CN112961245B

CN112961245B - Bispecific antibody targeting CD96 and preparation method and application thereof

Info

Publication number: CN112961245B
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: 2023-07-25
Anticipated expiration: 2041-02-24
Also published as: CN112961245A

Abstract

The invention belongs to the field of bio-pharmaceuticals, and particularly relates to a bispecific antibody targeting CD96, and a preparation method and application thereof. The bispecific antibody comprises anti-human CD96ScFv, anti-human PD1ScFv and IgG4Fc mutant, the configuration of which comprises: CD96ScFv eyes-PD 1ScFv knobes and CD96ScFv- (G4S) 4-PD1ScFv-Fc. According to the invention, CD96 and PD-1 are combined for the first time, and a CD96-PD1 bispecific antibody is constructed for the first time, and the bispecific antibody has higher affinity with PD1 and CD96, so that the CD96 is not only highly expressed in activated T cells, but also highly expressed in activated NK cells, and the expression quantity is increased along with the activation time, and the drug resistance problem caused by the PD1 single antibody in the treatment process can be greatly reduced by adding the CD 96.

Description

Bispecific antibody targeting CD96 and preparation method and application thereof

Technical Field

The invention belongs to the field of bio-pharmaceuticals, and particularly relates to a bispecific antibody targeting CD96, 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, and when it interacts with tumor cell surface ligands PD-L1 and PD-L2, it initiates downstream signaling pathways and inhibits T cell activation, and the use of antibodies to block PD1 interaction with its ligand has proven effective in the clinical treatment of various malignant tumors, but due to the single antibodies to PD1, drug resistance occurs during the course of treatment. Thus, the blocking of PD1 by light has not met clinical expectations and the development of bispecific antibodies for tumor therapy has become an urgent need. Bispecific antibodies possess diverse mechanisms of action and flexible combinations of targets due to simultaneous targeting of two antigens or different epitopes of the same antigen, more than 110 bispecific antibodies have entered different stages of clinical studies. Although bispecific antibodies directed against combined inhibition of immune checkpoint receptors such as KN046 targeting PD-1 x CTLA-4, MGD013 targeting PD1 x LAG3, RG7769 targeting PD1 and TIM3, and the like.

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 fragments scFv, 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 in tandem with 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 apoptosis protein 1 (PD 1) 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 uses thereof, the invention relates to an anti-PD 1 and CD19 bispecific antibody and uses thereof, the anti-PD-1 and CD19 bispecific antibody, or variants thereof, or functional fragments thereof, comprising: a domain that specifically recognizes and binds to immune cell surface antigen PD-1, comprising a heavy chain variable region of an anti-PD-1 specific antibody (anti-PD-1 VH); and a domain that specifically recognizes and binds CD19, including the heavy chain variable region of an anti-CD 19 specific antibody (anti-CD 19 VH).

The above bispecific antibodies have met with significant success in many cancers, but not all patients benefit from these therapies. Thus, finding new immune checkpoint receptors is a trend, stephen J Blake et al propose CD96, which is also an immune checkpoint, CD96 is highly expressed not only in activated T cells but also in activated NK cells, and increases the expression level with activation time, it competes with immune checkpoint molecule TIGIT and co-activator molecule CD226 for binding to tumor cell surface specific antigens CD155 and CD112, thus being an ideal immune checkpoint receptor for combination therapy, and currently, there are more than 30 mature bispecific antibody molecule platforms that promote correct matching of heavy and light chains. However, when two heavy and light chains of classical bispecific antibodies are expressed simultaneously in host cells, heavy and light chain mismatches occur and produce irremovable byproducts, and how to construct a stable bispecific antibody is also a problem, and no related literature currently discloses the use of CD96 in combination with other antibodies.

Disclosure of Invention

In view of this, it is an object of the present invention to provide a bispecific antibody that can specifically target CD96 and PD1, and a nucleic acid sequence encoding the same, 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 an Fc fragment of IgG 4; and n is more than or equal to 1.

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

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

Furthermore, one end of the anti-human CD96ScFv or anti-human PD1ScFv sequence is connected with a membrane-outlet peptide, and the amino acid sequence of the membrane-outlet peptide is shown as SEQ ID NO.12 or the amino acid sequence with homology more than or equal to 80%. The membrane-outlet peptide is a peptide chain capable of guiding 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 as follows: CD96ScFv Holes-PD1ScFv Knobes, wherein Holes and Knobes are different Fc mutant fragments of IgG4, respectively; the mutations T366W, S354C, S228P and R409K were introduced into PD1ScFv-Fc, and the mutations T366S, L368A, Y407V, Y349C, S228P and R409K were introduced into CD96 ScFv-Fc, respectively. Specifically, the linkage between two Fc mutations is a routine procedure for one skilled in the art, mutation site binding and disulfide bond binding of the upper segment of the Fc mutation.

