CN112961245A

CN112961245A - Bispecific antibody targeting CD96, and preparation method and application thereof

Info

Publication number: CN112961245A
Application number: CN202110206096.7A
Authority: CN
Inventors: 马佳兵; 赵文旭; 洪娟; 徐艳敏; 沈俊杰; 幺瑞娜
Original assignee: Chongqing Precision Biotech Co ltd
Current assignee: Chongqing Precision Biotech Co ltd
Priority date: 2021-02-24
Filing date: 2021-02-24
Publication date: 2021-06-15
Anticipated expiration: 2041-02-24
Also published as: CN112961245B

Abstract

The invention belongs to the field of biological pharmacy, and particularly relates to a CD 96-targeted bispecific antibody, and a preparation method and application thereof. The bispecific antibody comprises an anti-human CD96ScFv, an anti-human PD1ScFv and an IgG4Fc mutant, and the configuration of the bispecific antibody comprises: CD96ScFv holes-PD1ScFv knobs and CD96ScFv- (G4S)4-PD1 ScFv-Fc. The invention combines CD96 and PD-1 for the first time, and constructs the CD96-PD1 bispecific antibody for the first time, the bispecific antibody of the invention has higher affinity with PD1 and CD96, CD96 is highly expressed not only in activated T cells but also in activated NK cells, and the expression quantity is increased along with the activation time, and the addition of CD96 can greatly reduce the drug resistance problem caused by the single antibody of PD1 in the treatment process.

Description

Bispecific antibody targeting CD96, and preparation method and application thereof

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to a CD 96-targeted bispecific antibody, and a preparation method and application thereof.

Technical Field

Currently, immune checkpoint blockers are used in immunotherapy of various tumors. PD1 acts as an immune checkpoint on the surface of T cells, initiating downstream signaling pathways and inhibiting T cell activation when it interacts with the tumor cell surface ligands PD-L1 and PD-L2, and blocking the interaction of PD1 with its ligands using antibodies has proven effective in the clinical treatment of a variety of malignancies, but resistance occurs during treatment due to the PD1 single antibody. Therefore, light is blocking PD1, and clinical expectations have not been met, and the development of bispecific antibodies for tumor therapy has become an urgent need. Bispecific antibodies have more than 110 bispecific antibodies entering different stages of clinical research due to the simultaneous targeting of two antigens or different epitopes of the same antigen, possessing diverse mechanisms of action and flexible target combinations. Although directed against bispecific antibodies that jointly inhibit immune checkpoint receptors such as KN046 targeting PD-1 × CTLA-4, MGD013 targeting PD1 × LAG3, RG7769 targeting PD1 and TIM3, etc.

Patent CN111196856A discloses a bispecific antibody capable of specifically binding to HER2 and PD1, comprising an immunoglobulin antibody IgG and two identical single chain variable fragment scfvs, wherein each scFv comprises a variable region VH and a variable region VL, VH and VL being linked by a peptide linker L1, each scFv being linked in series to IgG by a linker peptide L2.

Patent CN110506059A discloses a bispecific antibody specifically binding to PD1 and LAG3, comprising a first antigen-binding domain specifically binding to programmed cell death protein 1(PD1) and a second antigen-binding domain specifically binding to lymphocyte activation gene-3 (LAG 3).

Patent CN106939050B discloses an anti-PD 1 and CD19 bispecific antibody and application thereof, the invention relates to an anti-PD 1 and CD19 bispecific antibody and application thereof, the anti-PD-1 and CD19 bispecific antibody, or a variant thereof, or a functional fragment thereof comprises: a domain that specifically recognizes and binds to an immune cell surface antigen PD-1, comprising the heavy chain variable region of an anti-PD-1 specific antibody (anti-PD-1 VH); and a domain that specifically recognizes and binds CD19, comprising the heavy chain variable region of an anti-CD 19 specific antibody (anti-CD 19 VH).

The above bispecific antibodies have had significant success in many cancers, but not all patients benefit from these therapies. Therefore, there is a trend to find new immune checkpoint receptors, Stephen J Blake et al propose CD96, also an immune checkpoint, CD96 is highly expressed not only in activated T cells but also in activated NK cells and increases in expression with activation time, it competes with the immune checkpoint molecule TIGIT and the co-activating molecule CD226 for binding to the tumor cell surface specific antigens CD155 and CD112, thus being an ideal immune checkpoint receptor for combination therapy, and more than 30 mature bispecific antibody molecule platforms promoting the correct matching of heavy and light chains have been used. However, when both heavy and light chains of a classical bispecific antibody are expressed in a host cell simultaneously, heavy and light chain mismatches occur and produce unremovable by-products, and how to construct a stable bispecific antibody is also a problem, and there is no disclosure of the use of CD96 in combination with other antibodies.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a bispecific antibody, which can specifically target CD96 and PD1, and a nucleic acid sequence encoding the bispecific antibody, an expression vector and a host cell comprising the nucleic acid sequence.

The bispecific antibody comprises n anti-human CD96ScFv, n anti-human PD1ScFv and Fc mutation segment of IgG 4; and n is more than or equal to 1.

