Bispecific antibodies useful for gene therapy

Abstract

The present disclosure provides bispecific antibodies useful in gene therapy. Specifically, the invention provides a bispecific antibody, and the bispecific antibody comprises a heavy chain variable region (VHVEGF) and a light chain variable region (VLVEGF) which are targeted to a vascular endothelial growth factor (VEGF), and a heavy chain variable region (VHPD-L1) and a light chain variable region (VLPD-L1) which are targeted to a programmed death receptor-ligand 1 (PD-L1). The bispecific antibody provided by the disclosure can be used for gene therapy, such as AAV or mRNA mediated gene therapy, and can effectively block PD-1 / PD-L1 and VEGF / VEGFR pathways, and inhibit tumors in vitro and in vivo.

Description

Technical Field

[0001] The present disclosure relates to the fields of gene therapy and antibodies, and particularly to bispecific antibodies that can be used in gene therapy. Background Art

[0002] Cancer is the second leading cause of death worldwide, accounting for nearly 10 million deaths in 2020[1]. Compared with traditional anti-cancer strategies, monoclonal antibodies are considered one of the most promising cancer treatments[2,3]. However, in the complex pathogenesis of cancer, different signaling pathways and cascade amplification effects lead to a significant reduction in the efficacy of monoclonal antibodies. In order to cope with tumor resistance and improve the therapeutic effect of monoclonal antibodies, the development of two antibody combination therapies may be a potential feasible solution. However, in practice, the development and application of such solutions are frequently hindered by problems such as high cost, insufficient effectiveness due to the difficulty in matching safety and pharmacokinetic properties[4].

[0003] With the development of genetic engineering technology, the therapeutic potential of bispecific antibodies (bsAbs) has received widespread attention[5]. Compared with combination therapy using two separate antibodies, bsAbs may potentially increase binding specificity by interacting with two molecules on target tissue cells; increase local concentrations in the tumor microenvironment; and reduce development and production costs[6]. Based on the presence or absence of a crystallizable fragment (Fc) in their structure, bsAbs can be divided into IgG-like and non-IgG types[5].

[0004] IgG-like bsAbs have longer half-lives, more convenient purification methods, and higher stability. This type of antibody retains a variety of Fc-mediated effector functions [5]. However, the Fc segment is a "double-edged sword" in bsAbs. On the one hand, it has great development potential, but on the other hand, it may cause unnecessary nonspecific immune responses during bsAb treatment, ultimately triggering a series of adverse reactions. For example, Catumaxomab, an IgG-like bsAb that targets CD3 and epithelial cell adhesion molecule (EpCAM), caused severe adverse reactions attributed to the off-target binding of the Fc segment to Kupffer cells expressing FcγR in the liver, resulting in strong cytokine release and T cell-mediated hepatotoxicity [7]. Other studies have shown that Fc-mediated effector mechanisms may hinder tumor-specific T cell redirection and tumor cell killing [8].

[0005] Non-IgG bsAbs have better tissue penetration ability and lower immunogenicity[9]. Bispecific T cell engager (BiTE) antibodies are typical non-IgG bsAbs, which are small and flexible molecules

[10] . Blinatumomab, a BiTE antibody targeting CD3 and CD19 that was approved for marketing in 2014, has a molecular weight of approximately 55 kDa and a short plasma half-life in vivo (1.25±0.63 hours). Blinatumomab has a significant complete remission rate of 43% in patients with relapsed / refractory precursor B-cell acute lymphoblastic leukemia

[11] . The drug's sales in 2021 reached US$500 million, but due to its short half-life, it needs to be administered through multiple intravenous drips, which puts great physical and financial pressure on patients. As a result, there are few applications of BiTE structures in the bispecific antibodies currently on the market and in the clinical stage.

[0006] Monoclonal antibodies targeting programmed cell death 1 (PD-1) or programmed cell death-ligand 1 (PD-L1) can reactivate suppressed T cells and thus block the immune escape of tumor cells [12,13]. Among them, atezolizumab is the first immunotherapy drug developed by Roche targeting PD-L1. Monoclonal antibodies targeting vascular endothelial growth factor (VEGF) or VEGF receptor (VEGFR) can also inhibit tumor growth

[14] . Bevacizumab is the first VEGF monoclonal antibody approved for marketing

[15] .

[0007] As mentioned above, the occurrence and development of malignant tumors involve multiple complex mechanisms, and it is impossible to achieve ideal therapeutic effects by targeting a single target. For example, both atezolizumab and bevacizumab can achieve objective tumor remission in the field of liver cancer as monotherapy

[16] , but the objective response rate (ORR) is less than 20%. A phase Ib clinical study (GO30140) of the combination of the two (hereinafter referred to as "T+A") for the treatment of newly diagnosed, unresectable hepatocellular carcinoma (HCC) showed an ORR of 36%. However, it is worth noting that in the IMbrave150 study, the incidence of serious adverse reactions in the "T+A" group (125 cases - 38.0%) was higher than that in sorafenib (48 cases - 30.8%). Another study showed that the combination of ipilimumab and nivolumab (anti-PD-1) improved survival outcomes in melanoma patients compared with ipilimumab (anti-cytotoxic T lymphocyte-associated protein 4) alone, however, the increased anti-tumor activity was associated with a significant increase in immune-related adverse events

[17] . Therefore, the development of bsAbs with lower immunogenicity and production costs is urgent compared with combination therapy.

[0008] Currently, the only marketed drug that simultaneously targets both the PD-1 / PD-L1 and VEGF / VEGFR signaling pathways is AK112 (PD-1 / VEGF). However, the immunogenicity of IgG-like antibodies and their limited infiltration ability against solid tumors limit their clinical application. Furthermore, IgG-like antibodies require multiple doses to maintain therapeutic effects. Long-term treatment increases the financial burden on patients, and repeated injections can increase pain and the likelihood of adverse reactions.

[0009] As mentioned above, the extremely low half-life of BiTE molecules limits the widespread use of this type of antibody. Some researchers have used adeno-associated virus (AAV) to mediate the expression of Blinatumomab in mice, effectively eliminating tumors and prolonging the overall survival of mice, and achieved sustained expression of the target protein after a single dose

[18] . The IND for the gene therapy drug "XMVA09" of Hefei Xingmou Biotechnology Co., Ltd. has been officially accepted. This drug is expressed by AAV-mediated BiTE-type antibodies (targeting VEGF and angiopoietin 2). After a single dose, patients with wet age-related macular degeneration showed significant clinical benefits and no drug-related adverse events

[19] . In the face of the demand for more economical, safe, longer-lasting and more effective treatment strategies in this field, gene therapy mediated by AAV or mRNA BiTE expression has good prospects.

[0010] In summary, there is an urgent need to develop bispecific antibodies against PD-L1 and VEGF that can be used for gene therapy.

[0011] Citations

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[0014] [3]Parakh S,King D,Gan HK,et al.Current development of monoclonalantibodies in cancer therapy.Recent Results In Cancer Research FortschritteDer Krebsforschung Progres Dans Les Recherches Sur Le Cancer.2020;214.

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[0016] [5]Li H,Er Saw P,Song E.Challenges and strategies for next-generationbispecificantibody-based antitumor therapeutics.Cellular&MolecularImmunology.2020;17(5):451-461.

[0017] [6]Cui X,Jia H,Xin H,et al.A novel bispecific antibody targeting pd-l1 and vegf withcombined anti-tumor activities.Frontiers In Immunology.2021;12:778978.

[0018] [7]Borlak J, F,Spanel R,et al.Immune-mediated liver injury ofthe cancertherapeutic antibody catumaxomab targeting epcam,cd3 and fcγreceptors.Oncotarget.2016;7(19):28059-28074.

[0019] [8]Labrijn A F,Janmaat M L,Reichert J M,et al.Bispecific antibodies:Amechanisticreview of the pipeline.Nature Reviews Drug Discovery.2019;18(8):585-608.

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[11] Topp M S, N,Stein A S,et al.Safety and activity ofblinatumomab foradult patients with relapsed or refractory b-precursor acutelymphoblastic leukaemia:Amulticentre,single-arm,phase 2 study.The LancetOncology.2015;16(1):57-66.

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[12] Rizvi N A,Hellmann M D,Snyder A,et al.Cancerimmunology.Mutationallandscape determines sensitivity to pd-1 blockade innon-small cell lung cancer.Science(NewYork,NY).2015;348(6230):124-128.

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[14] Atzori M G,Tentori L,Ruffini F,et al.The anti-vascularendothelial growth factorreceptor-1 monoclonal antibody d16f7 inhibits gliomagrowth and angiogenesis in vivo.TheJournal of Pharmacology and ExperimentalTherapeutics.2018;364(1):77-86.

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[15] Garcia J,Hurwitz H I,Sandler A B,et al.Bevacizumab incancertreatment:A review of 15 years of clinical experience and futureoutlook.Cancer TreatmentReviews.2020;86:102017.

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[17] Wolchok J D,Chiarion-Sileni V,Gonzalez R,et al.Overall survivalwith combinednivolumab and ipilimumab in advanced melanoma.The New EnglandJournal of Medicine.2017;377(14):1345-1356.

[0029]

[18] Cripe TP, Hutzen B, Currier MA, et al. Leveraging gene therapy to achieve long-term continuous or controllable expression of biotherapeutics. Science Advances. 2022; 8(28): eabm1890.

[0030]

[19] Cai Yuan, Ma Zhen, Zhou Peipei, et al. An AAV vector encoding anti-VEGF-A and ANG-2 bispecific antibodies. CN116925234A. [P]. 2023.10.24 Summary of the Invention

[0031] Problems to be solved by the invention

[0032] The present disclosure aims to provide an anti-PD-L1 and anti-VEGF bispecific antibody that can be used for gene therapy.

