Anti-PD-L1 and VEGF bispecific antibodies and their applications

Abstract

The present invention relates to a bispecific antibody targeting PD-L1 and VEGF and a preparation method thereof, and also relates to a pharmaceutical composition comprising the bispecific antibody and its use in treatment and diagnosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a bispecific antibody targeting PD-L1 and VEGF, and a preparation method and application thereof. Background Art

[0002] PD-L1 is a type of transmembrane protein on the cell membrane, expressed on immune cells such as T cells and B cells, as well as tumor cells. Tasuku Honjo et al. discovered and proved that when PD-L1 on the tumor cell membrane binds to PD-1 on immune cells such as T cells, the tumor cells send out inhibitory signals, and then T cells cannot recognize tumor cells and kill them, and the body's immune function is suppressed (Chamoto, K., Al-Habsi, M., & Honjo, T. (2017). Current topics in microbiology and immunology, 410, 75-97). Chen Lieping et al. first cured about 60% of head and neck cancer mice by combining PD-L1 blocking antibodies with T cells (Chen, L et al. (2003). Cancer research, 63 (19), 6501-6505.). Currently, 10 anti-PD-1 or PD-L1 monoclonal antibodies have been approved for marketing in China, with indications targeting various solid tumors such as PD-L1-positive NSCLC, head and neck squamous cell carcinoma, and melanoma (Radvanyi, et al, P. (2013). letter. Clinical cancer research, 19 (19), 5541). However, pathological detection indicators such as PD-L1 expression level and tumor mutation burden (TMB) indicate that the response rate of anti-PD-1 / PD-L1 antibodies is low. Currently, they are only effective for less than 40% of solid tumors, and 30% of patients develop a certain degree of drug resistance after taking the drug.

[0003] The most critical cytokine that promotes angiogenesis is VEGF-A (also known as VEGF). VEGF activates VEGFR2 (the main receptor tyrosine kinase receptor that mediates angiogenesis) to promote endothelial cell mitosis and increase vascular permeability, thereby promoting the sprouting of new blood vessels. At the same time, the high expression of VEGF in the tumor microenvironment can amplify the immunosuppressive effect of PD-(L)1. Therefore, targeting VEGF or VEGFR2 can effectively inhibit abnormal angiogenesis (Ferrara, N. (2010). Mol Biol Cell 21 (5): 687-690.). The anti-VEGF monoclonal antibody bevacizumab has good efficacy and target safety in tumor treatment and eye diseases related to abnormal vascular proliferation (Pfisterer, J., et al. (2020). Lancet Oncol 21(5):699-709. Bhandari, S., et al. (2020). Ophthalmology 127(5):608-615.).

[0004] In addition to its anti-angiogenic effect, anti-VEGF monoclonal antibodies can improve blood vessels, promote T cell infiltration into tumors, inhibit DC maturation, promote T cell priming and activation, improve tumor microenvironment, increase the response rate of anti-PD-1 / PD-L1 drugs, and further restore tumor immune function through T cell-mediated tumor killing (Socinski, MA, et al. (2018). N Engl J Med 378 (24): 2288-2301. N Engl J Med. 2018 Jun 14; 378 (24): 2288-2301. N Engl J Med 2020; 382: 1894-1905). The latest clinical trial results have confirmed that in the treatment of tumors such as liver cancer, lung cancer, endometrial cancer and renal cancer, anti-VEGF monoclonal antibodies combined with anti-PD-L1 monoclonal antibodies have shown superior efficacy than chemical drugs or anti-VEGF monoclonal antibodies or anti-PD-L1 monoclonal antibodies, achieving a very significant clinical breakthrough. Currently, both the anti-PD-L1 / VEGF monoclonal antibody combination and the anti-PD-1 / VEGF bispecific antibody have progressed to Phase III clinical trials, with treatment-related adverse reactions of 91% and 63.4%, respectively, and treatment-related adverse reactions of grade III or above of 40% and 19.5%, respectively. This shows that bispecific antibodies have greater potential in reducing toxic side effects and dosage and have better safety compared with monoclonal antibody combinations; at the same time, anti-PD-L1 can better target and enter the tumor microenvironment than anti-PD-1, and can improve toxic side effects on normal tissues.

[0005] In the development of bispecific antibody drugs, the biological activity of candidate antibodies is an important factor that needs to be taken into consideration. Although many bispecific antibodies have been proposed, due to the diversity of requirements for therapeutic functionality and therapeutic behavior of different therapeutic products, there is currently no theoretical prediction method that can be universally applied to most different desired molecular combinations. Therefore, it is often necessary to develop bispecific antibodies with good biological activity in a targeted manner based on specific targets and antibody compositions. Summary of the invention

[0006] The anti-PD-L1 and VEGF bispecific antibodies in the prior art are mainly anti-PD-L1 antibodies fused with VEGFR1 proteins (US20200172623A1, WO2020200210A1) or Mab-VHH type anti-PD-L1 and VEGF bispecific antibodies (WO2021147829A1). These bispecific antibodies all contain IgG1 type full-length antibodies, which have large molecular weights and drugability problems. Through in-depth research, the inventors have developed a bispecific nanoantibody that simultaneously targets VEGF and PD-L1 on the basis of the anti-PD-L1 nanoantibody and anti-VEGF nanoantibody developed. The molecular weight is only 107KDa, which has the advantages of strong potential tumor tissue infiltration and small dosage, and is superior to the main competitors in clinical dosage and safety. Furthermore, the in vitro VEGF neutralization activity of the anti-VEGF nanoantibody part in the bispecific antibody of the present invention is better than that of the marketed Bevacizumab, and in the animal model of angiogenesis, under the condition of equal mass administration, the in vivo angiogenesis inhibition activity is also better than that of Bevacizumab. At the same time, the anti-PD-L1 nanobody part of the bispecific antibody of the present invention is similar to the marketed Atezolizumab in terms of in vitro blocking activity and cell binding level. Furthermore, compared with monoclonal antibody combination and other prior art bispecific antibodies, the bispecific antibody of the present invention can simultaneously block the PD-1 / PD-L1 and VEGF / VEGFR signaling pathways, improve blood vessels and tumor microenvironment, and restore immunity, which has a more significant tumor inhibition effect and has a very broad application prospect.

[0007] Therefore, in one aspect, the present invention provides a bispecific antibody that specifically binds to PD-L1 and VEGF, wherein the antibody comprises one or two VHH domains (VHH PD-L1 ) and one or two VHH domains that specifically bind to VEGF (VHH VEGF ), wherein the VHH VEGF The CDR1-3 sequence of the VHH domain shown in SEQ ID NO: 2 is included, and preferably, wherein the VHH PD-L1The CDR1-3 sequence of the VHH domain shown in SEQ ID NO:1 is included.

[0008] In one embodiment, the VHH PD-L1 and VHH VEGF The domains can be in a tandem form contained on the same polypeptide chain (e.g., tandemly connected on one polypeptide chain with the aid of a linker and / or an Fc region); or in a Fab-like structure (e.g., Fab-like structure formed with the aid of immunoglobulin CH1 and CL constant domains), connected to the immunoglobulin Fc region.

[0009] In a preferred embodiment, the bispecific antibody comprises a polypeptide chain of a first VHH domain, an Fc region, a linker, and a second VHH domain from the N-terminus to the C-terminus, wherein the first VHH domain and the second VHH domain specifically bind to a first antigen and a second antigen, respectively, wherein the first antigen and the second antigen are different from each other and independently selected from PD-L1 and VEGF, preferably the first antigen is PD-L1 and the second antigen is VEGF. Preferably, the Fc region is an Fc region from IgG, especially IgG1, comprising CH2 and CH3 domain sequences from the N-terminus to the C-terminus. The Fc region can be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region. Preferably, the Fc region polypeptide is connected to the first VHH domain at the N-terminus through an immunoglobulin hinge region sequence. More preferably, the Fc region polypeptide is connected to the second VHH domain at the C-terminus through a flexible connecting peptide, such as (G4S)3. Preferably, the polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0010] In another preferred embodiment, the bispecific antibody comprises a polypeptide chain of a first VHH domain, a linker, a second VHH domain and an Fc region from the N-terminus to the C-terminus, wherein the first VHH domain and the second VHH domain specifically bind to a first antigen and a second antigen, respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably the first antigen is PD-L1 and the second antigen is VEGF. Preferably, the first and second VHH domains are connected in series through a linker, such as (G4S)3. Preferably, the Fc region is an Fc region from IgG, especially IgG1, comprising CH2 and CH3 domain sequences from the N-terminus to the C-terminus. The Fc region can be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region. Preferably, the Fc region polypeptide is connected to the second VHH domain at the N-terminus through an immunoglobulin hinge region sequence. Preferably, the polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0011] In yet another preferred embodiment, the bispecific antibody comprises a first and a second polypeptide chain, wherein:

[0012] The first polypeptide chain comprises, from N-terminus to C-terminus, a first VHH domain, a CH1 domain and an Fc region;

[0013] The second polypeptide chain comprises a second VHH domain and a CL domain from N-terminus to C-terminus;

[0014] wherein the first VHH domain and the CH1 domain in the first polypeptide chain are paired with the first VHH domain and the CL domain in the second polypeptide chain to form a Fab-like structure;

[0015] Wherein the first VHH domain and the second VHH domain specifically bind to the first antigen and the second antigen, respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably the first antigen is PD-L1 and the second antigen is VEGF. Preferably, the Fc region is an Fc region from IgG, especially IgG1, comprising CH2 and CH3 domain sequences from N-terminus to C-terminus. The Fc region can be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region. Preferably, the Fc region polypeptide is connected to the CH1 domain at the N-terminus by an immunoglobulin hinge region sequence. Preferably, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 9 or 11 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. Preferably, the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 10 or 12, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0016] In yet another aspect, the present invention also provides nucleic acids encoding the polypeptide chains of the bispecific antibodies of the present invention, and vectors and host cells comprising the same.

[0017] In another aspect, the present invention also provides a pharmaceutical composition comprising the bispecific antibody of the present invention, and in vitro and in vivo uses thereof, in particular, uses thereof in the treatment or diagnosis of a disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figures 1A-1D Schematic structures of candidate bispecific antibodies are shown. BsAb10 has Figure 1A BsAb11 has Figure 1B BsAb12 has Figure 1C BsAb13 has Figure 1D form.

[0019] Figure 2A-2B Gel electrophoresis images of candidate bispecific antibodies are shown. Figure 2A The samples were BsAb10 and reference IPI; Figure 2B The samples are BsAb11, BsAb12, BsAb13 and reference IPI.

[0020] Figures 3A-3D SEC-HPLC monomer detection profiles of candidate bispecific antibodies are shown. Figure 3A SEC-HPLC monomer detection spectrum of BsAb10; Figure 3B This is the SEC-HPLC monomer detection spectrum of BsAb11; Figure 3CSEC-HPLC monomer detection spectrum of BsAb12; Figure 3D This is the SEC-HPLC monomer detection spectrum of BsAb13.

[0021] Figure 4 The binding activity of the candidate bispecific antibodies to the recombinant protein VEGF-His was shown.

[0022] Figure 5 The binding activity of the candidate bispecific antibodies to the recombinant protein PD-L1-His was shown.

[0023] Figure 6 The binding activity of the candidate bispecific antibodies to huPD-L1-CHO-K cells is shown.

[0024] Figure 7A-7B The candidate bispecific antibodies showed simultaneous binding activity to VEGF and PD-L1. Fig. 7A The activity of the candidate bispecific antibody binding first to the recombinant protein VEGF-Fc and then to the recombinant protein PD-L1-Fc was shown; Figure 7B The activity of the candidate bispecific antibody binding to the recombinant protein PD-L1-Fc and then to the recombinant protein VEGF-Fc was shown.

[0025] Figure 8 The blocking activity of candidate bispecific antibodies against PD-1 / PD-L1 was detected by luciferase reporter gene method.

[0026] Fig. 9 The blocking activity of candidate bispecific antibodies against VEGF / VEGFR2 was detected by luciferase reporter gene method.

[0027] Fig.10 The candidate bispecific antibodies were shown to inhibit tumor growth in a mouse transplant model. DETAILED DESCRIPTION OF THE INVENTION

[0028] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods and examples described herein are only illustrative and are not intended to be restrictive. Other features, purposes and advantages of the present invention will be apparent from this specification and the accompanying drawings and from the appended claims.