Further, the anti-human CD96ScFv comprises a CD96 heavy chain with an amino acid sequence shown as SEQ ID NO.1 or an amino acid sequence with homology more than or equal to 95 percent; a CD96 light chain with amino acid shown as SEQ ID NO.2 or an amino acid sequence with homology more than or equal to 95 percent; the anti-human CD96ScFv is formed by connecting the CD96 heavy chain and the CD96 light chain through a Linker; the amino acid sequence of the Linker is shown as SEQ ID NO.5 or the amino acid sequence with homology more than or equal to 80%.

Further, the anti-human PD1ScFv comprises a PD1 heavy chain with an amino acid sequence shown as SEQ ID NO.3 or an amino acid sequence with homology more than or equal to 95 percent; PD1 light chain with amino acid sequence shown as SEQ ID NO.4 or amino acid sequence with homology more than or equal to 95%; the anti-human PD1ScFv is formed by connecting the PD1 heavy chain and the PD1 light chain through a Linker; the amino acid sequence of the Linker is shown as SEQ ID NO.5 or the amino acid sequence with homology more than or equal to 80%.

Furthermore, one end of the sequence of the anti-human CD96ScFv or anti-human PD1ScFv is connected with a membrane peptide, and the amino acid sequence of the membrane peptide is shown as SEQ ID NO.12 or the amino acid sequence with homology more than or equal to 80%. The membrane-out peptide is a peptide chain capable of directing the bispecific antibody to pass out of the cell membrane.

Further, the amino acid sequence of the Fc fragment of IgG4 is as follows:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

as one of the bispecific antibodies, the configuration of the bispecific antibody is: CD96ScFv Holes-PD1ScFv Knobes, wherein Holes and Knobes are respectively Fc mutation segments of different IgG 4; mutations of T366W, S354C, S228P and R409K are respectively introduced into PD1ScFv-Fc, and mutations of T366S, L368A, Y407V, Y349C, S228P and R409K are introduced into CD96 ScFv-Fc. Specifically, the linkage between the two Fc mutants is a routine operation for those skilled in the art, mutation site binding and disulfide bond binding of the upper part of the Fc mutant.

Further, the anti-human CD96ScFv comprises a CD96 heavy chain with an amino acid sequence shown as SEQ ID NO.1 or an amino acid sequence with homology more than or equal to 95 percent; the amino acid sequence is a CD96 light chain shown as SEQ ID NO.2 or an amino acid sequence with homology more than or equal to 95 percent; the anti-human CD96ScFv is formed by connecting the PD1 heavy chain and the CD96 light chain through a Linker; the amino acid sequence of the Linker is shown as SEQ ID NO.5 or the amino acid sequence with homology more than or equal to 80%.

Further, the amino acid sequence of the Fc mutation segment holes of IgG4 is shown as SEQ ID NO.6 or the amino acid sequence with homology more than or equal to 95%; the amino acid sequence of the CD96ScFv holes is shown as SEQ ID NO.7 or the amino acid sequence with homology more than or equal to 95 percent.

Furthermore, the amino acid sequence of the Fc mutant segment knobes of IgG4 is shown in SEQ ID NO.8 or the amino acid sequence with homology more than or equal to 95 percent; the amino acid sequence of the PD1ScFv knobes is shown in SEQ ID NO.9 or the amino acid sequence with the homology of more than or equal to 95 percent.

Further, a method for constructing a CD96 ScFholes-PD 1ScFv knobes bispecific antibody is also provided, which is characterized by comprising the following steps: respectively constructing a lentivirus expression plasmid of anti-human CD96 ScFv-holes and a lentivirus expression plasmid of anti-human PD1ScFv-knobes, respectively packaging the lentiviruses, simultaneously infecting CHO cells according to the same infection number and screening single clones to obtain a target cell strain.

Further, the construction method also comprises the steps of expanding the cultured target cell strain and collecting culture supernatant, purifying the target Protein through a Protein A affinity chromatography column (nano-micro), desalting a molecular exclusion chromatography column Superdex 200pg (GE), and detecting the purity by SDS-PAGE to obtain the CD96 ScFv-Holes-PD 1ScFv-Knobes bispecific antibody.

Further, a nucleic acid sequence encoding the CD96ScFv holes, which comprises the sequence shown in SEQ ID NO.13 or a sequence with homology of more than or equal to 90 percent; or a nucleic acid sequence encoding any one of the PD1ScFv knobes, wherein the nucleic acid sequence comprises the sequence shown in SEQ ID NO.14 or a sequence with homology of more than or equal to 90%.

Further, an expression vector comprising the nucleic acid sequence of any one of the above is also provided.

Further, the expression vector is a conventional expression vector in the art, and refers to an expression vector comprising appropriate regulatory sequences, such as a promoter sequence, a terminator sequence, a polyadenylation sequence, an enhancer sequence, a marker gene and/or sequence, and other appropriate sequences. The expression vector may be a virus or a plasmid, such as a suitable phage or phagemid.

Further, a host cell comprising a nucleic acid sequence or an expression vector as described in any of the preceding is also provided.

