Bispecific antibody for gene therapy

Abstract

The present disclosure provides a bispecific antibody for gene therapy. Specifically, the present disclosure provides a bispecific antibody. The bispecific antibody comprises: a heavy chain variable region (VHVEGF) and a light chain variable region (VLVEGF) that target vascular endothelial growth factor (VEGF), and a heavy chain variable region (VHPD-L1) and a light chain variable region (VLPD-L1) that target programmed death-ligand 1 (PD-L1). The bispecific antibody provided herein 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, thereby inhibiting tumors in vitro and in vivo.

Description

Bispecific antibodies that can be used for gene therapy

[0001] Priority and related applications

[0002] This disclosure claims priority to Chinese patent application 202510186654.6, filed on February 19, 2025, entitled "Bispecific Antibody for Gene Therapy". The entire contents of the above-cited patent application are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the fields of gene therapy and antibodies, specifically to bispecific antibodies that can be used for gene therapy. Background Technology

[0004] Cancer is the second leading cause of death worldwide, with nearly 10 million deaths in 2020 [1]. Compared with traditional anti-cancer strategies, monoclonal antibodies are considered one of the most promising cancer treatment methods [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 drug resistance and improve the therapeutic effect of monoclonal antibody drugs, developing a combination therapy of two antibodies may be a potential feasible option, but in practice, such options are often hindered in research and application due to high costs, safety and insufficient efficacy caused by the difficulty in matching pharmacokinetic characteristics [4].

[0005] With the development of genetic engineering technology, the therapeutic potential of bispecific antibodies (bsAbs) has attracted 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 concentration in the tumor microenvironment; and reduce development and production costs[6]. Based on the presence or absence of a structurally crystallizable fragment (Fc), bsAbs can be divided into IgG-like and non-IgG types[5].

[0006] IgG-like bsAbs have a longer half-life, easier purification methods, and higher stability. These antibodies retain multiple Fc-mediated effector functions [5]. However, the Fc segment is a double-edged sword among bsAbs. On the one hand, it has great development potential, but on the other hand, it may lead to unnecessary non-specific immune responses during bsAb treatment, ultimately causing a series of adverse reactions. For example, Catumaxomab, an IgG-like bsAb containing CD3 and epithelial cell adhesion molecule (EpCAM), caused serious adverse reactions due to the off-target binding of its 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 the Fc-mediated effector mechanism may hinder tumor-specific T cell redirection and tumor cell killing [8].

[0007] Non-IgG bsAbs have better tissue penetration and lower immunogenicity [9]. Bispecific T cell binder (BiTE) antibodies are typical non-IgG bsAbs, which are small and flexible molecules

[0010] . Blinatumomab, a BiTE antibody targeting CD3 and CD19 that was approved for marketing in 2014, has a molecular weight of about 55 kDa and a short plasma half-life (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

[0011] . The drug's sales reached $500 million in 2021, but due to its short half-life, it requires multiple intravenous infusions, which puts a huge burden on patients' physical and economic health. As a result, there are few BiTE structures used in currently marketed and clinical-stage bispecific antibody drugs.

[0008] Monoclonal antibodies targeting programmed cell death 1 (PD-1) or programmed cell death-ligand 1 (PD-L1) can reactivate suppressed T cells, thereby blocking the immune escape of tumor cells [12,13]. Among them, Atezolizumab is the first immunotherapy drug targeting PD-L1 developed by Roche. Monoclonal antibodies targeting vascular endothelial growth factor (VEGF) or VEGF receptor (VEGFR) can also inhibit tumor growth

[0014] , and Bevacizumab is the first VEGF monoclonal antibody to be approved for marketing

[0015] .

[0009] As mentioned earlier, the occurrence and development of malignant tumors involve multiple complex mechanisms, and ideal efficacy cannot be achieved by targeting a single point. For example, monotherapy with Atezolizumab and Bevacizumab in the field of liver cancer can achieve objective response

[0016] , but the objective response rate (ORR) is less than 20%. A phase Ib clinical trial (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 was higher in the "T+A" group (125 cases - 38.0%) than that of sorafenib (48 cases - 30.8%). Another study showed that treatment with ipilimumab plus navokumab (anti-PD-1) improved survival outcomes in melanoma patients compared to ipilimumab (anti-cytotoxic T-lymphocyte-associated protein 4) alone; however, the increased antitumor activity was associated with a significantly increased number of immune-related adverse events

[0017] . Therefore, the development of bsAbs with lower immunogenicity and production costs is urgently needed compared to combination therapy.

[0010] 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 invasiveness against solid tumors restrict the clinical application of this type of drug. In addition, IgG-like antibodies require multiple administrations to maintain the therapeutic effect, which increases the economic burden on patients with long-term treatment. Repeated injections also increase the patient's suffering and the possibility of adverse reactions.

[0011] As mentioned earlier, the extremely low half-life of BiTE molecules limits the widespread application of this type of antibody. 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 achieving sustained expression of the target protein after a single dose

[0018] . Hefei Xingmou Biotechnology Co., Ltd.'s gene therapy drug "XMVA09" has been formally accepted for IND approval. This drug is mediated by AAV to express BiTE-type antibodies (targeting VEGF and angiopoietin 2). A single dose showed significant clinical benefits in patients with wet age-related macular degeneration without any drug-related adverse events

[0019] . Given the need in this field for more economical, safer, longer-lasting, and more effective treatment strategies, gene therapy mediated by AAV or mRNA-mediated BiTE expression shows great promise.

[0012] In summary, there is an urgent need to develop bispecific antibodies against PD-L1 and VEGF that can be used in gene therapy.

[0013] References

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[0019] [6]Cui X,Jia H,Xin H,et al.A novel bispecific antibody targeting pd-l1 and vegf with combined anti-tumor activities.Frontiers In Immunology.2021;12:778978.

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[0011] Topp M S, N,Stein A S,et al.Safety and activity of blinatumomab for adult patients with relapsed or refractory b-precursor acute lymphoblastic leukaemia:A multicentre,single-arm,phase 2 study.The Lancet Oncology.2015;16(1):57-66.

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[0016] Jiang Y, Zhao X, Fu J, et al. Progress and challenges in precise treatment of tumors with pd-1 / pd-11 blockade. Frontiers In Immunology. 2020; 11:339.

[0030] Wolchok JD, Chiarion-Sileni V, Gonzalez R, et al. Overall survival with combined nivolumab and ipilimumab in advanced melanoma. The New England Journal of Medicine. 2017; 377 (14): 1345-1356.

[0031]

[0018] 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.

[0032]

[0019] Cai Yuan, Ma Zhen, Zhou Peipei, et al. An AAV vector encoding bispecific antibodies against VEGF-A and ANG-2. CN116925234A. [P]. 2023.10.24 Summary of the Invention

[0033] The problem the invention aims to solve

[0034] This disclosure aims to provide a bispecific antibody against PD-L1 and VEGF that can be used in gene therapy.

[0035] Solution for solving the problem

[0036] In a first aspect of this disclosure, a bispecific antibody is provided, wherein the bispecific antibody comprises: targeting the heavy chain variable region (VH) of vascular endothelial growth factor (VEGF). VEGF ) and light chain variable region (VL) VEGF ), and the heavy chain variable region (VH) targeting programmed death receptor-ligand 1 (PD-L1). PD-L1 ) and light chain variable region (VL)PD-L1 );in,

[0037] The VH VEGF Contains a sequence as shown in SEQ ID NO:1, or a sequence having at least 90% identity with it;

[0038] The VL VEGF Contains a sequence as shown in SEQ ID NO:2, or a sequence having at least 90% identity with it;

[0039] The VH PD-L1 Contains a sequence as shown in SEQ ID NO:3, or a sequence having at least 90% identity with it;

[0040] The VL PD-L1 It includes a sequence as shown in SEQ ID NO:4, or a sequence that is at least 90% identical to it.

[0041] In some implementations, the VH VEGF Includes 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 It includes 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 Includes 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 includes 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.

[0042] In some implementations, the VH VEGF VL VEGF VH PD-L1 and VL PD-L1 The connection between any two also includes a joint.