[0029] I. Definitions

[0030] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0031] As used herein, the term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. When referring to "comprising", unless the context clearly indicates otherwise, the expression also encompasses "consisting of". For example, when referring to a polypeptide chain comprising a specific amino acid sequence, it also encompasses the situation where the polypeptide chain consists of the specific amino acid sequence.

[0032] In this article, the term "VEGF" refers to vascular endothelial growth factor A (VEGF-A) protein. VEGF-A produces multiple isoforms through alternative splicing of exons during transcription, such as VEGF121, VEGF165, VEGF189 and VEGF206. In this article, VEGF refers in particular to isoform VEGF165, such as the human VEGF165 protein recorded under UniProtKB-P15692 (in particular, the amino acid sequence of amino acids 27-191 without a signal peptide).

[0033] VEGF165 is active in angiogenesis, vascularization and endothelial cell growth; it can induce endothelial cell proliferation, promote cell migration, inhibit apoptosis and induce vascular permeability. VEGF165 binds to the receptors FLT1 / VEGFR1 and KDR / VEGFR2; and binds to the protein of neuropilin-1 (NRP1). Studies have confirmed that blocking the binding of VEGF to the receptor VEGFR has good efficacy and target safety in tumor treatment and eye diseases related to abnormal vascular proliferation. Recent studies have also found that NRP1 protein plays an important role in inhibiting the immune response to cancer. Compared with normal mice, mice with NRP1 gene deletion on cytotoxic T cells have better prevention against secondary tumors (including relatively "cold" tumors such as B16.F10 mouse melanoma) and respond more positively to anti-PD1 immunotherapy (Chang Liu et al, Neuropilin-1 is a T cell memory checkpoint limiting long-term antitumor immunity, Nature Immunology (2020). DOI: 10.1038 / s41590-020-0733-2).

[0034] As used herein, "antigen binding specificity for VEGF" refers to a binding site or binding domain in a molecule that specifically binds to VEGF.

[0035] As used herein, the term "PD-L1" refers to programmed cell death 1 ligand 1. As a ligand for the inhibitory receptor PD-1, the protein regulates the activation threshold of T cells and limits the effector response of T cells. The PD1 / PD-L1-mediated immunosuppressive pathway can be used by tumors to weaken anti-tumor immunity and escape the clearance of the immune system, thereby promoting tumor survival. Blocking this pathway can reverse the exhausted T cell phenotype and normalize the anti-tumor response, promoting cancer immunotherapy. The term PD-L1 herein refers in particular to human PD-L1 protein, such as human PD-L1 protein under accession number UniProtKB-Q9NZQ7.

[0036] Herein, "antigen binding specificity for PD-L1" refers to a binding site or binding domain in a molecule that specifically binds to PD-L1.

[0037] As used herein, the terms "antigen binding site" and "antigen binding specificity" are used interchangeably to refer to the region where an antibody actually binds to an antigen. Preferably, in the bispecific antibodies of the present invention, the PD-L1 antigen binding site is provided by a VHH domain from anti-PD-L1; and the VEGF antigen binding site is provided by a VHH domain from anti-VEGF.

[0038] As used herein, the term "binding" or "specific binding" means that the binding effect is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of the antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, for example, by detecting the binding ability of the antibody to PD-L1 or VEGF protein or both by the ELISA assay described in Example 4.1, 4.2 or 4.4, or by detecting the binding ability of the antibody to PD-L1 expressed on the cell surface by the FACS assay described in Example 4.3.

[0039] Herein, the "blocking activity" of an antibody against PD-L1 refers to the function of the antibody to block the binding of PD-L1 to the receptor PD-1, and / or reduce the signal transduction of PD-L1 / PD-1. The assay that can be used to determine this blocking activity can be a FACS assay known in the art; or a reporter gene-based signal pathway blocking assay, such as the reporter gene-based detection method described in Example 5. The blocking activity of the antibody to be tested against PD-L1 can be determined by reference to the PD-L1 / PD-1 binding or PD-L1 / PD-1 signal transduction level in the absence of the antibody and / or in the presence of a positive antibody.

[0040] Herein, the "neutralizing activity" of an antibody against VEGF refers to the function of the antibody in blocking the signaling pathway between VEGF and the receptor VEGFR2. An assay that can be used to measure this activity can be, for example, a reporter gene-based signaling pathway blocking assay, such as the reporter gene-based detection method described in Example 6. The neutralizing activity of the antibody to be tested against VEGF can be determined with reference to the signaling level of VEGF / VEGFR2 in the absence of an antibody and / or in the presence of a positive antibody.

[0041] The term "variable region" or "variable domain" of an antibody refers to the domain of an antibody heavy chain or light chain that is involved in binding of the antibody to an antigen. The variable region of an antibody can be further subdivided into hypervariable regions (i.e., complementary determining regions (CDRs)) and relatively conservative regions (i.e., framework regions (FRs)) interposed between the hypervariable regions. In the case of heavy chain antibodies (also referred to herein as nanobodies), such as heavy chain antibodies from Camelidae, the antigen binding site is provided by a single heavy chain variable domain (also referred to herein as a VHH domain). Like the heavy chain variable region VH of an IgG antibody, the single heavy chain variable domain VHH comprises four FR regions and three CDR regions, and is arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0042] The "complementarity determining region" of an antibody, used interchangeably herein with "CDR region" or "CDR" or "hypervariable region", is a region in an antibody variable domain (e.g., VHH) that is highly variable in sequence and forms a structurally determined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes. CDRs located in the VHH variable region domain are referred to as CDR1, CDR2, and CDR3, starting with the N-terminus of the antibody chain. In a given variable region amino acid sequence, its CDR sequence can be determined using a variety of schemes known in the art, such as: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (International Immunogenetics Information System, World Wide Web imgt.cines.fr / ), and North CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Concepts", Journal of Molecular Biology, 406, 228-256 (2011)). Those skilled in the art can find out more about the CDR sequence of a variable region amino acid sequence. http: / / www.abysis.org / abysis / The CDR sequence range of any given antibody variable region amino acid sequence can be easily determined on the basis of the above, including the regional range of CDR defined by Kabat, AbM, Chothia, Contact and IMGT schemes and their combined ranges. Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" covers CDR sequences determined in any of the above ways and their combinations.

[0043] In the bispecific antibodies of the present invention, the antigen binding site that specifically binds to an antigen is provided by a VHH domain. The term "VHH" or "VHH domain" is used herein to refer to a heavy chain variable domain derived from a heavy chain antibody lacking a light chain (also sometimes referred to herein as a nanobody), also referred to as a single variable domain (sVD). Different from the conventional VH of a four-chain immunoglobulin, the VHH domain does not need to be paired with a light chain variable domain to form an antigen binding site. This VHH molecule can be derived from antibodies produced in camelid species (e.g., camels, alpacas, dromedaries, llamas and guanacos). In some cases, for the therapeutic application of VHH, it is desirable to reduce its immunogenicity. Therefore, preferably, in one embodiment, the present invention provides an antibody comprising a humanized VHH domain. In some embodiments according to the present invention, in the bispecific antibody according to the present invention, the VHH domain from anti-PD-L1 and the VHH domain from anti-VEGF are preferably connected to the opposite sides of the Fc region polypeptide of the antibody separately through an immunoglobulin hinge region or a linker; or in the form of a series of two (for example, from N-terminus to C-terminus, VHH PD-L1 -Linker-VHH VEGF ; or VHH VEGF -Linker-VHH PD-L1 ), connected to the N-terminal side of the Fc region polypeptide of the antibody.

[0044] "Fab-like structure" is used herein to refer to Figure 1C and Figure 1D As shown in , it is similar to the structure formed by the heavy chain variable region and heavy chain constant region CH1 paired with the complementary light chain variable region and light chain constant region CL in the conventional four-chain IgG antibody, but in this structure the heavy chain variable region is replaced by a VHH domain, and the light chain variable region is replaced by another different VHH domain. In one embodiment according to the present invention, the Fab-like structure comprises a VHH PD-L1 The first chain consists of the CH1 domain of the immunoglobulin heavy chain constant region and the VHH VEGF In another embodiment according to the present invention, the Fab-like structure comprises a second chain consisting of a VHH VEGF The first chain consists of the CH1 domain of the immunoglobulin heavy chain constant region and the VHH PD-L1 A second chain composed of a CL domain of an immunoglobulin light chain constant region. In some further embodiments according to the present invention, in the bispecific antibody according to the present invention, the VHH domain from anti-PD-L1 and the VHH domain from anti-VEGF are connected to the N-terminal side of the immunoglobulin Fc region polypeptide in a Fab-like structure, preferably through an immunoglobulin hinge region. Preferably, the Fab-like structure is connected to the Fc region polypeptide through its CH1 domain.

[0045] The term "immunoglobulin molecule" refers to a protein with the structure of a naturally occurring antibody. For example, an IgG class immunoglobulin is a heterotetrameric glycoprotein of about 150,000 daltons consisting of two light chains and two heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also known as a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also known as a light chain variable domain, followed by a light chain constant domain (CL). In an IgG molecule, the VH-CH1 of the heavy chain is usually paired with the VL-CL of the light chain to form a Fab fragment that specifically binds to an antigen. Therefore, an IgG immunoglobulin is essentially composed of two Fab molecules connected by an immunoglobulin hinge region and two dimerized Fc regions. The heavy chains of immunoglobulins can be classified into one of five categories based on the type of their constant regions, called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which can be further divided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chains of immunoglobulins can also be classified into one of two types, called κ and λ, based on the amino acid sequence of their constant domains.

[0046] "Immunoglobulin heavy chain constant region domain" refers to a constant region domain from, obtained from or derived from an immunoglobulin heavy chain, including heavy chain constant regions CH1, CH2, CH3 and optionally heavy chain constant region CH4 covalently linked sequentially from the N-terminus to the C-terminus. In most cases, the heavy chain constant regions CH1 and CH2 are connected by a heavy chain hinge region, but when appropriate, they can also be connected by a flexible connecting peptide. In some preferred embodiments of the present invention, the antibody of the present invention comprises a polypeptide chain consisting of an immunoglobulin heavy chain constant region CH1-hinge region-CH2-CH3. In other preferred embodiments of the present invention, the antibody of the present invention comprises an Fc region polypeptide chain consisting of an immunoglobulin heavy chain constant region CH2-CH3. In this article, the immunoglobulin constant domain can be selected according to the intended function of the antibody. For example, the constant domain can be an IgA, IgD, IgE, IgG or IgM domain, especially an immunoglobulin constant domain of human IgG, for example, a constant domain of human IgG1, IgG2, IgG3 or IgG4, preferably a constant domain of human IgG1. As an example, the CH1 and Fc fragments of an antibody may both be derived from IgG1.

[0047] "Immunoglobulin light chain constant region domain" refers to a constant region domain CL from, obtained from, or derived from an immunoglobulin light chain. The immunoglobulin light chain CL constant region, based on its amino acid sequence, can be a kappa light chain CL domain and a lambda light chain CL domain.

[0048] As used herein, the terms "Fc domain" or "Fc region" or "Fc region polypeptide" are used interchangeably to refer to a C-terminal region polypeptide containing at least a portion of a constant region derived from an immunoglobulin heavy chain. The term includes native sequence Fc region polypeptides and variant Fc region polypeptides. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of a heavy chain derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of a heavy chain derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, the term "Fc domain" or "Fc region" or "Fc polypeptide" does not include the heavy chain variable region VH and light chain variable region VL as well as the heavy chain constant region CH1 and light chain constant region CL of an immunoglobulin.

[0049] "Native sequence Fc region" polypeptides encompass naturally occurring various immunoglobulin Fc region sequences, such as various Ig subtypes and their allotype Fc region sequences (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389 / fimmu.2014.00520.). In some embodiments, the human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or from Pro230 to the heavy chain carboxyl end. However, the C-terminal terminal lysine (Lys447) of the Fc region may be present or absent. In some further embodiments, the human IgG heavy chain Fc region carries a hinge sequence or a partial hinge sequence of a natural immunoglobulin at the N-terminus, such as, according to EU numbering, a sequence from E216 to T225 or a sequence from D221 to T225.

