CN118290590B - A bispecific antibody, encoding gene and use thereof in preparing drugs for treating tumors - Google Patents

Abstract

The invention discloses a bispecific antibody, a coding gene and application thereof in preparing a medicament for treating tumors, and discloses application of a bispecific antibody protein gene sequence targeting GPC3/PD-L1 in preparing a medicament for treating tumors, belonging to the technical field of biological medicine. The invention proves that the bispecific antibody protein targeting GPC3/PD-L1 expressed by the bispecific antibody gene medicine targeting GPC3/PD-L1 can obviously inhibit the growth of tumor, prolong the survival period of tumor-bearing mice and improve the release level of serum IFN-gamma. The targeted GPC3/PD-L1 bispecific antibody gene medicine provided by the invention can be used for anti-tumor treatment.

Description

A bispecific antibody, encoding gene and its application in the preparation of drugs for treating tumors

Technical Field

The present invention relates to the field of biomedicine technology, and in particular to a bispecific antibody, an encoding gene and application thereof in preparing a drug for treating tumors.

Background Art

Cancer is the leading cause of death, with nearly 10 million people dying from cancer each year worldwide. Among them, hepatocellular carcinoma (HCC) is one of the most common malignant tumors of the digestive system and the third leading cause of cancer death worldwide, with an increasing incidence rate year by year. Due to the insidious onset of hepatocellular carcinoma and the difficulty in early diagnosis, most patients are already in the advanced or metastatic stage (such as extrahepatic spread or large blood vessel invasion) when diagnosed, and their prognosis is usually poor, with a short survival period and a high mortality rate. For patients with advanced hepatocellular carcinoma and patients with intermediate-stage HCC who have relapsed after local treatment, systemic therapy is the preferred treatment option to improve survival.

Since the SHARP trial in 2007 first demonstrated the effect of sorafenib in improving overall survival in unresectable HCC, it has become the first FDA-approved first-line treatment option for patients with advanced liver cancer. With the publication of the results of the IMbrave150 trial, targeted therapy based on tyrosine kinase inhibitors (TKIs) has become the first-line standard of care for advanced liver cancer. Although a variety of targeted drugs have emerged during this period, and the FDA has accelerated or fully approved a variety of first-line or second-line treatment options for advanced HCC, the overall survival of patients still needs to be improved, and there is an urgent need to explore new effective treatment methods. Since 2017, several Phase III clinical trials for first-line or second-line systemic treatment of advanced HCC have shown positive results. Based on the results of the Checkmate-040 and KEYNOTE-224 studies, the FDA conditionally approved PD-1 monoclonal antibodies Nivolumab and Pembrolizumab as second-line treatments for liver cancer. After more than a decade of tyrosine kinase inhibitor-dominated treatment of advanced liver cancer, immune checkpoint inhibitor (ICI)-based treatment regimens have become the main component of systemic treatment of advanced liver cancer, expanding the treatment options for advanced HCC patients and improving patient prognosis, thus ushering in a new era of liver cancer immunotherapy.

The IMbrave150 study demonstrated a significant survival benefit of atezolizumab combined with bevacizumab in patients with advanced HCC, leading to its FDA approval in 2020 as a first-line systemic treatment for unresectable HCC. In the latest survival analysis, the median overall survival of atezolizumab combined with bevacizumab was 19.2 months versus 13.4 months with sorafenib (P < 0.001), and patients achieved a significant survival advantage. In addition, ICI-based first-line systemic treatment combinations for advanced HCC also include dual immune checkpoint therapy with durvalumab + tremelimumab. In the HIMALAYA Phase III clinical trial, patients with advanced HCC who had not received systemic treatment had a median overall survival of 16.4 months with durvalumab plus tremelimumab, compared with 13.8 months with sorafenib, indicating that this regimen is superior to sorafenib. Currently, multiple phase III clinical trials of ICI-based systemic treatment regimens are being actively conducted, such as ORIENT-32, LEAP-002 and SHR-1210-III-310. However, the current combination therapy strategies still have limitations. Under different treatment strategies such as immune checkpoint inhibitors (ICIs) combined with TKI/bevacizumab, the objective response rate (ORR) is less than 50%, the median survival time (mOS) is less than 2 years, and the incidence of adverse reactions above grade 3-4 is as high as 30-90%. New treatment strategies are urgently needed to increase the efficacy of immunotherapy while reducing the incidence of adverse reactions.

The selection of tumor targets is a key factor in determining the therapeutic efficacy of bispecific antibodies. The selected tumor-specific antigens must be highly expressed in tumor tissues and not expressed or expressed at extremely low levels in important tissues.