[0043] In some embodiments, the bispecific antibody has the structure shown in any one of (i)-(iii):

[0044] (i) N-terminal-VL VEGF -Optional connector-VH VEGF -Optional connector-VL PD-L1 -Optional connector-VH PD-L1-C terminal;

[0045] (ii) N-terminus - VH VEGF -Optional connector-VL VEGF -Optional connector-VH PD-L1 -Optional connector-VL PD-L1 -C terminal;

[0046] (iii) N-terminal -VH PD-L1 -Optional connector-VL PD-L1 -Optional connector-VH VEGF -Optional connector-VL VEGF -C terminal.

[0047] In some preferred embodiments, the bispecific antibody comprises a sequence as shown in any one of SEQ ID NO:18 to 20, or a sequence having at least 90% identity with a sequence shown in any one of SEQ ID NO:18 to 20.

[0048] In some implementations, at least one disulfide bond is formed between the heavy chain variable region and the light chain variable region near the N-terminus.

[0049] In some 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.

[0050] In some embodiments, the N-terminus of the bispecific antibody also contains a signal peptide.

[0051] In some embodiments, the bispecific antibody comprises a sequence as shown in any one of SEQ ID NO:22-24, 30-32, or a sequence having at least 90% identity with the sequence shown in any one of SEQ ID NO:22-24, 30-32.

[0052] In a second aspect of this disclosure, an isolated polynucleotide is provided that encodes a bispecific antibody as described in the first aspect of this disclosure.

[0053] In a third aspect of this disclosure, a gene expression cassette is provided, comprising the polynucleotides described in the second aspect of this disclosure.

[0054] In a fourth aspect of this disclosure, a gene delivery vector is provided that includes a gene expression cassette as described in a third aspect of this disclosure.

[0055] In some implementations, the gene delivery vector is DNA or RNA.

[0056] In some implementations, the gene delivery vector is a viral vector derived from a virus.

[0057] In some implementations, the viral vector is a recombinant adeno-associated virus.

[0058] In some preferred embodiments, the gene delivery vector comprises a sequence as shown in any one of SEQ ID NO:34-36, or a sequence having at least 90% identity with a sequence shown in any one of SEQ ID NO:34-36.

[0059] In a fifth aspect of this disclosure, a cell is provided comprising a bispecific antibody as described in a first aspect of this disclosure, a polynucleotide as described in a second aspect of this disclosure, a gene expression cassette as described in a third aspect of this disclosure, or a gene delivery vector as described in a fourth aspect of this disclosure.

[0060] In a sixth aspect of this disclosure, a pharmaceutical composition is provided comprising a bispecific antibody as described in the first aspect of this disclosure, a polynucleotide as described in the second aspect of this disclosure, a gene expression cassette as described in the third aspect of this disclosure, a gene delivery vector as described in the fourth aspect of this disclosure, or a cell as described in the fifth aspect of this disclosure; and optionally, a pharmaceutically acceptable vector.

[0061] In a seventh aspect of this disclosure, a method for treating or preventing a tumor in a subject of need is provided, the method comprising administering to the subject of need an effective amount of a bispecific antibody as described in a first aspect of this disclosure, a polynucleotide as described in a second aspect of this disclosure, a gene expression cassette as described in a third aspect of this disclosure, a gene delivery vector as described in a fourth aspect of this disclosure, a cell as described in a fifth aspect of this disclosure, or a pharmaceutical composition as described in a sixth aspect of this disclosure.

[0062] In some optional embodiments, the tumor is a tumor involving PD-L1 and / or VEGF.

[0063] In some alternative implementations, the tumor is selected from breast cancer, gastric cancer, colorectal 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, rhabdomyosarcoma, and lung cancer.

[0064] This disclosure also provides bispecific antibodies as described in the first aspect of this disclosure, polynucleotides as described in the second aspect of this disclosure, gene expression cassettes as described in the third aspect of this disclosure, gene delivery vectors as described in the fourth aspect of this disclosure, cells as described in the fifth aspect of this disclosure, or pharmaceutical compositions as described in the sixth aspect of this disclosure for the treatment of tumors.

[0065] In some optional embodiments, the tumor is a tumor involving PD-L1 and / or VEGF.

[0066] In some alternative implementations, the tumor is selected from breast cancer, gastric cancer, colorectal 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, rhabdomyosarcoma, and lung cancer.

[0067] This disclosure also provides the use of bispecific antibodies as described in the first aspect of this disclosure, polynucleotides as described in the second aspect of this disclosure, gene expression cassettes as described in the third aspect of this disclosure, gene delivery vectors as described in the fourth aspect of this disclosure, and cells as described in the fifth aspect of this disclosure in the preparation of medicaments for treating tumors.

[0068] In some optional embodiments, the tumor is a tumor involving PD-L1 and / or VEGF.

[0069] In some alternative implementations, the tumor is selected from breast cancer, gastric cancer, colorectal 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, rhabdomyosarcoma, and lung cancer.

[0070] The effects of the invention

[0071] In some embodiments of this disclosure, a bispecific antibody against PD-L1 and VEGF is provided.

[0072] In some embodiments of this disclosure, the bispecific antibodies provided herein can be used for gene therapy, such as AAV or mRNA-mediated gene therapy.

[0073] In some embodiments of this disclosure, the bispecific antibody provided herein can effectively block the PD-1 / PD-L1 and VEGF / VEGFR pathways.

[0074] In some embodiments of this disclosure, the bispecific antibody provided herein can inhibit tumor cell viability in vitro.

[0075] In some embodiments of this disclosure, the bispecific antibody provided herein possesses antitumor activity in vivo. Attached Figure Description

[0076] Figure 1 is a schematic diagram of BiTE-type bsAbs. The antibody design employs a tandem scfv approach, with the specific order as follows:

[0077] BiTE1: VL VEGF -3×(G4S)-VH VEGF -SGGGGS-VL PD-L1 -3×(G4S)-VH PD-L1 -HHHHHH;

[0078] BiTE2:VH VEGF -3×(G4S)-VL VEGF -SGGGGS-VH PD-L1 -3×(G4S)-VL PD-L1 -HHHHHH;

[0079] BiTE3: VH PD-L1 -3×(G4S)-VL PD-L1 -SGGGGS-VH VEGF -3×(G4S)-VL VEGF -HHHHHH;

[0080] BiTE4: VL PD-L1 -3×(G4S)-VH PD-L1 -SGGGGS-VL VEGF -3×(G4S)-VH VEGF -HHHHHH.

[0081] Figure 2 shows a schematic diagram of the SDS-PAGE results of the one-step purified antibody. Loading: Lanes 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.

[0082] Figure 3 shows a schematic diagram of the antibody ELISA results. In Figure 3, A represents human programmed death receptor-ligand 1 (PD-L1), with atezolizumab as a positive control and human IgG as a negative control. In Figure 3, B represents vascular endothelial growth factor 110 (VEGF-110), with bevacizumab as a positive control and human IgG as a negative control. In Figure 3, C represents vascular endothelial growth factor 121 (VEGF-121), with bevacizumab as a positive control and human IgG as a negative control. In Figure 3, D represents vascular endothelial growth factor 165 (VEGF-165), with bevacizumab as a positive control and human IgG as a negative control.

[0083] Figure 4 shows the results of the in vitro PD-L1 / PD-1 blockade experiment, with atezolizumab as a positive control and human IgG as a negative control.

[0084] Figure 5 shows the results of the in vitro VEGF / VEGFR blocking experiment, with bevacizumab as a positive control and human IgG as a negative control.

[0085] Figure 6 shows the results of cell viability testing.

[0086] Figure 7 shows the body weight change curves of glioma patients in vivo, with the AAV-isotype control serving as a negative control.

[0087] Figure 8 shows the tumor volume change curves in glioma patients during the in vivo drug efficacy experiment. The AAV-isotype control is a negative control.

[0088] Figure 9 shows the tumor volume change curve in lung cancer experimental animals, with AAV-isotype control serving as a negative control.

[0089] Figure 10 shows the body weight change curves of lung cancer patients in vivo, with the AAV-isotype control serving as a negative control. Detailed Implementation

[0090] Various exemplary embodiments, features, and aspects of this disclosure will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0091] Furthermore, to better illustrate this disclosure, numerous specific details are set forth in the following detailed description. Those skilled in the art will understand that this disclosure can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the main points of this disclosure.