[0050] The terms "variant Fc region" or "Fc region variant" polypeptide, as used interchangeably herein, refer to an Fc region polypeptide comprising modifications relative to a native sequence Fc region polypeptide. The Fc region variant polypeptides of the invention are defined according to the amino acid modifications that comprise them. Thus, for example, L234A is an Fc region variant with alanine replacing leucine at position 234 relative to the parent polypeptide, wherein numbering is according to the EU index. Modifications may be additions, deletions or substitutions. Substitutions may include naturally occurring amino acids and non-naturally occurring amino acids. Variants may comprise non-natural amino acids. The purpose of the modification may be to alter the binding of the Fc region to its receptors and the effector functions elicited thereby.

[0051] The term "effector function" refers to those biological activities attributed to the Fc region of an immunoglobulin that vary with the immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Depending on the intended use of the antibody, the antibodies of the present invention may have altered effector functions relative to antibodies having a wild-type Fc region, such as reduced or eliminated ADCC activity, etc.

[0052] As used herein, the term "flexible connecting peptide" or "connector" or "connecting peptide" is used interchangeably and refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T), used alone or in combination, or from the hinge region of an immunoglobulin. In one embodiment, the connecting peptide has a length of 5-50 amino acids, for example, 10, 15, 20, 25, 30 amino acids. In one embodiment, the connecting peptide comprises the amino acid sequence (G4S) n , wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7. In one embodiment, the connecting peptide comprises the amino acid sequence TS(G4S) n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7. In yet another embodiment, the connecting peptide is a hinge region from an immunoglobulin, for example, a hinge region amino acid sequence comprising "CPPC", for example, an amino acid sequence "EPKSCDKTHTCPPCP" or "EPKSSDKTHTCPPCP". The connecting peptide that can be used to connect the domains of the antibody of the present invention can also be, for example but not limited to, the following amino acid sequences: GGG; DGGGS; TGEKP; GGRR; EGKSSGSGSESKVD; KESGSVSSEQLAQFRSLD; GGRRGGGS; LRQRDGERP; LRQKDGGGSERP and GSTSGSGKPGSGEGSTKG. Alternatively, a computer program can be used to simulate the three-dimensional structure of proteins and peptides, or by phage display methods, to rationally design suitable flexible connecting peptides.

[0053] The term "chimeric antibody" is an antibody in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site is linked to a constant region of a different or altered class, effector function and / or species, or to an entirely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug, etc.) that confers new properties to the chimeric antibody; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigenic specificity.

[0054] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., an alpaca heavy chain antibody) while being less immunogenic when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen binding site and replacing the remainder of the antibody with their human counterparts (i.e., the constant region and the portion of the variable region that does not participate in binding are the corresponding portions of a human antibody).

[0055] The "percentage (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a specific amino acid sequence shown in this specification, after comparing the candidate sequence to the specific amino acid sequence shown in this specification and, if necessary, introducing gaps to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibodies of the present invention that have a considerable degree of identity, such as at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibodies and sequences specifically disclosed herein. The variants may comprise conservative modifications.

[0056] For polypeptide sequences, "conservative modifications" include substitutions, deletions or additions to the polypeptide sequence that result in substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude the polymorphic variants, interspecies homologs and alleles of the present invention. The following eight groups contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine ​​(C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.

[0057] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibodies of the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells, and also include cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0058] The term "expression vector" refers to a vector comprising a recombinant polynucleotide, which comprises an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) into which the recombinant polynucleotide is incorporated.

[0059] The terms "individual" or "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

[0060] The term "treatment" refers to a clinical intervention intended to change the natural course of a disease in an individual being treated. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the development of the disease or to slow the progression of the disease. In the case of tumor or cancer treatment, "treatment" covers anti-tumor biological effects that can be caused by human intervention means (e.g., the administration of drugs such as antibodies of the present invention), including but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0061] The terms "cancer" and "tumor" are used interchangeably to refer to or describe the physiological condition in mammals that is generally characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, solid tumors, and liquid tumors.

[0062] Various aspects of the present invention are described in detail below.

[0063] I. Bispecific Antibodies of the Present Invention

[0064] In one aspect, the present invention provides a bispecific antibody based on a VHH domain that specifically binds to PD-L1 and VEGF, wherein the antibody comprises a VHH domain that specifically binds to PD-L1 (also abbreviated herein as VHH PD-L1 ) and a VHH domain that specifically binds to VEGF (also abbreviated herein as VHH VEGF ).

[0065] In some specific embodiments, in the bispecific antibodies of the present invention, the VHH PD-L1 and VHH VEGF The domains may be in a tandem form contained on the same polypeptide chain (e.g., tandemly linked on one polypeptide chain with the aid of a linker and / or Fc region); or may form a Fab-like structure (e.g., forming a Fab-like structure with the aid of immunoglobulin CH1 and CL constant domains).

[0066] In some specific embodiments, the bispecific antibody according to the present invention further comprises an Fc region. PD-L1 and VHH VEGF The domain may comprise a VHH PD-L1 and VHH VEGF A tandem form of the two domains (e.g. Figure 1A and Figure 1B as shown), or to include VHH PD-L1 and VHH VEGFThe Fab-like structure of both domains (e.g. Figure 1C and Figure 1D As shown in the figure), it is connected to the Fc region. Since the antibody polypeptide chains containing the Fc region can be associated through the dimerization of the Fc region, in some embodiments, the bispecific antibody according to the present invention can be a tetravalent two-chain antibody that specifically binds to PD-L1 and VEGF, or can be a tetravalent four-chain antibody that specifically binds to PD-L1 and VEGF.

[0067] The components of the bispecific antibodies of the present invention and the bispecific antibodies of the present invention composed thereof are described in detail below. It will be understood by those skilled in the art that any combination of any technical features of these components is within the scope of the present invention unless the context clearly indicates otherwise. Furthermore, it will be understood by those skilled in the art that the antibodies of the present invention (including antibodies of any form, such as two-chain antibodies and four-chain antibodies) may contain any such combination features unless the context clearly indicates otherwise.

[0068] VHHs according to the invention VEGF Domains and VHH PD-L1 Domain

[0069] In the bispecific antibodies according to the present invention (including the diabody of the present invention and the tetrabody of the present invention), the antigen binding site that specifically binds to PD-L1 is composed of the VHH according to the present invention. PD-L1 Provided; and the antigen binding site that specifically binds to VEGF is composed of the VHH according to the present invention VEGF supply.

[0070] VHHs according to the invention PD-L1 The domain comprises a VHH domain from an anti-PD-L1 nanobody. Preferably, the VHH comprises a CDR1, CDR2 and CDR3 sequence having a variable region as shown in SEQ ID NO: 1. The CDR sequence range of the amino acid sequence of the variable region SEQ ID NO: 1 can be defined according to the Kabat, AbM, Chothia, Contact or IMGT schemes, or can be defined according to any two or more or all of these definition schemes. A person skilled in the art can easily define the CDR sequence range according to the Kabat, AbM, Chothia, Contact or IMGT schemes. http: / / www.abysis.org / abysis / The CDR sequences defined by these definitions are known. In a preferred embodiment, the VHH according to the invention PD-L1 The domain comprises a CDR1 sequence, a CDR2 sequence and a CDR3 sequence defined according to AbM in the variable region having SEQ ID NO:1.

[0071] In another preferred embodiment, the VHH according to the invention PD-L1 Domain contains

[0072] (i) a CDR1 comprising or consisting of SEQ ID NO: 13;

[0073] (ii) a CDR2 comprising or consisting of SEQ ID NO: 14; and

[0074] (iii) a CDR3 comprising or consisting of SEQ ID NO:15.

[0075] In one embodiment, the VHH PD-L1 The domain comprises the amino acid sequence shown in SEQ ID NO: 1. In yet another embodiment, the VHH PD-L1 The VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1 and retains the ability to specifically bind to PD-L1. PD-L1 The domain comprises an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 1 and retaining the ability to specifically bind to PD-L1. Preferably, the addition, deletion and / or substitution of amino acids does not occur in the CDR region. Most preferably, in the bispecific antibody according to the present invention, the VHH according to the present invention PD-L1 The domain comprises the amino acid sequence of SEQ ID NO:1, or consists of the amino acid sequence shown in SEQ ID NO:1.

[0076] VHHs according to the invention VEGF The domain comprises a VHH domain from an anti-VEGF nanobody. Preferably, the VHH comprises a CDR1, CDR2 and CDR3 sequence having a variable region as shown in SEQ ID NO: 2. The CDR sequence range of the amino acid sequence of the variable region SEQ ID NO: 2 can be defined according to the Kabat, AbM, Chothia, Contact or IMGT schemes, or can be defined according to any two or more or all of these definition schemes. A person skilled in the art can easily define the CDR sequence range according to the Kabat, AbM, Chothia, Contact or IMGT schemes. http: / / www.abysis.org / abysis / The CDR sequences defined by these definitions are known. In a preferred embodiment, the VHH according to the invention VEGF The domain comprises the CDR1 sequence, CDR2 sequence and CDR3 sequence defined according to AbM in the variable region having SEQ ID NO:2.

[0077] In another preferred embodiment, the VHH according to the invention VEGF Domain contains

[0078] (i) a CDR1 comprising or consisting of SEQ ID NO: 16;

[0079] (ii) a CDR2 comprising or consisting of SEQ ID NO: 17; and

[0080] (iii) a CDR3 comprising or consisting of SEQ ID NO:18.

[0081] In one embodiment, the VHH VEGF The domain comprises the amino acid sequence shown in SEQ ID NO: 2. In yet another embodiment, the VHH VEGF The VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 2 and retains the ability to specifically bind to VEGF. VEGF The domain comprises an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 2 and retaining the ability to specifically bind to VEGF. Preferably, the amino acid additions, deletions and / or substitutions do not occur in the CDR region. Most preferably, in the bispecific antibody according to the present invention, the VHH according to the present invention VEGF The domain comprises the amino acid sequence of SEQ ID NO:2, or consists of the amino acid sequence shown in SEQ ID NO:2.

[0082] Immunoglobulin constant domain

[0083] According to the bispecific antibody of the present invention, in addition to the aforementioned VHH VEGF Domains and VHH PD-L1 In addition to the immunoglobulin domain, in some embodiments, it may also include an immunoglobulin constant domain, such as an Fc region composed of immunoglobulin constant regions CH2 and CH3, and / or a CH1 and CL constant domain located in a Fab-like structure.

[0084] In some embodiments of the present invention, therefore, the present invention provides a bispecific antibody comprising an Fc region polypeptide chain consisting of an immunoglobulin heavy chain constant region CH2-CH3. In other embodiments of the present invention, the present invention provides a bispecific antibody comprising an Fc region and also comprising an immunoglobulin CH1 domain and a CL domain, preferably, wherein the CH1 domain is connected to the Fc region polypeptide by an immunoglobulin hinge region, and more preferably, the antibody comprises a polypeptide chain consisting of an immunoglobulin heavy chain constant region CH1-hinge region-CH2-CH3. The immunoglobulin constant domains contained in the bispecific antibody according to the present invention, including the CH2 and CH3 constant domains in the Fc region and the CH1 and CL constant domains in the Fab-like structure (if present), can be independently of each other immunoglobulin constant domains from human IgG, for example, constant domains of human IgG1, IgG2, IgG3 or IgG4, preferably constant domains of human IgG1. Furthermore, the immunoglobulin constant domains contained in the bispecific antibodies according to the present invention, including the constant regions CH2 and CH3 as well as CH1 and CL (if present), may independently of one another be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof, preferably native sequence constant domains.

[0085] In a preferred embodiment, the Fc region contained in the bispecific antibody according to the present invention is an Fc region sequence from human IgG1. In a more preferred embodiment, the Fc region has an amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having at least 90%, 95% or 99% identity thereto, or an amino acid sequence having no more than 1-10 (preferably 1-5) amino acid residues changed therefrom.