[0033] Solutions for solving problems

[0034] In a first aspect of the present disclosure, a bispecific antibody is provided, wherein the bispecific antibody comprises: a heavy chain variable region (VH) targeting vascular endothelial growth factor (VEGF); VEGF ) and light chain variable region (VL VEGF ), and the heavy chain variable region (VH PD-L1 ) and light chain variable region (VL PD-L1 );in,

[0035] The VH VEGF comprising the sequence shown in SEQ ID NO: 1, or a sequence having at least 90% identity thereto;

[0036] The VL VEGF comprising the sequence shown in SEQ ID NO: 2, or a sequence having at least 90% identity thereto;

[0037] The VH PD-L1 comprising the sequence shown in SEQ ID NO: 3, or a sequence having at least 90% identity thereto;

[0038] The VL PD-L1 The sequence comprises SEQ ID NO: 4, or a sequence having at least 90% identity thereto.

[0039] In some embodiments, the VH VEGFcomprising HCDR1 as shown in SEQ ID NO: 5, HCDR2 as shown in SEQ ID NO: 6, and HCDR3 as shown in SEQ ID NO: 7; the VL VEGF comprising LCDR1 as shown in SEQ ID NO: 8, LCDR2 as shown in SEQ ID NO: 9, and LCDR3 as shown in SEQ ID NO: 10; the VH PD-L1 comprising HCDR1 as shown in SEQ ID NO: 11, HCDR2 as shown in SEQ ID NO: 12, and HCDR3 as shown in SEQ ID NO: 13; the VL PD-L1 It comprises LCDR1 as shown in SEQ ID NO:14, LCDR2 as shown in SEQ ID NO:15 and LCDR3 as shown in SEQ ID NO:16.

[0040] In some embodiments, the VH VEGF 、VL VEGF 、VH PD-L1 and VL PD-L1 A linker is also included between any two.

[0041] In some embodiments, the bispecific antibody has a structure as shown in any one of the following (i)-(iii):

[0042] (i) N-terminal-VL VEGF -Optional linker-VH VEGF -Optional Linker-VL PD-L1 -Optional linker-VH PD-L1 -C-terminus;

[0043] (ii) N-terminal-VH VEGF -Optional Linker-VL VEGF -Optional linker-VH PD-L1 -Optional Linker-VL PD-L1 -C-terminus;

[0044] (iii) N-terminal-VH PD-L1 -Optional Linker-VL PD-L1 -Optional linker-VH VEGF -Optional Linker-VL VEGF -C-terminus.

[0045] In some preferred embodiments, the bispecific antibody comprises a sequence as shown in any one of SEQ ID NOs: 18 to 20, or a sequence that is at least 90% identical to a sequence as shown in any one of SEQ ID NOs: 18 to 20.

[0046] In some embodiments, at least one disulfide bond is formed between the heavy chain variable region and the light chain variable region near the N-terminus.

[0047] In some embodiments, the amino acid at position 44 of the heavy chain variable region near the N-terminus is mutated to cysteine ​​(C), and the amino acid at position 100 of the light chain variable region near the N-terminus is mutated to cysteine ​​(C), thereby forming a disulfide bond between the heavy chain variable region and the light chain variable region near the N-terminus.

[0048] In some embodiments, the bispecific antibody further comprises a signal peptide at the N-terminus.

[0049] In some embodiments, the bispecific antibody comprises a sequence as shown in any one of SEQ ID NOs: 22-24, 30-32, or a sequence that is at least 90% identical to a sequence as shown in any one of SEQ ID NOs: 22-24, 30-32.

[0050] In a second aspect of the present disclosure, an isolated polynucleotide is provided, which encodes the bispecific antibody as described in the first aspect of the present disclosure.

[0051] In a third aspect of the present disclosure, a gene expression cassette is provided, comprising the polynucleotide as described in the second aspect of the present disclosure.

[0052] In a fourth aspect of the present disclosure, a gene delivery vector is provided, comprising the gene expression cassette as described in the third aspect of the present disclosure.

[0053] In some embodiments, the gene delivery vector is DNA or RNA.

[0054] In some embodiments, the gene delivery vector is a viral vector derived from a virus.

[0055] In some embodiments, the viral vector is a recombinant adeno-associated virus.

[0056] In some preferred embodiments, the gene delivery vector comprises a sequence as shown in any one of SEQ ID NOs: 34-36, or a sequence having at least 90% identity to a sequence as shown in any one of SEQ ID NOs: 34-36.

[0057] In the fifth aspect of the present disclosure, a cell is provided, which comprises the bispecific antibody as described in the first aspect of the present disclosure, the polynucleotide as described in the second aspect of the present disclosure, the gene expression cassette as described in the third aspect of the present disclosure, or the gene delivery vector as described in the fourth aspect of the present disclosure.

[0058] In the sixth aspect of the present disclosure, a pharmaceutical composition is provided, comprising the bispecific antibody as described in the first aspect of the present disclosure, the polynucleotide as described in the second aspect of the present disclosure, the gene expression cassette as described in the third aspect of the present disclosure, the gene delivery vector as described in the fourth aspect of the present disclosure, or the cell as described in the fifth aspect of the present disclosure; and, optionally, a pharmaceutically acceptable carrier.

[0059] In the seventh aspect of the present disclosure, a method for treating or preventing a tumor in a subject in need thereof is provided, the method comprising administering to a subject in need thereof an effective amount of the bispecific antibody as described in the first aspect of the present disclosure, the polynucleotide as described in the second aspect of the present disclosure, the gene expression cassette as described in the third aspect of the present disclosure, the gene delivery vector as described in the fourth aspect of the present disclosure, the cell as described in the fifth aspect of the present disclosure, or the pharmaceutical composition as described in the sixth aspect of the present disclosure.

[0060] In some optional embodiments, the tumor is a tumor involving PD-L1 and / or VEGF.

[0061] In some optional embodiments, the tumor is selected from breast cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, leukemia, lymphoma, myeloma, esophageal cancer, liver cancer, biliary tract cancer, pancreatic cancer, head and neck cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, osteosarcoma, soft tissue sarcoma, neuroblastoma, endocrine organ tumors, bladder cancer, skin cancer, nasopharyngeal cancer, and rhabdomyosarcoma.

[0062] The present disclosure also provides the bispecific antibody as described in the first aspect of the present disclosure, the polynucleotide as described in the second aspect of the present disclosure, the gene expression cassette as described in the third aspect of the present disclosure, the gene delivery vector as described in the fourth aspect of the present disclosure, the cell as described in the fifth aspect of the present disclosure, or the pharmaceutical composition as described in the sixth aspect of the present disclosure for use in treating tumors.

[0063] In some optional embodiments, the tumor is a tumor involving PD-L1 and / or VEGF.

[0064] In some optional embodiments, the tumor is selected from breast cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, leukemia, lymphoma, myeloma, esophageal cancer, liver cancer, biliary tract cancer, pancreatic cancer, head and neck cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, osteosarcoma, soft tissue sarcoma, neuroblastoma, endocrine organ tumors, bladder cancer, skin cancer, nasopharyngeal cancer, and rhabdomyosarcoma.

[0065] The present disclosure also provides use of the bispecific antibody as described in the first aspect of the present disclosure, the polynucleotide as described in the second aspect of the present disclosure, the gene expression cassette as described in the third aspect of the present disclosure, the gene delivery vector as described in the fourth aspect of the present disclosure, and the cell as described in the fifth aspect of the present disclosure in the preparation of a medicament for treating tumors.

[0066] In some optional embodiments, the tumor is a tumor involving PD-L1 and / or VEGF.

[0067] In some optional embodiments, the tumor is selected from breast cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, leukemia, lymphoma, myeloma, esophageal cancer, liver cancer, biliary tract cancer, pancreatic cancer, head and neck cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, osteosarcoma, soft tissue sarcoma, neuroblastoma, endocrine organ tumors, bladder cancer, skin cancer, nasopharyngeal cancer, and rhabdomyosarcoma.

[0068] Effects of the Invention

[0069] In some embodiments of the present disclosure, a bispecific antibody against PD-L1 and VEGF is provided.

[0070] In some embodiments of the present disclosure, the bispecific antibodies provided by the present disclosure can be used for gene therapy, such as AAV or mRNA-mediated gene therapy.

[0071] In some embodiments of the present disclosure, the bispecific antibodies provided by the present disclosure can effectively block the PD-1 / PD-L1 and VEGF / VEGFR pathways.

[0072] In some embodiments of the present disclosure, the bispecific antibodies provided by the present disclosure can inhibit tumor cell viability in vitro.

[0073] In some embodiments of the present disclosure, the bispecific antibodies provided by the present disclosure have anti-tumor activity in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 Schematic diagram of BiTE-type bsAbs. The antibody design adopts the tandem scFv approach, and the specific order is:

[0075] BiTE1:VL VEGF -3×(G4S)-VH VEGF -SGGGGS-VL PD-L1 -3×(G4S)-VH PD-L1 -HHHHHH;

[0076] BiTE2:VH VEGF -3×(G4S)-VLVEGF -SGGGGS-VH PD-L1 -3×(G4S)-VL PD-L1 -HHHHHH;

[0077] BiTE3:VH PD-L1 -3×(G4S)-VL PD-L1 -SGGGGS-VH VEGF -3×(G4S)-VL VEGF -HHHHHH;

[0078] BiTE4:VL PD-L1 -3×(G4S)-VH PD-L1 -SGGGGS-VL VEGF -3×(G4S)-VH VEGF -HHHHHH.