Further, the host cell is a variety of host cells which are conventional in the art, as long as the host cell is capable of stably replicating itself in the above-mentioned recombinant expression vector and carrying the nucleotide which can be efficiently expressed. Wherein the host cell comprises prokaryotic expression cells and eukaryotic expression cells, the expression vector preferably comprises: COS, CHO (chinese hamster ovary), NS0, sf9, sf21, DH5 α, BL21(DE3) or TG1, more preferably e.coli tg1, BL21(DE3) cells (expressing single chain or Fab antibodies) or CHO-K1 cells (expressing full length IgG antibodies). The recombinant expression transformant of the present invention can be obtained by transforming the expression vector into a host cell.

As another scheme of the bispecific antibody, the bispecific antibody is constructed by serially connecting Fc mutations of anti-human CD96ScFv, anti-human PD1ScFv and IgG4, wherein S228P and R409K mutations are introduced into the Fc of the CD96 ScFv-Holes and the Fc of the PD1ScFv-Knobes, other parts have no mutation, and the same antibodies at the left end and the right end are naturally combined together, wherein disulfide bonds are also formed.

The configuration is as follows: CD96ScFv- (G4S)4-PD 1ScFv-Fc, wherein the (G4S)4 sequence is shown as SEQ ID NO.10 or an amino acid sequence with homology more than or equal to 80 percent; the amino acid sequence of the Fc mutant segment of the IgG4 is shown as SEQ ID NO.11 or the amino acid sequence with homology more than or equal to 95 percent.

Further, a method for constructing the CD96ScFv- (G4S)4-PD 1ScFv-F bispecific antibody is also provided, and the method for constructing the CD96ScFv- (G4S)4-PD 1ScFv-F bispecific antibody comprises the following steps: a target cell line was obtained by constructing a CD96ScFv- (G4S)4-PD 1ScFv-Fc lentiviral plasmid by linking the ScFv of CD96 with the ScFv of PD1 using a linker peptide (G4S)4 in Fc mutation S228P, R409K, packaging into a lentivirus, infecting CHO cells and screening for single clones.

Further, the construction method also comprises the steps of culturing the obtained target cell strain and collecting culture supernatant, purifying the target Protein through a Protein A affinity chromatography column (nano-micro), desalting a molecular exclusion chromatography column Superdex 200pg (GE), and detecting the purity by using SDS-PAGE to obtain the CD96ScFv- (G4S)4-PD 1ScFv-Fc bispecific antibody.

Further, a nucleic acid sequence encoding the CD96ScFv- (G4S)4-PD 1ScFv-Fc bispecific antibody is also provided, wherein the nucleic acid sequence comprises a sequence shown as SEQ ID NO.15 or a sequence with homology of more than or equal to 90%.

Further, an expression vector comprising a nucleic acid sequence encoding a CD96ScFv- (G4S)4-PD 1ScFv-Fc bispecific antibody is also provided.

The second object of the present invention is to provide a use of the bispecific antibody of any of the above.

Further, an application of the bispecific antibody in preparation of a medicament for treating tumor cells expressing PD1 and/or CD96 is provided.

Further, also provides an application of the bispecific antibody in the preparation of a detection reagent for detecting PD1 and/or CD 96.

Further, there is also provided the use of a bispecific antibody as described above in modifying a CAR or CAR-T cell.

The third objective of the present invention is to provide an anti-tumor pharmaceutical composition, which comprises the bispecific antibody or the nucleic acid sequence or the expression vector or the host cell described in any of the above.

The marketed PD1 monoclonal antibody medicament can be used for treating small cell lung cancer, colorectal cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, urothelial cancer, squamous cell carcinoma of head and neck, classical Hodgkin lymphoma, squamous non-cell lung cancer and the like.

Furthermore, the anti-tumor pharmaceutical composition also comprises one or more pharmaceutically acceptable carriers, diluents or excipients, and the pharmaceutical composition can be prepared into suspension, water injection, freeze-drying and other preparations commonly used in the pharmaceutical field. The bispecific antibody and pharmaceutically acceptable carrier formulation together constitute a pharmaceutical composition to more stably exert therapeutic effects, and these carrier formulations can ensure the conformation integrity of the amino acid core sequence of the bispecific antibody disclosed in the present invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation).

Further, the antitumor pharmaceutical composition can be used in combination with chemicals for improving the effect of treating tumors, wherein the chemicals comprise: opdivo, Keytruda, Tecntriq, Bavencio, Imfinzi |, Carrillizumab for injection, Tereprilumab injection, and Cedilizumab injection.

Further, the anti-tumor drug composition includes, but is not limited to: lung cancer, bone cancer, stomach cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, rectal cancer, colon cancer, cancer of the anal region, breast cancer, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, cancer of the urinary tract, cancer of the penis, prostate cancer, pancreatic cancer, brain cancer, testicular cancer, lymphatic cancer, transitional cell cancer, bladder cancer, kidney or ureter cancer, renal cell cancer, renal pelvis cancer, hodgkin's disease, non-hodgkin's lymphoma, soft tissue sarcoma, solid tumor of childhood, lymphocytic lymphoma, Central Nervous System (CNS) tumor, primary central nervous system lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma, pituitary adenoma, melanoma, kaposi's sarcoma, and cervical cancer, Epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, chronic or acute leukemia, and combinations thereof.