[0092] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this disclosure should be understood to include systematic errors that are unavoidable in industrial production.

[0093] I. Definition of Terms

[0094] Unless otherwise stated, the following terms have the following meanings in this disclosure.

[0095] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0096] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0097] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0098] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0099] The "antibody" described in this disclosure is an immunoglobulin. A complete antibody is typically a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain can further divide it into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ or λ chain.

[0100] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0101] The term "antibody framework" or "FR region" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0102] The terms “complementarity-determining region,” “CDR,” or “hypervariant region” refer to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitates antigen binding. Typically, three CDRs (HCDR1, HCDR2, HCDR3) exist in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) exist in each light chain variable region. The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (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 rule (see Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering rule (Lefranc). 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 Kabat rules, 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 Chothia rules, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acids in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acids in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acids in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3). The amino acid residues are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the CDR definitions from Kabat and Chothia, the CDR consists of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) from human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) from human VL. Following the IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), while those in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3).Following the IMGT rules, the CDR region of an antibody can be determined using the IMGT / DomainGap Align procedure. Unless otherwise stated, the variable region and CDR sequence of the antibody described in the specific embodiments of this disclosure are subject to the "Kabat" numbering rules.

[0103] The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments that enable the antibody to maintain its specific ability to bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by disulfide bridges on their hinge regions; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains on a single arm of the antibody; (v) dsFv, stable antigen-binding fragments formed by interchain disulfide bonds between VH and VL; and (vi) bispecific, bispecific, and multispecific antibodies containing fragments such as scFv, dsFv, and Fab. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, thereby enabling the production of a single protein chain (called a single-chain Fv (scFv) in which the VL and VH regions pair to form a monovalent molecule; 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 fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0104] The terms “single-chain antibody,” “single-chain Fv,” or “scFv” refer to molecules containing a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example, using variants with 1–4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444–6448). Other connectors that may be used in this 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.

[0105] Biantibodies are antigen-binding fragments in which scFv or Fab is dimerized, exhibiting bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens can be the same or different.

[0106] Bispecific antibodies and multispecific antibodies are antibodies that can bind to two or more antigens or antigenic determinants simultaneously, including scFv or Fab fragments that can bind to antigens.

[0107] The biantibody disclosed herein can be produced by the following steps: obtaining cDNA encoding VH and VL of monoclonal antibodies that specifically recognize and bind to the antigen; constructing DNA encoding scFv such that the amino acid sequence length of the peptide linker is 8 residues or less; inserting the DNA into a prokaryotic or eukaryotic expression vector; and then introducing the expression vector into a prokaryote or eukaryote to express the biantibody.

[0108] The term "epitope" or "antigenic determinant" refers to the site on an antigen where 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-consecutive amino acids. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996).

[0109] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -7 M, for example, approximately less than 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12M Or bind with a lower affinity (KD).

[0110] The term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, antibodies dissociate at a rate less than approximately 10-1. -7 M, for example, less than approximately 10 -8 M or 10 -9 The dissociation equilibrium constant (KD) of M binds to the antigen, for example, determined in a BIACORE instrument using surface plasmon resonance (SPR) technology. The smaller the KD value, the greater the affinity.

[0111] The term "amino acid difference" or "amino acid mutation" refers to an amino acid change or mutation in a variant protein or polypeptide compared to the original protein or polypeptide, including the insertion, deletion, or substitution of one, two, three, or more amino acids.

[0112] "Conservative modification" or "conservative substitution" 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), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will appreciate that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity. Exemplary conserved substitutions are described below.

[0113] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, 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.

[0114] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0115] "Treatment" means administering an oral or topical therapeutic agent, such as a composition comprising any antibody of the present disclosure or an antigen-binding fragment thereof, to a patient who has one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more symptoms of a disease, inducing the regression of such symptoms or inhibiting their progression to any clinically measured extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0116] "Effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired preventive or therapeutic outcome. For preventive use, beneficial or desired outcomes include eliminating or reducing risk, mitigating severity, or delaying the onset of a condition, including the condition, its complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes presented during the development of the condition. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various target antigen-related conditions of this disclosure or improving one or more symptoms of said conditions, reducing the dosage of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-related condition of this disclosure in a patient.

[0117] "Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are 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 of 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 multiplied by 100. For example, in optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences match or are homologous, then the two sequences are 95% homologous. Typically, comparisons are made when aligning two sequences to give the maximum percentage of identity. For example, comparisons can be performed using the BLAST algorithm, where the algorithm's parameters are chosen to give the maximum match between the sequences over the entire length of each reference sequence. The following references relate to the BLAST algorithm commonly used in 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 common BLAST algorithms, such as those provided by NCBI BLAST, are also well-known to those skilled in the art.

[0118] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include progeny. Therefore, the terms “transformation” and “transformed cell” include primary test cells and cultures derived from them, regardless of the number of transfections. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in terms of DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are intended, the context will be clear.

[0119] "Separated" 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 media. Generally, the term "separated" is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compound as described herein.

[0120] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, and the description includes the circumstances under which the event or circumstances may or may not occur.

[0121] "Pharmaceutical composition" refers to a mixture containing one or more of the bispecific antibodies described herein, along with other chemical components, such as physiological / pharmaceutical-grade carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0122] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in formulations for the delivery of bispecific antibodies, polynucleotides, gene expression cassettes, or gene delivery vectors. Carriers can be anti-adhesion agents, adhesives, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delay agents, wetting agents, emulsifiers, buffers, etc. Examples of 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 solutions, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0123] II. Bispecific antibodies

[0124] In some aspects of this disclosure, a bispecific antibody is provided, comprising: targeting the heavy chain variable region (VH) of vascular endothelial growth factor (VEGF). VEGF ) and light chain variable region (VL) VEGF ), and the heavy chain variable region (VH) targeting programmed death receptor-ligand 1 (PD-L1). PD-L1 ) and light chain variable region (VL) PD-L1 ).

[0125] In some implementations, the VH VEGF It includes a sequence as 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 with it.

[0126] In some implementations, the VL VEGF It includes a sequence as 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 with it.

[0127] In some implementations, the VHPD-L1 It includes a sequence as 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 with it.

[0128] In some implementations, the VL PD-L1 It includes a sequence as 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 with it.

[0129] In some implementations, the VH VEGF It includes 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.

[0130] In some implementations, the VL VEGF It includes 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.

[0131] In some implementations, the VH PD-L1 It includes 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.

[0132] In some implementations, the VL PD-L1 It includes 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.

[0133] In some implementations, the VH VEGF VL VEGF VH PD-L1 and VL PD-L1 The connection between any two also includes a joint.

[0134] The term "connector" refers to the link between two molecules or parts, such as a heavy chain variable region and a light chain variable region. Typically, a connector is located between or on the flank of two groups, molecules, or other parts and is covalently bonded to each, thereby connecting them. In some embodiments, the connector is an organic molecule, group, polymer, or chemical part.

[0135] In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker of this disclosure comprises an 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.

[0136] In some specific embodiments, the connector comprises (GGGGS)n, where n is an integer from 1 to 32. In some more specific embodiments, the connector comprises GGGGSGGGGSGGGGS (SEQ ID NO:43).

[0137] In some specific implementations, the connector includes SGGGGS (SEQ ID NO:44).

[0138] In some specific embodiments, the bispecific antibody has the structure shown in any one of (i)-(iv):

[0139] (i) N-terminal-VL VEGF -Optional connector-VH VEGF -Optional connector-VL PD-L1 -Optional connector-VH PD-L1 -C terminal;

[0140] (ii) N-terminus - VH VEGF -Optional connector-VL VEGF -Optional connector-VH PD-L1 -Optional connector-VL PD-L1 -C terminal;

[0141] (iii) N-terminal -VH PD-L1 -Optional connector-VL PD-L1 -Optional connector-VH VEGF -Optional connector-VL VEGF -C terminal;

[0142] (iv) N-terminal-VL PD-L1 -Optional connector-VH PD-L1 -Optional connector-VL VEGF -Optional connector-VH VEGF -C terminal.