[0079] Figure 2 Schematic diagram of the one-step antibody purification SDS-PAGE results. Loading: Lanes, Lane 1: Reduced BiTE1, Lane 2: Reduced BiTE2, Lane 3: Reduced BiTE3, Lane 4: Reduced BiTE4, Lane 5: Non-reduced BiTE1, Lane 6: Non-reduced BiTE2, Lane 7: Non-reduced BiTE3, Lane 8: Non-reduced BiTE4, Lane 9: Human IgG.

[0080] Figure 3 Schematic diagram of antibody ELISA results. Figure 3 A in the figure: human programmed death receptor-ligand 1 (human PD-L1), atezolizumab as positive control, and human IgG as negative control. Figure 3 B in the figure: vascular endothelial growth factor 110 (VEGF-110), bevacizumab as positive control, human IgG as negative control, Figure 3 C in the figure: vascular endothelial growth factor 121 (VEGF-121), bevacizumab as positive control, human IgG as negative control, Figure 3 D in the figure: vascular endothelial growth factor 165 (VEGF-165), bevacizumab as a positive control, and human IgG as a negative control.

[0081] Figure 4 Figure 5. Results of an in vitro PD-L1 / PD-1 blocking experiment. Atezolizumab was used as a positive control, and human IgG was used as a negative control.

[0082] Figure 5 Figure 4 shows the results of an in vitro VEGF / VEGFR blocking experiment. Bevacizumab was used as a positive control and human IgG was used as a negative control.

[0083] Figure 6 Cell viability test results.

[0084] Figure 7 This is the body weight change curve of animals in the in vivo efficacy experiment. The AAV-isotype control is the negative control.

[0085] Figure 8 This is the curve of tumor volume changes in animals during the in vivo efficacy experiment. The AAV-isotype control is the negative control. DETAILED DESCRIPTION

[0086] Various exemplary embodiments, features, and aspects of the present disclosure will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0087] In addition, numerous specific details are provided in the following detailed description to better illustrate the present disclosure. Those skilled in the art will appreciate that the present disclosure can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present disclosure.

[0088] Unless otherwise stated, the units used in this specification are international standard units, and the numerical values ​​and numerical ranges appearing in this disclosure should be understood to include the inevitable systematic errors in industrial production.

[0089] I. Definition of Terms

[0090] Unless otherwise stated, the following terms have the following meanings in this disclosure.

[0091] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0092] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0093] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0094] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0095] The "antibody" described in this disclosure is an immunoglobulin. Typically, a complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and order of the constant region of the immunoglobulin heavy chain differ, resulting in different antigenicity. Based on this, immunoglobulins can be divided into five classes, or immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Within the same class of Ig, there are different subclasses based on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either κ or λ chains based on differences in the constant region. Each of the five classes of Ig can have either κ or λ chains.

[0096] The approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains vary greatly in sequence and constitute the variable region (Fv region). The remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region. The variable region comprises three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged in the following order from amino to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0097] The term "antibody framework" or "FR region" refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

[0098] The term "complementarity determining region," "CDR," or "hypervariable region" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of the CDRs can be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al., (1997) JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering convention (see Lefranc et al., (1998) JMB 274:927-948). MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003) etc. For example, for the classical format, following the Kabat rule, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). Following the Chothia rule, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the CDR amino acid residues in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3). Amino acid residues are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of Kabat and Chothia, the CDRs are composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Following the IMGT rule, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3).Following the IMGT rules, the CDR regions of antibodies can be determined using the program IMGT / DomainGap Align. Unless otherwise specified, the antibody variable region and CDR sequences described in the specific examples of this disclosure are all numbered using the "Kabat" convention.

[0099] The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a dsFv, a stable antigen-binding fragment formed by VH and VL via an interchain disulfide bond; and (vi) diabodies, bispecific antibodies, and multispecific antibodies comprising fragments such as scFv, dsFv, and Fab. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, so that they can be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883). Such single-chain antibodies are also included in the term "antigen-binding fragment" of an antibody. Such antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of intact immunoglobulins. The antibodies can be of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.

[0100] The term "single-chain antibody", "single-chain Fv" or "scFv" refers to a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example, variants using 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0101] Diabodies are antigen-binding fragments in which scFv or Fab is dimerized, and are antigen-binding fragments with bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens can be the same or different.

[0102] Bispecific antibodies and multispecific antibodies are antibodies that can simultaneously bind to two or more antigens or antigenic determinants, and contain scFv or Fab fragments that can bind to antigens.

[0103] The diabodies disclosed herein can be produced by the following steps: obtaining cDNA encoding the VH and VL of a monoclonal antibody that specifically recognizes and binds to an antigen, constructing a DNA encoding scFv so that the amino acid sequence length of the peptide linker is 8 residues or less, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryotic or eukaryotic organism to express the diabody.

[0104] The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes include linear epitopes and conformational epitopes. For example, conformational epitopes typically have a unique spatial conformation and include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-continuous amino acids. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0105] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example, less than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12M or less affinity (KD) binding.

[0106] The term "KD" refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. -7 M, for example, less than about 10 -8 M or 10 -9 The dissociation equilibrium constant (KD) of M binding to the antigen is determined, for example, using surface plasmon resonance (SPR) technology in a BIACORE instrument. The smaller the KD value, the greater the affinity.

[0107] The term "amino acid difference" or "amino acid mutation" refers to the presence of amino acid changes or mutations in a variant protein or polypeptide compared to the original protein or polypeptide, including insertions, deletions or substitutions of 1, 2, 3 or more amino acids based on the original protein or polypeptide.

[0108] "Conservative modification" or "conservative substitution or replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. It is known to those skilled in the art that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th ed.)). In addition, replacement of amino acids with similar structure or function is unlikely to destroy biological activity. Exemplary conservative replacements are set forth below.

[0109] Original residue Conservative substitution Ala(A) Gly; Ser Arg(R) Lys; His Asn(N) Gln; His; Asp Asp(D) Glu; Asn Cys(C) Ser; Ala; Val Gln(Q) Asn;Glu Glu(E) Asp; Gln Gly(G) Ala His(H) Asn; Gln Ile(I) Leu; Val Leu(L) Ile; Val Lys(K) Arg; His Met(M) Leu; Ile; Tyr Phe(F) Tyr;Met;Leu Pro(P) Ala Ser(S) Thr Thr(T) Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe Val(V) Ile; Leu

[0110] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacteria, microorganisms, plants, or animal cells. Easily transformed bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line), 293 cells, and NSO cells.

[0111] "Administer," "give," and "treat" as applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administer," "give," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "give," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0112] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments thereof disclosed herein, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable extent. The amount of a therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0113] "Effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired prophylactic or therapeutic results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the onset of a condition, including the biochemical, histological, and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes that present during the progression of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-associated conditions of the present invention or ameliorating one or more symptoms of the condition, reducing the dose of another agent required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-associated condition of the present invention in a patient.

[0114] "Identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in the two sequences match or are homologous, then the two sequences are 95% homologous. Typically, when two sequences are aligned, the comparison is made to give the maximum percentage identity. For example, the comparison can be performed using the BLAST algorithm, where the parameters of the algorithm are selected to give the maximum match between each sequence over the entire length of each reference sequence. The following references relate to the BLAST algorithm commonly used for sequence analysis: BLAST ALGORITHMS: Altschul, SF et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W. et al., (1993) Nature Genet. 3: 266-272; Madden, TL et al., (1996) Meth. Enzymol. 266: 131-141; Altschul, SF et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. et al., (1997) Genome Res. 7: 649-656. Other conventional BLAST algorithms, such as those provided by NCBI BLAST, are also well known to those skilled in the art.

[0115] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It will also be understood that, due to deliberate or unintentional mutations, all progeny may not be precisely identical in DNA content. Mutant progeny that possess the same function or biological activity as screened for in the originally transformed cell are included. Where a different designation is intended, this is clear from the context.

[0116] "Isolated" refers to a purified state and, in this context, means that the specified molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. Generally, the term "isolated" is not intended to imply the complete absence of such materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with experimental or therapeutic uses of the compounds as described herein.

[0117] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0118] A "pharmaceutical composition" refers to a mixture containing one or more bispecific antibodies described herein and other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient, and thereby exert its biological activity.

[0119] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering a bispecific antibody, polynucleotide, gene expression cassette, or gene delivery vector. The carrier can be an antiadhesive, adhesive, coating, disintegrant, filler, or diluent, preservative (such as an antioxidant, antibacterial, or antifungal agent), sweetener, absorption delaying agent, wetting agent, emulsifier, buffer, etc. Suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffer, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0120] 2. Bispecific Antibodies

[0121] In some aspects of the present disclosure, a bispecific antibody is provided, comprising: a heavy chain variable region (VH) targeting vascular endothelial growth factor (VEGF); VEGF ) and light chain variable region (VL VEGF ), and the heavy chain variable region (VH PD-L1 ) and light chain variable region (VL PD-L1 ).

[0122] In some embodiments, the VH VEGF comprising the sequence shown in SEQ ID NO: 1, or a sequence having at least 90%, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0123] In some embodiments, the VL VEGF comprising the sequence shown in SEQ ID NO: 2, or a sequence having at least 90%, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0124] In some embodiments, the VHPD-L1 comprising the sequence shown in SEQ ID NO: 3, or a sequence having at least 90%, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0125] In some embodiments, the VL PD-L1 comprising the sequence shown in SEQ ID NO: 4, or a sequence having at least 90%, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0126] In some embodiments, the VH VEGF It comprises HCDR1 as shown in SEQ ID NO: 5, HCDR2 as shown in SEQ ID NO: 6 and HCDR3 as shown in SEQ ID NO: 7.