The invention has the beneficial effects that:

(1) the bispecific antibody of the invention adopts ScFv rather than Fab structure, so that the construction does not need to consider the light chain mismatch problem, the construction difficulty and the generation of byproducts are greatly reduced, and the introduction of S228P and R409K in the Fc region avoids Fab-arm Exchange caused by adopting IgG4 framework, so that the finally formed bispecific antibody is more stable.

(2) The invention combines CD96 and PD-1 for the first time, and constructs the CD96-PD1 bispecific antibody for the first time, the bispecific antibody of the invention has higher affinity with PD1 and CD96, CD96 is highly expressed not only in activated T cells but also in activated NK cells, and the expression quantity is increased along with the activation time, and the addition of CD96 can greatly reduce the drug resistance problem caused by the single antibody of PD1 in the treatment process.

(3) The invention provides a simple and efficient construction method of a CD96 and PD1 bispecific antibody.

(4) The bispecific antibody of the invention has smaller molecular weight than the classical bispecific antibody, can reach the action site more easily, and the CD96ScFv- (G4S)4-PD 1ScFv-Fc type bispecific antibody is tetravalent, namely, one antibody can respectively recognize two CD96 and PD1, and can play stronger action effect.

(5) The bispecific antibody has the potential of being developed into antitumor drugs or being applied to the antitumor field.

Drawings

FIG. 1 shows the structure of CD96 ScFv-Holes-PD 1ScFv-Knobes bispecific antibody.

FIG. 2 shows the structure of CD96ScFv- (G4S)4-PD 1ScFv-Fc bispecific antibody.

FIG. 3 is a graph of the results of SDS-PAGE for bispecific antibodies.

Figure 4 is a graph of the results of size exclusion chromatography column of bispecific antibody.

FIG. 5 shows the binding of PD1 and CD96 monoclonal antibodies to target on T cells.

FIG. 6 shows the binding of PD1 and CD96 monoclonal antibodies to targets on exogenously constructed cells.

FIG. 7 is a graph showing the result of the Fortebio measurement of PD 1.

FIG. 8 is a graph showing the results of the Fortebio assay of CD 96.

FIG. 9 shows the binding of CD96 ScFv-Holes-PD 1ScFv-Knobes to targets on T cells and on foreign constructs.

FIG. 10 shows the binding of CD96ScFv- (G4S)4-PD 1ScFv-Fc to the target on T cells and on exogenously constructed cells.

In FIG. 3, Line 1 is CD96 ScFv-Holes-PD 1ScFv-Knobes, and Line 2 is CD96ScFv- (G4S)4-PD1 ScFv-Fc.

Detailed Description

The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

In the examples of the present invention, RMPI1640 medium, DMEM medium, F12 medium were purchased from gibco; flow antibodies APC-CD96, APC-PD1, Percp/Cy5.5-PD1, Percp/Cy5.5-CD226, BV650-PD1, FITC-IgG, FITC-CD3, PE/Cy7-CD3 and isotype control antibodies were purchased from Biolegend; restriction enzymes Nhe I, Xho I, Sal I, EcoR I from Thermo fisher; SFM-Z04 serum-free medium was obtained from Chongqing institute of precision Biotechnology technology, Inc.

Example 1 construction and production of CD96 ScFv-Holes-PD 1ScFv-Knobes and CD96ScFv- (G4S)4-PD 1ScFv-Fc

(1) Construction and production of CD96 ScFv-Holes-PD 1ScFv-Knobes

Introducing mutations of T366W, S354C, S228P and R409K into PD1ScFv-Fc, introducing mutations of T366S, L368A, Y407V, Y349C, S228P and R409K into CD96 ScFv-Fc, respectively, constructing a lentivirus expression plasmid resisting human CD96 ScFv-Holes and a lentivirus expression plasmid resisting human PD1ScFv-Knobes, respectively, packaging the plasmids into lentiviruses, simultaneously infecting CHO cells according to the same infection number and screening monoclones to obtain a target cell strain, culturing the target cell strain and collecting culture supernatant, purifying the target Protein by a Protein A affinity chromatography column (nanomicr), desalting the molecular exclusion chromatography column Superdex 200pg (GE), and detecting purity by SDS-PAGE to obtain the bispecific antibody with the structure shown in figure 1, wherein the antibody is CD96 ScFv-Holes-1 ScFv-Knobes specific antibody.

The primers used for the Knobes-into-holes mutation are shown in Table 1 below:

TABLE 1 primers used for Knobes-intoholes mutations

(2) Construction and production of CD96ScFv- (G4S)4-PD 1ScFv-Fc

A CD96ScFv and PD1ScFv were ligated using a linker peptide (G4S)4 at Fc mutation S228P, R409K to construct a CD96ScFv- (G4S)4-PD 1ScFv-Fc lentiviral plasmid, which was packaged into lentiviruses, and CHO cells were infected and monoclonals were selected. Obtaining a target cell strain, culturing the target cell strain, collecting culture supernatant, purifying a target Protein through a Protein A affinity chromatography column (nano-micro), desalting through a molecular exclusion chromatography column Superdex 200pg (GE), and detecting the purity by using SDS-PAGE to obtain the CD96ScFv- (G4S)4-PD 1ScFv-Fc bispecific antibody, wherein the structure of the bispecific antibody is shown in figure 2.