Further, the anti-human CD96ScFv comprises an amino acid sequence of which the amino acid sequence is shown as SEQ ID NO.1, wherein the homology of the amino acid sequence is more than or equal to 95 percent of a CD96 heavy chain; the amino acid sequence is shown as SEQ ID NO.2, and the homology of the CD96 light chain or the amino acid sequence is more than or equal to 95%; 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 in 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 mutant segment holes of IgG4 is shown in SEQ ID NO.6 or the amino acid sequence with the homology of more than or equal to 95 percent; the amino acid sequence of the CD96ScFv holes is shown as SEQ ID NO.7 or the amino acid sequence with the homology of more than or equal to 95 percent.

Further, the amino acid sequence of the Fc mutant knobes of IgG4 is shown in SEQ ID NO.8 or the amino acid sequence with the homology of more than or equal to 95%; the amino acid sequence of PD1ScFv knobes is shown as 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 CD96ScFv holes-PD1ScFv knobes bispecific antibody is provided, which is characterized in that the method comprises the following steps: respectively constructing lentiviral expression plasmids of anti-human CD96 ScFv-holes and anti-human PD1ScFv-knobes, respectively packaging the lentiviruses into lentiviruses, simultaneously infecting CHO cells according to the same infection complex number, and screening monoclonal to obtain a target cell strain.

Further, the construction method further comprises the steps of culturing the obtained target cell strain, performing amplification culture, collecting culture supernatant, purifying the target Protein by a Protein A affinity chromatography column (nano-micro), desalting by 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 of any one of the preceding claims is provided, wherein the nucleic acid sequence comprises a sequence shown as SEQ ID NO.13 or a sequence with homology of more than or equal to 90%; or the nucleic acid sequence of PD1ScFv knobes, which codes for any one of the former, and comprises a sequence shown as SEQ ID NO.14 or a sequence with the homology of more than or equal to 90 percent.

Further, there is provided an expression vector comprising the nucleic acid sequence of any one of the preceding claims.

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

Further, there is provided a host cell comprising the nucleic acid sequence or expression vector of any one of the preceding claims.

Further, the host cell is a variety of host cells conventional in the art, as long as it is capable of stably self-replicating the above recombinant expression vector and the nucleotide carried thereby can be expressed efficiently. Wherein the host cell comprises a prokaryotic expression cell and a eukaryotic expression cell, the expression vector preferably comprising: COS, CHO (chinese hamster ovary), NS0, sf9, sf21, DH 5. Alpha., BL21 (DE 3) or TG1, more preferably E.coli TG1, BL21 (DE 3) cells (expressing single chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full length IgG antibodies). The expression vector is transformed into a host cell to obtain the preferred recombinant expression transformant of the invention.

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

The configuration is as follows: CD96ScFv- (G4S) 4-PD1ScFv-Fc, wherein the (G4S) 4 sequence is shown in SEQ ID NO.10 or the amino acid sequence with homology more than or equal to 80%; the amino acid sequence of the Fc mutant section of the IgG4 is shown as SEQ ID NO.11 or the amino acid sequence with the homology of more than or equal to 95 percent.

Further, a construction method of the CD96ScFv- (G4S) 4-PD1ScFv-F bispecific antibody is also provided, and the construction method comprises the following steps: a fragment of connecting peptide (G4S) 4 is used to connect ScFv of CD96 and ScFv of PD1 in Fc mutation S228P, R409K, so as to construct CD96ScFv- (G4S) 4-PD1ScFv-Fc lentiviral plasmid, after packaging into lentivirus, CHO cells are infected, and monoclonal is selected to obtain a target cell strain.

Further, the construction method further comprises culturing the obtained target cell strain, collecting culture supernatant, purifying the target Protein by a Protein A affinity chromatography column (nano-micro), desalting by a size exclusion chromatography column Superdex 200pg (GE), and detecting the purity by SDS-PAGE to obtain the CD96ScFv- (G4S) 4-PD1ScFv-Fc bispecific antibody.

Further, a nucleic acid sequence for encoding the CD96ScFv- (G4S) 4-PD1ScFv-Fc bispecific antibody is provided, wherein the nucleic acid sequence comprises a sequence shown as SEQ ID NO.15 or a sequence with the homology of more than or equal to 90%.

