2x2 bifactor experiment, including T cell + PBS group, T cell + CD3×B7H6-14 group, HCT-15 + T cell + PBS group, and HCT-15 + T cell + CD3×B7H6-14 group. As described in Examples 4.1 and 4.2, T cells were isolated and purified, and the purified T cells were marked with 5 μM cell trace CFSE (Invitrogen, catalog number C34554). Subsequently, the marked T cells and human colon cancer cells (HCT-15 cells) were mixed in a target ratio of 2:1, and PBS or a bispecific antibody was added. After 3 days, the cells were collected, and the T cell proliferation status was detected by flow cytometry.

[0165] The specific experimental results, as shown in the figure, show that the CD3×B7H6-14 bispecific antibody significantly promotes T cell proliferation in the presence of HCT-15 (Figure 5A), and that the effect of promoting T cell proliferation becomes significantly greater with increasing concentration of the bispecific antibody CD3×B7H6-14 (Figure 5B).

[0166] Example 7: Detection of T cell activation and degranulation levels in the presence of B7H6-positive tumor cells. As described in Examples 4.1 and 4.2, T cells were isolated and purified. In the experiment, a total of two groups were set up: a control group consisting of T cells and an experimental group. T cells and HCT-15 target cells were added to a 96-well plate in a target ratio of 1:1, and CD3×B7H6 bispecific antibody was added at a gradient concentration and mixed homogeneously. After incubation at 37°C for 24 hours, the cells were collected by centrifugation, resuspended in PBS, mouse serum was added and the mixture was closed at room temperature for 15 minutes, a flow cytometry antibody to detect surface molecules was added, and the mixture was incubated in the dark at 4°C for 30 minutes, then washed with PBS, and further fixed with a transmembrane fixation buffer (Invitrogen, catalog number 00512343) for at least half an hour at room temperature. Further washing was done with transmembrane buffer (Invitrogen, catalog number 00833356), mouse serum was added and the mixture was closed at room temperature for 15 minutes, then the corresponding intracellular molecule-marking antibody was added and incubated in the dark at room temperature for 30 minutes, and further washed twice with PBS before detection by flow cytometry.

[0167] The specific experimental results, as shown in Figures 6A and 6B, indicate that when (+HCT-15) is present in B7H6+ cells, the expression of the CD69 molecule (Figure 6A) on the surface of CD4+ T cells and CD8+ T cells, and the expression of CD107a (Figure 6B) within the cells, gradually increases with increasing concentration of the CD3×B7H6-14 bispecific antibody. When B7H6+ tumor cells (-HCT-15) are absent, the activation and degranulation ability of T cells after incubation with the bispecific antibody is very limited. This demonstrates that the bispecific antibody can specifically target T cells and B7H6+ tumor cells and promote T cell activation and degranulation.

[0168] Example 8: Detection of T cell secretion factors in the presence of B7H6-positive tumor cells. As described in Examples 4.1 and 4.2, T cells were isolated and purified. The control group consisted only of T cells, while the experimental group contained T cells and HCT-15 cells, totaling 5 × 10⁶ cells. 5 T cells, 5 × 10 5Individual T cells and 5 × 10 5 Each HCT-15 cell was added to a 24-well plate, with a total volume of 500 μL. Furthermore, CD3×B7H6 bispecific antibody (0, 10) was added. 0 , 10 1 , 10 2 , 10 3 The ) was added at a gradient concentration and mixed uniformly. After incubation at 37°C for 24 hours, the mixture was centrifuged, the supernatant was collected, and the concentrations of various cellular factors in the supernatant were detected using the CBA Human Th1 / Th2 / Th17 Cell Factor Detection Kit (BD, catalog number 560484), and finally detected by flow cytometry.

[0169] The specific experimental results, as shown in Figures 7A-D, show the concentrations of cells co-incubating IFN-γ, IL-2, IL-10, and IL-17A cellular factors in the supernatant. When HCT-15 tumor cells are present, the levels of T cells secreting these four cellular factors gradually increase with increasing concentrations of CD3×B7H6-14 antibody, indicating that this bispecific antibody can promote T cell activation and secrete cellular factors.

[0170] Example 9: Study on the effects of in vivo xenotransplantation. To detect the in vivo antitumor activity of the CD3×B7H6 bispecific antibody molecule against HCT-15 tumor cells, this example uses a total of three groups: a buffer group, a human immunoglobulin (IGg) group, and a CD3×B7H6-14 bispecific antibody molecule group. As described in Examples 4.1 and 4.2, T cells were isolated and purified, and then expanded for 7 days using a T cell expansion kit (Gibco, catalog no. 11131D). HCT-15 colorectal cancer cells (1×10⁶) 6 ) and dilated T cells (2 × 10⁻¹⁰ 6After mixing (sc), (sc) was injected subcutaneously into male NOD-Prkdcscid Il2rgem1 / Smoc mice. After inoculation, CD3×B7H6 bispecific antibody (1 mg / kg, soluble in PBS), IgG control antibody (sigma, catalog number I4506), or buffer (control solvent) was intravenously injected into the mice every two days for a total of three injections. The length and width of the tumor were detected by external caliper measurement, and the tumor volume was calculated using a standard formula.