Example 2 identification of the size of the antibodies to CD96 ScFv-Holes-PD 1ScFv-Knobes and CD96ScFv- (G4S)4-PD 1ScFv-Fc

The purified antibody of example 1 was confirmed by SDS-PAGE (shown in FIG. 3), and the band was correctly positioned, and the theoretical size of CD96 ScFv-Holes-PD 1ScFv-Knobes was 103.79kDa, and the monomers were 51.49kDa and 52.32kDa, respectively.

The theoretical size of the CD96ScFv- (G4S)4-PD 1ScFv-Fc monomer was 79.40kDa, and the purified antibody was verified by SDS-PAGE (validation shown in figure 3) to find the correct band position, and then determined by size exclusion (determination shown in figure 4), the bispecific antibody was highly pure (> 95%) and formed dimers.

Example 3 biological Activity validation of antibodies

(1) Biological Activity of monoclonal antibodies

1) Binding of antibodies to antigens

The CHO-PD1 cell line is exogenously constructed, the expression of target antigen and the combination of the antibody and the target antigen are detected by flow, and the PD1 antibody can specifically recognize the foreign constructed cell line CHO-PD1 and PD1 antigen molecules on T cells. The 293T-CD96 cell line is constructed exogenously, the expression of target antigen and the combination of antibody and target antigen are detected by flow, and the CD96 antibody can specifically recognize CD96 antigen molecules on the 293T-CD96 cell line and T cells which are constructed exogenously.

As shown in FIGS. 5 and 6, the expression rates of the target antigens of the exogenously constructed CHO-PD1 and 293T-CD96 were 95.42% and 98.55%, respectively. After adding 1.25. mu.g of purified antibodies to CD96 and PD1, the expression rates of FITC-IgG were 86.68% and 96.26%, respectively, demonstrating that the purified antibodies can recognize the target antigen. As shown in the following figure, while 1.25. mu.g of purified antibody was also added to CHO and 293T cells, there was no expression rate of FITC-IgG, demonstrating that ScFv-Fc antibody expressing purified CD96 and PD1 specifically binds to the target antigen. The expression level of CD96 is gradually increased in the process of activating and culturing T cells, while the expression rate of PD1 is about 10% under the condition of no stimulation of target cells. On day 9, the expression ratio of PD1 was 11.57%, the expression of FITC-IgG was 10.61% after the addition of PD1ScFv-Fc antibody, and on day 11, the expression ratio of CD96 was 96.05% after the addition of CD96 ScFv-Fc antibody. Expression of FITC-IgG was detected at 96.09%. ScFv-Fc antibodies to PD1 and CD96 were shown to recognize and bind to PD1 and CD96 antigens on the surface of T cells.

2) Affinity of

After the PD1-His and CD96-His antigens were immobilized, their binding dissociation and affinity were measured using Fortebio, and the data results are shown in table 2 and table 3 below (plotted according to the data, and the Fortebio measurement results are shown in fig. 7 and fig. 8), the KD of PD1ScFv-Fc to PD1-His was 8.897E-09M, and the KD of CD96 ScFv-Fc to CD96-His was 3.081E-08M, both having higher affinity.

TABLE 2 PD1 Fortebio assay results

TABLE 3 CD96 Fortebio assay results

(2) Biological Activity of bispecific antibodies

Flow cytometry to verify antibody binding to antigen: a CHO-PD1 cell line is exogenously constructed, the positive rate of the cell line is detected by flow cytometry, purified CD96 ScFv-Holes-PD 1ScFv-Knobes and CD96ScFv- (G4S)4-PD 1ScFv-Fc antibodies are respectively incubated with CHO-PD1, and the binding of the antibodies and the antigens is verified by flow cytometry. A293T-CD 96 cell line is constructed exogenously, and the positive rate is detected by flow cytometry. The purified CD96 ScFv-Holes-PD 1ScFv-Knobes antibody and CD96ScFv- (G4S)4-PD 1ScFv-Fc antibody were respectively incubated with 293T-CD96, and the binding of the antibody and the antigen was verified by flow cytometry, and as a result, as shown in FIGS. 9 and 10, the bispecific antibody bound to two target cells well, respectively, and it was found that both the bispecific antibody and the antigen had high affinity.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

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Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 7

<211> 478

<212> PRT

<213> Artificial Sequence

<400> 7

Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Glu Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Ala