Further, an expression vector comprising a nucleic acid sequence encoding a CD96ScFv- (G4S) 4-PD1ScFv-Fc bispecific antibody is provided.

The second object of the present invention is to provide a use of the bispecific antibody according to any one of the preceding claims.

Further, there is provided an application of the bispecific antibody in preparing a medicament for treating tumor cells expressing PD1 and/or CD 96.

Further, the application of the bispecific antibody in preparing a detection reagent for detecting PD1 and/or CD96 is also provided.

Further, there is also provided the use of the aforementioned bispecific antibody for modifying a CAR or CAR-T cell.

The third object of the present invention is to provide an antitumor pharmaceutical composition comprising the bispecific antibody or nucleic acid sequence or expression vector or host cell of any one of the preceding.

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

Further, the anti-tumor pharmaceutical composition also comprises one or more pharmaceutically acceptable carriers, diluents or excipients, and the pharmaceutical composition can be a suspension, a water injection, a freeze-drying preparation and the like which are commonly used in the pharmaceutical field. Bispecific antibodies are formulated into pharmaceutical compositions with pharmaceutically acceptable carrier formulations that ensure the conformational integrity of the amino acid core sequences of the disclosed bispecific antibodies while also protecting the multifunctional groups of the proteins from degradation (including but not limited to aggregation, deamination or oxidation) to provide more stable therapeutic efficacy.

Further, the antitumor pharmaceutical composition can be used in combination with chemicals for improving the effect of treating tumors, the chemicals comprising: opdivo, keytruda, tecentriq, bavencio, imfinzi and Carilizumab for injection, terlipressin Li Shan anti-injection, xindi Li Shan anti-injection.

Further, the antitumor pharmaceutical 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, vulvar cancer, rectal cancer, colon cancer, anal region cancer, breast cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urinary tract cancer, penile cancer, prostate cancer, pancreatic cancer, brain cancer, testicular cancer, lymphoma, transitional cell carcinoma, bladder cancer, renal cancer or ureter cancer, renal cell carcinoma, renal pelvis cancer, hodgkin's disease, non-hodgkin's lymphoma, soft tissue sarcoma, childhood solid tumors, lymphocytic lymphomas, central Nervous System (CNS) tumors, primary central nervous system lymphomas, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, melanoma, kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma, chronic or acute leukemia, combinations thereof, and the like.

The invention has the beneficial effects that:

(1) The bispecific antibody adopts an ScFv rather than a Fab structure, so that the problem of mismatch of a light chain is not considered in construction, the construction difficulty and the production of byproducts are greatly reduced, S228P and R409K are introduced into an Fc region, fab-arm Exchange caused by adopting an IgG4 framework is avoided, and the finally formed bispecific antibody is more stable.

(2) According to the invention, CD96 and PD-1 are combined for the first time, and a CD96-PD1 bispecific antibody is constructed for the first time, and the bispecific antibody has higher affinity with PD1 and CD96, so that the CD96 is not only highly expressed in activated T cells, but also highly expressed in activated NK cells, and the expression quantity is increased along with the activation time, and the drug resistance problem caused by the PD1 single antibody in the treatment process can be greatly reduced by adding the CD 96.

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

(4) The bispecific antibody has smaller molecular weight than the classical bispecific antibody, can reach an action site more easily, and the CD96ScFv- (G4S) 4-PD1ScFv-Fc type bispecific antibody is tetravalent, namely one antibody can respectively recognize two CD96 and PD1 and can play a stronger action effect.

(5) The bispecific antibody of the invention has the potential of being developed into an anti-tumor drug or being applied to the anti-tumor field.

Drawings

FIG. 1 is a diagram showing the structure of CD96 ScFv-Holes-PD 1ScFv-Knobes bispecific antibody.

FIG. 2 is a diagram showing the structure of a CD96ScFv- (G4S) 4-PD1ScFv-Fc bispecific antibody.

FIG. 3 is a diagram showing the result of SDS-PAGE of bispecific antibodies.

FIG. 4 is a graph of the results of a size exclusion chromatography column for bispecific antibodies.

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

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

FIG. 7 is a graph of PD1 Fortebio assay results.

FIG. 8 is a graph of CD96 Fortebio assay results.

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

FIG. 10 shows the binding of CD96ScFv- (G4S) 4-PD1ScFv-Fc to a target on T cells and on foreign construct cells.

In FIG. 3, line 1 is CD96 ScFv-Holes-PD 1ScFv-Knobes, and Line 2 is CD96ScFv- (G4S) 4-PD1ScFv-Fc.

Detailed Description

The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.