[0171] Figure 8 shows the in vivo antitumor activity of the CD3×B7H6-14 bispecific antibody molecule against HCT-15 cells. Treatment with the CD3×B7H6-14 bispecific antibody significantly promotes regression of HCT-15 tumors, while the use of control IgG or control solvent fails to achieve the same therapeutic effect, demonstrating that the CD3×B7H6 bispecific antibody possesses significant in vivo antitumor activity.

[0172] In this specification, reference terms such as “one embodiment,” “several embodiments,” “example,” “specific example,” or “several examples” refer to specific features, structures, materials, or characteristics described in combination with such embodiments or examples, which are included in at least one embodiment or example of the present invention. In this specification, the above-mentioned exemplary descriptions do not necessarily apply to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described can be combined in an appropriate manner in any or more embodiments or examples. Notwithstanding the fact that they do not conflict with each other, those skilled in the art can combine and combine different embodiments or examples and features of different embodiments or examples described herein.

[0173] Although embodiments of the present invention have been presented and described, these embodiments are illustrative and should not be understood as limiting the present invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations are possible in the above embodiments within the scope of the present invention. Cross-reference to related applications.

[0174] This application claims priority and rights to the Chinese patent application no. 202111485404.0, which was submitted to the China Intellectual Property Administration on December 7, 2021, and the entire contents of the aforementioned Chinese patent application are incorporated herein by reference.

Claims

1. An antibody that binds to CD3 and B7H6, CD3 antibody light chain variable region CDR1 sequence: SEQ ID NO: 1: CD3 antibody light chain variable region CDR2 sequence: SEQ ID NO: 2: CD3 antibody light chain variable region CDR3 sequence: SEQ ID NO: 3: CD3 antibody heavy chain variable region CDR1 sequence: SEQ ID NO: 4: CD3 antibody heavy chain variable region CDR2 sequence: SEQ ID NO: 5: CD3 antibody heavy chain variable region CDR3 sequence: SEQ ID NO: 6: B7H6 antibody light chain variable region CDR1 sequence: SEQ ID NO: 7: B7H6 antibody light chain variable region CDR2 sequence: SEQ ID NO: 8: B7H6 antibody light chain variable region CDR3 sequence: SEQ ID NO: 9: B7H6 antibody heavy chain variable region CDR1 sequence: SEQ ID NO: 10: B7H6 antibody heavy chain variable region CDR2 sequence: SEQ ID NO: 11: and B7H6 antibody heavy chain variable region CDR3 sequence: SEQ ID NO: 12: Recombinant antibodies containing [specific antibodies].

2. The recombinant antibody according to claim 1, comprising a variable region of a CD3 antibody, wherein the variable region of the CD3 antibody has a light chain variable region indicated by SEQ ID NO: 13 and a heavy chain variable region indicated by SEQ ID NO:

14.

3. The antibody includes a variable region of the B7H6 antibody, and the variable region of the B7H6 antibody is The amino acid sequence containing the light chain variable region of the amino acid sequence indicated by SEQ ID NO: 20, and the amino acid sequence containing the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO: 23, The amino acid sequence containing the light chain variable region of the amino acid sequence indicated by SEQ ID NO: 16, and the amino acid sequence containing the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO: 22, The amino acid sequence containing the light chain variable region of the amino acid sequence indicated by SEQ ID NO: 16, and the amino acid sequence containing the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO: 23, The amino acid sequence containing the light chain variable region of the amino acid sequence indicated by SEQ ID NO: 16, and the amino acid sequence containing the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO: 24, The amino acid sequence containing the light chain variable region of the amino acid sequence indicated by SEQ ID NO: 16, and the amino acid sequence containing the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO: 25, The amino acid sequence containing the light chain variable region of the amino acid sequence indicated by SEQ ID NO: 17, and the amino acid sequence containing the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO: 22, The amino acid sequence containing the light chain variable region of the amino acid sequence indicated by SEQ ID NO: 17, and the amino acid sequence containing the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO: 23, The amino acid sequence containing the light chain variable region of the amino acid sequence indicated by SEQ ID NO: 17, and the amino acid sequence containing the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO: 24, The amino acid sequence containing the light chain variable region of the amino acid sequence indicated by SEQ ID NO: 17, and the amino acid sequence containing the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO: 25, The amino acid sequence containing the light chain variable region indicated by SEQ ID NO: 19, and the amino acid sequence containing the heavy chain variable region indicated by SEQ ID NO: 22, The amino acid sequence containing the light chain variable region of the amino acid sequence indicated by SEQ ID NO: 19, and the amino acid sequence containing the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO: 23, The amino acid sequence containing the light chain variable region of the amino acid sequence indicated by SEQ ID NO: 19, and the amino acid sequence containing the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO: 24, The amino acid sequence containing the light chain variable region of the amino acid sequence indicated by SEQ ID NO: 19, and the amino acid sequence containing the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO: 25, The amino acid sequence containing the light chain variable region of the amino acid sequence indicated by SEQ ID NO: 20, and the amino acid sequence containing the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO: 22, A sequence containing the light chain variable region of the amino acid sequence indicated by SEQ ID NO: 20, and the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO: 24, or The amino acid sequence containing the light chain variable region of the amino acid sequence indicated by SEQ ID NO: 20, and the amino acid sequence containing the heavy chain variable region of the amino acid sequence indicated by SEQ ID NO: 25, The recombinant antibody according to claim 1.