Bispecific antibodies (BsAbs) are a family of proteins that can simultaneously recognize two different antigens or epitopes. With the continuous advancement of drug development and technology platforms, BsAbs have received widespread attention as a new strategy for anti-tumor immunotherapy in recent years. With the development of advanced technologies, a variety of BsAb construction forms have been proposed. According to the different Fc domains, BsAbs can be roughly divided into two types: IgG format and non-IgG format. Fc-free BsAbs include TandAb, TandscFv, DART, Diabody, F(ab')2 and ImmTAC. IgG-like antibody molecules containing Fc domains retain Fc-mediated effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP). Although there are many structural forms of bispecific antibodies, BiTE and Triomabs are one of the more mature forms of BsAb molecules. BiTE is a fusion protein composed of a single-chain variable fragment (scFv) connected by a short peptide linker. It can target two different targets at the same time, usually by recruiting T cells to mediate the killing of tumor cells. In 2014, Blinatumomab became the first anti-CD19/CD3 BiTE molecule approved by the FDA for the treatment of relapsed/refractory B-precursor acute lymphoblastic leukemia (ALL), and was approved in different types of hematological tumors. The success of the Blinatumomab bispecific antibody has promoted the development of bispecific antibody immunotherapy in solid tumors. Triomabs is another form of antibody construction. As an IgG-like molecule, it has a longer serum clearance half-life compared to BiTE molecules. It can interact with the FcR of innate immune cells through the Fc segment domain, thereby mediating the recruitment of immune cells and the activation and proliferation of T cells, and producing an effective anti-tumor effect by combining the killing mechanism of innate immune response and adaptive immune response. There are currently no reports on liver cancer immunotherapy through bispecific antibodies targeting GPC3/PD-L1.

Summary of the invention

The purpose of the present invention is to provide a bispecific antibody, an encoding gene and use thereof in the preparation of a drug for treating tumors, so as to solve the problems existing in the above-mentioned prior art.

To achieve the above object, the present invention provides the following solutions:

The present invention provides a bispecific antibody, which is a bispecific antibody targeting GPC3/PD-L1.

Preferably, its amino acid sequence is as shown in SEQ ID NO.1.

The present invention also provides a DNA molecule encoding the bispecific antibody, and its nucleotide sequence is shown in SEQ ID NO.2.

The present invention also provides an expression vector of the bispecific antibody, wherein the expression vector uses CMV as a promoter, and the Igκ secretory peptide encoding gene, the GPC3 single-chain variable region fragment GPC3-scFv, Hinge, Fc, P2A, the Igκ secretory peptide encoding gene, the PD-L1 single-chain variable region fragment PD-L1-scFv, Hinge and Fc are sequentially connected downstream of the CMV promoter.

The present invention also provides a method for constructing the expression vector, comprising the following steps:

(1) Based on the light and heavy chain sequences of GPC3, the light and heavy chain sequences of PD-L1 and the Fc fragment, the GPC3-scFv-Fc fragment and the PD-L1-scFv-Fc fragment were amplified by overlapping PCR, with the cloning sites being Hind III/ Eco R I and Eco R I/ Xho I, respectively;

(2) The GPC3 light and heavy chain sequences and the PD-L1 light and heavy chain sequences were connected by (G ₄ S) ₃ , and the amplified GPC3-scFv and PD-L1-scFv were connected to the Fc segment by Hinge;

(3) During the primer design process, the P2A cleavage peptide was introduced, and the target sequence fragment and the vector were double-enzyme-cut to remove the sticky ends, and then the bispecific antibody plasmid was constructed. The promoter was CMV.

The present invention also provides use of the bispecific antibody, the DNA molecule or the expression vector in preparing a drug for treating liver cancer.

The present invention also provides a gene medicine for treating liver cancer, comprising the bispecific antibody, the DNA molecule or the expression vector.

The present invention also provides a pharmaceutical preparation for treating liver cancer, comprising the bispecific antibody, the DNA molecule, the expression vector or the gene drug and a pharmaceutically acceptable carrier.

Preferably, the pharmaceutically acceptable carrier is suitable for liquid dosage form, solid dosage form or paste dosage form.

Based on the above technical solution, the present invention has the following technical effects:

The present invention discloses the use of a bispecific antibody gene drug targeting GPC3/PD-L1 in the preparation of a drug for treating tumors, and discloses the use of a bispecific antibody protein gene sequence targeting GPC3/PD-L1 in the preparation of a drug for treating tumors, and confirms that the bispecific antibody protein targeting GPC3/PD-L1 expressed by the bispecific antibody gene drug targeting GPC3/PD-L1 can significantly inhibit tumor growth, prolong the survival period of tumor-bearing mice, and increase the release level of serum IFN-γ. It shows that the bispecific antibody gene drug targeting GPC3/PD-L1 provided by the present invention can be used for anti-tumor treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

Figure 1 is a schematic diagram of the DNA structure encoding a bispecific antibody gene drug, wherein A. is a connection diagram of the construction of a bispecific antibody gene drug; B. is a schematic diagram of the unilateral structure of a bispecific antibody; C. is a schematic diagram of the structure of a bispecific antibody;