In the embodiment of the invention, RMPI1640 medium, DMEM medium and F12 medium are purchased from gibco company; streaming 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 were purchased from Thermo fisher company; SFM-Z04 serum free medium was from Chongqing precision Biotechnology institute of technology Co.

EXAMPLE 1 construction and production of CD96 ScFv-Holes-PD 1ScFv-Knobes and CD96ScFv- (G4S) 4-PD1ScFv-Fc

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

T366W, S354C, S228P and R409K mutations are respectively introduced into PD1ScFv-Fc, T366S, L368A, Y407V, Y349C, S228P and R409K mutations are introduced into CD96 ScFv-Fc, respectively, a lentiviral expression plasmid for resisting human CD96 ScFv-Holes and a lentiviral expression plasmid for resisting human PD1ScFv-Knobes are respectively constructed, respectively, after being packaged into lentiviruses, CHO cells are simultaneously infected according to the same infection complex number, monoclonal is selected, a target cell strain is obtained, the target cell strain is cultured, a culture supernatant is collected, a target Protein is purified through a Protein A affinity chromatography column (nano-micro), a molecular exclusion chromatography column Superdex 200pg (GE) is used for desalting treatment, and purity is detected by SDS-PAGE, so that the CD96 ScFv-Holes-PD1 ScFv-knobs bispecific antibody is obtained, and the structure of the antibody is shown in figure 1.

Primers used for Knobes-into-holes mutation are shown in Table 1 below:

TABLE 1 primers used for Knobes-into-holes mutation

(2) Construction and production of CD96ScFv- (G4S) 4-PD1ScFv-Fc

The ScFv of CD96 and ScFv of PD1 were ligated using a fragment of linker peptide (G4S) 4 at Fc mutations S228P, R409K to construct a CD96ScFv- (G4S) 4-PD1ScFv-Fc lentiviral plasmid, which was packaged into lentiviruses, CHO cells were infected and screened for monoclonal. Obtaining target cell strain, culturing target cell strain, collecting culture supernatant, purifying target Protein by Protein A affinity chromatography column (nanometer), desalting by molecular exclusion chromatography column Superdex 200pg (GE), and detecting purity by SDS-PAGE to obtain CD96ScFv- (G4S) 4-PD1ScFv-Fc bispecific antibody, the structure of which is shown in figure 2.

Example 2 sizing of CD96 ScFv-Holes-PD 1ScFv-Knobes and CD96ScFv- (G4S) 4-PD1ScFv-Fc antibodies

The purified antibody of example 1 was confirmed by SDS-PAGE (the result of which is shown in FIG. 3), and the position of the band was found to be correct, 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-PD1ScFv-Fc monomer was 79.40kDa, and the purified antibody was confirmed by SDS-PAGE (the result of the confirmation is shown in FIG. 3), and the position of the band was found to be correct, and then the bispecific antibody was high in purity (> 95%) and formed into a dimer by size exclusion assay (the result of the measurement is shown in FIG. 4).

Example 3 validation of biological Activity of antibodies

(1) Biological Activity of monoclonal antibodies

1) Binding of antibodies to antigens

Exogenously constructed CHO-PD1 cell lines, the expression of the target antigen and the binding of the antibody to the target antigen were flow detected, demonstrating that PD1 antibodies can specifically recognize the exogenously constructed cell lines CHO-PD1 and PD1 antigen molecules on T cells. Exogenous construction of 293T-CD96 cell lines, flow detection of target antigen expression and antibody binding to target antigen demonstrated that CD96 antibodies can specifically recognize exogenous construction of 293T-CD96 cell lines and CD96 antigen molecules on T cells.

As shown in FIGS. 5 and 6, the expression rates of the target antigens were 95.42% and 98.55%, respectively, for the exogenously constructed CHO-PD1 and 293T-CD 96. 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 recognize the target antigen. As shown in the following graph, 1.25. Mu.g of purified antibody was added to CHO and 293T cells as well, and FITC-IgG was not expressed, demonstrating that ScFv-Fc antibodies expressing purified CD96 and PD1 specifically bound to the target antigen. The expression level of CD96 gradually increases during the activation culture of T cells, while the expression rate of PD1 is about 10% in the absence of stimulation by target cells. On day 9, the expression ratio of PD1 was detected as 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 detected as 96.05%, after the addition of CD96 ScFv-Fc antibody. FITC-IgG expression was examined to be 96.09%. It was demonstrated that ScFv-Fc antibodies to PD1 and CD96 can recognize and bind to PD1 and CD96 antigens on the surface of T cells.