[0086] In some specific embodiments, the bispecific antibody according to the present invention may comprise a polypeptide chain having two Fc regions, for example, a two-chain antibody and a four-chain antibody of the present invention as shown in FIG1 , wherein the Fc regions of the two polypeptide chains are capable of pairing and dimerization. In such an embodiment, the first Fc region polypeptide and the second Fc region polypeptide constituting the dimerization Fc region of the antibody may both be from IgG, such as the Fc domain of human IgG1, IgG2, IgG3 or IgG4, preferably the Fc domain of human IgG1; and may be independently of each other the native sequence Fc region and the variant Fc region defined herein, provided that the first Fc region polypeptide and the second Fc region polypeptide may be paired and dimerized. Preferably, the first Fc region and the second Fc region are native sequence Fc regions, especially human IgG1 native sequence Fc regions. More preferably, the first Fc region and the second Fc region have the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 90%, 95% or 99% identity thereto, or an amino acid sequence having no more than 1-10 (preferably 1-5) amino acid residues changed therefrom.

[0087] In another embodiment, the antibody according to the present invention (e.g., the four-chain antibody of the present invention) may comprise a Fab-like structure having a heavy chain constant region CH1 domain and a light chain constant region CL domain, wherein the CH1 domain is preferably connected to the C-terminus of the first VHH domain, and the light chain constant region CL domain is connected to the C-terminus of the second VHH domain. In such a bispecific antibody, the heavy chain constant region CH1 and CL constituting the antibody may be from IgG, such as a constant domain of human IgG1, IgG2, IgG3 or IgG4, preferably a constant domain of human IgG1. The CL domain of the antibody may be a Kappa light chain CL domain or a Lamda light chain CL domain, preferably a Kappa light chain CL domain. In such a bispecific antibody, the CH1 and CL domains may be independently of each other native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof, provided that an antibody polypeptide chain comprising the CH1 domain and a first VHH domain connected at its N-terminus, and an antibody polypeptide chain comprising the CL domain and a second VHH domain connected at its N-terminus can be paired with each other to form a Fab-like structure according to the present invention. Preferably, the CH1 and CL domains are native sequence constant domains, especially human IgG1 native sequence CH1 domain and CL domain. More preferably, the CH1 domain has the amino acid sequence shown in SEQ ID NO:4. More preferably, the CL domain has the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6.

[0088] Linker

[0089] In the bispecific antibody according to the present invention, the antibody components can be connected by a linker. For example, the VHH domain according to the present invention can be connected to the N-terminus or C-terminus of the Fc region through a linker, or can be connected to another VHH domain according to the present invention through a linker. There is no specific limitation on the linker that can be used in the antibody of the present invention. Those skilled in the art can easily determine the available linker sequence according to the components to be connected and the connection position.

[0090] In some embodiments, in a bispecific antibody according to the invention, the VHH domains according to the invention are in tandem (e.g. Figure 1A and 1B ) or in a Fab-like structure (e.g., Figure 1C and 1D As shown in FIG. 1 , the VHH is connected to the polypeptide chain of the Fc region through a linker. Those skilled in the art can select different linkers according to the connection position of the VHH, that is, connected to the N-terminus or C-terminus of the Fc region.

[0091] In one embodiment, the Fc region polypeptide chain is connected to a single VHH domain or two VHH domains in series, or a Fab-like structure at the N-terminus by a linker, wherein the linker preferably comprises (one or two) cysteine ​​residues, thereby being able to form (a pair or two pairs) of disulfide bonds when two antibody polypeptide chains comprising the Fc region are paired to stabilize the structure of the bispecific antibody thus formed. For example, the two-chain antibody structure and the four-chain antibody structure shown in Figure 1. Therefore, in a preferred embodiment, the Fc region polypeptide chain contained in the bispecific antibody of the present invention carries a linker sequence comprising "CPPC" at the N-terminus. In another preferred embodiment, the Fc region polypeptide chain contained in the bispecific antibody of the present invention carries a hinge region from an immunoglobulin at the N-terminus, such as a hinge region amino acid sequence comprising "CPPC", for example, the amino acid sequence "EPKSCDKTHTCPPCP" or "EPKSSDKTHTCPPCP"; more preferably, the amino acid sequence "EPKSCDKTHTCPPCP".

[0092] In another embodiment, the Fc region polypeptide chain is connected to the VHH domain at the C-terminus via a linker, wherein the linker is preferably a flexible linker peptide of 5-50 amino acids, preferably a linker peptide comprising glycine (G) and / or serine (S) and / or threonine residues (T). In one embodiment, the linker has a length of 5-50 amino acids, for example, 10, 15, 20, 25 or 30 amino acids in length, or has an amino acid length falling between any two integers. In one embodiment, the linker comprises the amino acid sequence (G4S) n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7. In a preferred embodiment, the linker consists of the amino acid sequence (G4S)3.

[0093] In some further embodiments, in the bispecific antibodies according to the invention, the VHH according to the invention VEGF Domains and VHH PD-L1 The domains form a tandem VHH domain through a connector. In the bispecific antibody according to the present invention having such a tandem VHH domain, preferably, the first and second domains are connected in series through a connector, wherein the connector is a flexible connecting peptide of 5-50 amino acids. In one embodiment, the connector has a length of 5-50 amino acids, for example, 10, 15, 20, 25 or 30 amino acids, or has an amino acid length falling between any two integers. In one embodiment, the connector is a connecting peptide comprising glycine (G) and / or serine (S) and / or threonine residues (T). In another embodiment, the connector comprises the amino acid sequence (G4S) n , wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7. In a preferred embodiment, the linker consists of the amino acid sequence (G4S)3.

[0094] Bispecific Antibody Embodiments of the Invention

[0095] In one aspect, the present invention provides a bispecific antibody that specifically binds to PD-L1 and VEGF, wherein the antibody comprises a VHH domain that specifically binds to a first antigen and a VHH domain that specifically binds to a second antigen, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF.

[0096] In one embodiment, the VHH domain that specifically binds to VEGF comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO: 2. In another embodiment, the VHH domain that specifically binds to PD-L1 comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO: 1.

[0097] In another embodiment, the VHH domain that specifically binds to VEGF comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 2, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acids added, deleted and / or substituted compared to SEQ ID NO: 2. Most preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 2, or consists of the amino acid sequence shown in SEQ ID NO: 2.

[0098] In yet another embodiment, the VHH domain that specifically binds to PD-L1 comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 1, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) additions, deletions and / or substitutions of amino acids compared to SEQ ID NO: 1. Most preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 1, or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0099] In some embodiments, the bispecific antibody according to the present invention further comprises a linker, which comprises a length of 10-20 amino acids, preferably, comprises an amino acid sequence (G4S) 3. In some further embodiments, the bispecific antibody according to the present invention further comprises an immunoglobulin Fc region, wherein the Fc region is an IgG Fc region, for example, an Fc domain of human IgG1, IgG2, IgG3 or IgG4, preferably an Fc domain of human IgG1, and more preferably, the Fc region carries an immunoglobulin hinge region sequence at the N-terminus.

[0100] In some preferred embodiments, the bispecific antibody according to the present invention comprises a polypeptide chain comprising a VHH connected in series via an Fc region and a linker. PD-L1 and VHH VEGFDomain. Preferably, the bispecific antibody comprises a polypeptide chain comprising, from N-terminus to C-terminus, a first VHH domain, an Fc region, a linker, and a second VHH domain, wherein the first VHH domain and the second VHH domain specifically bind to a first antigen and a second antigen, respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably, the first antigen is PD-L1 and the second antigen is VEGF. Preferably, the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0101] In other preferred embodiments, the bispecific antibody according to the present invention comprises a polypeptide chain comprising a VHH connected in series via a linker. PD-L1 and VHH VEGF Domain. Preferably, the bispecific antibody comprises a polypeptide chain comprising, from N-terminus to C-terminus, a first VHH domain, a linker, a second VHH domain and an Fc region, wherein the first VHH domain and the second VHH domain specifically bind to a first antigen and a second antigen, respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably, the first antigen is PD-L1 and the second antigen is VEGF. Preferably, the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0102] In some further preferred embodiments, the bispecific antibody according to the present invention comprises a first and a second polypeptide chain forming a Fab-like structure. Preferably, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first VHH domain, a CH1 domain and an Fc region from the N-terminus to the C-terminus; the second polypeptide chain comprises a second VHH domain and a CL domain from the N-terminus to the C-terminus; wherein the first VHH domain and the CH1 domain in the first polypeptide chain are paired with the second VHH domain and the CL domain in the second polypeptide chain to form a Fab-like structure; wherein the first VHH domain and the second VHH domain specifically bind to the first antigen and the second antigen, respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably, the first antigen is PD-L1 and the second antigen is VEGF. In such an embodiment, preferably, the bispecific antibody comprises a first and a second polypeptide chain, wherein the first antigen is PD-L1 and the second antigen is VEGF, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:10, and an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0103] Two-chain bispecific antibody embodiments of the present invention

[0104] In the VHH with tandem arrangement PD-L1 and VHH VEGF In some embodiments of the bispecific antibodies of the present invention comprising a domain, the antibody may comprise two polypeptide chains comprising an Fc region, wherein the two polypeptide chains can associate to form a dimer through dimerization of the Fc region. As will be appreciated by those skilled in the art, the two polypeptide chains that associate to form a dimer do not need to be completely identical. For example, asymmetric modifications may be introduced into one or both polypeptide chains (e.g., in the Fc region of the polypeptide chain) without affecting the desired target activity. However, in some aspects, preferably, the two polypeptide chains are identical. For example, Figure 1A and 1B shown.

[0105] Therefore, in one aspect, the present invention provides a bispecific antibody that specifically binds to PD-L1 and VEGF, wherein the antibody comprises two polypeptide chains, which may be the same or different, wherein:

[0106] Each polypeptide chain comprises, from N-terminus to C-terminus, a first VHH domain, an Fc region, a linker, and a second VHH domain (e.g. Figure 1Aor each polypeptide chain comprises, from N-terminus to C-terminus, a first VHH domain, a linker, a second VHH domain and an Fc region (as shown); Figure 1B shown);

[0107] The Fc region polypeptides of the two polypeptide chains are paired and form a dimerized Fc region, wherein the first VHH domain and the second VHH domain specifically bind to a first antigen and a second antigen, respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF. In one embodiment, the first antigen is PD-L1 and the second antigen is VEGF; in another embodiment, the first antigen is VEGF and the second antigen is PD-L1.

[0108] In some embodiments of this aspect, the present invention provides a bispecific antibody that specifically binds to PD-L1 and VEGF, wherein the antibody comprises two polypeptide chains, which may be the same or different, wherein:

[0109] Each polypeptide chain comprises, from N-terminus to C-terminus, a first VHH domain, an Fc region, a linker, and a second VHH domain,

[0110] The Fc region polypeptides of the two polypeptide chains are paired and form a dimerized Fc region, wherein the first VHH domain and the second VHH domain specifically bind to the first antigen and the second antigen, respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably the first antigen is PD-L1 and the second antigen is VEGF. Preferably, the Fc region is an Fc region from IgG, especially IgG1, comprising CH2 and CH3 domain sequences from N-terminus to C-terminus. The Fc region can be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region. Preferably, the Fc region polypeptide is connected to the first VHH domain at the N-terminus through an immunoglobulin hinge region sequence. More preferably, the Fc region polypeptide is connected to the second VHH domain at the C-terminus through a flexible connecting peptide of 10-20 amino acids in length, such as (G4S)3. Preferably, the first VHH domain is a VHH according to the present invention. PD-L1 The second VHH domain preferably comprises the CDR1-3 sequence of the variable region shown in SEQ ID NO: 1, more preferably comprises the CDR1-3 sequence of SEQ ID NOs: 13-15; most preferably comprises the variable region sequence shown in SEQ ID NO: 1; and the second VHH domain is a VHH according to the present invention. VEGFThe structural domain preferably comprises the CDR1-3 sequence of the variable region shown in SEQ ID NO:2, more preferably comprises the CDR1-3 sequence of SEQ ID NOs:16-18; most preferably comprises the variable region sequence shown in SEQ ID NO:2. Preferably, the two polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. Most preferably, the two polypeptide chains are identical and respectively comprise the amino acid sequence of SEQ ID NO:7.