85 90 95

Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

115 120 125

Lys Gly Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro

130 135 140

Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Asp

145 150 155 160

Thr Phe Gly Met Gly Val Gly Trp Ile Arg Gln Ser Ser Gly Lys Gly

165 170 175

Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Phe Tyr Asn

180 185 190

Pro Ala Leu Lys Ser Arg Leu Thr Val Ser Lys Asp Thr Ser Lys Asn

195 200 205

Gln Val Phe Leu Lys Ile Ala Asn Val Asp Thr Ala Asp Thr Ala Thr

210 215 220

Tyr Tyr Cys Ala His Tyr Tyr Gly Ser Leu Ser Phe Asp Val Trp Gly

225 230 235 240

Thr Gly Thr Thr Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro

245 250 255

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

260 265 270

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

275 280 285

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

290 295 300

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

305 310 315 320

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

325 330 335

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

340 345 350

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

355 360 365

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

370 375 380

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

385 390 395 400

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

405 410 415

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

420 425 430

Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

435 440 445

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

450 455 460

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

465 470 475

<210> 8

<211> 229

<212> PRT

<213> Artificial Sequence

<400> 8

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 9

<211> 467

<212> PRT

<213> Artificial Sequence

<400> 9

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln

115 120 125

Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg

130 135 140

Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser Gly Met His

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile

165 170 175

Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys Gly Arg

180 185 190

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Thr Asn

210 215 220

Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Ser

225 230 235 240

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

275 280 285

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys Asn Gln Val Ser

370 375 380

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Leu Gly Lys

465

<210> 10

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 10

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 11

<211> 229

<212> PRT

<213> Artificial Sequence

<400> 11

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 12

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 12

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 13

<211> 1500

<212> DNA

<213> Artificial Sequence

<400> 13

atggcactgc ctgtgaccgc cctgctgctg ccactggccc tgctgctgca cgcagcaagg 60

ccagacgtgg tcatgaccca gacacctctg accctgtccg tgacaatcgg ccagcctgcc 120

agcatctcct gcaagagctc ccagtctctg ctggatagcg acggcaagac atacctgaac 180

tggctgctgc agaggccagg agagtctcct aagctgctga tctatctggt gtccaagctg 240

gattctggag tgccagaccg gttcaccgga tctggaagcg gaaccgactt caccctgaag 300

atcagcagag tggaggccga ggacctgggc gtgtactatt gcctgcaggc cacccacttc 360

ccatggacat ttggcggcgg caccaagctg gagatcaagg gctccacatc tggaagcggc 420

aagccaggaa gcggagaggg ctccacaaag ggacaggtga ccctgaagga gagcggacca 480

ggcatcctgc agccatctca gaccctgagc ctgacatgtt ccttctctgg cttttccctg 540

gacaccttcg gcatgggcgt gggctggatc aggcagtcta gcggcaaggg actggagtgg 600

ctggcccaca tctggtggga cgatgacaag ttctacaatc ccgccctgaa gagccgcctg 660

acagtgtcca aggatacctc taagaaccag gtgtttctga agatcgccaa tgtggatacc 720

gccgacaccg ccacatacta ttgtgcccac tactatggca gcctgtcctt tgacgtgtgg 780

ggcacaggca ccacagtgac cgtgtcctct gagagcaagt acggccctcc ctgcccccct 840

tgccctgccc ccgagttcct gggcggaccc agcgtgttcc tgttcccccc caagcccaag 900

gacaccctga tgatcagccg gacccccgag gtgacctgtg tggtggtgga cgtgtcccag 960

gaggaccccg aggtccagtt caactggtac gtggacggcg tggaggtgca caacgccaag 1020

accaagcccc gggaggagca gttcaatagc acctaccggg tggtgtccgt gctgaccgtg 1080

ctgcaccagg actggctgaa cggcaaggaa tacaagtgta aggtgtccaa caagggcctg 1140

cccagcagca tcgagaaaac catcagcaag gccaagggcc agcctcggga gccccaggtg 1200

tgcaccctgc cccctagcca agaggagatg accaagaatc aggtgtccct gtcctgcgcg 1260