[0143] In some preferred embodiments, the bispecific antibody has the structure shown in any one of (i)-(iii):

[0144] (i) N-terminal-VL VEGF -Optional connector-VH VEGF -Optional connector-VL PD-L1 -Optional connector-VH PD-L1 -C terminal;

[0145] (ii) N-terminus - VH VEGF -Optional connector-VL VEGF -Optional connector-VH PD-L1 -Optional connector-VL PD-L1 -C terminal;

[0146] (iii) N-terminal -VH PD-L1 -Optional connector-VL PD-L1 -Optional connector-VH VEGF -Optional connector-VL VEGF -C terminal.

[0147] From a permutation and combination perspective, there are theoretically 16 different combinations of the VH (heavy chain variable region) and VL (light chain variable region) of two antibodies. Through in-depth research and reasoning, we selected four combinations from these 16 possibilities for experimentation. Unexpectedly, the experimental results showed that three of these four bispecific antibodies exhibited superior biological activity.

[0148] In some preferred embodiments, the bispecific antibody has the following structure:

[0149] (i) N-terminal-VL VEGF -Optional connector-VH VEGF -Optional connector-VL PD-L1 -Optional connector-VH PD-L1 -C terminal; or

[0150] (ii) N-terminus - VH VEGF -Optional connector-VL VEGF -Optional connector-VH PD-L1 -Optional connector-VL PD-L1 -C terminal.

[0151] In some implementations, at least one disulfide bond is formed between the heavy chain variable region and the light chain variable region near the N-terminus.

[0152] In some specific implementations, 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.

[0153] In some embodiments of this disclosure, a signal peptide is included at the N-terminus of the bispecific antibody.

[0154] The terms "signal peptide," "signal sequence," or "signal peptide sequence" refer to short peptides that, when fused with a target protein (such as the bispecific antibody disclosed herein), promote the secretion of the target protein expressed by the cell onto the cell membrane or extracellularly. Signal peptides are typically located at the N-terminus of the target protein, and various signal peptides are known to those skilled in the art, such as, but not limited to, the human CD5 signal peptide, erythroglobulin signal sequence, human insulin signal sequence, human interleukin-2 (IL2) signal peptide sequence, albumin signal sequence, etc.

[0155] In some preferred embodiments, the N-terminus of the bispecific antibody contains a human CD5 signal peptide or a human interleukin-2 signal peptide sequence.

[0156] In some specific embodiments, the human CD5 signal peptide comprises an amino acid sequence as shown in SEQ ID NO:17.

[0157] In some specific embodiments, the human interleukin-2 signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO:33.

[0158] In some specific embodiments, the bispecific antibody comprises a sequence as shown in any one of SEQ ID NO:18 to 21, or a sequence having at least 90% identity with the sequence shown in any one of SEQ ID NO: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%.

[0159] In some preferred embodiments, the bispecific antibody comprises a sequence as shown in any one of SEQ ID NO:18-20, or a sequence having at least 90% identity with the sequence shown in any one of SEQ ID NO:18-20, 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%.

[0160] In some preferred embodiments, the bispecific antibody comprises a sequence as shown in SEQ ID NO:18 or 19, or a sequence having at least 90% identity with 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%.

[0161] In some embodiments of this disclosure, the bispecific antibody also includes a tag at its N-terminus and / or C-terminus.

[0162] According to this disclosure, the term "tag" refers to a short peptide that is fused or linked to a target protein (e.g., a bispecific antibody of this disclosure) and thereby facilitates the soluble expression, detection, and / or purification of the recombinant protein. The tag may be fused to or linked to the N-terminus and / or C-terminus of the target protein (optionally via a linker or protease cleavage site). Such tags are well known to those skilled in the art and, for example, 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, priming factor tags, ubiquitin tags, Myc tags, Flag tags, fluorescent protein (e.g., GFP) tags, biotin tags, and avidin tags.

[0163] III. Polynucleotides

[0164] In some aspects of this disclosure, an isolated polynucleotide is provided that encodes the aforementioned bispecific antibody.

[0165] The polynucleotides disclosed herein can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0166] The polynucleotides encoding the bispecific antibodies disclosed herein include: a coding sequence that encodes 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.

[0167] The term "polynucleotide encoding a bispecific antibody" can be a polynucleotide that includes encoding the bispecific antibody, or it can include polynucleotides that also include additional coding and / or non-coding sequences.

[0168] This disclosure also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. This disclosure particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In this disclosure, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine 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%. Furthermore, the polypeptides encoded by the hybridizable polynucleotides have the same biological function and activity as bispecific antibodies.

[0169] In some implementations, the polynucleotide encoding a bispecific antibody is codon-optimized. This type of optimization may require mutations in the nucleotide sequence encoding the bispecific antibody to mimic the codon preferences of the intended host organism or cell that simultaneously encodes the same protein.

[0170] IV. Gene Expression Kit

[0171] In some aspects of this disclosure, a gene expression cassette is provided that contains the polynucleotides provided in this disclosure, namely, polynucleotides encoding bispecific antibodies provided in this disclosure.

[0172] V. Gene Delivery Vectors

[0173] In some aspects of this disclosure, the gene expression cassette is used to deliver a gene (encoding a bispecific antibody) into animal cells, for example, to determine the effect of the gene on cell viability and / or function, to treat cellular diseases, etc. Therefore, in some aspects of this disclosure, a gene delivery vector comprising the gene expression cassette of this disclosure is provided. In some preferred embodiments, the gene delivery vector is used to express a transgene (encoding a bispecific antibody) in mammalian cells.

[0174] The gene delivery vectors disclosed herein encompass any convenient gene delivery vector for delivering polynucleotide sequences into mammalian cells. For example, the vector may comprise a single-stranded or double-stranded nucleic acid, such as single-stranded or double-stranded DNA. For example, the gene delivery vector may be DNA, such as naked DNA, such as plasmids or microcircles. The vector may comprise single-stranded or double-stranded RNA, containing a modified form of RNA. In another instance, the gene delivery vector may be RNA, such as mRNA or modified mRNA.

[0175] As another example, the gene delivery vector can be a viral vector derived from a virus, such as adenovirus, adeno-associated virus (AAV), lentivirus, herpesvirus, alpha virus, or retrovirus, such as Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibberish leukemia virus (GaLV), feline leukemia virus (FLV), foam virus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), or lentivirus. While embodiments covering the use of adeno-associated viruses are described in more detail below, it is expected that those skilled in the art will recognize that similar knowledge and skills in the art can also be applied to non-AAV gene delivery vectors.

[0176] In some embodiments, the gene delivery vector is a recombinant adeno-associated virus (rAAV). In this embodiment, the gene expression cassette has functional AAV inverted terminal repeat (ITR) sequences flanked at the 5' and 3' ends. A “functional AAV ITR sequence” refers to an ITR sequence intended for rescuing, replicating, and packaging AAV viral particles. Therefore, the AAV ITR used in the gene delivery vector of this disclosure does not need to have a wild-type nucleotide sequence and can be altered by nucleotide insertion, deletion, or substitution, or the AAV ITR can be derived from any of several AAV serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. Preferred AAV vectors have all or part of the wild-type Rep and Cap genes deleted, but retain the functional flanked ITR sequences. In certain embodiments, the AAV viral vector is an AAV variant. In some embodiments, the AAV variant is an AAV viral vector containing a variant AAV capsid (or, more specifically, an AAV capsid protein variant).

[0177] In some embodiments, the gene expression cassette is capsidated within 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., and any of these adeno-associated virus serotypes can serve as a gene delivery vector. For example, the AAV capsid can be a wild-type capsid or a natural capsid. However, similar to ITRs, the capsid does not need to have a wild-type nucleotide sequence, but can be altered relative to the wild-type sequence by inserting, deleting, or substituting nucleotides in the VP1, VP2, or VP3 sequences, as long as the capsid is capable of transducing mammalian cells. In other words, the AAV capsid can be a variant AAV capsid comprising one or more amino acid substitutions, deletions, or insertions relative to the derived parental capsid protein or AAV capsid protein.

[0178] 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, omitting the N-terminal amino acids of the VP1 protein. By convention, the capsid protein modification sites are named according to the amino acid sequence of the VP1 protein.

[0179] AAV capsid protein variants can be AAV capsid protein variants containing targeting peptides that target specific tissues.

[0180] Preferably, rAAV is replication-defective because AAV vectors cannot independently replicate and package their genome further.