[0111] In other embodiments of this aspect, the present invention provides a bispecific antibody that specifically binds to PD-L1 and VEGF, wherein the antibody comprises two polypeptide chains, which may be the same or different, wherein:

[0112] Each polypeptide chain comprises, from N-terminus to C-terminus, a first VHH domain, a linker, a second VHH domain and an Fc region,

[0113] The Fc region polypeptides of the two polypeptides are paired and form a dimerized Fc region, wherein the first VHH domain and the second VHH domain specifically bind to the first antigen and the second antigen, respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF, preferably the first antigen is PD-L1 and the second antigen is VEGF. Preferably, the linker is a flexible connecting peptide with a length of 10-20 amino acids, such as (G4S)3. More preferably, the Fc region is an Fc region from IgG, especially IgG1, comprising CH2 and CH3 domain sequences from N-terminus to C-terminus. The Fc region can be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region. Preferably, the Fc region polypeptide is connected to the tandem VHH domains at the N-terminus through an immunoglobulin hinge region sequence. Preferably, the first VHH domain is a VHH according to the present invention. PD-L1 The second VHH domain preferably comprises the CDR1-3 sequence of the variable region shown in SEQ ID NO: 1, more preferably comprises the CDR1-3 sequence of SEQ ID NOs: 13-15; most preferably comprises the variable region sequence shown in SEQ ID NO: 1; and the second VHH domain is a VHH according to the present invention. VEGFThe structural domain preferably comprises the CDR1-3 sequence of the variable region shown in SEQ ID NO:2, more preferably comprises the CDR1-3 sequence of SEQ ID NOs:16-18; most preferably comprises the variable region sequence shown in SEQ ID NO:2. Preferably, the two polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. Most preferably, the two polypeptide chains are identical and respectively comprise the amino acid sequence of SEQ ID NO:8.

[0114] Four-chain bispecific antibody embodiments of the present invention

[0115] In the case of VHHs contained in a Fab-like structure PD-L1 and VHH VEGF In some embodiments of the bispecific antibodies of the present invention comprising a domain, the antibody may comprise two first polypeptide chains comprising an Fc region and two second polypeptide chains, wherein the two first polypeptide chains can form a dimer by dimerization association of the Fc region. As understood by those skilled in the art, the two first polypeptide chains comprising the bispecific antibody do not need to be completely identical; and similarly, the two second polypeptide chains do not need to be completely identical. For example, asymmetric modifications can be introduced into one or both of the first polypeptide chains and / or the second polypeptide chains without affecting the desired target activity. However, in some aspects, preferably, the two first polypeptide chains are identical, and the two second polypeptide chains are also identical.

[0116] Therefore, in yet another aspect, the present invention provides a bispecific antibody that specifically binds to PD-L1 and VEGF, wherein the antibody comprises two first polypeptide chains and two second polypeptide chains, wherein the two first polypeptide chains may be the same or different, and the two second polypeptide chains may be the same or different, wherein:

[0117] The two first polypeptide chains each comprise a first VHH domain, a CH1 domain and an Fc region from the N-terminus to the C-terminus;

[0118] The two second polypeptide chains each comprise a second VHH domain and a CL domain from N-terminus to C-terminus;

[0119] wherein the Fc region polypeptides in the two first polypeptide chains pair up to form a dimerized Fc region, and

[0120] The first VHH domain and the CH1 domain in the two first polypeptide chains are paired with the second VHH domain and the CL domain in the two second polypeptide chains to form two Fab-like structures;

[0121] wherein the first VHH domain and the second VHH domain specifically bind to a first antigen and a second antigen, respectively, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF. Figure 1C and Figure 1D In one embodiment, the first antigen is PD-L1 and the second antigen is VEGF; or in another embodiment, the first antigen is VEGF and the second antigen is PD-L1. Preferably, the Fc region is an Fc region from IgG, especially IgG1, comprising CH2 and CH3 domain sequences from N-terminus to C-terminus. The Fc region may be a native sequence Fc region or a variant Fc region, preferably a native sequence Fc region. Preferably, the Fc region polypeptide is connected to the CH1 domain at the N-terminus via an immunoglobulin hinge region sequence. Preferably, the first VHH domain is a VHH according to the present invention. PD-L1 The second VHH domain preferably comprises the CDR1-3 sequence of the variable region shown in SEQ ID NO: 1, more preferably comprises the CDR1-3 sequence of SEQ ID NOs: 13-15; most preferably comprises the variable region sequence shown in SEQ ID NO: 1; and the second VHH domain is a VHH according to the present invention. VEGF The domain preferably comprises the CDR1-3 sequence of the variable region shown in SEQ ID NO: 2, more preferably comprises the CDR1-3 sequence of SEQ ID NOs: 16-18; most preferably comprises the variable region sequence shown in SEQ ID NO: 2.

[0122] In one embodiment of this aspect, the bispecific antibody of the invention comprises two first polypeptide chains and two second polypeptide chains, wherein the first antigen is PD-L1 and the second antigen is VEGF, wherein:

[0123] The two first polypeptide chains each comprise the amino acid sequence of SEQ ID NO:9, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and

[0124] The two second polypeptide chains respectively comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 10.

[0125] Most preferably, the two first polypeptide chains are identical and each comprises the amino acid sequence of SEQ ID NO: 9; and the two second polypeptide chains are identical and each comprises the amino acid sequence of SEQ ID NO: 10.

[0126] In one embodiment of this aspect, the bispecific antibody of the invention comprises two first polypeptide chains and two second polypeptide chains, wherein the first antigen is VEGF and the second antigen is PD-L1, wherein:

[0127] The two first polypeptide chains each comprise the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and

[0128] The two second polypeptide chains respectively comprise an amino acid sequence represented by SEQ ID NO: 12 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0129] Most preferably, the two first polypeptide chains are identical and each comprises the amino acid sequence of SEQ ID NO: 11; and the two second polypeptide chains are identical and each comprises the amino acid sequence of SEQ ID NO: 12.

[0130] In the above-mentioned embodiment of the four-chain antibody according to the present invention, the present invention also considers the embodiment in which the CH1 and CL domains in the first and second polypeptide chains are exchanged. In this embodiment with CH1 / CL domain exchange, preferably, the two Fc regions are connected to the CL domain of the Fab-like structure through the immunoglobulin hinge region.

[0131] II.. Production and purification of antibodies of the invention

[0132] In another aspect, the present invention provides a method for producing an antibody of the present invention. To produce an antibody of the present invention, the polypeptide chains of the antibody of the present invention can be obtained, for example, by solid-state peptide synthesis (eg, Merrifield solid phase synthesis) or recombinant production and assembled under suitable conditions.

[0133] For recombinant production, the polynucleotide encoding any one polypeptide chain and / or multiple polypeptide chains of the antibody can be separated and inserted into one or more vectors for further cloning and / or expression in a host cell. Using conventional methods, the polynucleotide can be easily isolated and sequenced. In one embodiment, a polynucleotide encoding one or more polypeptide chains of an antibody of the present invention is provided. In another embodiment, the present invention provides a vector comprising one or more polynucleotides of the present invention, preferably an expression vector. Therefore, in one embodiment, the present invention provides a method for producing an antibody of the present invention, the method comprising: culturing a host cell comprising the polypeptide chain encoding the antibody under conditions suitable for expressing the polypeptide chain of the antibody; and assembling the polypeptide chains to produce the antibody under conditions suitable for the assembly of the polypeptide chains into the antibody.

[0134] Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0135] Once an expression vector comprising one or more polynucleotides of the invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.

[0136] In one embodiment, the present invention also provides host cells comprising one or more polynucleotides of the present invention. In some embodiments, host cells comprising expression vectors of the present invention are provided. As used herein, the term "host cell" refers to any type of cell system that can be engineered to produce antibodies of the present invention. Host cells suitable for replication and support of antibody expression of the present invention are well known in the art. As required, such cells can be transfected or transduced with specific expression vectors, and a large number of cells containing vectors can be cultivated for inoculating large-scale fermenters to obtain sufficient amounts of antibodies of the present invention for clinical applications. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (HEK293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), CHO cells, NSO cells, myeloma cell lines such as YO, NS0, P3X63 and Sp2 / 0, etc. For a review of mammalian host cell lines suitable for producing proteins, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO, HEK293 or NSO cell.

[0137] The antibodies prepared by the methods described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. After purification, the purity of the antibodies of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc. The physical / chemical properties and / or biological activities of the antibodies provided herein can be identified, screened or characterized by a variety of assays known in the art.

[0138] In a preferred embodiment, the bispecific antibodies of the present invention exhibit good production properties in recombinant production in mammalian host cells such as CHO cells, in particular, good expression yield and good by-product spectrum. In one embodiment, the transient expression amount of the bispecific antibody of the present invention reaches 50 μg / mL or more, preferably 150 μg / mL or more; in another embodiment, the bispecific antibody of the present invention is purified by one-step protein A affinity chromatography, and the product purity is determined by SEC-HPLC, showing a purity of more than 95%, preferably 96%, 97%, 98% or 99%. Preferably, the determination of the transient expression amount is determined according to the method described in Example 3; the purity of the product after one-step protein A purification is determined according to the SEC-HPLC monomer purity identification method described in Example 3.4.

[0139] III. Pharmaceutical Compositions, Drug Combinations, and Kits

[0140] In one aspect, the present invention provides compositions, e.g., pharmaceutical compositions, comprising an antibody as described herein formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are physiologically compatible. The pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion). In some embodiments, the antibody of the present invention is the only active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise an antibody as described herein with more than one therapeutic agent.

[0141] In another aspect, the invention also provides a pharmaceutical combination comprising an antibody described herein and one or more therapeutic agents.

[0142] The therapeutic agent suitable for use in the pharmaceutical compositions and drug combinations of the present invention can be a therapeutic agent selected from any of the following categories (i)-(iv): (i) drugs that enhance antigen presentation (e.g., tumor antigen presentation); (ii) drugs that enhance effector cell responses (e.g., B cell and / or T cell activation and / or mobilization); (iii) drugs that reduce immunosuppression; (iv) drugs that have tumor suppressor effects.

[0143] The compositions of the present invention can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. Common preferred compositions are in the form of injectable solutions or infusible solutions. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, peritoneal (ip), intramuscular) injection. In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular, peritoneal or subcutaneous injection.

[0144] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intradermal, intraperitoneal, transtracheal, subcutaneous injection and infusion.

[0145] Therapeutic compositions should generally be sterile and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, dispersion, liposome or lyophilized form. Sterile injectable solutions can be prepared by adding the active compound (i.e., antibody) to a suitable solvent in the required amount, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile solvent containing a basic dispersion medium and other components. Coating agents such as lecithin etc. can be used. In the case of dispersions, the appropriate fluidity of the solution can be maintained by using a surfactant. Prolonged absorption of injectable compositions can be caused by including delayed absorption substances such as monostearate and gelatin in the composition.

[0146] The pharmaceutical composition of the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody of the present invention. "Therapeutically effective amount" refers to an amount that effectively achieves the desired therapeutic result at the required dosage and for the required period of time. The therapeutically effective amount can be varied according to a variety of factors such as disease state, age, sex and weight of the individual. The therapeutically effective amount is an amount in which any toxic or harmful effect is inferior to the therapeutically beneficial effect. Relative to untreated subjects, a "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80%. The ability of the antibodies of the present invention to inhibit measurable parameters (e.g., tumor volume) can be evaluated in an animal model system that predicts efficacy in human tumors.

[0147] A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the required dosage and for the required period of time. Typically, since a prophylactic dose is used in a subject before or at an earlier stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.

[0148] Kits comprising the antibodies described herein are also within the scope of the present invention. The kit may include one or more other elements, such as instructions for use; other reagents, such as markers or reagents for coupling; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

[0149] IV. Uses and methods of the molecules of the invention

[0150] In one aspect, the present invention provides in vivo and in vitro uses and methods of using the antibodies of the present invention.

[0151] In some embodiments, the uses and methods of the invention involve applying the antibodies of the invention in vivo and / or in vitro to:

[0152] - binds to human PD-L1 and human VEGF; or

[0153] - Blocking of human PD-L1 / PD-1 signaling pathway; or

[0154] - Neutralizes VEGF activity; or

[0155] - Inhibit tumor cell growth,

[0156] Or for preparing a medicament for any of the above uses.