Figure 2 shows the gene drug sequence (A) and schematic diagram (B) encoding the BsAb bispecific antibody; C. DNA gel electrophoresis (Marker: 5 kb DNA Ladder); D. Gene drug sequencing results;

Figure 3 is a Western blotting result diagram: in vitro expression verification of hGPC3/PD-L1 BsAb (A) and mGPC3/PD-L1 BsAb (B) gene drugs;

Figure 4 is an ELISA binding ability test of the antibodies expressed in the cell supernatant after gene drug (expression plasmid) transfection with GPC3 and PD-L1 target proteins; A: phBsAb supernatant binds to human GPC3 protein, B: phBsAb supernatant binds to human PD-L1 protein, C: mhBsAb supernatant binds to mouse PD-L1 protein, D: phBsAb supernatant binds to human GPC3 protein under dilution gradient; E: phBsAb supernatant binds to human PD-L1 protein under dilution gradient, F: pmBsAb supernatant binds to mouse PD-L1 protein under dilution gradient;

Figure 5 is a binding test of the antibody expressed in the cell supernatant after transfection of the gene drug (expression plasmid) with the GPC3 target protein on the surface of the human liver cancer cell line; wherein, A: pmBsAb and HepG2 cell binding ability test, B: pmBsAb and Huh7 cell binding ability test, C: pmBsAb and Hep3B cell binding ability test, D is the binding percentage of pmBsAb and HepG2 cells; E: pmBsAb and Huh7 cell binding percentage, F: pmBsAb and Hep3B cell binding percentage;

Figure 6 shows the tumor-suppressing effect of gene drugs in the subcutaneous Huh 7 liver cancer cell xenograft model of humanized immune system NSG mice, including: A. Schematic diagram of the construction of Huh 7 subcutaneous liver cancer model and treatment plan of humanized immune system NSG mice; B. Average tumor volume growth curve of Huh 7 subcutaneous liver cancer model of humanized immune system NSG mice after gene drug treatment; C. Tumor volume growth curve of tumor-bearing mice in each treatment group; ns, no statistical difference; *p<0.05; **p<0.01; ***p<0.001;

Figure 7 shows the survival curves of mice in different treatment groups in the Huh 7 tumor-bearing model of NSG mice with humanized immune system; ns, no statistical difference; *p<0.05; **p<0.01; ***p<0.001;

Figure 8 shows the results of ELISA quantitative analysis of IFN-γ in the serum of each treatment group on the 30th day after bispecific antibody gene drug (expression plasmid) delivery treatment.

DETAILED DESCRIPTION

Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

Example 1

Construction of bispecific antibody gene drug (expression plasmid) targeting GPC3

The amino acid sequence of the bispecific antibody gene drug targeting GPC3 is shown in SEQ ID NO.1, and the encoding gene is shown in SEQ ID NO.2:

SEQ ID NO.1: METDTLLLWVLLLWVPGSTGDDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMH WVKQTPVHGLKWIGALDPKTGDTAYSQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRFYSY type SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKEFGSGATNFSL LKQAGDVEENPGPMELPVRLFWIPASLSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFY ADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR IKLGTVTTVDYWGQGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*;

SEQ ID NO.2: ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACGACGTGGTGATGACCCAGACCCCCCTGAGCCTGCCCGTGAGCCTGGGCGACCAGGCCAGCATCAGCTGCAGGAGCAGCCAGCCTGGTGCACAGCAACGGCAACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTGAGCAACAGGTT CAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTTCTGCAGCCAGAACACCCACGTGCCCCCCACCTTCGGCA GCGGCACCAAGCTGGAGATCAAGGGCGGCGGAGGTAGCGGAGGTGGTGGCAGTGGTGGAGGAGGCTCACAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGAGGCCCGGCGCCAGCGTGAAGCTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGACTACGAGATGCACTGGGTGAAGCAGACCCCCGTGCACGGGCCTGAAGTGGATCGGCGCCCTGGACCCCAAGACCGGCGACACCGCCTACAGC CAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGGAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCACCAGGTTCTACAGCTACACCTACTG GGGCCAGGGCACCCTGGTGACCGTGAGCGCCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGA GGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATC GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGAATTCGGAAGCGGAGCTACTAACTTCAGCCTGCTGA AGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGGAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCTTAAGCCAGTCCGCCCTGACCCAGCCTGCCTCCGTGTCTGGCTCCCCTGGCCAGTCCATCACCATCAGCTGCACCGGCACCTCCAGCGACGTGGGCGGCTACAACTACGTGTCCTGGTATCAGCAGCACCCCGGCAAGGCCCCAAGCTGATGATCTACGACGTGTCCA ACCGGCCCTCCGGCGTGTCCAACAGATTCTCCGGCTCCAAGTCCGGCAACACCGCCTCCCTGACCATCAGCGGACTGCAGGCAGAGGACGAGGCCGACTACTGCTCCCCTACACCTCCTCCA GCACCAGAGTGTTCGGCACCGGCACAAAAGTGACCGTGCTGGGCGGAGGCGGAAGCGGAGGCGGAGGAAGCGGCGCGGCAGCGAGGTGCAGCTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCAGCTACATCATGATGTGGGTGCGACAGGCCCTGGCAAGGGCCTGGAATGGGTGTCCTCCATCTACCCCTCCGGCGG CATCACCTTCTACGCCGACACCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCCGGAT CAAGCTGGGCACCGTGACCACCGTGGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGG AGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTCCTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTCCTCGTAAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA.