4. The recombinant antibody according to claim 1, further comprising a linked peptide.

5. The recombinant antibody according to claim 4, wherein the linked peptide has the amino acid sequence indicated by SEQ ID NO:

46.

6. The recombinant antibody according to claims 4 to 5, wherein the N-terminus of the linked peptide is linked to the C-terminus of the CD3 antibody light chain variable region, and the C-terminus of the linked peptide is linked to the N-terminus of the CD3 antibody heavy chain variable region.

7. The recombinant antibody according to claims 4 to 5, wherein the N-terminus of the linked peptide is linked to the C-terminus of the B7H6 light chain variable region, and the C-terminus of the linked peptide is linked to the N-terminus of the B7H6 heavy chain variable region.

8. The recombinant antibody according to claim 1, further comprising a first Fc region and a second Fc region, wherein at least a portion of the first Fc region and the second Fc region is derived from at least one of a mouse-derived antibody, a human-derived antibody, a primate-derived antibody, or a variant thereof.

9. The recombinant antibody according to claim 8, wherein at least a portion of the first Fc region and the second Fc region is derived from human IgG or a variant thereof.

10. The recombinant antibody according to claim 8, wherein at least a portion of the first Fc region and the second Fc region is derived from human IgG1 or a variant thereof.

11. The recombinant antibody according to claim 8, wherein the first Fc region has at least one of the S384C mutation and the T396W mutation compared to the Fc region of wild-type IgG1.

12. The recombinant antibody according to claim 9, wherein the second Fc region has at least one of the Y380C mutation, T397S mutation, L399A mutation, and Y408V mutation compared to the Fc region of wild-type IgG1.

13. The recombinant antibody according to any one of claims 8 to 12, wherein the first antibody Fc region has an amino acid sequence indicated by SEQ ID NO: 47, and the second antibody Fc region has an amino acid sequence indicated by SEQ ID NO:

48.

14. A recombinant antibody that binds to CD3 and B7H6, having amino acid sequences indicated by SEQ ID NO: 49 and 63.

15. A nucleic acid encoding a recombinant antibody according to claim 14.

16. The nucleic acid according to claim 15, having at least one of the nucleotide sequences represented by SEQ ID NO: 69 and the nucleotide sequences represented by SEQ ID NO: 70-87.

17. An expression vector supporting a nucleic acid molecule as described in claim 15.

18. Recombinant cells carrying a nucleic acid molecule according to claim 15 or 16, an expression vector according to claim 17, or a recombinant antibody according to claim 14.

19. A composition comprising the recombinant antibody described in claim 14, the nucleic acid molecule described in claim 15, or the expression vector described in claim 17.

20. Use of the recombinant antibody according to claim 14, the nucleic acid molecule according to claim 15 or 16, or the expression vector according to claim 17 in the preparation of a drug for treating or preventing cancer.

21. The use according to claim 20, wherein the cancer comprises at least one of rectal cancer, non-small cell lung cancer, breast cancer, and liver cancer.

22. A drug used for the treatment and prevention of cancer, comprising a recombinant antibody according to claim 14, a nucleic acid molecule according to claim 15 or 16, or an expression vector according to claim 17.

23. The drug according to claim 22, wherein the cancer comprises at least one of rectal cancer, non-small cell lung cancer, breast cancer, and liver cancer.

24. A kit comprising the recombinant antibody described in claim 14.

25. The kit according to claim 24, used for diagnosing at least one of rectal cancer, non-small cell lung cancer, breast cancer, and liver cancer.

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