[0157] In some embodiments, the antibodies of the present invention or pharmaceutical compositions comprising the antibodies of the present invention are used as drugs for treating and / or preventing diseases in individuals or as diagnostic tools for diseases, preferably, the individuals are mammals, more preferably humans.

[0158] In some embodiments, the present invention provides methods and uses of using the antibodies of the present invention, especially bispecific antibodies, to treat cancer, wherein the cancer may be a PD-L1 positive solid tumor or a blood cancer, for example, selected from head and neck squamous cell carcinoma, melanoma, renal cell carcinoma, non-small cell lung cancer, bladder cancer, urothelial carcinoma, gastric cancer, colon cancer, colorectal cancer, ovarian cancer, breast cancer, lung cancer, cervical cancer, glioblastoma, pancreatic cancer, prostate cancer, esophageal cancer, lymphoma, liver cancer, microsatellite unstable solid tumor. Preferably, the cancer is colorectal cancer, liver cancer, ovarian cancer, breast cancer, lung cancer.

[0159] In one aspect, the invention provides a diagnostic method for detecting the presence of a relevant antigen in a biological sample, such as serum, semen or urine or a tissue biopsy sample (e.g., from a hyperproliferative or cancerous lesion) in vitro or in vivo. The diagnostic method comprises: (i) contacting the sample (and optionally a control sample) with an antibody as described herein or administering the antibody to a subject under conditions that allow the interaction to occur and (ii) detecting the formation of a complex between the antibody and the sample (and optionally a control sample). The formation of a complex indicates the presence of the relevant antigen and can indicate the suitability or need for treatment and / or prevention as described herein.

[0160] In one embodiment, the invention provides a diagnostic kit comprising an antibody described herein and instructions for use.

[0161] The following examples are described to assist understanding of the present invention. The examples are not intended to and should not be interpreted in any way as limiting the scope of protection of the present invention. Example

[0162] Example 1 Preparation of raw materials

[0163] 1.1 Antigen preparation

[0164] The extracellular region of human PD-L1 (UniProtKB-Q9NZQ7), the extracellular region of human PD-1 (UniProtKB-Q15116) and human VEGF (UniProtKB-P15692) were synthesized by Universal Biosystems (Anhui) Co., Ltd. Each target fragment was amplified by PCR, and a His tag or hIgG1 Fc (amino acid sequence shown in SEQ ID NO: 3) was introduced at the C-terminus by primers, and then constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) by homologous recombination. The constructed recombinant protein expression vectors were transformed into Escherichia coli DH5α, cultured overnight at 37°C, and then the plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids for eukaryotic expression.

[0165] Recombinant protein VEGF-His, recombinant protein VEGF-Fc, recombinant protein PD-L1-His, recombinant protein PD-L1-Fc, recombinant protein PD-1-His and recombinant protein PD-1-Fc were all expressed by Expi293 transient expression system (ThermoFisher, A14635). For transient expression method, see Expi293 TMInstructions for the expression system kit. After 5-7 days of transfection, the cell expression supernatant was centrifuged at 15000g for 10 minutes. The obtained His-tagged recombinant protein expression supernatant was affinity purified with Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was eluted with gradient concentrations of imidazole. The eluted proteins were replaced into PBS buffer through ultrafiltration concentration tubes (Millipore, UFC901096); the obtained Fc-tagged recombinant protein expression supernatant was filtered through a 0.22μm filter membrane and purified by affinity chromatography using a Protein A / G affinity chromatography column. After purification, the target protein was eluted with 100mM glycine (pH3.0), then concentrated and replaced, and frozen at -80°C after SDS-PAGE identification and activity identification.

[0166] 1.2 Preparation of control antibodies

[0167] In this example, the anti-human VEGF antibody P30-10-26 (VHH VEGF -hIgG1 Fc, VHH VEGF The amino acid sequence is shown in SEQ ID NO: 2) from patent application CN202110995278.7; anti-human PD-L1 antibody D21-4 (VHH PD-L1 -hIgG1 Fc, VHH PD-L1 The amino acid sequence (shown in SEQ ID NO: 1) is derived from patent application PCT / CN2020 / 125301; the sequence of the anti-VEGF control antibody bevacizumab (Bevacizumab, abbreviated as Beva) is derived from Drug Bank (Drug Bank No: DB00112); the sequence of the anti-PD-L1 control antibody atezolizumab (Ate, abbreviated as Ate) is derived from Drug Bank (Drug Bank No: DB11595); the sequence of the control bispecific antibody W3256 against PD-L1 and VEGF is derived from WO2021147829A1; the sequence of the control bispecific antibody AK112 against PD-1 and VEGF is derived from the VP101 sequence in US20210340239A1. After the above amino acid sequences were converted into gene sequences, the target fragment gene was synthesized by General Biotechnology Co., Ltd. Each target fragment was amplified by PCR and then constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) by homologous recombination.

[0168] The antibodies were expressed using the transient transfection system (ExpiCHO). For transient transfection methods, see ExpiCHO. TMExpression System Kit User Guide. The expressed cell suspension was centrifuged at high speed and the supernatant was taken. The supernatant was filtered through a 0.22μm filter membrane and purified by affinity chromatography using a Protein A / G affinity column. After purification, the target protein was eluted with 100mM glycine salt (pH3.0), then concentrated and replaced, and frozen at -80℃ after SDS-PAGE identification and activity identification.

[0169] 1.3 Cell line construction

[0170] 1.3.1 Construction of huPD-L1-CHO-K cell line

[0171] CHO-K cells (Thermo, A1461801) were passaged to 5×10 6 / mL, and the next day, the constructed plasmid containing the full-length human PD-L1 (UniProtKB-Q9NZQ7) gene sequence was introduced into CHO-K cells using an electroporation kit (Invitrogen, MPK10096) and an electroporator (Invitrogen, MP922947). The electroporated cells were transferred to CD-CHO medium (Gibco, 10743029) and placed in a 37°C cell culture incubator for 48 hours. Then 2000 cells / well were plated in a 96-well plate, and a final concentration of 30 μM MSX (Millipore, GSS-1015-F) and GS supplement (Sigma, 58672C) were added for pressure screening. The single cell clone grown in the 96-well plate was picked, and the huPD-L1-CHO-K cell line was obtained by FACS identification after expansion culture.

[0172] 1.3.2 Construction of HEK293-VEGFR2-NFAT cell line

[0173] The pGL4.30 plasmid (Promega, #E8481) containing the NF-AT-re nucleic acid sequence was electroporated into HEK293 cells. HEK293-NFAT cell line was obtained. HEK293-NFAT cells were passaged to 2×10 5 / mL, the next day, the constructed plasmid containing the full-length human VEGFR2 gene sequence (NCBI Gene ID: 3791) was introduced into HEK293-NFAT cells using an electroporation kit (Invitrogen, MPK10096) and an electroporator (Invitrogen, MP922947). The electroporated cells were moved to DMEM culture medium (Gibco, 12634010) and placed in a 37°C cell culture incubator for 48h. Then 1000 cells / well were spread into a 96-well plate, and puromycin was added at a final concentration of 2μg / mL for pressure screening. The single cell clone grown in the 96-well plate was picked, and after expansion culture, the fluorescent signal was detected by adding a catalytic substrate of luciferase to identify the HEK293-VEGFR2-NFAT cell line.

[0174] 1.3.3 Construction of Jurkat-PD-1-NFAT cell line

[0175] The pGL4.30 plasmid (Promega, #E8481) containing the NF-AT-re nucleic acid sequence was electroporated into Jurkat cells. The Jurkat-NFAT cell line was obtained. On this basis, the full-length expression gene sequence of PD-1 (NCBIGene ID: 5133) was stably transfected, and the method was consistent with 1.3.2. A final concentration of 2 μg / mL of puromycin and 0.5 μg / mL of hygromycin B solution was added to select monoclonal cell lines. After expansion culture, the Jurkat-PD-1-NFAT cell line was obtained by FACS identification.

[0176] 1.3.4 Construction of CHO-PD-L1-CD3L cell line

[0177] The scFv sequence of OKT-3 (Drug Bank No: DB00075) was electroporated into CHO cells to obtain a CHO-CD3L stable cell line. On this basis, the full-length expression gene sequence of PD-L1 (NCBI Gene ID: 29126) was stably transfected. The method was consistent with 1.3.2. A final concentration of 30 mM methionine iminosulfone and 8 μg / mL puromycin were added to select monoclonal cell lines. After expanded culture, the CHO-PD-L1-CD3L cell line was obtained by FACS identification.

[0178] Example 2 Construction of anti-PD-L1 and VEGF bispecific antibodies

[0179] This example describes the structure and expression vector construction of an exemplary anti-PD-L1 and VEGF bispecific antibody (BsAb). Four constructs were designed and constructed: VHH PD-L1The amino acid sequence is from D21-4, and the amino acid sequence of its variable region is shown in SEQ ID NO: 1; VHH VEGF The amino acid sequence is from P30-10-26, and its variable region amino acid sequence is shown in SEQ ID NO: 2; the linker amino acid sequence is GGGGSGGGGSGGGGS. Exemplary BsAb constructs are shown in Table 1, and the corresponding amino acid sequences are provided in Table 2.

[0180] Construct BsAb10: Contains two identical polypeptide chains, including the anti-PD-L1 VHH domain, IgG1 heavy chain hinge region, IgG1 heavy chain Fc, linker and anti-VEGF VHH domain from N-terminus to C-terminus. BsAb10 has Figure 1A form.

[0181] Construct BsAb11: Contains two identical polypeptide chains, including the anti-PD-L1 VHH domain, linker, anti-VEGF VHH domain, IgG1 heavy chain hinge region and IgG1 heavy chain Fc from N-terminus to C-terminus. BsAb11 has Figure 1B form.

[0182] Construct BsAb12: Contains two identical first polypeptide chains, including the anti-PD-L1 VHH domain and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Fc) from the N-terminus to the C-terminus; contains two identical second polypeptide chains, including the anti-VEGF VHH domain and the antibody kappa light chain CL domain from the N-terminus to the C-terminus. BsAb12 has Figure 1C form.

[0183] Construct BsAb13: Contains two identical first polypeptide chains, including the anti-VEGF VHH domain and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Fc) from the N-terminus to the C-terminus; contains two identical second polypeptide chains, including the anti-PD-L1 VHH domain and the antibody kappa light chain CL domain from the N-terminus to the C-terminus. BsAb13 has Figure 1D form.

[0184] According to the structure of the construct, the fragments of each antibody variable region and constant region were amplified by PCR, and the fragments were connected by overlapping extension PCR, and then constructed into the modified eukaryotic expression vector plasmid pcDNA3.4 (Invitrogen) by homologous recombination to form a complete construct polypeptide chain full-length gene. The constructed vectors containing the full-length gene of the construct polypeptide chain were transformed into Escherichia coli DH5α and cultured overnight at 37°C. The plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain an endotoxin-free construct polypeptide chain plasmid for eukaryotic expression.