gtgaagggct tctaccccag cgacatcgcc gtggagtggg agagcaacgg ccagcccgag 1320

aacaactaca agaccacccc ccctgtgctg gacagcgacg gcagcttctt cctggtcagc 1380

aaactgaccg tggacaagag ccggtggcag gagggcaacg tctttagctg ctccgtgatg 1440

cacgaggccc tgcacaacca ctacacccag aagagcctgt ccctgagcct gggcaagtga 1500

<210> 14

<211> 1464

<212> DNA

<213> Artificial Sequence

<400> 14

atggccctgc cagtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccagg 60

cctgagatcg tgctgaccca gtcccctgcc acactgtctc tgagcccagg cgagcgggcc 120

acactgtctt gcagagcctc ccagtctgtg agctcctacc tggcctggta tcagcagaag 180

ccaggacagg cacctaggct gctgatctac gacgccagca acagggcaac cggcatccca 240

gcacgcttca gcggatccgg atctggcaca gactttaccc tgacaatctc tagcctggag 300

cccgaggatt tcgccgtgta ctattgccag cagtcctcta attggcctcg gacctttggc 360

cagggcacaa aggtggagat caagggcagc acctccggat ctggcaagcc aggatctgga 420

gagggcagca caaagggcca ggtgcagctg gtggagagcg gaggaggagt ggtgcagcca 480

ggccggtctc tgagactgga ttgtaaggcc agcggcatca ccttcagcaa ctccggcatg 540

cactgggtgc ggcaggcacc aggcaaggga ctggagtggg tggccgtgat ctggtacgac 600

ggcagcaaga gatactatgc cgattccgtg aagggcaggt tcaccatctc ccgcgacaac 660

tctaagaata cactgtttct gcagatgaac tccctgagag ccgaggatac cgccgtgtac 720

tattgtgcca caaatgacga ttattggggc cagggcaccc tggtgacagt gagctccgag 780

agcaagtacg gccctccctg ccccccttgc cctgcccccg agttcctggg cggacccagc 840

gtgttcctgt tcccccccaa gcccaaggac accctgatga tcagccggac ccccgaggtg 900

acctgtgtgg tggtggacgt gtcccaggag gaccccgagg tccagttcaa ctggtacgtg 960

gacggcgtgg aggtgcacaa cgccaagacc aagccccggg aggagcagtt caatagcacc 1020

taccgggtgg tgtccgtgct gaccgtgctg caccaggact ggctgaacgg caaggaatac 1080

aagtgtaagg tgtccaacaa gggcctgccc agcagcatcg agaaaaccat cagcaaggcc 1140

aagggccagc ctcgggagcc ccaggtgtac accctgcccc cttgccaaga ggagatgacc 1200

aagaatcagg tgtccctgtg gtgcctggtg aagggcttct accccagcga catcgccgtg 1260

gagtgggaga gcaacggcca gcccgagaac aactacaaga ccaccccccc tgtgctggac 1320

agcgacggca gcttcttcct gtacagcaaa ctgaccgtgg acaagagccg gtggcaggag 1380

ggcaacgtct ttagctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag 1440

agcctgtccc tgagcctggg caag 1464

<210> 15

<211> 2271

<212> DNA

<213> Artificial Sequence

<400> 15

atggcactgc ctgtgaccgc cctgctgctg ccactggccc tgctgctgca cgcagcaagg 60

ccagacgtgg tcatgaccca gacacctctg accctgtccg tgacaatcgg ccagcctgcc 120

agcatctcct gcaagagctc ccagtctctg ctggatagcg acggcaagac atacctgaac 180

tggctgctgc agaggccagg agagtctcct aagctgctga tctatctggt gtccaagctg 240

gattctggag tgccagaccg gttcaccgga tctggaagcg gaaccgactt caccctgaag 300

atcagcagag tggaggccga ggacctgggc gtgtactatt gcctgcaggc cacccacttc 360

ccatggacat ttggcggcgg caccaagctg gagatcaagg gctccacatc tggaagcggc 420

aagccaggaa gcggagaggg ctccacaaag ggacaggtga ccctgaagga gagcggacca 480

ggcatcctgc agccatctca gaccctgagc ctgacatgtt ccttctctgg cttttccctg 540

gacaccttcg gcatgggcgt gggctggatc aggcagtcta gcggcaaggg actggagtgg 600

ctggcccaca tctggtggga cgatgacaag ttctacaatc ccgccctgaa gagccgcctg 660

acagtgtcca aggatacctc taagaaccag gtgtttctga agatcgccaa tgtggatacc 720

gccgacaccg ccacatacta ttgtgcccac tactatggca gcctgtcctt tgacgtgtgg 780

ggcacaggca ccacagtgac cgtgtcctct ggaggcggcg gttcaggtgg tggcggatct 840

ggcggaggtg gttccggagg tggaggttca atcgtgctga cccagtcccc tgccacactg 900

tctctgagcc caggcgagcg ggccacactg tcttgcagag cctcccagtc tgtgagctcc 960

tacctggcct ggtatcagca gaagccagga caggcaccta ggctgctgat ctacgacgcc 1020

agcaacaggg caaccggcat cccagcacgc ttcagcggat ccggatctgg cacagacttt 1080

accctgacaa tctctagcct ggagcccgag gatttcgccg tgtactattg ccagcagtcc 1140

tctaattggc ctcggacctt tggccagggc acaaaggtgg agatcaaggg cagcacctcc 1200

ggatctggca agccaggatc tggagagggc agcacaaagg gccaggtgca gctggtggag 1260

agcggaggag gagtggtgca gccaggccgg tctctgagac tggattgtaa ggccagcggc 1320

atcaccttca gcaactccgg catgcactgg gtgcggcagg caccaggcaa gggactggag 1380

tgggtggccg tgatctggta cgacggcagc aagagatact atgccgattc cgtgaagggc 1440

aggttcacca tctcccgcga caactctaag aatacactgt ttctgcagat gaactccctg 1500

agagccgagg ataccgccgt gtactattgt gccacaaatg acgattattg gggccagggc 1560

accctggtga cagtgagctc cgagagcaag tacggccctc cctgcccccc ttgccctgcc 1620

cccgagttcc tgggcggacc cagcgtgttc ctgttccccc ccaagcccaa ggacaccctg 1680

atgatcagcc ggacccccga ggtgacctgt gtggtggtgg acgtgtccca ggaggacccc 1740

gaggtccagt tcaactggta cgtggacggc gtggaggtgc acaacgccaa gaccaagccc 1800

cgggaggagc agttcaatag cacctaccgg gtggtgtccg tgctgaccgt gctgcaccag 1860

gactggctga acggcaagga atacaagtgt aaggtgtcca acaagggcct gcccagcagc 1920

atcgagaaaa ccatcagcaa ggccaagggc cagcctcggg agccccaggt gtacaccctg 1980