In this example, a bispecific antibody gene drug targeting GPC3/PD-L1 was constructed, hereinafter referred to as pBsAb. The GPC3 heavy chain and light chain variable region coding sequences used in this example are as follows:

Igκ secretory peptide encoding gene sequence:

SEQ ID NO.3: ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC;

VL of α-hGPC3 (Patent No.: US20140044714A1):

SEQ ID NO.4: GACGTGGTGATGACCCAGACCCCCCTGAGCCTGCCCGTGAGCCTGGGCCGACCAGGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGGTGCACAGCAACGGCAACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTGAGCAACAGGTTCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGAT CAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTTCTGCAGCCAGAACACCCACGTGCCCCCCACCTTCGGCAGCGGCACCAAGCTGGAGATCAAG;

VH of α-hGPC3 (Patent No.: US20140044714A1):

SEQ ID NO.5: CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGAGGCCCGGCGCCAGCGTGAAGCTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGACTACGAGATGCACTGGGTGAAGCAGACCCCCGTGCACGGCCTGAAGTGGATCGGCGCCCTGGACCCCAAGACCGGCGACACCGCCTACAGCCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCG CCTACATGGAGCTGAGGAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCACCAGGTTCTACAGCTACACCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC;

VL of α-hPD-L1:

SEQ ID NO.6: GAGTTGCCTGTTAGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCTTAAGCCAGTCCGCCCTGACCCAGCCTGCCTCCGTGTCTGGCTCCCCTGGCCAGTCCATCACCATCAGCTGCACCGGCACCTCCAGCGACGTGGGCGGCTACAACTACGTGTCCTGGTATCAGCAGCACCCCGGCAAGGCCCCCAAGCTGATGATCTACGACGTGTCCAACCGGCCCTCCGGCGTGTCCAA CAGATTCTCCGGCTCCAAGTCCGGCAACACCGCCTCCCTGACCATCAGCGGACTGCAGGCAGAGGACGAGGCGACTACTACTGCTCCCCTACACCTCCTCCAGCACCAGAGTGTTCGGCACCGGCACAAAAGTGACCGTGCTG;

VH of α-hPD-L1:

SEQ ID NO.7: GAGGTGCAGCTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTTCCAGCTACATCATGATGTGGGTGCGACAGGCCCCTGGCAAGGGCCTGGAATGGGTGTCCTCCATCTACCCCTCCGGCGGCATCACCTTCTACGCCGACACCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTG CAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCCGGATCAAGCTGGGCACCGTGACCACCGTGGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCC;

VL of α-mPD-L1:

SEQ ID NO.8: GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGCAGTGGATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAG ACTTGGCAGATTATTTCTGTCAGCAGTATAACAGCTATCCTCTCACGTTCGGTGCTGGGTCCAAGCTGGAGCTGAAA;

VH of α-mPD-L1:

SEQ ID NO.9: CAGGTCCACCTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGTTACTGGATGTACTGGGTGAAACAGGGGCCTGGACGAGGCCTTGAGTGGATTGGAAGGATTGATCCTAATAGTGGGAGTACTAAGTACAATGAGAAGTTCAAGAACAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGC CTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTGCAAGGGACTATAGAAAGGGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA;

Fc (hIgG1):

SEQ ID NO.10: TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAACAA GTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAG CCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGGTGATGCATGA GGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA;

Fc (mIgG 2a):

SEQ ID NO.11: GCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCCATCCAGCACCAGGACTGG ATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCATCGAGAGAACCATCTCAAAA CCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTT CAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAATGA;

(G ₄ S) ₃ flexible linker encoding gene sequence:

SEQ ID NO.12: GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC;

Hinge (SEQ ID NO. 13): GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCG;

Cleavage peptide P2A encoding gene sequence:

SEQ ID NO. 14: GGAACGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT.