[0181] Gene delivery vectors (e.g., rAAV viral particles) that capsidate the gene expression cassettes of this disclosure can be generated using standard methods. For example, in the case of rAAV viral particles, the AAV expression vector according to this disclosure can be introduced into production cells, followed by the introduction of an AAV helper construct, wherein the helper construct contains an AAV coding region capable of expression in the production cells and said AAV coding region supplements AAV helper functions not present in the AAV vector. A helper virus and / or additional vectors are then introduced into the production cells, wherein the helper virus and / or additional vectors provide helper functions capable of supporting efficient rAAV viral production. The production cells are then cultured to produce rAAV. These steps are performed using standard methods. Replication-deficient AAV viral particles capsidating the recombinant AAV vectors of this disclosure are prepared using AAV packaging cells and packaging techniques known in the art.

[0182] Viral particles of any concentration suitable for effective transduction into mammalian cells can be prepared for contact with mammalian cells in vitro or in vivo. Similarly, any total quantity of viral particles suitable for providing appropriate cellular transduction to confer the desired effect or treat disease can be administered to mammals. Any suitable quantity of the vector can be administered to the eyes of mammals or primates.

[0183] A viral vector can be formulated into a pharmaceutical composition comprising any suitable unit dose of the vector, and the pharmaceutical composition can be administered to a subject to produce changes in the subject or to treat the subject's disease.

[0184] In some cases, the multiple of infection (MOI) can be used to measure the unit dose of a drug composition. MOI refers to the ratio or multiple of the vector or viral genome to the nucleic acid that can be delivered to cells.

[0185] In preparing the rAAV composition, any host cell used to produce 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 and cap genes are stably maintained within 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. The AAV vector is purified and formulated using standard techniques known in the art.

[0186] In some preferred embodiments, the gene delivery vector comprises a sequence as shown in any one of SEQ ID NO:34-36, or a sequence having at least 90% identity with a sequence shown in any one of SEQ ID NO:34-36.

[0187] VI. Cells

[0188] In some aspects, this disclosure provides a cell comprising one or more of the following:

[0189] (a) The aforementioned bispecific antibodies;

[0190] (b) the aforementioned polynucleotides;

[0191] (c) the gene expression cassettes described above; and,

[0192] (d) The gene delivery vectors described above.

[0193] The cell can be any of a variety of cells, including, for example, in vitro cells, in vivo cells, isolated cells, primary cells, cancer cells, animal cells, plant cells, algal cells, fungal cells, etc.

[0194] In some implementations, non-limiting examples of cells include: prokaryotic cells, eukaryotic cells, bacterial cells, archaea cells, cells of unicellular eukaryotes, protozoan cells, cells derived from plants, algae cells, fungal cells, animal cells, cells derived from invertebrates, cells derived from vertebrates, and cells derived from mammals (e.g., ungulates; rodents; non-human primates; humans; felines; dogs, etc.). In some cases, the cell is not derived from a natural organism (e.g., the cell may be a synthetic cell; also known as an artificial cell).

[0195] VII. Pharmaceutical Compositions

[0196] In some aspects of this disclosure, a pharmaceutical composition is provided comprising a bispecific antibody, a polynucleotide, a gene expression cassette, or a gene delivery vector provided in this disclosure, and optionally, a pharmaceutically acceptable vector.

[0197] In some embodiments, the pharmaceutical compositions described herein contain the above-described bispecific antibody, polynucleotide, gene expression cassette, or gene delivery vector as active ingredients.

[0198] The individual dose is generally not less than the amount required to produce a measurable effect in the subject and can be determined based on the pharmacokinetics and pharmacology of the absorption, distribution, metabolism, and excretion (“ADME”) of the pharmaceutical composition or its byproducts, and therefore on the disposal of the composition within the subject. This includes consideration of the route of administration and dosage. Effective doses and / or dosing 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.

[0199] As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvent, dispersion medium, coating, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents that are compatible with drug administration.

[0200] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, intravenous, intra-arterial, subcutaneous, intraperitoneal, intrathecal, intramuscular, or injection or infusion administration. Thus, delivery can be systemic or local.

[0201] Methods for preparing 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).

[0202] VIII. Applications and Methods

[0203] In some aspects of this disclosure, the bispecific antibodies, polynucleotides, gene expression cassettes, gene delivery vectors, cells, or pharmaceutical compositions provided herein are applied to:

[0204] Combining PD-L1 and human VEGF; or

[0205] Blocking the PD-L1 / PD-1 pathway and / or the VEGF / VEGFR pathway; or

[0206] Inhibit tumor cell growth,

[0207] Or it may be used to prepare a drug for any of the above uses.

[0208] In some aspects of this disclosure, this disclosure provides methods for treating or preventing a disease (e.g., tumor) in a subject of need, said methods comprising administering to the subject of need an effective amount of a bispecific antibody, polynucleotide, gene expression cassette, gene delivery vector, cell, or pharmaceutical composition of this disclosure.

[0209] In other aspects of this disclosure, the use of the bispecific antibodies, polynucleotides, gene expression cassettes, gene delivery vectors, or cells of this disclosure in the preparation of medicaments for treating diseases (e.g., tumors) is also provided.

[0210] In other aspects of this disclosure, bispecific antibodies, polynucleotides, gene expression cassettes, gene delivery vectors, cells, or pharmaceutical compositions thereof are provided for the treatment of diseases (e.g., tumors).

[0211] In some specific implementations, the tumor can be a variety of tumors involving PD-L1 and / or VEGF, whether malignant or benign, and whether primary or secondary. These tumors can be solid carcinomas or hematologic malignancies.

[0212] In some specific implementation schemes, the tumor is selected from breast cancer, gastric cancer, colorectal 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, rhabdomyosarcoma, and lung cancer.

[0213] Preferably, the neuroepithelial tumor is selected from astrocytoma, anaplastic astrocytoma, glioblastoma, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, oligodendroglioma, ependymal cell tumor, mixed glioma, choroid plexus tumor, pineal cell tumor, embryonal tumor, with glioblastoma being the most preferred.

[0214] Preferably, the lung cancer includes non-small cell lung cancer.

[0215] Example

[0216] The embodiments of this disclosure will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this disclosure. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0217] Example 1: Antibody Design and Plasmid Vector Construction

[0218] This disclosure provides a bispecific antibody containing binding domains specific to PD-L1 and VEGF, intended for use in AAV or mRNA-mediated gene therapy. Due to the limited capacity of AAV vectors (less than 4.7 kb) and the need to minimize the immunogenicity of antibody drugs, this disclosure constructs BiTE-type bsAbs using tandem single-chain variable domain (scFv) fragments. Theoretically, there are 16 different combinations of the VH (heavy chain variable domain) and VL (light chain variable domain) of the two antibodies. Through in-depth research and reasoning, we selected four combinations from these 16 possibilities for experimental testing. In the bispecific antibody construct, the heavy chain variable domain (VH) and light chain variable domain (VL) are arranged from the N-terminus to the C-terminus in the following order:

[0219] BiTE1: VL VEGF -3×(G4S)-VH VEGF -SGGGGS-VLPD-L1 -3×(G4S)-VH PD-L1 -HHHHHH

[0220] BiTE2:VH VEGF -3×(G4S)-VL VEGF -SGGGGS-VH PD-L1 -3×(G4S)-VL PD-L1 -HHHHHH

[0221] BiTE3: VH PD-L1 -3×(G4S)-VL PD-L1 -SGGGGS-VH VEGF -3×(G4S)-VL VEGF -HHHHHH

[0222] BiTE4: VL PD-L1 -3×(G4S)-VH PD-L1 -SGGGGS-VL VEGF -3×(G4S)-VH VEGF -HHHHHH

[0223] The tandem sequence of BiTE-type antibodies is shown in Figure 1. Additional disulfide bonds are expressed on the N-terminus 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).

[0224] The vector construction was commissioned to Jiangsu GenScript Biotech Co., Ltd.

[0225] The specific sequence of the antibody expressed and purified in vitro is as follows:

[0226] BiTE1 (SEQ ID NO:22):

[0227] In this diagram, a single underscore indicates a signal peptide sequence, while a double underscore indicates a tag sequence.