[0127] In some embodiments, the VL VEGF It comprises LCDR1 as shown in SEQ ID NO:8, LCDR2 as shown in SEQ ID NO:9 and LCDR3 as shown in SEQ ID NO:10.

[0128] In some embodiments, the VH PD-L1 It comprises HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:12 and HCDR3 as shown in SEQ ID NO:13.

[0129] In some embodiments, the VL PD-L1 It comprises LCDR1 as shown in SEQ ID NO:14, LCDR2 as shown in SEQ ID NO:15 and LCDR3 as shown in SEQ ID NO:16.

[0130] In some embodiments, the VH VEGF 、VL VEGF 、VH PD-L1 and VL PD-L1 A linker is also included between any two.

[0131] The term "linker" refers to a linker that connects two molecules or moieties, such as a heavy chain variable region and a light chain variable region. Typically, a linker is located between or flanking two groups, molecules, or other moieties and is connected to each by a covalent bond, thereby connecting the two. In some embodiments, a linker is an organic molecule, group, polymer, or chemical moiety.

[0132] In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker of the present disclosure comprises the amino acid sequence (GGGS)n (SEQ ID NO: 38), (GGGGS)n (SEQ ID NO: 39), (G)n, (EAAAK)n (SEQ ID NO: 40), (GGS)n, (SGGS)n (SEQ ID NO: 41), SGGGGS (SEQ ID NO: 42), or (XP)n motif or a combination thereof, wherein n is independently an integer from 1 to 32, and wherein X is any amino acid.

[0133] In some specific embodiments, the linker comprises (GGGGS)n, where n is an integer from 1 to 32. In some more specific embodiments, the linker comprises GGGGSGGGGSGGGGS (SEQ ID NO: 43).

[0134] In some specific embodiments, the linker comprises SGGGGS (SEQ ID NO: 44).

[0135] In some specific embodiments, the bispecific antibody has a structure shown in any one of the following (i)-(iv):

[0136] (i) N-terminal-VL VEGF -Optional linker-VH VEGF -Optional Linker-VL PD-L1 -Optional linker-VH PD-L1 -C-terminus;

[0137] (ii) N-terminal-VH VEGF -Optional Linker-VL VEGF -Optional linker-VH PD-L1 -Optional Linker-VL PD-L1 -C-terminus;

[0138] (iii) N-terminal-VH PD-L1 -Optional Linker-VL PD-L1 -Optional linker-VH VEGF -Optional Linker-VL VEGF -C-terminus;

[0139] (iv) N-terminal-VL PD-L1 -Optional linker-VH PD-L1 -Optional Linker-VL VEGF -Optional linker-VH VEGF -C-terminus.

[0140] In some preferred embodiments, the bispecific antibody has a structure shown in any one of the following (i)-(iii):

[0141] (i) N-terminal-VL VEGF -Optional linker-VH VEGF -Optional Linker-VL PD-L1 -Optional linker-VH PD-L1 -C-terminus;

[0142] (ii) N-terminal-VH VEGF -Optional Linker-VL VEGF -Optional linker-VH PD-L1 -Optional Linker-VL PD-L1 -C-terminus;

[0143] (iii) N-terminal-VH PD-L1 -Optional Linker-VL PD-L1 -Optional linker-VH VEGF -Optional Linker-VL VEGF -C-terminus.

[0144] From a combinatorial perspective, there are theoretically 16 different combinations of the VH (heavy chain variable region) and VL (light chain variable region) of two antibodies. After in-depth research and reasoning, we screened four of these 16 possible combinations for experimentation. Surprisingly, the results showed that three of these four bispecific antibodies exhibited superior biological activity.

[0145] In some more preferred embodiments, the bispecific antibody has the following structure:

[0146] (i) N-terminal-VL VEGF -Optional linker-VH VEGF -Optional Linker-VL PD-L1 -Optional linker-VH PD-L1 -C-terminus; or

[0147] (ii) N-terminal-VH VEGF -Optional Linker-VL VEGF -Optional linker-VH PD-L1 -Optional Linker-VL PD-L1 -C-terminus.

[0148] In some embodiments, at least one disulfide bond is formed between the heavy chain variable region and the light chain variable region near the N-terminus.

[0149] In some specific embodiments, the 44th amino acid in the heavy chain variable region near the N-terminus is mutated to cysteine ​​(C), and the 100th amino acid in the light chain variable region near the N-terminus is mutated to cysteine ​​(C), thereby forming a disulfide bond between the heavy chain variable region and the light chain variable region near the N-terminus.

[0150] In some embodiments of the present disclosure, a signal peptide is included at the N-terminus of the bispecific antibody.

[0151] The terms "signal peptide," "signal sequence," or "signal peptide sequence" refer to a short peptide that, when fused to a protein of interest (e.g., a bispecific antibody of the present disclosure), promotes secretion of the expressed protein of interest onto the cell membrane or outside the cell. Signal peptides are typically located at the N-terminus of the protein of interest, and various signal peptides are known to those skilled in the art, such as, but not limited to, the human CD5 signal peptide, the hemagglutinin signal sequence, the human insulin signal sequence, the human interleukin-2 (IL2) signal peptide sequence, and the albumin signal sequence.

[0152] In some preferred embodiments, the N-terminus of the bispecific antibody comprises a human CD5 signal peptide or a human interleukin-2 signal peptide sequence.

[0153] In some specific embodiments, the human CD5 signal peptide comprises the amino acid sequence shown in SEQ ID NO:17.

[0154] In some specific embodiments, the human interleukin-2 signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO:33.

[0155] In some specific embodiments, the bispecific antibody comprises a sequence as shown in any one of SEQ ID NOs: 18 to 21, or a sequence that is at least 90% identical to a sequence as shown in any one of SEQ ID NOs: 18 to 21, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0156] In some preferred embodiments, the bispecific antibody comprises a sequence as shown in any one of SEQ ID NOs: 18 to 20, or a sequence that is at least 90% identical to the sequence shown in any one of SEQ ID NOs: 18 to 20, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0157] In some more preferred embodiments, the bispecific antibody comprises a sequence as shown in SEQ ID NO: 18 or 19, or a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 18 or 19, preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0158] In some embodiments of the present disclosure, the bispecific antibody further comprises a tag at the N-terminus and / or C-terminus.

[0159] According to the present disclosure, the term "tag" refers to a short peptide that is fused or connected to a protein of interest (e.g., a bispecific antibody of the present disclosure) and thereby promotes soluble expression, detection, and / or purification of the recombinant protein. The tag can be fused or connected to the N-terminus and / or C-terminus of the protein of interest (optionally via a linker or protease cleavage site). Such tags are well known to those skilled in the art, and for example, such tags include, but are not limited to, histidine tags, glutathione transferase (GST) tags, maltose binding protein (MBP) tags, thioredoxin (Trx) tags, NusA tags, disulfide isomerase DsbA tags, DsbC tags, SUMO tags, msyB tags, TF tags, initiation factor tags, ubiquitin tags, Myc tags, Flag tags, fluorescent proteins (e.g., GFP) tags, biotin tags, and avidin tags.

[0160] 3. Polynucleotides

[0161] In some aspects of the present disclosure, an isolated polynucleotide encoding the above-mentioned bispecific antibody is provided.

[0162] The polynucleotides disclosed herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0163] The polynucleotide encoding the bispecific antibody of the present disclosure includes: a coding sequence encoding only the bispecific antibody; a coding sequence for the bispecific antibody and various additional coding sequences; a coding sequence for the bispecific antibody (and optional additional coding sequences) and non-coding sequences.

[0164] The term "polynucleotide encoding a bispecific antibody" may include a polynucleotide encoding the bispecific antibody, and may also include additional coding and / or non-coding sequences.

[0165] The present disclosure also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present disclosure particularly relates to polynucleotides that can hybridize to the polynucleotides described in the present disclosure under stringent conditions. In the present disclosure, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the bispecific antibody.

[0166] In some embodiments, the polynucleotide encoding the bispecific antibody is codon-optimized. This type of optimization may require mutation of the nucleotide sequence encoding the bispecific antibody to mimic the codon preferences of the intended host organism or cell while encoding the same protein.

[0167] 4. Gene Expression Cassette

[0168] In some aspects of the present disclosure, a gene expression cassette is provided, comprising a polynucleotide provided by the present disclosure, that is, comprising a polynucleotide encoding a bispecific antibody provided by the present disclosure.

[0169] 5. Gene Delivery Vectors

[0170] In some aspects of the present disclosure, the gene expression cassette of the present disclosure is used to deliver genes (encoding bispecific antibodies) to animal cells, for example, to determine the effects of genes on cell viability and / or function, to treat cell disorders, etc. Therefore, in some aspects of the present disclosure, a gene delivery vector is provided, which comprises the gene expression cassette of the present disclosure. In some preferred embodiments, the gene delivery vector is used to express a transgene (encoding bispecific antibodies) in mammalian cells.

[0171] The gene delivery vectors disclosed herein encompass any convenient gene delivery vector for delivering a polynucleotide sequence to a mammalian cell. For example, the vector can include single-stranded nucleic acid or double-stranded nucleic acid, such as single-stranded DNA or double-stranded DNA. For example, the gene delivery vector can be DNA, such as naked DNA, such as a plasmid or minicircle, etc. The vector can include single-stranded RNA or double-stranded RNA, including modified forms of RNA. In another example, the gene delivery vector can be RNA, such as mRNA or modified mRNA.