The above sequence was delivered to Shanghai Bioengineering for synthesis by our laboratory in the early stage. In this example, the light and heavy chain sequences of GPC3 were amplified from the plasmid pcDNA3.1-GPC3 × CD3 constructed in the early stage of our laboratory by using the PCR method to construct a bispecific antibody gene drug (expression plasmid) targeting GPC3/PD-L1. The remaining required PD-L1 fragments and Fc fragments were obtained by PCR amplification from the plasmid pcDNA3.1-PD-L1 constructed in the early stage of our laboratory. Then, the GPC3scFv-Fc fragment and the PD-L1scFv-Fc fragment were amplified by overlapping PCR, and the cloning sites were Hind III/ Eco RI; Eco RI/ Xho I, respectively. The light and heavy chains of GPC3 and PD-L1 are connected by (G ₄ S) ₃ , and the amplified GPC3 scFv and PD-L1 scFv are connected to the Fc segment by Hinge, wherein the Fc segment is not subjected to base mutation to retain the ADCC and other effects of the Fc segment. Secondly, the P2A shearing peptide is introduced in the primer design process to facilitate the successful expression of the target plasmid and to effectively shear and smoothly combine into a bispecific antibody structure. In this embodiment, the double enzyme digestion method is used to double enzyme digest the target sequence fragment and the vector to remove the sticky ends and then construct the bispecific antibody plasmid, and the promoter is CMV. The specific information and schematic diagram of the construction of the GPC3/PD-L1 bispecific antibody gene drug (expression plasmid) are shown in Table 1.

Table 1 Plasmid construction information

,

At the same time, this example uses the protein structure prediction website Alphafold2 to predict and analyze the structure of the bispecific antibody expressed by the designed gene drug, and its structure is shown in B in Figure 1.

After the bispecific antibody gene drug was constructed, the correct construction of the target plasmid was verified by plasmid double enzyme digestion and sequencing (Figure 2). The target antibody gene sequence fragment was cut out by Hind III/ Eco R I and then analyzed by DNA gel electrophoresis. As shown in Figure 2, C, the target band size encoding hBsAb and mBsAb was about 1500 bp, and the target band size of αhPD-L1+ gene sequence vector and αmPD-L1+ gene sequence vector was about 4500 bp, which was consistent with expectations. In addition, the plasmid sequencing results (Figure 2, D) were correctly identified and compared.

Example 2

1. In vitro expression of bispecific antibody gene therapy targeting GPC3/PD-L1

HEK293T cells were transiently transfected with target gene drugs (expression plasmids) pαhGPC3/PD-L1 (human) and pαmGPC3/PD-L1 (mouse) to detect the expression of target gene drugs.

A. HEK293T cell recovery and passaging.

B. 24 h before transfection, HEK293T cells that are in good growth condition and cover more than 80% of the total area of the culture dish after attachment are digested and passaged. An appropriate amount of digested cells are transferred to a 6-well plate so that the cell growth confluence density reaches 70-80% before transfection. The cells are cultured in a cell culture incubator in preparation for transfection.

C. Replace complete culture medium 1 h before transfection.

D. Preparation of target gene drug (expression plasmid) transfection mixture: Add 125 mL serum-free Opti-DMEM, 2.5 mg target plasmid (phBsAb, pmBsAb), and 4 mL Lipo8000 to a sterile 1.5 mL EP tube. Mix thoroughly and let stand at room temperature for 10 min to allow DNA and transfection reagent to combine to form a stable transfection complex.

E. Take out the culture dish and carefully add the prepared DNA-transfection reagent mixture dropwise into the culture dish containing 180 mL of culture medium. Mix gently and place back into the incubator to continue culturing after marking.

F. After 6-8 h, observe the growth status of the transfected 293T cells, remove the culture medium, wash once with 3 mL PBS, and add 2 mL of fresh complete culture medium to continue culturing.

G. Collect the culture supernatant 48 h after transfection into a 2 mL centrifuge tube, mark it and place it on ice for later use.

2. Western blot (WB) of target antibody to detect antibody expression in vitro

The supernatant of the hBsAb and mBsAb-expressing 293T cells collected above was centrifuged at low speed for a short time, and 30 mL of the supernatant was taken out of each of 2 tubes. 10 mL of 4 × Loading Buffer was added to each tube. One tube was placed in a 98°C metal bath for 10 min to detect the antibody reduction state, and the other tube was mixed and placed on ice to wait for sample loading for protein immunoblotting detection.

The pαhGPC3/PD-L1 BsAb and pαmGPC3/PD-L1 BsAb gene drugs (expression plasmids) that were successfully constructed and correctly identified were transiently expressed in 293T cells by liposome transient transfection technology. The supernatant was harvested 48 hours after transient transfection, and the in vitro expression of the bispecific antibody gene drug (expression plasmid) was detected by protein immunoblotting. The Western Blot results showed (as shown in Figure 3) that the bispecific antibody proteins expressed by the expressed bispecific gene drugs (human and mouse) were detected in both reduced and non-reduced states, and in the non-reduced state, the bispecific antibody could show specific bands between 140-150 kDa.