[0185] Table 1 Constructs of anti-PD-L1 and VEGF bispecific antibodies

[0186] Antibody Name First polypeptide chain Second polypeptide chain BsAb10 <![CDATA[VHH PD-L1 -Hinge-CH2-CH3-Linker-VHH VEGF ]]> NA BsAb11 <![CDATA[VHH PD-L1 -Linker-VHH VEGF -Hinge-CH2-CH3]]> NA BsAb12 <![CDATA[VHH PD-L1 -CH1-Hinge-CH2-CH3]]> <![CDATA[VHH VEGF -CL]]> BsAb13 <![CDATA[VHH VEGF -CH1-Hinge-CH2-CH3]]> <![CDATA[VHH PD-L1 -CL]]>

[0187] Table 2 Amino acid sequences of anti-PD-L1 and VEGF bispecific antibodies

[0188] Antibody Name First polypeptide chain amino acid sequence Second polypeptide chain amino acid sequence BsAb10 SEQ ID NO:7 NA BsAb11 SEQ ID NO:8 NA BsAb12 SEQ ID NO:9 SEQ ID NO:10 BsAb13 SEQ ID NO:11 SEQ ID NO:12

[0189] Example 3 Expression, purification, and physicochemical property analysis of anti-PD-L1 and VEGF bispecific antibodies

[0190] 3.1 Expression and purification of anti-PD-L1 and VEGF bispecific antibodies

[0191] The construct of Example 2 was expressed using the ExpiCHO transient expression system (Thermo Fisher, A29133) as follows: On the day of transfection, the cell density was confirmed to be 7×10 6 Up to 1×10 7 The cell viability was >98%. At this time, the cells were adjusted to a final concentration of 6×10 6 OptiPRO pre-cooled at 4°C TM The target plasmid was diluted in SFM (1 μg of plasmid was added to 1 mL of the culture medium) and OptiPRO TM Dilute ExpiFectamine in SFM TM CHO, and then mix the two in equal volumes and gently pipette to mix to prepare ExpiFectamine TM Incubate the CHO / plasmid DNA mixture at room temperature for 1-5 minutes, slowly add it to the prepared cell suspension while gently shaking, and finally place it in a cell culture shaker and culture it at 37°C and 8% CO2. 18-22 hours after transfection, add ExpiCHO to the culture medium. TMEnhancer and ExpiCHO TM Feed, place the shake flask in a 32°C shaker and continue culturing under 5% CO2 conditions. On the 5th day after transfection, add the same volume of ExpiCHO TM Feed, slowly add and gently mix the cell suspension. 7-15 days after transfection, the cell culture supernatant expressing the target protein was centrifuged at 15000g for 10 minutes, and the resulting supernatant was affinity purified using MabSelect SuRe LX (GE, 17547403), and then the target protein was eluted with 100mM sodium acetate (pH3.0), followed by neutralization with 1M Tris-HCl, and finally the resulting protein was replaced into PBS buffer through an ultrafiltration concentration tube (Millipore, UFC901096).

[0192] 3.2 Concentration determination of anti-PD-L1 and VEGF bispecific antibodies

[0193] The purified bispecific antibody of Example 3.1 was measured for concentration using a validated ultra-micro spectrophotometer (Hangzhou Aosheng Instrument Co., Ltd., Nano-300). The measured A280 value was divided by the theoretical extinction coefficient of the antibody as the antibody concentration value for subsequent studies. After passing the quality inspection, the antibody was packaged and stored at -80°C.

[0194] 3.3 SDS-PAGE identification of anti-PD-L1 and VEGF bispecific antibodies

[0195] Preparation of non-reducing solution: 1 μg of candidate bispecific antibody and reference product IPI (IPI is the abbreviation of Ipilimumab, prepared by the method of Example 3.1) was added with 5×SDS loading buffer and 40 mM iodoacetamide, heated in a dry bath at 75°C for 10 min, cooled to room temperature, centrifuged at 12000 rpm for 5 min, and the supernatant was taken. Preparation of reducing solution: 2 μg of candidate bispecific antibody and reference product IPI were added with 5×SDS loading buffer and 5 mM DTT, heated in a dry bath at 100°C for 10 min, cooled to room temperature, centrifuged at 12000 rpm for 5 min, and the supernatant was taken. The supernatant was added to Bis-tris 4-15% gradient gel (purchased from GenScript), and electrophoresis was performed at a constant voltage of 110V. When Coomassie brilliant blue migrated to the bottom of the gel, the operation was stopped, and the gel slice was taken out and placed in Coomassie brilliant blue staining solution for 1-2 hours. The staining solution was discarded, and a destaining solution was added. The destaining solution was replaced 2-3 times as needed. After destaining until the gel background was transparent, it was stored in deionized water. After destaining, it was scanned with an EPSON V550 color scanner, and the purity of the reduced and non-reduced bands was calculated by ImageJ according to the peak area normalization method.

[0196] The results are as follows Figure 2A-2BAs shown: the bands of the candidate bispecific antibody and the reference IPI reduced gel and non-reduced gel are consistent with the expected size, and the purity of the reduced gel is greater than 95%.

[0197] 3.4 SEC-HPLC Monomer Purity Identification of Anti-PD-L1 and VEGF Bispecific Antibodies

[0198] Material preparation: 1. Mobile phase: 150mmol / L phosphate buffer, pH 7.4; 2. Sample preparation: Candidate bispecific antibodies were diluted to 0.5mg / mL with mobile phase solution. Agilent HPLC 1100 column (XBridge BEH SEC 3.5μm, 7.8mm ID×30cm, Waters) was used with a flow rate of 0.8mL / min, an injection volume of 20μL, and a VWD detector wavelength of 280nm and 214nm.

[0199] The SEC-HPLC results of the candidate bispecific antibody are as follows: The percentages of high molecular weight polymers, antibody monomers and low molecular weight substances in the sample were calculated by the area normalization method, and the results are shown in Figures 3A-3D and Table 3.

[0200] Table 3 Physicochemical data of anti-PD-L1 and VEGF bispecific antibodies

[0201]

[0202] Example 4 Affinity activity analysis of anti-PD-L1 and VEGF bispecific antibodies

[0203] 4.1 Detection of the binding ability of candidate bispecific antibodies to recombinant protein VEGF-His based on ELISA method

[0204] The recombinant protein VEGF-His was coated on a 96-well ELISA plate at 4°C overnight. The next day, the plate was washed three times with PBST and blocked with 5% skim milk for 2 hours. After washing the plate three times with PBST, different concentrations of candidate bispecific antibodies, control antibodies bevacizumab and P30-10-26 were added and incubated for 1 hour. After that, after washing three times with PBST, the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab79225) was added and incubated for 1 hour. After the incubation was completed, the plate was washed six times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the plate was read at OD450 using an enzyme reader (Molecular Devices, SpecterMax190). Using PRISM TM The data were analyzed by GraphPad Software, San Diego, CA, and the EC was calculated.50 value.

[0205] ELISA binding assay results Figure 4 As shown in Table 4, the bispecific antibodies BsAb11, BsAb12 and BsAb13 showed an ability to bind to VEGF that was superior to that of the control antibody bevacizumab, and was comparable to that of the control antibody P30-10-26.

[0206] 4.2 Detection of the binding ability of candidate bispecific antibodies to recombinant protein PD-L1-His based on ELISA method

[0207] The recombinant protein PD-L1-His was coated on a 96-well ELISA plate at 4°C overnight. The next day, the plate was washed three times with PBST and blocked with 5% skim milk for 2 hours. After washing the plate three times with PBST, different concentrations of candidate bispecific antibodies and control antibody D21-4 were added and incubated for 1 hour. After that, after washing three times with PBST, the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab79225) was added and incubated for 1 hour. After the incubation was completed, the plate was washed six times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the plate was read at OD450 using an enzyme reader (Molecular Devices, SpecterMax 190). Using PRISM TM The data were analyzed by GraphPad Software, San Diego, CA, and the EC was calculated. 50 value.

[0208] ELISA binding assay results Figure 5 As shown in Table 4, the candidate bispecific antibodies all showed comparable ability to bind to PD-L1 as the control antibody D21-4.

[0209] 4.3 Detection of the Binding Ability of Candidate Bispecific Antibodies to huPD-L1-CHO-K Based on FACS Method

[0210] The huPD-L1-CHO-K cells in the exponential growth phase were collected and centrifuged at 300 g to remove the supernatant. The cells were resuspended in FACS buffer (PBS containing 1% BSA), counted, and the cell suspension density was adjusted to 2×10 6 / mL. Subsequently, huPD-L1-CHO-K cells were added to a 96-well round-bottom plate at 100 μL per well, and the supernatant was removed by centrifugation at 300g. Different concentrations of candidate bispecific antibodies and control antibody D21-4 dilutions were added to the corresponding wells, the cells were resuspended and incubated at 4°C for 30 minutes. After washing the incubated cell mixture three times, PE-labeled anti-human-IgG-Fc flow cytometry antibody (Abcam, 98596) was added, and the mixture was resuspended and incubated at 4°C for 30 minutes. After washing the incubated cell mixture three times, 200 μL of FACS buffer was added to resuspend the cells, and the cells were detected and analyzed by flow cytometer (Beckman, CytoFLEX AOO-1-1102). Using PRISM TM The data were analyzed by GraphPad Software, San Diego, CA, and the EC was calculated. 50 value.

[0211] FACS binding assay results Figure 6 As shown in Table 4, the bispecific antibodies BsAb10 and BsAb12 exhibited better binding ability to PD-L1 expressed on cells than the control antibody D21-4.

[0212] 4.4 ELISA-based detection of the ability of candidate bispecific antibodies to simultaneously bind to VEGF and PD-L1

[0213] The recombinant protein VEGF-Fc was coated on a 96-well ELISA plate at 4°C overnight. The next day, the plate was washed three times with PBST and blocked with 5% skim milk for 2 hours. After washing the plate three times with PBST, different concentrations of candidate bispecific antibodies were added and incubated for 1 hour. After that, the recombinant protein PD-L1-His was added after washing three times with PBST. After washing the plate three times with PBST, Anti-6×His-HRP (Proteintech, HRP-660050) was added and incubated at room temperature for 50 minutes. After completion, the plate was washed 12 times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the plate was read at OD450 using an enzyme reader (Molecular Devices, SpecterMax 190). Using PRISM TM The data were analyzed by GraphPad Software, San Diego, CA, and the EC was calculated. 50 value.

[0214] The recombinant protein PD-L1-Fc was coated on a 96-well ELISA plate at 4°C overnight. The next day, the plate was washed three times with PBST and blocked with 5% skim milk for 2 hours. After washing the plate three times with PBST, different concentrations of candidate bispecific antibodies were added and incubated for 1 hour. After that, the recombinant protein VEGF-His was added after washing three times with PBST. After washing the plate three times with PBST, Anti-6×His-HRP (Proteintech, HRP-660050) was added and incubated at room temperature for 50 minutes. After completion, the plate was washed 12 times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the plate was read at OD450 using an enzyme reader (Molecular Devices, SpecterMax 190). Using PRISM TM The data were analyzed by GraphPad Software, San Diego, CA, and the EC was calculated. 50 value.

[0215] ELISA binding assay results Figure 7A-7B , the bispecific antibody BsAb10 exhibited the ability to bind to PD-L1 and VEGF simultaneously.

[0216] Table 4 Antigen binding data of anti-PD-L1 and VEGF bispecific antibodies

[0217]

[0218] Example 5 Detection of PD-1 / PD-L1 blocking activity of anti-PD-L1 and VEGF bispecific antibodies based on reporter gene method

[0219] This example uses a luciferase reporter gene system to detect the activity of candidate anti-PD-L1 and VEGF bispecific antibodies in blocking the PD-1 / PD-L1 signaling pathway. The binding of PD-1 to PD-L1 can block CD3 downstream signal transduction, thereby inhibiting the expression of luciferase. When anti-PD-L1 and VEGF bispecific antibodies are added, the blocking effect is reversed, and luciferase expression is stimulated with antibodies of different concentration gradients. A fluorescence reading curve with antibody concentration dependence is obtained, thereby evaluating the blocking activity of the antibody. This example uses a luciferase reporter gene system to detect the activity of candidate anti-PD-L1 and VEGF bispecific antibodies in blocking the PD-1 / PD-L1 signaling pathway. The specific method is as follows:

[0220] Add 50 μL of 2×10 4 CHO-PD-L1-CD3L cells and 1×10 5Jurkat-PD-1-NFAT cells were added per well. 50 μL of the candidate bispecific antibody and control antibody D21-4 were added and incubated at 37°C for 6 hours. 50 μL Bright-Lite (vazyme, catalog number: DD1204-03) was added to each well, incubated in the dark for 10 minutes, and the fluorescence signal was detected.

[0221] The results of the blocking activity test are as follows: Figure 8 As shown, both bispecific antibodies BsAb10 and BsAb12 exhibited PD-1 / PD-L1 blocking activity comparable to that of the control antibody D21-4.