[0172] As another example, the gene delivery vector can be a viral vector derived from a virus, such as an adenovirus, adeno-associated virus (AAV), a lentivirus, a herpes virus, an alphavirus, or a retrovirus, such as Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), Gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), foamy virus, Friedman's murine leukemia virus, murine stem cell virus (MSCV), and Rous' sarcoma virus (RSV), or a lentivirus. Although embodiments encompassing the use of adeno-associated viruses are described in more detail below, it is expected that those of ordinary skill will recognize that similar knowledge and skills in the art can also be applied to non-AAV gene delivery vectors.

[0173] In some embodiments, the gene delivery vector is a recombinant adeno-associated virus (rAAV). In this embodiment, the gene expression cassette is flanked by functional AAV inverted terminal repeat (ITR) sequences at the 5' and 3' ends. "Functional AAV ITR sequence" refers to the ITR sequence for rescuing, replicating, and packaging AAV virus particles as expected. Therefore, the AAV ITR for the gene delivery vector of the present disclosure does not need to have a wild-type nucleotide sequence and can be changed by insertion, deletion, or substitution of nucleotides, or the AAV ITR can be derived from any AAV serotype in several AAV serotypes, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10. Preferred AAV vectors have wild-type Rep genes and Cap genes that are all or partially deleted, but retain functional flanking ITR sequences. In specific embodiments, the AAV viral vector is an AAV variant. In some embodiments, the AAV variant is an AAV viral vector comprising a variant AAV capsid (or referred to as an AAV capsid protein variant).

[0174] In some embodiments, the gene expression cassette is encapsidated in an AAV capsid, which can be derived from any adeno-associated virus serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc., any of which can serve as a gene delivery vector. For example, the AAV capsid can be a wild-type capsid or a natural capsid. However, like ITRs, the capsid does not need to have a wild-type nucleotide sequence, but as long as the capsid is capable of transducing mammalian cells, it can be changed relative to the wild-type sequence by insertion, deletion or substitution of nucleotides in the VP1, VP2 or VP3 sequence. In other words, the AAV capsid can be a variant AAV capsid that includes one or more amino acid substitutions, deletions or insertions relative to the parent capsid protein or AAV capsid protein from which it is derived.

[0175] The AAV capsid is an icosahedron composed of 60 VP capsid protein monomers, including 5 VP1 monomers, 5 VP2 monomers, and 50 VP3 monomers. VP1, VP2, and VP3 monomers are all transcribed and translated from the AAV Cap gene. VP1 is the longest, containing approximately 735 amino acids. VP2 and VP3 are "truncated" versions of VP1, excluding the N-terminal amino acids of the VP1 protein. By convention, capsid protein modification sites are named according to the amino acid sequence of the VP1 protein.

[0176] The AAV capsid protein variant may be an AAV capsid protein variant containing a targeting peptide that is targeted to a specific tissue.

[0177] Preferably, rAAV is replication-defective, as AAV vectors are incapable of further replication and packaging of their genome independently.

[0178] Standard methods can be used to produce gene delivery vectors (e.g., rAAV virus particles) that encapsidate the gene expression cassette of the present disclosure. For example, in the case of rAAV virus particles, the AAV expression vector according to the present disclosure can be introduced into production cells, and then an AAV helper construct is introduced, wherein the helper construct comprises an AAV coding region that can be expressed in production cells and the AAV coding region can supplement the AAV auxiliary functions that do not exist in the AAV vector. Helper virus and / or another vector are then introduced into the production cells, wherein the helper virus and / or another vector provide auxiliary functions that can support effective rAAV virus production. The production cells are then cultured to produce rAAV. These steps are performed using standard methods. AAV packaging cells and packaging technology are used to prepare replication-defective AAV virus particles that encapsidate the recombinant AAV vector of the present disclosure by standard techniques known in the art.

[0179] Any concentration of viral particles suitable for effective transduction of mammalian cells can be prepared for contact with mammalian cells in vitro or in vivo. Similarly, any total amount of viral particles suitable for providing adequate cell transduction to impart the desired effect or treat a disease can be administered to a mammal. Any suitable amount of vector can be administered to the eye of a mammal or primate.

[0180] The viral vector can be formulated into a pharmaceutical composition comprising any suitable unit dosage of the vector, which can be administered to a subject to produce a change in the subject or to treat a disease in the subject.

[0181] In some cases, the multiplicity of infection (MOI) can be used to measure the unit dose of a pharmaceutical composition. MOI refers to the ratio or number of vector or viral genomes to the number of cells to which the nucleic acid can be delivered.

[0182] When preparing rAAV compositions, any host cell for producing rAAV viral particles can be used, including but not limited to mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. The host cell can also be a packaging cell or a production cell, in which the AAV rep gene and cap gene are stably maintained in the host cell, and the AAV vector genome is stably maintained and packaged in the production cell. Exemplary packaging and production cells are derived from SF-9, 293, A549, or HeLa cells. Standard techniques known in the art are used to purify and formulate the AAV vector.

[0183] In some preferred embodiments, the gene delivery vector comprises a sequence as shown in any one of SEQ ID NOs: 34-36, or a sequence having at least 90% identity to a sequence as shown in any one of SEQ ID NOs: 34-36.

[0184] 6. Cells

[0185] In some aspects, the present disclosure provides a cell comprising one or more of the following:

[0186] (a) the bispecific antibody described above;

[0187] (b) the polynucleotide described above;

[0188] (c) the gene expression cassette described above; and,

[0189] (d) The above-mentioned gene delivery vector.

[0190] The cell can be any of a variety of cells, including, for example, in vitro cells, in vivo cells, ex vivo cells, primary cells, cancer cells, animal cells, plant cells, algae cells, fungal cells, and the like.

[0191] In some embodiments, non-limiting examples of cells include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoan cells, cells from plants, algal cells, fungal cells, animal cells, cells from invertebrates, cells from vertebrates, cells from mammals (e.g., ungulates; rodents; non-human primates; humans; felines; dogs, etc.), etc. In some cases, the cell is a cell that is not derived from a natural organism (e.g., the cell can be a synthetic cell; also known as an artificial cell).

[0192] VII. Pharmaceutical Composition

[0193] In some aspects of the present disclosure, a pharmaceutical composition is provided, comprising the bispecific antibody, polynucleotide, gene expression cassette or gene delivery vector provided by the present disclosure, and, optionally, a pharmaceutically acceptable carrier.

[0194] In some embodiments, the pharmaceutical composition described herein contains the aforementioned bispecific antibody, polynucleotide, gene expression cassette, or gene delivery vector as an active ingredient.

[0195] Individual doses are generally not less than the amount required to produce a measurable effect on the subject and can be determined based on the pharmacokinetics and pharmacology of absorption, distribution, metabolism and excretion ("ADME") of the pharmaceutical composition or its by-products and therefore based on the disposition of the composition within the subject. This includes consideration of route of administration and dosage. Effective doses and / or dosage regimens can be readily determined empirically based on preclinical assays, safety and escalation and dose range trials, individual clinician-patient relationships, and in vitro and in vivo assays.

[0196] As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0197] The pharmaceutical composition is typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intraarterial, subcutaneous, intraperitoneal, intrathecal, intramuscular, or injection or infusion. Thus, delivery can be systemic or local.

[0198] Methods of formulating suitable pharmaceutical compositions are known in the art; see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY).

[0199] 8. Application and Method

[0200] In some aspects of the present disclosure, the bispecific antibodies, polynucleotides, gene expression cassettes, gene delivery vectors, cells, or pharmaceutical compositions provided herein are used in:

[0201] binds to PD-L1 and human VEGF; or

[0202] Blockade of the PD-L1 / PD-1 pathway and / or the VEGF / VEGFR pathway; or

[0203] Inhibit tumor cell growth,

[0204] Or for preparing a medicament for any of the above uses.

[0205] In some aspects of the present disclosure, the present disclosure provides a method for treating or preventing a disease (e.g., a tumor) in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of the bispecific antibody, polynucleotide, gene expression cassette, gene delivery vector, cell, or pharmaceutical composition of the present disclosure.

[0206] In other aspects of the present disclosure, the present disclosure also provides use of the bispecific antibodies, polynucleotides, gene expression cassettes, gene delivery vectors, or cells of the present disclosure in preparing a medicament for treating a disease (eg, a tumor).

[0207] In other aspects of the present disclosure, the present disclosure also provides the bispecific antibodies, polynucleotides, gene expression cassettes, gene delivery vectors, cells, or pharmaceutical compositions of the present disclosure, which are used for treating diseases (eg, tumors).

[0208] In some specific embodiments, the tumor can be a variety of tumors involving PD-L1 and / or VEGF, whether malignant or benign, and primary or secondary. These tumors can be solid cancers or hematological malignancies.

[0209] In some specific embodiments, the tumor is selected from breast cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, leukemia, lymphoma, myeloma, esophageal cancer, liver cancer, biliary tract cancer, pancreatic cancer, head and neck cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, osteosarcoma, soft tissue sarcoma, neuroblastoma, endocrine organ tumors, bladder cancer, skin cancer, nasopharyngeal cancer, and rhabdomyosarcoma.

[0210] Preferably, the neuroepithelial tissue tumor is selected from astrocytoma, anaplastic astrocytoma, glioblastoma, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, oligodendroglioma, ependymoma, mixed glioma, choroid plexus tumor, pineal cell tumor, embryonal tumor, most preferably glioblastoma.