3. In vitro binding biological activity analysis of bispecific antibodies after expression of GPC3/PD-L1-targeted bispecific antibody gene drugs

3.1 Detection of the binding ability of the bispecific antibody targeting GPC3/PD-L1 with GPC3 protein and PD-L1 protein in the supernatant

In vitro biological function analysis of bispecific antibodies targeting GPC3:

After transient transfection of gene drug (expression plasmid) (Vector, pαGPC3, pαPD-L1 (human; mouse), phBsAb, pmBsAb) plasmid, collect the cell supernatant expressing the target antibody and place it on ice for antibody binding activity detection. ELISA is used to detect the binding activity of bispecific antibodies to target proteins. The steps are as follows:

A. Coating: Use 1 × ELISA coating solution to dilute the target protein (mouse GPC3 protein, human GPC3 protein, human PD-L1 protein, mouse PD-L1 protein) to 10 ng/well, and add 100 mL per well to a 96-well plate. After sealing with sealing film, incubate at 4°C overnight.

B. Washing: After the incubation is completed the next day, discard the liquid in the wells and wash each well of the 96-well plate with 300 mL of washing buffer PBST for 1 min, a total of 5 times. After discarding the liquid in the well each time, place the plate on absorbent paper and tap it repeatedly several times to completely remove the remaining liquid in the well.

C. Blocking: Add 100 mL of 1% BSA (prepared in PBS T) to each well and place in a 37°C incubator for 2 hours for blocking.

D. Discard the blocking solution and wash the plate 5 times with PBST, 30 s each time.

E. Sample addition: Add 100 mL/well of the supernatant to be tested and the supernatant diluted in PBS, and incubate in a 37°C incubator for 1 h.

F. Washing: After incubation, discard the liquid in the wells and wash each well of the 96-well plate with 300 mL of washing buffer PBS T for a total of 5 times, each time for 1 min.

G. Add HRP enzyme-labeled antibody: Add 100 mL/well of enzyme-labeled antibody (goat anti-human IgG Fc (HRP), goat anti-mouse IgG Fc (HRP)) diluted with 1% BSA to each of the above reaction wells and incubate at 37°C for 1 hour.

H. Washing: Wash the plate 7-8 times with PBS T, 1 min each time, and tap it on absorbent filter paper to remove the liquid in the wells.

I. Add 100 mL of the prepared TMB colorimetric solution to each well and incubate in a 37°C incubator for 15 min in the dark.

J. Stop: Add 100 mL/well of 1M sulfuric acid to stop color development and read the absorbance of the 96-well plate using an ELISA reader at a wavelength of 450 nm.

After transient transfection of HEK293T cells, the supernatant was collected after the gene drug (expression plasmid) (Vector, pαGPC3, pαPD-L1 (human; mouse), phBsAb, pmBsAb), and the binding ability of the expressed antibody in the cell supernatant to the target protein was detected and analyzed by direct ELISA. As shown in Figure 4, compared with αGPC3 monoclonal antibody and αPD-L1 monoclonal antibody, the expressed hBsAb and mBsAb can effectively bind to human GPC3 protein, human PD-L1 protein, and mouse PD-L1 protein. And the binding ability shows a gradient change of OD value with the increase of dilution ratio, but BsAb still maintains effective binding activity. The results show that the bispecific antibody expressed by the bispecific antibody gene drug constructed by the present invention has good binding biological activity with the corresponding antigen. At the same time, compared with αGPC3 monoclonal antibody and αPD-L1 monoclonal antibody, the bispecific antibody still has a strong binding ability with the corresponding antigen after dilution 30 times.

3.2 Detection of the binding ability of the GPC3-targeting bispecific antibody expressed in the supernatant after HEK293T cells were transfected with gene drugs (expression plasmid) to the GPC3 protein on the surface of liver cancer cells

Flow cytometry was used to detect the binding activity of the bispecific antibody to the target protein on the cell surface. The steps are as follows:

A. Digest HepG2, Hepa3b, and Huh7 cells that are growing well and have a confluence of more than 80%, and centrifuge the whole blood cell suspension at 1200 rpm for 5 min.

B. Remove the supernatant, resuspend with pre-cooled PBS, wash and centrifuge the cells twice at 1200 rpm for 5 min to remove any serum; during this operation, divide each cell type into 5 clean 1.5 mL centrifuge tubes for later use, specifically grouped as follows: blank tube, vector tube, commercial positive control antibody tube (anti-human GPC3 antibody), fluorescent secondary antibody negative control tube (α-mouse PE) and mBsAb experimental tube.

C. Prepare 0.5% BSA: Add 0.5 g BSA to 100 mL PBS, mix well and place on ice until dissolved.

D. Take 500 mL of the above collected cell supernatant and add them to the corresponding tubes of the above three types of cells respectively; add 100 μL of 0.5% BSA to the remaining three tubes: blank tube, positive commercial antibody tube, and fluorescent secondary antibody tube, resuspend the cells, and add appropriate amount of antibody according to the dilution ratio in the antibody manual; and incubate on ice or away from light for 1 h.

E. Add 200 mL of pre-cooled PBS to wash the cells, centrifuge at 1400 rpm for 5 min, remove the supernatant, and repeat the above steps for 2 more times.