2) Affinity for

After the PD1-His and CD96-His antigens are solidified, the binding dissociation and the affinity of the PD1-His and the CD96-His antigens are measured by using Fortebio, the data are shown in the following table 2 and the following table 3 (according to the data graphs, the Fortebio measurement results are shown in fig. 7 and fig. 8), the KD of the PD1ScFv-Fc and the PD1-His is 8.897E-09M, and the KD of the CD96 ScFv-Fc and the CD96-His is 3.081E-08M, and the PD has higher affinity.

TABLE 2 PD1 Fortebio assay results

TABLE 3 CD96 Fortebio assay results

(2) Biological Activity of bispecific antibodies

Flow cytometry verifies binding of antibodies to antigens: exogenously constructing a CHO-PD1 cell line, detecting the positive rate by flow cytometry, incubating purified CD96 ScFv-Holes-PD 1ScFv-Knobes and CD96ScFv- (G4S) 4-PD1ScFv-Fc antibodies with CHO-PD1 respectively, and verifying the combination of the antibodies and antigens by flow cytometry. Exogenous construction of 293T-CD96 cell line, and detection of positive rate by flow cytometry. The purified CD96 ScFv-Holes-PD 1ScFv-Knobes antibody and CD96ScFv- (G4S) 4-PD1ScFv-Fc antibody were incubated with 293T-CD96, respectively, and the binding of the antibodies to the antigen was confirmed by flow cytometry, and as shown in FIGS. 9 and 10, the bispecific antibody bound well to two target cells, respectively, and it was found that both antibodies had higher affinity to both antigens.

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and 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 and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

Sequence listing

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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 Val 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> 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, wherein the bispecific antibody has the configuration: CD96ScFv holes-PD1ScFv knobs, wherein the amino acid sequence of the CD96ScFv holes is shown as SEQ ID NO.7, and the amino acid sequence of the PD1ScFv knobs is shown as SEQ ID NO. 9; or a CD96ScFv- (G4S) 4-PD1ScFv-Fc, wherein the CD96ScFv comprises a CD96 heavy chain with an amino acid sequence shown as SEQ ID NO.1, a CD96 light chain with an amino acid sequence shown as SEQ ID NO.2, the CD96ScFv is formed by connecting the CD96 heavy chain and the CD96 light chain through a Linker, the PD1ScFv comprises a PD1 heavy chain with an amino acid sequence shown as SEQ ID NO.3, a PD1 light chain with an amino acid sequence shown as SEQ ID NO.4, the 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, and the (G4S) 4 sequence is shown as SEQ ID NO. 10. The amino acid sequence of the Fc is shown as SEQ ID NO. 11.

2. The method of constructing a bispecific antibody of claim 1, comprising:

respectively constructing lentiviral expression plasmids of anti-human CD96 ScFv-holes and anti-human PD1 ScFv-knobs, respectively packaging the lentiviruses, simultaneously infecting CHO cells according to the same infection complex number, and screening monoclonal to obtain a target cell strain.

3. The method of constructing a bispecific antibody of claim 2, comprising: a fragment of connecting peptide (G4S) 4 is used to connect the ScFv of CD96 and the ScFv of PD1, so as to construct CD96ScFv- (G4S) 4-PD1ScFv-Fc lentiviral plasmid, after packaging into lentivirus, CHO cells are infected, and monoclonal cells are screened to obtain the target cell strain.

4. Nucleic acid encoding the bispecific antibody according to claim 1, characterized in that it comprises a nucleic acid sequence as shown in SEQ ID No.13 and SEQ ID No.14, wherein the amino acid sequence encoded by the nucleic acid sequence as shown in SEQ ID No.13 comprises an amino acid sequence as shown in SEQ ID No.7, and wherein the amino acid sequence encoded by the nucleic acid sequence as shown in SEQ ID No.14 comprises an amino acid sequence as shown in SEQ ID No. 9.

5. An expression vector comprising the nucleic acid of claim 4.

6. A host cell comprising the nucleic acid of claim 4 or comprising the expression vector of claim 5.

7. Use of the bispecific antibody of claim 1 for the preparation of a medicament for the treatment of PD 1-expressing and/or CD 96-expressing tumor cells.

8. Use of a bispecific antibody according to claim 1 for the preparation of a detection reagent for the detection of PD1 and/or CD 96.

9. Use of the bispecific antibody of claim 1 in the preparation of a CAR or a modifier of a CAR-T cell.

10. An anti-neoplastic pharmaceutical composition comprising the bispecific antibody of claim 1 or the nucleic acid of claim 4 or the expression vector of claim 5 or the host cell of claim 6.