[0211] Example

[0212] The embodiments of the present disclosure will be described in detail below with reference to the examples. However, those skilled in the art will appreciate that the following examples are intended only to illustrate the present disclosure and should not be construed as limiting the scope of the present disclosure. Where specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0213] Example 1. Antibody Design and Plasmid Vector Construction

[0214] The present disclosure provides a bispecific antibody comprising binding domains specific for PD-L1 and VEGF, which is intended to be used in AAV or mRNA-mediated gene therapy. Because the capacity of AAV vectors is limited (less than 4.7kb) and the immunogenicity of antibody drugs should be avoided as much as possible, the present disclosure constitutes BiTE-type bsAbs by serially connecting single-chain variable region fragments (Single-Chain Fragment Variable, scFv). The combination of VH (heavy chain variable region) and VL (light chain variable region) of two antibodies theoretically has 16 different combinations. After in-depth research and reasoning, we screened out 4 of these 16 possible combinations for experiments. The heavy chain variable domain (VH) and light chain variable domain (VL) in the bispecific antibody construct are arranged in the following order from N-terminus to C-terminus:

[0215] BiTE1:VL VEGF -3×(G4S)-VH VEGF -SGGGGS-VL PD-L1 -3×(G4S)-VH PD-L1 -HHHHHH

[0216] BiTE2:VH VEGF -3×(G4S)-VL VEGF -SGGGGS-VH PD-L1 -3×(G4S)-VL PD-L1 -HHHHHH

[0217] BiTE3:VH PD-L1 -3×(G4S)-VL PD-L1 -SGGGGS-VH VEGF -3×(G4S)-VL VEGF -HHHHHH

[0218] BiTE4:VL PD-L1 -3×(G4S)-VH PD-L1 -SGGGGS-VL VEGF -3×(G4S)-VH VEGF -HHHHHH

[0219] BiTE antibody tandem sequence Figure 1 Additional disulfide bonds were expressed on the N-terminal end of the antibody to stabilize the antibody structure (amino acid 44 of the heavy chain variable region and amino acid 100 of the light chain variable region near the N-terminus).

[0220] The vector construction was entrusted to Jiangsu GenScript Pengbo Biotechnology Co., Ltd.

[0221] The specific sequences of the antibodies expressed and purified in vitro are as follows:

[0222] BiTE1 (SEQ ID NO: 22):

[0223]

[0224] Among them, the single underline is the signal peptide sequence; the double underline is the tag sequence.

[0225] BiTE2 (SEQ ID NO: 23):

[0226]

[0227] Among them, the single underline is the signal peptide sequence; the double underline is the tag sequence.

[0228] BiTE3 (SEQ ID NO: 24):

[0229]

[0230] Among them, the single underline is the signal peptide sequence; the double underline is the tag sequence.

[0231] BiTE4 (SEQ ID NO: 25):

[0232]

[0233] Among them, the single underline is the signal peptide sequence; the double underline is the tag sequence.

[0234] The sequence of the bispecific antibody is as follows:

[0235] BiTE1 bispecific antibody (SEQ ID NO: 18):

[0236] DIQMTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGCGTKVEIKGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS SGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKGGGGSGGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS

[0237] BiTE2 bispecific antibody (SEQ ID NO: 19):

[0238] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKCLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGCGTKVEIKSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK

[0239] BiTE3 bispecific antibody (SEQ ID NO:20):

[0240] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKCLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGCGTKVEIKSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK

[0241] BiTE4 bispecific antibody (SEQ ID NO:21):

[0242] DIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGCGTKVEIKGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKCLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSSGGGG SDIQMTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS

[0243] In the above-mentioned bispecific antibodies, the heavy chain variable region targeting VEGF (anti-VEGF VH region, referred to as VH VEGF ) and light chain variable region (anti-VEGF VL region, referred to as VL VEGF ) and HCDR1-3 and LCDR1-3 (CDR was determined using the Kabat definition scheme) sequences are as follows:

[0244] Anti-VEGF VH region (SEQ ID NO: 1)

[0245] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS

[0246] anti-VEGF VL region (SEQ ID NO: 2)

[0247] DIQMTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK

[0248] HCDR1 (SEQ ID NO: 5): NYGMN

[0249] HCDR2(SEQ ID NO:6):WINTYTGEPTYAADFKR

[0250] HCDR3(SEQ ID NO:7):YPHYYGSSHWYFDV

[0251] LCDR1(SEQ ID NO:8):SASQDISNYLN

[0252] LCDR2 (SEQ ID NO: 9): FTSSLHS

[0253] LCDR3(SEQ ID NO:10):QQYSTVPWT

[0254] In the above-mentioned bispecific antibodies, the heavy chain variable region targeting PD-L1 (anti-PD-L1 VH region, referred to as VH PD-L1 ) and light chain variable region (anti-PD-L1 VL region, referred to as VL PD-L1 ) and HCDR1-3 and LCDR1-3 (CDR was determined using the Kabat definition scheme) sequences are as follows:

[0255] anti-PD-L1 VH region (SEQ ID NO: 3)

[0256] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS

[0257] anti-PD-L1 VL region (SEQ ID NO: 4)

[0258] DIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK

[0259] HCDR1 (SEQ ID NO: 11): DSWIH

[0260] HCDR2(SEQ ID NO:12):WISPYGGSTYYADSVKG

[0261] HCDR3(SEQ ID NO:13):RHWPGGFDY

[0262] LCDR1(SEQ ID NO:14):RASQDVSTAVA

[0263] LCDR2 (SEQ ID NO: 15): SASFLYS

[0264] LCDR3(SEQ ID NO:16):QQYLYHPAT

[0265] In the above bispecific antibody, the human CD5 signal peptide sequence (SEQ ID NO: 17):

[0266] MPMGSLQPLATLYLLGMLVASCLG

[0267] Target antigen sequence:

[0268] PD-L1 (SEQ ID NO: 26):

[0269] MRIFAVFFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVD PVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET

[0270] VEGF-110 (SEQ ID NO: 27):

[0271] APMA EGGGQNHHEV VKFMDVYQRS YCHPIETLVDIFQEYPDEIE YIFKPSCVPLMRCGGCCNDEGLECVPTEES NITMQIMRIK PHQGQHIGEMSFLQHNKCEC RPKKDR

[0272] VEGF-121 (SEQ ID NO: 28):

[0273] APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKCDKPRR

[0274] VEGF-165 (SEQ ID NO: 29):

[0275] APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRDKPRR

[0276] Example 2. Antibody expression and target protein binding ability determination (ELISA)

[0277] Antibody expression and purification: 24 hours before transfection, ExpiCHO-S cells (Gibco) were cultured normally with ExpiCHO expression medium (Gibco) and then cultured on a shaker. During transfection, the plasmid prepared in Example 1 was mixed with transfection reagent (ExpiFectamine CHO Reagent, Gibco) and incubated at room temperature to obtain a complex. The complex was added to the ExpiCHO-S cells, and the shake flask was returned to the shaker for continued culture. On the first day of transfection, enhancer (ExpiFectamine CHO Enhancer, Gibco) and feed (ExpiCHO Feed, Gibco) were added to the cells, and the cells were placed on a shaker for continued culture. On the fifth day of transfection, feed was added to the cells, and the cells were placed on a shaker for continued culture. The supernatant was collected by centrifugation, and the purified protein was obtained using a His affinity column (Genscript).

[0278] Enzyme-linked immunosorbent assay (ELISA): To evaluate the ability of BiTEs to bind to PD-L1 in vitro, ELISA experiments were performed using atezolizumab (Pengbo Biotechnology, RD230509144) as a positive control. To evaluate the ability of BiTEs to bind to VEGF-110, VEGF-121, and VEGF-165 in vitro, ELISA experiments were performed using bevacizumab (Pengbo Biotechnology, RD230203109) as a positive control. The specific steps involved were:

[0279] a. Dilute human PD-L1 (Baipusaisi, PD1-H5229), human VEGF-110 (Baipusaisi, VE0-H52H3), human VEGF-121 (Baipusaisi, VE1-H5246), and human VEGF-145 (Baipusaisi, VE5-H5248) proteins to appropriate concentrations using CBS buffer (Genscript). Coat 96-well plates at 100 μL / well at 4°C overnight.

[0280] b. Discard the liquid in the 96-well plate, add 300 μL PBST to each well, wash three times, and absorb the residual liquid in the 96-well plate with absorbent paper for the last time.

[0281] c. Add the prepared 5% skim milk powder solution to a 96-well plate at 200 μL / well and incubate at 37°C for 2 hours.

[0282] d. Repeat step b.

[0283] e. Dilute the test article and positive control to the appropriate concentration in PBS. Add 100 μL / well to a 96-well plate. Add 100 μL of PBS to the blank control well. Repeat the test twice. Incubate at room temperature for 2 hours.

[0284] f. Repeat step b.

[0285] g. Prepare anti-His-HRP (Genscript) solution with PBS at a 1:1 ratio. Add 100 μL / well to a 96-well plate. Incubate at room temperature for 1 hour.

[0286] h. Repeat step b.

[0287] i. TMB solution (Genscript) was added to a 96-well plate at 100 μL / well and incubated at room temperature for 15 minutes in the dark.

[0288] j. Add 50 μL of ELISA stop buffer to each well of a 96-well plate. Read the OD450 nm value using a microplate reader.

[0289] Antibody expression, purification, and ELISA experiments were commissioned to Jiangsu GenScript Pengbo Biotechnology Co., Ltd.

[0290] Experimental results: Effective expression of PD-L1 / VEGF antibody (ELISA)

[0291] The SDS-PAGE results showed that the position of the target antibody band was consistent with the expected position (such as Figure 2 The median effective concentration (EC50) of the purified antibody binding to the target protein was calculated based on the ELISA test results. The results showed that the purified antibody could bind to PD-L1 and VEGF proteins (as shown in Table 1 and Figure 3 shown).