F. Add pre-cooled 0.5% BSA to the cells after centrifugation to remove PBS and gently pipette to resuspend. Add fluorescent secondary antibody to the positive commercial antibody tube and mBsAb, incubate on ice and away from light for 1 h, and proceed with subsequent staining operations; keep the remaining tubes on ice and away from light waiting for flow cytometry.

G. Add 200 mL of pre-cooled PBS to wash the cells. Centrifuge at 1400 rpm for 5 min, remove the supernatant, and repeat the above steps for 2 more times.

H. Finally, resuspend all cells in 200 mL pre-cooled PBS and proceed to the machine.

In this example, bispecific antibodies (BsAb) were produced by plasmid transfection technology, and the binding activity of the bispecific antibody protein secreted in the supernatant of HEK293T cells to the cell surface GPC3 protein was determined. As shown in Figure 5, compared with the negative control group of the empty plasmid and the positive control group targeting GPC3, the BsAb group showed a significant positive peak shift, indicating that the bispecific antibody protein expressed by the bispecific antibody gene drug targeting GPC3 designed by the present invention can specifically bind to the target protein on the surface of liver cancer cells.

Example 3

Treatment effect detection:

1. phBsAb inhibits tumor growth in humanized immune system NSG Huh 7 liver cancer-bearing mice

1.1 Construction of hPBMC-NSG mouse model

A. Purchase 5-6 week old female NSG mice from Huafukang Biological Company and feed them adaptively for one week;

B. Collect fresh blood samples from healthy volunteers, separate human peripheral blood mononuclear cells (PBMCs), and count them;

C. After one week of adaptive feeding, each mouse was injected with 5×10 ⁶ cells/100 mL human PBMC cells through the tail vein to complete the construction of the humanized immune system mouse model;

D. Spleens of NSG mice were obtained 30 days after PBMC injection for flow cytometry analysis of immune cell composition;

1.2 Construction of Huh 7 liver cancer model in NSG mice with humanized immune system

A. Purchase 5-6 week old female NSG mice from Huafukang Biological Company and feed them for one week;

B. Collect fresh blood samples from healthy volunteers, isolate human PBMCs, and complete the construction of a humanized immune system mouse model;

C. One week after PBMC injection, 2.5×10 ⁶ Huh 7 cells were subcutaneously injected into the right rib cage of each mouse, and the inoculation day was set as Day 0;

D. On the second day after tumor inoculation, the LPL efficient skeletal muscle in situ gene expression delivery system established in our laboratory was used to inject 100 μg of therapeutic plasmid into the anterior tibialis muscle of the right hind leg of mice according to different treatment groups (Vector, pαhGPC3, pαhPD-L1, pαhGPC3 + pαhPD-L1, phBsAb);

E. After Huh 7 cells were inoculated, the growth of mouse tumors was observed every other day. The short diameter (width) and long diameter (length) of tumors of tumor-bearing mice were measured with a vernier caliper, and the weight of mice was recorded using an electronic balance. The tumor volume was calculated using the following formula: V = (length × width ² ) / 2. Data statistical analysis was performed using the statistical software GraphPadprism 9.5.0.

In the in vivo tumor inhibition experimental model of bispecific antibodies, the present invention selected Huh7 cell line (2.5 × 10 ⁶ ) for CDX model construction to evaluate the tumor inhibition effect of bispecific antibodies. One week after the injection of PBMC in severely immunodeficient mice, the mice were randomly divided into 5 groups, and a Huh7 subcutaneous tumor model was constructed on the right axillary skin side of the mice, recorded as D0. On the second day after modeling, 100 μg of therapeutic plasmid was injected intramuscularly into the right posterior tibialis anterior muscle of the mice, and LPL and EGCG materials were used for plasmid delivery. Tumor growth was observed regularly, and the weight changes of mice during treatment were monitored. As shown in Figure 6, on the 25th day after the treatment of 100 μg plasmid delivery, the phBsAb group and the Control group began to show significant statistical differences (P). Continuous monitoring revealed that the tumor in the phBsAb group continued to maintain a relatively slow growth level, showing a significant tumor inhibition trend compared with the pαhGPC3 group and the pαhPD-L1 group. The mice in the phBsAb group and the pαhGPC3 combined with pαhPD-L1 group showed significantly prolonged survival, which was statistically different from the Control group, the pαhGPC3 group, and the pαhPD-L1 group (P) (Figure 7).