ccccctagcc aagaggagat gaccaagaat caggtgtccc tgacctgcct ggtgaagggc 2040

ttctacccca gcgacatcgc cgtggagtgg gagagcaacg gccagcccga gaacaactac 2100

aagaccaccc cccctgtgct ggacagcgac ggcagcttct tcctgtacag caggctgacc 2160

gtggacaaga gccggtggca ggagggcaac gtctttagct gctccgtgat gcacgaggcc 2220

ctgcacaacc actacaccca gaagagcctg tccctgagcc tgggcaagtg a 2271

Claims

1. A bispecific antibody comprising n anti-human CD96ScFv, n anti-human PD1ScFv, and an Fc mutant of IgG 4; and n is more than or equal to 1.

2. The bispecific antibody of claim 1, wherein the anti-human CD96ScFv comprises a CD96 heavy chain having an amino acid sequence as shown in SEQ ID No. 1; a CD96 light chain having an amino acid sequence as set forth in SEQ ID No. 2; the anti-human CD96ScFv is formed by connecting the CD96 heavy chain and the CD96 light chain through a Linker; the amino acid sequence of the Linker is shown as SEQ ID NO. 5.

3. The bispecific antibody of claim 1, wherein the anti-human PD1ScFv comprises a PD1 heavy chain having an amino acid sequence as shown in SEQ ID No. 3; a PD1 light chain with an amino acid sequence shown as SEQ ID NO. 4; the anti-human PD1ScFv is formed by connecting the PD1 heavy chain and the PD1 light chain through a Linker; the amino acid sequence of the Linker is shown as SEQ ID NO. 5.

4. The bispecific antibody of any one of claims 1-3, wherein the bispecific antibody has the configuration: CD96ScFv Holes-PD1ScFv Knobes, wherein Holes and Knobes are respectively Fc mutation segments of different IgG 4; mutations of T366W, S354C, S228P and R409K are respectively introduced into PD1ScFv-Fc, and mutations of T366S, L368A, Y407V, Y349C, S228P and R409K are introduced into CD96 ScFv-Fc.

5. The bispecific antibody of claim 4, wherein the amino acid sequence of the Fc mutant holes of IgG4 is represented by SEQ ID No. 6; the amino acid sequence of the CD96ScFv holes is shown in SEQ ID NO. 7.

6. The bispecific antibody of claim 4, wherein the amino acid sequence of the Fc mutant knob of IgG4 is set forth in SEQ ID No. 8; the amino acid sequence of the PD1ScFv knobes is shown in SEQ ID NO. 9.

7. The bispecific antibody of any one of claims 1-3, wherein said bispecific antibody is configured as: CD96ScFv- (G4S)4-PD 1ScFv-Fc, wherein the (G4S)4 sequence is shown as SEQ ID NO. 10; the amino acid sequence of the Fc mutant segment (Fc) of the IgG4 is shown in SEQ ID NO. 11.

8. The method of constructing a bispecific antibody according to any one of claims 4 to 6, wherein said method comprises: respectively constructing a lentivirus expression plasmid of anti-human CD96 ScFv-holes and a lentivirus expression plasmid of anti-human PD1ScFv-knobes, respectively packaging the lentiviruses, simultaneously infecting CHO cells according to the same infection number and screening single clones to obtain a target cell strain.

9. The method of constructing a bispecific antibody according to claim 7, wherein the method comprises: a target cell line was obtained by constructing a CD96ScFv- (G4S)4-PD 1ScFv-Fc lentiviral plasmid by linking the ScFv of CD96 with the ScFv of PD1 using a linker peptide (G4S)4 in Fc mutation S228P, R409K, packaging into a lentivirus, infecting CHO cells and screening for single clones.

10. A nucleic acid sequence comprising the nucleic acid sequence shown as SEQ ID No.13 or SEQ ID No.14 or SEQ ID No. 15; the nucleic acid sequence containing SEQ ID NO.13 codes the amino acid sequence shown in SEQ ID NO. 7; the nucleic acid sequence containing SEQ ID NO.14 codes the amino acid sequence shown in SEQ ID NO. 9; the bispecific antibody of claim 7 is encoded by a nucleic acid sequence comprising SEQ ID No. 15.

11. An expression vector comprising the nucleic acid sequence of claim 10.

12. A host cell comprising the nucleic acid sequence of claim 10 or comprising the expression vector of claim 11.

13. Use of a bispecific antibody of any one of claims 1-7 for the preparation of a medicament for the treatment of tumor cells expressing PD1 and/or expressing CD 96.

14. Use of the bispecific antibody of any one of claims 1 to 7 in the preparation of a detection reagent for detecting PD1 and/or CD 96.

15. Use of a bispecific antibody of any of claims 1-7 to modify a CAR or CAR-T cell.

16. An anti-neoplastic pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 7 or a nucleic acid sequence according to claim 10 or an expression vector according to claim 11 or a host cell according to claim 12.