[0292] Table 1 ELISA assay to detect the ability of BiTEs to bind to PD-L1 / VEGF (EC50) (unit: μg / mL)

[0293]

[0294] Example 3: Surface Plasmon Resonance (SPR) Detection

[0295] SPR experiment:

[0296] a. Antibody immobilization was performed at 25°C using HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% P20, pH 7.4) as the running buffer.

[0297] b. Activate the sensor chips of flow cells 1 and 2 with a fresh mixture of 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).

[0298] c. The antibody was diluted with 10 mmol / L NaAC and injected into flow cell 2, while flow cell 1 was set as a blank. After the coupling reaction was completed, 1 M ethanolamine was used to block the remaining active coupling sites on the chip surface.

[0299] d. For single-target assays, each antigen was diluted and injected into flow cells 1 and 2 for binding, followed by HBS-EP+ for dissociation. For dual-target assays, a fixed concentration of human PD-L1 was injected first (first injection), followed by human VEGF-110 (second injection), followed by HBS-EP+ for dissociation. All data were processed using Biacore8K Evaluation Software version 4.0.

[0300] The SPR test was commissioned by Jiangsu GenScript Pengbo Biotechnology Co., Ltd.

[0301] Experimental results: PD-L1 / VEGF antibody effective expression (SPR)

[0302] The structure of bispecific antibodies combines the characteristics of fusion proteins, and the two antigen-binding domains may affect each other's binding to the target antigen. However, surprisingly, the single-target SPR results (Table 2) and dual-target SPR results (Table 3) in this example showed that BiTE1, BiTE2, and BiTE3 can simultaneously bind to both PD-L1 and VEGF proteins.

[0303] Table 2 BiTE antibody SPR results (single target)

[0304]

[0305] In this specification, "E" is generally used to represent "exponent", that is, the power of 10. For example, 2.48E-02 means 2.48×10 -2 ; 6.94E+04 means 6.94×10 4 .

[0306] Table 3 BiTE antibody SPR results (dual targets)

[0307]

[0308] In Tables 2 and 3:

[0309] Chi 2 (RU2) indicates: Chi-squared test, determination coefficient (R-squared, R 2 )

[0310] ka(1 / Ms) means: association constant per mole per second (M-1s-1)

[0311] kd(1 / s) means: dissociation rate constant per second (s-1)

[0312] KD(M) stands for equilibrium dissociation constant. "M" stands for molar concentration.

[0313] Rmax (RU) means: Response Unit of the maximum binding capacity on the chip surface

[0314] Example 4: In vitro blocking experiment

[0315] PD-L1 / PD-1 blocking experiment in vitro: resuscitation of Jurkat-PD-1-NFAT-Luc (Pengbo Bio, RD00871) reporter gene cell line and aAPC CHO-PDL1 cells (Pengbo Biotechnology, RD00703) were subcultured to the logarithmic growth phase and seeded in 96-well plates at 2 × 10 4 Effector cells Jurkat-PD-1-NFAT-Luc were added to target cells at a ratio of 1:1. aAPC CHO-PDL1. A gradient dilution of the detection antibody (positive control: Atezolizumab) was added to the corresponding wells. The antibody was diluted 5-fold over 8 consecutive dilutions, with final concentrations of 100 μg / mL, 20 μg / mL, 4 μg / mL, 0.8 μg / mL, 0.16 μg / mL, 0.032 μg / mL, 0.0064 μg / mL, and 0.00128 μg / mL. After 18 hours of co-culture, 25 μL of luciferase assay reagent working solution (Nanjing Novozymes) was added to each well, and the luciferase activity in the wells was measured using a microplate reader.

[0316] VEGF / VEGFR blocking experiment in vitro: cells expressing VEGFR-NFAT-Luc (Pengbo Bio, RD00704) were cultured at 1×10 6 Cells were seeded into a 96-well plate at a density of 100 μL per well and incubated at 37°C overnight. A serial dilution of the detection antibody (positive control: Bevacizumab) was added to the corresponding wells. The antibody was diluted 3-fold over 8 consecutive dilutions, with final concentrations of 10 μg / mL, 3.3333 μg / mL, 1.1111 μg / mL, 0.3704 μg / mL, 0.1235 μg / mL, 0.0412 μg / mL, 0.0137 μg / mL, and 0.0046 μg / mL. After 18 hours of co-incubation, 25 μL of luciferase assay reagent working solution (Nanjing Novozymes) was added to each well, and the luciferase activity in the wells was measured using a microplate reader.

[0317] Experimental results: BiTE dual antibodies effectively block PD-1 / PD-L1 and VEGF / VEGFR pathways in vitro

[0318] The results of in vitro blocking experiments showed that Atezolizumab, as a PD-L1 positive control antibody, had an EC50 of 0.07193 μg / mL. BiTE1, BiTE2, and BiTE3 could block the binding of PD-L1 to PD-1. The EC50 value of BiTE1 was 2.112 μg / mL, and the EC50 value of BiTE2 was 1.537 μg / mL. The EC50 value of BiTE3 was 0.2494 μg / ml (e.g. Figure 4 Bevacizumab, as a positive control antibody, can block the binding of VEGF-165 to its receptor VEGFR2 with an IC50 of 0.2699 μg / ml. BiTE1, BiTE2, and BiTE3 can all block the binding of VEGF-165 to its receptor VEGFR2. The IC50 value of BiTE1 is 0.3167 μg / ml, and the IC50 value of BiTE2 is 0.9284 μg / ml. The IC50 value of the dose-effect curve of BiTE3 is 1.001 μg / ml (e.g. Figure 5 The negative control antibody (human IgG) in the above experiment had no blocking effect.

[0319] Example 5: In vitro efficacy experiment

[0320] U87 cells (human glioblastoma cell line) (Wuhan Punosai) were cultured to the third generation using MEM complete medium (Wuhan Punosai), and 10,000 cells were plated per well in a 96-well plate. After culturing for 24 hours, BiTE1 protein (Pengbo Bio) was added at final concentrations of 0, 6.25, 12.5, 25, 50, and 100 μg / mL, and the culture was continued for 96 hours. 10 μL of CCK8 solution (Japan Tongren) was added to each well, and the absorbance was measured at 450 nm using a microplate reader (SynergyH1) after culturing for 1 hour.

[0321] Experimental results: BITE1 bispecific antibody inhibits U87 cell viability in vitro in a concentration-dependent manner

[0322] The results of the CCK8 assay showed that after 96 hours of culture of U87 cells in complete medium containing 0, 6.25, 12.5, 25, 50, and 100 μg / mL BiTE1 protein, the cell viability was 100%, 95%, 93%, 79%, 68%, and 53%, respectively. The above experimental results show that the BITE1 bispecific antibody inhibits the viability of U87 cells in vitro in a concentration-dependent manner (e.g. Figure 6 ).

[0323] Experimental Example 6: AAV packaging

[0324] AAV virus packaging was carried out according to the commonly used three-plasmid transfection method. AAV2 capsid gene plasmid (Shandong Weizhen), Helper plasmid (Shandong Weizhen) and shuttle plasmids (BITE1-AAV plasmid, BITE2-AAV plasmid, BITE3-AAV plasmid and isotype control AVV plasmid, the specific sequences are shown below) were co-transfected into HEK293 cells. AAV virus particles were separated and purified from HEK293T cells using an iodixanol concentration gradient. The titer of AAV was determined by the commonly used qPCR method. The control antibody Isotype and BITE1 were packaged as AAV-isotype control (Isotype) and AAV-BITE1, respectively. The packaging and titer determination of AAV virus were completed by Shandong Weizhen Biotechnology Co., Ltd.

[0325] The specific amino acid sequence of the antibody expressed by the shuttle plasmid is as follows:

[0326] BiTE1 bispecific antibody (SEQ ID NO: 30):

[0327] MYRMQLLSCIALSLALVTNS DIQMTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQK

[0328] PGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFG

[0329] CGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGM

[0330] NWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDT

[0331] AVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSSGGGGSDIQMTQSPSSSLSASVGDRV

[0332] TITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQP

[0333] EDFATYYCQQYLYHPATFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPG

[0334] GSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISAD

[0335] TSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS

[0336] Among them, the single underline is the signal peptide sequence.

[0337] BiTE2 bispecific antibody (SEQ ID NO: 31):

[0338] MYRMQLLSCIALSLALVTNS EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKCLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGCGTKVEIK SGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK

[0339] Among them, the single underline is the signal peptide sequence.

[0340] BiTE3 bispecific antibody (SEQ ID NO: 32):

[0341] MYRMQLLSCIALSLALVTNSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKCLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGCGTKVEIKSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK

[0342] Among them, the single underlined part is the signal peptide sequence.

[0343] Human interleukin-2 signal peptide sequence (SEQ ID NO: 33):

[0344] MYRMQLLSCIALSLALVTNS

[0345] BITE1-AAV plasmid (SEQ ID NO: 34):

[0346]

[0347]

[0348] Among them, the double underline is the signal peptide coding sequence, and the single underline is the bispecific antibody coding sequence.

[0349] BITE2-AAV plasmid (SEQ ID NO: 35):

[0350]

[0351]

[0352]

[0353] Among them, the double underline is the signal peptide coding sequence, and the single underline is the bispecific antibody coding sequence.

[0354] BITE3-AAV plasmid (SEQ ID NO: 36):

[0355]

[0356]

[0357]

[0358] Among them, the double underline is the signal peptide coding sequence, and the single underline is the bispecific antibody coding sequence.