2. ELISA was used to detect the release level of IFN-γ in the serum of humanized immunodeficient NSG mouse Huh 7 liver cancer model:

According to the instructions of the ELISA kit manufacturer (Dayou: Cat. No.: 1110002), the collected mouse serum was stored in a -80°C refrigerator and the detection operation was performed according to the following steps:

A. According to the kit instructions, equilibrate the kit at room temperature for 20-30 minutes before use; then mix all reagents thoroughly to avoid foaming;

B. Preparation of standard products: Dissolve the lyophilized standard products according to the instructions and prepare them into different concentrations such as 400 pg/mL, 200 pg/mL, 100 pg/mL, 50 pg/mL, 25 pg/mL, 12.5 pg/mL, and 6.25 pg/mL in sequence;

C. Sample addition: Add 100 mL/well of diluted Cytokine Standard to the standard well, 100 mL/well of serum sample to the sample well, and 100 mL/well of diluent to the blank control well;

D. Add detection antibody: Add 50 mL/well of Biotinylated Antibody working solution. Mix well, cover with sealing film, and incubate at 37℃ for 2 hours;

E. Washing: Remove the liquid in the wells and add 1 × Washing Buffer working solution at 300 mL/well; leave for 1 minute and discard the liquid in the wells. Repeat 3 times, and dry on filter paper each time;

F. Add enzyme: Add 100 mL/well of Streptavidin-HRP working solution. Cover with sealing film and incubate at 37°C for 20 minutes;

G. Wash the plate: Repeat step E;

H. Color development: Add TMB to 100 mL/well and incubate at 37°C in the dark for 10-20 minutes. The reaction is terminated based on the color depth (dark blue) in the well.

I. Stop the reaction: quickly add 100 mL/well of Stop Solution to stop the reaction;

J. Plate reading: Within 10 minutes after termination, read the absorbance of the plate on a microplate reader at a detection wavelength of 450 nm. Draw a standard curve based on the absorbance of the standard sample, and calculate the concentration of IFN-γ in the serum sample through the curve.

IFN-γ, also known as type II interferon, is mainly secreted by T lymphocytes and NK cells. It has antiviral and anti-tumor effects, regulates cell proliferation and apoptosis, and secondly plays an important role in tumor immune surveillance. However, studies have shown that in the tumor microenvironment of primary liver cancer, T lymphocyte infiltration is blocked and function is suppressed, resulting in reduced IFN-γ release during the anti-tumor process, inhibiting the anti-tumor efficacy. Therefore, this example further evaluates the IFN-γ content in serum to evaluate the ability of cytotoxic T lymphocytes (CTLs) to kill tumor cells. As shown in Figure 8, the release of serum IFN-γ in the phBsAb treatment group increased significantly, which was statistically different from the empty group, pαhGPC3 group, pαhPD-L1 group and pαhGPC3 + pαhPD-L1 group. It suggests that the phBsAb treatment group has a stronger tumor inhibition effect.

Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims (9)

1. A bispecific antibody, characterized in that the bispecific antibody is a bispecific antibody targeting GPC3/PD-L1; and its amino acid sequence is shown in SEQ ID NO.1. 2. A DNA molecule encoding the bispecific antibody according to claim 1, characterized in that its nucleotide sequence is shown in SEQ ID NO.2. 3. The expression vector of the bispecific antibody according to claim 1, characterized in that the expression vector uses CMV as a promoter, and the Igκ secretory peptide encoding gene, the GPC3 single-chain variable region fragment GPC3-scFv encoding gene, the Hinge encoding gene, the Fc encoding gene, the P2A encoding gene, the Igκ secretory peptide encoding gene, the PD-L1 single-chain variable region fragment PD-L1-scFv encoding gene, the Hinge encoding gene and the Fc encoding gene are sequentially connected downstream of the CMV promoter. 4. The method for constructing the expression vector according to claim 3, characterized in that it comprises the following steps: (1) Based on the light and heavy chain nucleotide sequences of GPC3, the light and heavy chain nucleotide sequences of PD-L1, and the Fc encoding gene, the GPC3-scFv-Fc gene fragment and the PD-L1-scFv-Fc gene fragment were amplified by overlapping PCR, and the cloning sites were HindIII / Eco RI and Eco RI/ Xho I, respectively; (2) The GPC3 light and heavy chain nucleotide sequences and the PD-L1 light and heavy chain nucleotide sequences are connected through the ( _G4S ) ₃ flexible linker encoding gene, and the amplified GPC3-scFv gene fragment and PD-L1-scFv gene fragment are connected to the Fc encoding gene through the Hinge encoding gene; (3) The P2A cleavage peptide encoding gene was introduced during the primer design process, and the target sequence fragment and the vector were double-enzyme cut to remove the sticky ends to construct a bispecific antibody plasmid. The promoter was CMV. 5. Use of the bispecific antibody according to claim 1, the DNA molecule according to claim 2 or the expression vector according to claim 3 in the preparation of a drug for treating liver cancer. 6. A gene medicine for treating liver cancer, characterized in that it comprises the DNA molecule according to claim 2 or the expression vector according to claim 3. 7. A pharmaceutical preparation for treating liver cancer, characterized in that it comprises the bispecific antibody according to claim 1, the DNA molecule according to claim 2, the expression vector according to claim 3 or the gene drug according to claim 6 and a pharmaceutically acceptable carrier. 8. The pharmaceutical preparation according to claim 7, characterized in that the pharmaceutically acceptable carrier is suitable for liquid dosage form, solid dosage form or paste dosage form. 9. A drug for treating liver cancer, comprising the bispecific antibody according to claim 1.