[0228] BiTE2 (SEQ ID NO:23):

[0229] In this diagram, a single underscore indicates a signal peptide sequence, while a double underscore indicates a tag sequence.

[0230] BiTE3 (SEQ ID NO:24):

[0231] In this sequence, a single underscore indicates the signal peptide sequence; double underscores indicate the tag sequence. BiTE4 (SEQ ID NO:25):

[0232] In this diagram, a single underscore indicates a signal peptide sequence, while a double underscore indicates a tag sequence.

[0233] The sequence of the bispecific antibody is as follows:

[0234] BiTE1 bispecific antibody (SEQ ID NO:18):

[0235] BiTE2 bispecific antibody (SEQ ID NO:19):

[0236] BiTE3 bispecific antibody (SEQ ID NO:20):

[0237] BiTE4 bispecific antibody (SEQ ID NO:21):

[0238] Among the aforementioned bispecific antibodies, those targeting the heavy chain variable region of VEGF (anti-VEGF VH region, or VH for short) VEGF ) and light chain variable region (anti-VEGF VL region, abbreviated as VL) VEGF The sequences of HCDR1-3 and LCDR1-3 (CDR is determined to use the Kabat definition scheme) are as follows:

[0239] anti-VEGF VH region (SEQ ID NO:1)

[0240] anti-VEGF VL region (SEQ ID NO:2)

[0241] HCDR1 (SEQ ID NO:5): NYGMN

[0242] HCDR2(SEQ ID NO:6):WINTYTGEPTYAADFKR

[0243] HCDR3(SEQ ID NO:7):YPHYYGSSHWYFDV

[0244] LCDR1(SEQ ID NO:8):SASQDISNYLN

[0245] LCDR2 (SEQ ID NO:9): FTSSLHS

[0246] LCDR3(SEQ ID NO:10):QQYSTVPWT

[0247] Among the aforementioned bispecific antibodies, the heavy chain variable region targeting PD-L1 (anti-PD-L1 VH region, abbreviated as VH) is targeted. PD-L1 ) and light chain variable region (anti-PD-L1 VL region, abbreviated as VL) PD-L1 The sequences of HCDR1-3 and LCDR1-3 (CDR is determined to use the Kabat definition scheme) are as follows:

[0248] anti-PD-L1VH region (SEQ ID NO:3)

[0249] anti-PD-L1VL region (SEQ ID NO:4)

[0250] HCDR1 (SEQ ID NO:11): DSWIH

[0251] HCDR2(SEQ ID NO:12):WISPYGGSTYYADSVKG

[0252] HCDR3(SEQ ID NO:13):RHWPGGFDY

[0253] LCDR1(SEQ ID NO:14):RASQDVSTAVA

[0254] LCDR2 (SEQ ID NO:15): SASFLYS

[0255] LCDR3(SEQ ID NO:16):QQYLYHPAT

[0256] In the above-mentioned bispecific antibodies, the human CD5 signal peptide sequence (SEQ ID NO:17) is:

[0257] Target antigen sequence:

[0258] PD-L1 (SEQ ID NO:26):

[0259] VEGF-110 (SEQ ID NO:27):

[0260] VEGF-121 (SEQ ID NO:28):

[0261] VEGF-165 (SEQ ID NO:29):

[0262] Example 2: Antibody Expression and Target Protein Binding Ability Assay (ELISA)

[0263] Antibody Expression and Purification: 24 hours before transfection, ExpiCHO-S cells (Gibco) were cultured normally on ExpiCHO expression medium (Gibco) and then cultured in a shaker. For transfection, the plasmid prepared in Example 1 was mixed with the transfection reagent (ExpiFectamine CHO Reagent, Gibco) and incubated at room temperature to obtain the complex. The complex was added to ExpiCHO-S cells, and the shake flask was returned to the shaker for further culture. On day 1 of transfection, the enhancer (ExpiFectamine CHO Enhancer, Gibco) and feed (ExpiCHO Feed, Gibco) were added to the cells, and the cells were then placed in the shaker for further culture. On day 5 of transfection, the feed was added to the cells, and the cells were then placed in the shaker for further culture. The supernatant was collected by centrifugation. The purified protein was obtained using a His affinity column (Genscript).

[0264] Enzyme-linked immunosorbent assay (ELISA): To assess the ability of BiTE to bind PD-L1 in vitro, Atezolizumab (Pengbo Biotechnology, RD230509144) was used as a positive control in the ELISA experiment. To assess the ability of BiTE to bind VEGF-110, VEGF-121, and VEGF-165 in vitro, Bevacizumab (Pengbo Biotechnology, RD230203109) was used as a positive control in the ELISA experiment. The specific steps included:

[0265] a. Dilute human PD-L1 (Bepsys, PD1-H5229), human VEGF-110 (Bepsys, VE0-H52H3), human VEGF-121 (Bepsys, VE1-H5246), and human VEGF-145 (Bepsys, VE5-H5248) proteins to appropriate concentrations using CBS buffer (Genscript), and coat 96-well plates with 100 μL / well. Incubate overnight at 4°C.

[0266] b. Discard the liquid in the 96-well plate, add 300 μL of PBST to each well, wash 3 times, and finally blot the remaining liquid in the 96-well plate with absorbent paper.

[0267] c. Add the prepared 5% skim milk powder solution to the 96-well plate at a rate of 200 μL / well and incubate at 37°C for 2 hours.

[0268] d. Repeat step b.

[0269] e. Dilute the test sample and positive control with PBS to appropriate conditions. Add 100 μL / well to each well of a 96-well plate. Add 100 μL of PBS to each blank control well. Repeat the test twice. Incubate at room temperature for 2 hours.

[0270] f. Repeat step b.

[0271] g. anti-His-HRP (Genscript) solution was prepared with PBS at a 1:X ratio. 100 μL was added to each well of a 96-well plate. The plate was incubated at room temperature for 1 hour.

[0272] h. Repeat step b.

[0273] i. Add 100 μL of TMB solution (Genscript) to each well of a 96-well plate and incubate at room temperature for 15 minutes, protected from light.

[0274] j. Add 50 μL of ELISA stop solution to each well of a 96-well plate. Read the OD value at 450 nm using a microplate reader.

[0275] Antibody expression, purification, and ELISA experiments were outsourced to Jiangsu GenScript Biotech Co., Ltd.

[0276] Experimental results: Effective expression of PD-L1 / VEGF antibody (ELISA)

[0277] SDS-PAGE results showed that the target antibody band position was consistent with expectations (Figure 2). Using ELISA results, the median effective concentration (EC50) of the purified antibody was calculated, showing that the purified antibody could bind to both PD-L1 and VEGF proteins (as shown in Table 1 and Figure 3).

[0278] Table 1. ELISA assay for the ability of BiTE to bind PD-L1 / VEGF (EC50) (unit: μg / mL)

[0279] Example 3: Surface Plasmon Resonance (SPR) Detection

[0280] SPR experiment:

[0281] a. Antibody fixation was performed at 25°C, using HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% P2O, pH 7.4) as the running buffer.

[0282] b. Activate the sensor chips of flow units 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).

[0283] 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, the remaining active coupling sites on the chip surface were blocked with 1M ethanolamine.

[0284] d. For single-target detection, each antigen was diluted and injected into flow cell 1 and flow cell 2 for binding, followed by HBS-EP+ injection for dissociation. For dual-target detection, a fixed concentration of human PD-L1 was injected first (first injection), followed by human VEGF-110 (second injection), and then HBS-EP+ injection for dissociation. All data were processed using Biacore 8K evaluation software version 4.0.

[0285] SPR testing was commissioned to Jiangsu GenScript Biotech Co., Ltd.

[0286] Experimental results: Effective expression of PD-L1 / VEGF antibody (SPR)

[0287] Bispecific antibodies possess the structural characteristics of fusion proteins, where the two antigen-binding domains may influence each other's binding to the target antigen. However, unexpectedly, the single-target SPR (as shown in Table 2) and dual-target SPR results in this embodiment show that BiTE1, BiTE2, and BiTE3 can all bind to both PD-L1 and VEGF proteins simultaneously.