[0359] Isotype-AAV plasmid (SEQ ID NO: 37):

[0360]

[0361]

[0362] Among them, the double underline is the signal peptide coding sequence, and the single underline is the bispecific antibody coding sequence.

[0363] Experimental Example 7: In vivo pharmacodynamic study in a U87 subcutaneous xenograft tumor model

[0364] This example uses 6-8 week old female NOG mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). U87 cells (ATCC, HTB-14) were cultured in vitro at 37°C and 5% CO2 using EMEM medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% double antibody; routine digestion and passage were performed with trypsin (Gibco) twice a week. When the cells reached the required amount, the cells were collected, counted and inoculated, and the grouping information is shown in Table 4; on the day of tumor cell inoculation, the mice were randomly grouped according to their weight, U87 cells were collected, and the cells were resuspended in EMEM medium without fetal bovine serum. The test substance and the cell suspension were mixed according to the table below, and 5E6 U87 cells and the suspension of the test substance were subcutaneously inoculated above the right shoulder blade of the mouse (Day 0); the mouse tumor volume was then monitored using a vernier caliper. When the tumor volume reached 70mm 3 At the same time, mice in the AAV-Isotype group and the AAV-BiTE1 group were inoculated with 0.1 mL containing 3.5×10 6 PBMC (Sai ​​Li, XW0801120W) cells were cultured in DPBS and the bevacizumab group was administered twice weekly. Mice were euthanized on day 36. This experiment was commissioned by Nantong WuXi AppTec Pharmaceutical Technology Co., Ltd.

[0365] Table 4 Grouping information of in vivo efficacy experiments

[0366]

[0367] Experimental results

[0368] This example evaluated the anti-tumor efficacy of the test substance in a humanized NOG mouse U87 cell subcutaneous xenograft tumor model. The results showed that the AAV-BITE1 group exhibited significant anti-tumor activity, while there was no statistical difference in the other groups.

[0369] During the experiment, the weight of mice in each group maintained an upward trend (e.g. Figure 7 ), no abnormal body weight was observed, indicating that the mice tolerated the test drugs well.

[0370] Starting from tumor cell inoculation, 36 days after inoculation, the average tumor volume of the tumor-bearing mice in the isotype control group (AAV-Isotype) reached 3043 mm 3 Compared with the isotype control group, the Bevacizumab group did not show significant tumor inhibition, with tumor volumes of 2,121 mm 3 (TGI=31%, p=0.114) Compared with the isotype control group, the AAV-BiTE1 group showed a significant tumor inhibition effect, with a tumor volume of 1,334 mm 3(TGI=56%, p=0.005) (as shown in Table 5 and Figure 8 ).

[0371] Table 5 In vivo efficacy test results

[0372]

[0373] It should be noted that, although the technical solutions of the present disclosure are described with specific examples, those skilled in the art will appreciate that the present disclosure should not be limited thereto.

[0374] While various embodiments of the present disclosure have been described above, the foregoing description is intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or technological improvements in the marketplace, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A bispecific antibody, wherein: The bispecific antibody comprises: a heavy chain variable region (VH VEGF ) and light chain variable region (VL VEGF ), and a heavy chain variable region (VH PD-L1 ) and light chain variable region (VL PD-L1 );in, The VH VEGF comprising the sequence shown in SEQ ID NO: 1, or a sequence having at least 90% identity thereto; The VL VEGF comprising the sequence shown in SEQ ID NO: 2, or a sequence having at least 90% identity thereto; The VH PD-L1 comprising the sequence shown in SEQ ID NO:3, or a sequence having at least 90% identity thereto; The VL PD-L1 The sequence comprises the sequence shown in SEQ ID NO: 4, or a sequence having at least 90% identity thereto.

2. The bispecific antibody according to claim 1, wherein The VH VEGF comprising HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:6, and HCDR3 as shown in SEQ ID NO:7; The VL VEGF comprising LCDR1 as shown in SEQ ID NO:8, LCDR2 as shown in SEQ ID NO:9, and LCDR3 as shown in SEQ ID NO:10; The VH PD-L1 comprising HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:12, and HCDR3 as shown in SEQ ID NO:13; The VL PD-L1 It comprises LCDR1 as shown in SEQ ID NO:14, LCDR2 as shown in SEQ ID NO:15 and LCDR3 as shown in SEQ ID NO:

16.

3. The bispecific antibody according to claim 1 or 2, wherein: The VH VEGF , VL VEGF , VH PD-L1 and VL PD-L1 A linker is also included between any two.

4. The bispecific antibody according to any one of claims 1 to 3, wherein: The bispecific antibody has a structure shown in any one of the following (i)-(iii): (i) N-terminal-VL VEGF -Optional linker-VH VEGF -Optional connector-VL PD-L1 -Optional linker-VH PD-L1 -C-terminus; (ii) N-terminal-VH VEGF -Optional connector-VL VEGF -Optional linker-VH PD-L1 -Optional connector-VL PD-L1 -C-terminus; (iii) N-terminal-VH PD-L1 -Optional connector-VL PD-L1 -Optional linker-VH VEGF -Optional connector-VL VEGF -C-terminus; Preferably, the bispecific antibody comprises a sequence as shown in any one of SEQ ID NOs: 18 to 20, or a sequence having at least 90% identity with a sequence as shown in any one of SEQ ID NOs: 18 to 20.

5. The bispecific antibody according to any one of claims 1 to 4, wherein At least one disulfide bond is formed between the heavy chain variable region and the light chain variable region near the N-terminus.

6. The bispecific antibody according to claim 5, wherein The 44th amino acid in the heavy chain variable region near the N-terminus is mutated to cysteine ​​(C), and the 100th amino acid in the light chain variable region near the N-terminus is mutated to cysteine ​​(C), thereby forming a disulfide bond between the heavy chain variable region and the light chain variable region near the N-terminus.

7. The bispecific antibody according to any one of claims 1 to 6, wherein: The bispecific antibody also contains a signal peptide at the N-terminus.

8. The bispecific antibody according to any one of claims 1 to 7, wherein: The bispecific antibody comprises a sequence as shown in any one of SEQ ID NOs: 22-24, 30-32, or a sequence having at least 90% identity with the sequence as shown in any one of SEQ ID NOs: 22-24, 30-32.

9. An isolated polynucleotide encoding the bispecific antibody according to any one of claims 1 to 8.

10. A gene expression cassette comprising the polynucleotide according to claim 9.

11. A gene delivery vector comprising the gene expression cassette according to claim 10.

12. The gene delivery vector according to claim 11, wherein The gene delivery vector is DNA or RNA.

13. The gene delivery vector according to claim 11, wherein The gene delivery vector is a viral vector derived from a virus.

14. The gene delivery vector according to claim 13, wherein The viral vector is a recombinant adeno-associated virus; Preferably, the gene delivery vector comprises a sequence as shown in any one of SEQ ID NOs: 34-36, or a sequence having at least 90% identity to the sequence as shown in any one of SEQ ID NOs: 34-36.

15. A cell comprising the bispecific antibody of any one of claims 1 to 8, the polynucleotide of claim 9, the gene expression cassette of claim 10, or the gene delivery vector of any one of claims 11 to 14.

16. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-8, the polynucleotide of claim 9, the gene expression cassette of claim 10, the gene delivery vector of any one of claims 11-14, or the cell of claim 15; and, optionally, a pharmaceutically acceptable carrier.

17. A method for treating or preventing a tumor in a subject in need thereof, the method comprising administering to a subject in need thereof an effective amount of the bispecific antibody of any one of claims 1 to 8, the polynucleotide of claim 9, the gene expression cassette of claim 10, the gene delivery vector of any one of claims 11 to 14, the cell of claim 15, or the pharmaceutical composition of claim 16; Optionally, the tumor is a tumor involving PD-L1 and / or VEGF; Optionally, the tumor is selected from breast cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, leukemia, lymphoma, myeloma, esophageal cancer, liver cancer, biliary tract cancer, pancreatic cancer, head and neck cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, osteosarcoma, soft tissue sarcoma, neuroblastoma, endocrine organ tumors, bladder cancer, skin cancer, nasopharyngeal carcinoma, and rhabdomyosarcoma.

18. The bispecific antibody according to any one of claims 1 to 8, the polynucleotide according to claim 9, the gene expression cassette according to claim 10, the gene delivery vector according to any one of claims 11 to 14, the cell according to claim 15, or the pharmaceutical composition according to claim 16 for use in treating tumors; Optionally, the tumor is a tumor involving PD-L1 and / or VEGF; Optionally, the tumor is selected from breast cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, leukemia, lymphoma, myeloma, esophageal cancer, liver cancer, biliary tract cancer, pancreatic cancer, head and neck cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, osteosarcoma, soft tissue sarcoma, neuroblastoma, endocrine organ tumors, bladder cancer, skin cancer, nasopharyngeal carcinoma, and rhabdomyosarcoma.

19. Use of the bispecific antibody according to any one of claims 1 to 8, the polynucleotide according to claim 9, the gene expression cassette according to claim 10, the gene delivery vector according to any one of claims 11 to 14, and the cell according to claim 15 in the preparation of a medicament for treating a tumor; Optionally, the tumor is a tumor involving PD-L1 and / or VEGF; Optionally, the tumor is selected from breast cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, leukemia, lymphoma, myeloma, esophageal cancer, liver cancer, biliary tract cancer, pancreatic cancer, head and neck cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, osteosarcoma, soft tissue sarcoma, neuroblastoma, endocrine organ tumors, bladder cancer, skin cancer, nasopharyngeal carcinoma, and rhabdomyosarcoma.

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