[0288] Table 2. SPR results of BiTE antibody (single target)

[0289] In this specification, "E" is generally used to represent "exponent," that is, a power of 10. For example, 2.48E-02 represents 2.48 × 10⁻⁶. -2 6.94E+04 represents 6.94 × 10⁻⁴. 4 .

[0290] Table 3. SPR results of BiTE antibody (dual target)

[0291] In Tables 2 and 3:

[0292] Chi 2 (RU2) represents: Chi-squared test, coefficient of determination (R-squared, R²). 2 )

[0293] ka(1 / Ms) represents: association constant per mole per second (Ms) -1 s -1 )

[0294] kd(1 / s) represents the dissociation rate constant in seconds. - 1)

[0295] KD(M) represents the dissociation constant, where "M" represents the molar concentration.

[0296] Rmax(RU) represents the response unit of the maximum bonding capacity on the chip surface.

[0297] Example 4: In vitro blocking experiment

[0298] PD-L1 / PD-1 in vitro blockade experiment: resuscitation of Jurkat-PD-1-NFAT-Luc (Pengbo Biotechnology, RD00871) reporter gene cell line and aAPC CHO-PDL1 cells (Pengbo Biotechnology, RD00703) were continuously subcultured to the logarithmic growth phase and seeded at 2 × 10⁶ cells per well in 96-well plates. 4 One effector cell, Jurkat-PD-1-NFAT-Luc, was added to the target cells at a 1:1 ratio. aAPC CHO-PDL1. Add serially diluted detection antibody (positive control: Atezolizumab) to the corresponding wells, performing 5-fold serial dilutions for 8 consecutive wells, resulting in 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 co-culturing for 18 hours, add 25 μL of luciferase detection reagent working solution (Nanjing Novizan) to each well and use a microplate reader to detect the luciferase activity in the wells.

[0299] VEGF / VEGFR in vitro blockade experiment: Cells expressing VEGFR-NFAT-Luc (Pengbo Biotechnology, RD00704) were injected one day in advance with 1×10 6100 μL of the assay antibody (positive control: Bevacizumab) was seeded at a density of 1 cell / mL in 96-well plates and incubated overnight at 37°C. Serially diluted detection antibody (positive control: Bevacizumab) was added to the corresponding wells, with eight consecutive 3-fold dilutions resulting in 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 working solution (Nanjing Novizan) was added to each well, and the luciferase activity was measured using a microplate reader.

[0300] Experimental results: BiTE double antibody effectively blocks the PD-1 / PD-L1 and VEGF / VEGFR pathways in vitro.

[0301] In vitro blocking assays showed that Atezolizumab, as a positive control antibody for PD-L1, had an EC50 of 0.07193 μg / mL. BiTE1, BiTE2, and BiTE3 could block the binding of PD-L1 to PD-1. The EC50 values ​​of BiTE1, BiTE2, and BiTE3 were 2.112 μg / mL, 1.537 μg / mL, and 0.2494 μg / mL (Figure 4). Bevacizumab, as a positive control antibody, could block the binding of VEGF-165 to its receptor VEGFR2, with an IC50 of 0.2699 μg / mL. BiTE1, BiTE2, and BiTE3 could all block the binding of VEGF-165 to its receptor VEGFR2. The IC50 value of BiTE1 was 0.3167 μg / mL, and that of BiTE2 was 0.9284 μg / mL. The IC50 value of the BiTE3 dose-response curve was 1.001 μg / ml (Figure 5). The negative control antibody (human IgG) in the above experiment had no blocking effect.

[0302] Example 5: In vitro pharmacodynamic experiment

[0303] U87 cells (human glioblastoma cell line) (Wuhan Pronosai) were cultured to the third generation using MEM complete medium (Wuhan Pronosai). 10,000 cells per well were seeded into 96-well plates. After 24 hours of culture, BiTE1 protein (Pengbo Biotechnology) was added at final concentrations of 0, 6.25, 12.5, 25, 50, and 100 μg / mL. The cells were cultured for another 96 hours. 10 μL of CCK8 solution (Japan Tongjin) was added to each well. After 1 hour of culture, the absorbance was measured at 450 nm using a SynergyH1 microplate reader.

[0304] Experimental results: The BITE1 double antibody showed a concentration-dependent trend in inhibiting the viability of U87 cells.

[0305] The CCK8 experiment results showed that after culturing U87 cells in complete culture media containing 0, 6.25, 12.5, 25, 50, and 100 μg / mL BiTE1 protein for 96 hours, the cell viability was 100%, 95%, 93%, 79%, 68%, and 53%, respectively. These results indicate that the BITE1 double antibody in vitro inhibits U87 cell viability in a concentration-dependent manner (Figure 6).

[0306] Experiment Example 6: AAV Packaging

[0307] AAV virus packaging was performed using a common three-plasmid transfection method. HEK293 cells were co-transfected with the AAV2 capsid gene plasmid (Shandong Weizhen), the Helper plasmid (Shandong Weizhen), and shuttle plasmids (BITE1-AAV, BITE2-AAV, BITE3-AAV, and the isotype control AVV plasmid, with specific sequences shown below). AAV virus particles were purified from HEK293T cells using a gradient of iodixanol concentrations. AAV titers were determined using a common qPCR method. Control antibodies Isotype and BITE1 were packaged as AAV-isotype control (Isotype) and AAV-BITE1, respectively. AAV virus packaging and titer determination were performed by Shandong Weizhen Biotechnology Co., Ltd.

[0308] The specific amino acid sequence of the antibody expressed by the shuttle plasmid is as follows:

[0309] BiTE1 bispecific antibody (SEQ ID NO:30):

[0310] In this context, a single underscore indicates a signal peptide sequence.

[0311] BiTE2 bispecific antibody (SEQ ID NO:31):

[0312] In this context, a single underscore indicates a signal peptide sequence.

[0313] BiTE3 bispecific antibody (SEQ ID NO:32):

[0314] In this context, a single underscore indicates a signal peptide sequence.

[0315] Human interleukin-2 signal peptide sequence (SEQ ID NO:33):

[0316] BITE1-AAV plasmid (SEQ ID NO:34):

[0317] In this context, double underscores represent signal peptide coding sequences, while single underscores represent bispecific antibody coding sequences.

[0318] BITE2-AAV plasmid (SEQ ID NO:35):

[0319] In this context, double underscores represent signal peptide coding sequences, while single underscores represent bispecific antibody coding sequences.

[0320] BITE3-AAV plasmid (SEQ ID NO:36):

[0321] In this diagram, double underscores represent signal peptide coding sequences, while single underscores represent bispecific antibody coding sequences.

[0322] Isotype-AAV plasmid (SEQ ID NO:37):

[0323] In this diagram, double underscores represent signal peptide coding sequences, while single underscores represent bispecific antibody coding sequences.

[0324] Experimental Example 7: In vivo pharmacodynamic study in the U87 subcutaneous xenograft tumor model

[0325] This embodiment used 6-8 week old female NOG mice (Beijing Vital River Laboratory 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 (FBS) and 1% penicillin-dextrose antibody. Cells were passaged twice a week using trypsin (Gibco). Cells were collected, counted, and seeded when the required volume was reached. Grouping information is shown in Table 4. On the day of tumor cell seeding, mice were randomly grouped according to body weight, and U87 cells were collected and resuspended in EMEM medium without FBS. The test substance and cell suspension were mixed according to the table below. 5E6 U87 cells and the test substance suspension were subcutaneously seeded above the right scapula of the mouse (Day 0). Tumor volume was then monitored using calipers. When the tumor volume reached 70 mm², the tumor was seeded. 3At that time, mice in the AAV-Isotype group and the AAV-BiTE1 group were injected via tail vein with 0.1 mL of a solution containing 3.5 × 10⁻⁶ mg / L of iodine. 6 Mice were administered DPBS to PBMC (SaiLi, XW0801120W) cells while simultaneously receiving Bevacizumab (twice weekly). Mice were euthanized on day 36. This experiment was conducted by Nantong WuXi AppTec Pharmaceutical Technology Co., Ltd.

[0326] Table 4. Grouping information for in vivo pharmacodynamic experiments

[0327] Experimental results

[0328] This embodiment evaluated the antitumor 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 antitumor activity, while there were no statistically significant differences in other groups.

[0329] During the experiment, the overall weight of mice in each group maintained an upward trend (as shown in Figure 7), and no abnormal weight was observed, indicating that the mice tolerated the test drug well.

[0330] Starting with tumor cell inoculation, 36 days post-inoculation, the average tumor volume in the isotype control group (AAV-Isotype) tumor-bearing mice reached 3043 mm. 3 Compared with the isotype control group, the Bevacizumab group did not show significant tumor-suppressive activity, 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-suppressive effect, with a tumor volume of 1,334 mm. 3 (TGI = 56%, p = 0.005) (as shown in Table 5 and Figure 8).

[0331] Table 5 Results of in vivo pharmacodynamic experiments

[0332] Experimental Example 8: In vivo pharmacodynamic study of a subcutaneous xenograft tumor model of human non-small cell lung cancer NCI-H358 cells.

[0333] This embodiment tested the antitumor effect of AAV-BiTE1 in a subcutaneous xenograft model of human non-small cell lung cancer (NSCLC) NCI-H358 cells (Cyagen Biosciences Co., Ltd.) in female PBMC-NKG mice (Cyagen Biosciences Co., Ltd.). NCI-H358 cells were cultured in vitro at 37°C and 5% CO2 using 1640 medium (Gibco) containing 10% fetal bovine serum (FBS) and 1% penicillin-dextrose antibody. The cells were passaged twice weekly using routine digestion with trypsin (Gibco). One day before tumor inoculation, each animal received a tail vein injection of 0.1 mL (5E6) of PBMC cells. Once the required NCI-H358 cell volume was reached, the cells were collected and grouped according to mouse weight. 5E6 NCI-H358 cells (containing matrix gel) were subcutaneously inoculated into the right anterior dorsal region of the mice. The average tumor volume reached approximately 100-150 mm². 3 On day 3 (D3), mice in groups G2, G3, G4, and G6 were administered the drug as planned. Tumor volume was then monitored using calipers. This experiment was conducted by Cyagen (Suzhou) Biotechnology Co., Ltd.

[0334] Table 6 Grouping Information

[0335] The solvent used was phosphate buffer saline (PBS) (Gibco).

[0336] Experimental results:

[0337] This embodiment evaluated the antitumor efficacy of the test substance in a PBMC-NKG mouse NCI-H358 cell subcutaneous xenograft tumor model. The results showed that the AAV-BITE1 group exhibited significant antitumor activity (Table 7, Figure 9).

[0338] At the experimental endpoint, the average tumor volume in the G1AAV-Isotype group was 671 mm. 3 The average tumor volume in the G3 AAV-BiTE1 group was 79 mm. 3 The TGI was 88%. The mean tumor volume in the G6 Vehicle group was 1011 mm. 3 The average tumor volume in the G2 Atezolizumab group was 391 mm. 3 The TGI was 61%; the mean tumor volume in the G4 bevacizumab group was 458 mm. 3 The TGI was 55%; the mean tumor volume in the G5 group treated with a combination of Atezolizumab and Bevacizumab was 283 mm. 3The TGI was 72%. Compared with the G6 Vehicle group, the tumor volume of the G2, G4, and G5 groups was significantly reduced (p<0.05), and the combination of Atezolizumab and Bevacizumab in the G5 group may have a better effect on controlling tumor growth than the single drug; compared with the G1 group, the tumor volume of the G3 AAV-BiTE1 group was significantly reduced (p<0.0001), indicating that AAV-BiTE1 has a good effect on controlling tumor growth in the NCI-H358 tumor model. Throughout the experiment, the tumor-bearing mice tolerated the test drugs AAV-BiTE1, Bevacizumab, Atezolizumab, and the combination of Bevacizumab well overall (Figure 10).

[0339] Table 7 Experimental Results

[0340] It should be noted that although the technical solutions of this disclosure have been described with specific examples, those skilled in the art will understand that this disclosure should not be limited thereto.

[0341] The various embodiments of this disclosure have been described above. These descriptions are exemplary and not exhaustive, nor are they 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 chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A bispecific antibody, wherein, The bispecific antibody includes a target for the heavy chain variable region (VH) of vascular endothelial growth factor (VEGF). VEGF ) and light chain variable region (VL) VEGF ), and the heavy chain variable region (VH) targeting programmed death receptor-ligand 1 (PD-L1). PD-L1 ) and light chain variable region (VL) PD-L1 );in, The VH VEGF Contains a sequence as shown in SEQ ID NO:1, or a sequence having at least 90% identity with it; The VL VEGF Contains a sequence as shown in SEQ ID NO:2, or a sequence having at least 90% identity with it; The VH PD-L1 Contains a sequence as shown in SEQ ID NO:3, or a sequence having at least 90% identity with it; The VL PD-L1 It includes a sequence as shown in SEQ ID NO:4, or a sequence that is at least 90% identical to it.

2. The bispecific antibody according to claim 1, wherein, The VH VEGF Includes 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 It includes 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 Includes 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 includes 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 The connection between any two also includes a joint.

4. The bispecific antibody according to any one of claims 1-3, wherein, The bispecific antibody has any one of the following structures (i)-(iii): (i) N-terminal-VL VEGF -Optional connector-VH VEGF -Optional connector-VL PD-L1 -Optional connector-VH PD-L1 -C terminal; (ii) N-terminus - VH VEGF -Optional connector-VL VEGF -Optional connector-VH PD-L1 -Optional connector-VL PD-L1 -C terminal; (iii) N-terminal -VH PD-L1 -Optional connector-VL PD-L1 -Optional connector-VH VEGF -Optional connector-VL VEGF -C terminal; Preferably, the bispecific antibody comprises a sequence as shown in any one of SEQ ID NO:18 to 20, or a sequence having at least 90% identity with the sequence shown in any one of SEQ ID NO:18 to 20.

5. The bispecific antibody according to any one of claims 1-4, 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 1-6, wherein, The N-terminus of the bispecific antibody also contains a signal peptide.

8. The bispecific antibody according to any one of claims 1-7, wherein, The bispecific antibody comprises a sequence as shown in any one of SEQ ID NO:22-24, 30-32, or a sequence having at least 90% identity with a sequence shown in any one of SEQ ID NO:22-24, 30-32.

9. An isolated polynucleotide encoding a bispecific antibody as described in any one of claims 1-8.

10. A gene expression cassette comprising the polynucleotide as described in claim 9.

11. A gene delivery vector comprising the gene expression cassette as described in 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 NO:34-36, or a sequence having at least 90% identity with the sequence shown in any one of SEQ ID NO:34-36.

15. A cell comprising a bispecific antibody as claimed in any one of claims 1-8, a polynucleotide as claimed in claim 9, a gene expression cassette as claimed in claim 10, or a gene delivery vector as claimed in any one of claims 11-14.

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

17. A method for treating or preventing tumors in a subject in need, the method comprising administering to the subject in need an effective amount of a bispecific antibody as claimed in any one of claims 1-8, a polynucleotide as claimed in claim 9, a gene expression cassette as claimed in claim 10, a gene delivery vector as claimed in any one of claims 11-14, a cell as claimed in claim 15, or a pharmaceutical composition as claimed in claim 16. Optionally, the tumor is a tumor involving PD-L1 and / or VEGF; Optionally, the tumor may be selected from breast cancer, gastric cancer, colorectal 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, rhabdomyosarcoma, and lung cancer.

18. The bispecific antibody as described in any one of claims 1-8, the polynucleotide as described in claim 9, the gene expression cassette as described in claim 10, the gene delivery vector as described in any one of claims 11-14, the cell as described in claim 15, or the pharmaceutical composition as described in claim 16, for the treatment of tumors; Optionally, the tumor is a tumor involving PD-L1 and / or VEGF; Optionally, the tumor may be selected from breast cancer, gastric cancer, colorectal 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, rhabdomyosarcoma, and lung cancer.

19. The use of the bispecific antibody as described in any one of claims 1-8, the polynucleotide as described in claim 9, the gene expression cassette as described in claim 10, the gene delivery vector as described in any one of claims 11-14, and the cell as described in claim 15 in the preparation of a medicament for treating tumors; Optionally, the tumor is a tumor involving PD-L1 and / or VEGF; Optionally, the tumor may be selected from breast cancer, gastric cancer, colorectal 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, rhabdomyosarcoma, and lung cancer.

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