[0222] Example 6 Detection of VEGF Neutralization Activity of Anti-PD-L1 and VEGF Bispecific Antibodies Based on Reporter Gene Method

[0223] In this embodiment, in order to determine the function of the candidate bispecific antibody in neutralizing the VEGF-VEGFR2 signaling pathway, the HEK293-VEGFR2-NFAT cell line (VEGF recombinant protein is added to the cell line culture system, the intracellular NFAT luciferase reporter gene transcription and expression is activated through the VEGF-VEGFR2 signal axis, and the catalytic substrate of luciferase is added to generate a fluorescent signal) is used as a material to detect the ability of the candidate bispecific antibody to neutralize VEGF-VEGFR2 binding and thus block the expression of the downstream NFAT luciferase reporter gene. The specific implementation method is as follows:

[0224] HEK293-VEGFR2-NFAT cell line was adjusted to 4×10 5 / mL, add 100μL per well to a new 96-well cell culture plate and place it in a 37°C cell culture incubator. At the same time, use DMEM medium to gradient dilute the candidate bispecific antibody and control antibody P30-10-26, add 60ng / mL VEGF-Fc, mix and incubate at room temperature for 30min. Subsequently, the gradient diluted antibody and VEGF-Fc mixture after co-incubation is added to the 96-well cell culture plate and cultured in a 37°C incubator for 18h. After the incubation, add 30μL of luciferase substrate Bright-Lite (Vazyme, DD1204-03) to each well, shake for 2min and detect the fluorescence value of the 96-well plate.

[0225] The results are as follows Fig. 9 As shown in Table 5, the effect of bispecific antibody BsAb10 in neutralizing VEGF is significantly better than that of control antibody P30-10-26, and the effect of bispecific antibodies BsAb11, BsAb12 and BsAb13 in neutralizing VEGF is comparable to that of control antibody P30-10-26.

[0226] Table 5 VEGF neutralizing activity of anti-PD-L1 and VEGF bispecific antibodies

[0227] Antibody Name <![CDATA[IC 50 (nM)]]> BsAb10 0.8656 BsAb11 1.3760 BsAb12 1.0900 BsAb13 1.0290 P30-10-26 0.9824

[0228] Example 7 In vivo tumor inhibition experiment of anti-PD-L1 and VEGF bispecific antibodies

[0229] Female NOG mice (purchased from Shanghai Weitong Lihua, strain: NOD.Cg-Prkdc) were used at 6-8 weeks of age. scid Iq tm1Sug / JicCrl), the experimental mice were kept in an independent ventilation box with constant temperature and humidity, the temperature of the breeding room was 21-24°C, and the humidity was 30-53%. huPD-L1-COLO205 cells (a stable cell line overexpressing human PD-L1 obtained by stably transfecting PD-L1 (Gene ID: 29126) into COLO205 cells (purchased from Beina Biotechnology, catalog number: BNCC338682)) were inoculated with 3×10 6 Each mouse was injected subcutaneously on the right back (day 0), and then randomly divided into groups (6 mice in each group): PBS treatment group, Atezolizumab administration group, Bevacizumab administration group, Atezolizumab + Bevacizumab combined administration group, W3256 administration group, AK112 administration group and bispecific antibody BsAb10 administration group. Each administration group was set up with a high-dose group, and the bispecific antibody BsAb10 administration group and AK112 administration group also had medium and low-dose groups. On the second day after tumor loading, PBMC (C2106025) was injected through the tail vein, and each mouse was injected with 3×10 6 PBMC cells were added, and the first administration was performed 2 hours later. The administration was then performed twice a week by intraperitoneal injection (ip) for a total of 3 weeks.

[0230] Observe and record the length (mm) and width (mm) of the tumor at any time, and calculate its tumor volume (V). The calculation method is V = (length × width 2 ) / 2, tumor inhibition rate TGI (%) = (1-average tumor volume of drug administration group / average tumor volume of PBS treatment group) × 100%. The tumor inhibition results are shown in Fig.10As shown in Table 6. From the results, it can be seen that all the drug-treated groups showed tumor growth inhibition compared with the PBS-treated group; the Atezolizumab + Bevacizumab combination group was superior to the Atezolizumab monotherapy group and the Bevacizumab monotherapy group, proving that in this model, anti-PD-L1 and anti-VEGF can show combined efficacy; at a high dose of 1.87 μM, all mice in the bispecific antibody BsAb10-treated group showed a trend of complete tumor remission on day 13, and the tumor inhibition rate was 89.75%, which was significantly better than the control antibody W3256-treated group (tumor inhibition rate was 80.74%) and the control antibody AK112-treated group (tumor inhibition rate was 81.7%). drug group (tumor inhibition rate of 54.79%) and Atezolizumab + Bevacizumab combination administration group (tumor inhibition rate of 76.52%); at the middle dose of 0.47 μM, on the 13th day, all mice in the bispecific antibody BsAb10 administration group also showed a trend of complete tumor remission, and the tumor inhibition rate was 83.68%, which was significantly better than the control antibody AK112 administration group (tumor inhibition rate of 35.69%); at the low dose of 0.094 μM, the bispecific antibody BsAb10 showed a tumor inhibition effect of 36.26% on the 23rd day, which was significantly better than the control bispecific antibody AK112 (tumor inhibition rate of 14.25%).

[0231] Table 6 Tumor inhibition rate TGI (%) of different drug administration groups

[0232]

[0233] Sequence Listing Overview

[0234] This application is accompanied by a sequence listing containing many nucleic acid and amino acid sequences. The following table provides an overview of the sequences contained.

[0235]

[0236]

[0237] Sequence Listing <110> Sanyou Biopharmaceuticals (Shanghai) Co., Ltd. <120> Anti-PD-L1 and VEGF bispecific antibodies and their applications <130> PF 210783CNI <160> 18 <170> PatentIn version 3.3 <210> 1 <211> 116 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Thr Asp Arg Asn Ile Asn 20 25 30 Thr Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val 35 40 45 Gly Thr Ile Phe Ile Asp Leu Asn Thr Ile Val Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser 115 <210> 2 <211> 129 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly 100 105 110 Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser <210> 3 <211> 232 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 3 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 4 <211> 98 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 4 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val <210> 5 <211> 107 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 5 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 6 <211> 106 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 6 Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 7 <211> 492 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Thr Asp Arg Asn Ile Asn 20 25 30 Thr Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val 35 40 45 Gly Thr Ile Phe Ile Asp Leu Asn Thr Ile Val Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 115 120 125 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 130 135 140 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 145 150 155 160 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 165 170 175 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 180 185 190 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 195 200 205 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 210 215 220 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 225 230 235 240 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 245 250 255 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 260 265 270 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 275 280 285 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 290 295 300 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 305 310 315 320 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 325 330 335 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly 340 345 350 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 355 360 365 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 370 375 380 Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr Ala Ile Gly Trp Phe 385 390 395 400 Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ser Cys Ile Gly Ser 405 410 415 Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 420 425 430 Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser 435 440 445 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ala Gly Ser Pro 450 455 460 Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly Leu Tyr Glu Tyr Asp 465 470 475 480 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 485 490 <210> 8 <211> 492 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Thr Asp Arg Asn Ile Asn 20 25 30 Thr Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val 35 40 45 Gly Thr Ile Phe Ile Asp Leu Asn Thr Ile Val Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu 145 150 155 160 Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 165 170 175 Glu Gly Val Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala 180 185 190 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 195 200 205 Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp 225 230 235 240 His Tyr Gly Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val 245 250 255 Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 260 265 270 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 275 280 285 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 290 295 300 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 305 310 315 320 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 325 330 335 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 340 345 350 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 355 360 365 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 370 375 380 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 385 390 395 400 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 405 410 415 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 420 425 430 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 435 440 445 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 450 455 460 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 465 470 475 480 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 485 490 <210> 9 <211> 446 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Thr Asp Arg Asn Ile Asn 20 25 30 Thr Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val 35 40 45 Gly Thr Ile Phe Ile Asp Leu Asn Thr Ile Val Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 10 <211> 236 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly 100 105 110 Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 11 <211> 459 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly 100 105 110 Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 130 135 140 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 145 150 155 160 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 165 170 175 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 180 185 190 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 195 200 205 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 210 215 220 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 225 230 235 240 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 245 250 255 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 260 265 270 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 275 280 285 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 290 295 300 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 305 310 315 320 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 325 330 335 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 340 345 350 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 355 360 365 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 370 375 380 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 385 390 395 400 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 405 410 415 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 420 425 430 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 435 440 445 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 12 <211> 223 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Thr Asp Arg Asn Ile Asn 20 25 30 Thr Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val 35 40 45 Gly Thr Ile Phe Ile Asp Leu Asn Thr Ile Val Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 115 120 125 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 130 135 140 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 145 150 155 160 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 165 170 175 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 180 185 190 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 195 200 205 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 13 <211> 10 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 13 Arg Thr Asp Arg Asn Ile Asn Thr Met His 1 5 10 <210> 14 <211> 9 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 14 Thr Ile Phe Ile Asp Leu Asn Thr Ile 1 5 <210> 15 <211> 8 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 15 Asp Val Ser Gly Tyr Gly Arg Ala 1 5 <210> 16 <211> 10 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 16 Gly Phe Gly Leu Asp Tyr Tyr Ala Ile Gly 1 5 10 <210> 17 <211> 10 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 17 Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn 1 5 10 <210> 18 <211> 20 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 18 Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly Leu Tyr 1 5 10 15 Glu Tyr Asp Tyr 20

Claims

1. A bispecific antibody that specifically binds to PD-L1 and VEGF, wherein the antibody comprises a VHH domain that specifically binds to a first antigen and a VHH domain that specifically binds to a second antigen, wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF; in, The VHH domain that specifically binds to VEGF comprises a CDR1 sequence shown in SEQ ID NO: 16, a CDR2 sequence shown in SEQ ID NO: 17, and a CDR3 sequence shown in SEQ ID NO: 18; And the VHH domain that specifically binds to PD-L1 comprises the CDR1 sequence shown in SEQ ID NO: 13, the CDR2 sequence shown in SEQ ID NO: 14, and the CDR3 sequence shown in SEQ ID NO:

15.

2. The bispecific antibody according to claim 1, wherein The VHH domain that specifically binds to VEGF comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2, or an amino acid sequence that has 1-10 amino acid additions, deletions and / or substitutions compared to SEQ ID NO:

2.

3. The bispecific antibody according to claim 1, wherein The VHH domain that specifically binds VEGF comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

2.

4. The bispecific antibody according to claim 1, wherein The VHH domain that specifically binds VEGF comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

2.

5. The bispecific antibody according to claim 1, wherein The VHH domain that specifically binds VEGF comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

2.

6. The bispecific antibody according to claim 1, wherein The VHH domain that specifically binds VEGF comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:

2.

7. The bispecific antibody according to claim 1, wherein The VHH domain that specifically binds to VEGF comprises the amino acid sequence of SEQ ID NO:

2.

8. The bispecific antibody according to claim 1, wherein The VHH domain that specifically binds to VEGF consists of the amino acid sequence shown in SEQ ID NO:

2.

9. The bispecific antibody according to claim 1, wherein The VHH domain that specifically binds to PD-L1 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, or an amino acid sequence that has 1 to 10 amino acid additions, deletions and / or substitutions compared to SEQ ID NO:

1.

10. The bispecific antibody according to claim 1, wherein The VHH domain that specifically binds to PD-L1 comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:

1.

11. The bispecific antibody according to claim 1, wherein The VHH domain that specifically binds to PD-L1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

1.

12. The bispecific antibody according to claim 1, wherein The VHH domain that specifically binds to PD-L1 comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

1.

13. The bispecific antibody according to claim 1, wherein The VHH domain that specifically binds to PD-L1 comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:

1.

14. The bispecific antibody according to claim 1, wherein The VHH domain that specifically binds to PD-L1 comprises the amino acid sequence of SEQ ID NO:

1.

15. The bispecific antibody according to claim 1, wherein The VHH domain that specifically binds to PD-L1 consists of the amino acid sequence shown in SEQ ID NO:

1.

16. The bispecific antibody according to claim 1, wherein The bispecific antibody comprises a linker, and the linker comprises 10-20 amino acids in length.

17. The bispecific antibody according to claim 16, wherein The linker comprises the amino acid sequence (G4S)3.

18. The bispecific antibody according to claim 1, wherein the bispecific antibody comprises an Fc region of an immunoglobulin, wherein: The Fc region is an IgG Fc region. The bispecific antibody according to claim 18 , wherein the Fc region is the Fc domain of human IgG1, IgG2, IgG3 or IgG4.

20. The bispecific antibody according to claim 18, wherein the Fc region carries an immunoglobulin hinge region sequence at the N-terminus.

21. The bispecific antibody according to claim 1, wherein the bispecific antibody comprises a polypeptide chain comprising, from N-terminus to C-terminus, a first VHH domain, an Fc region, a linker and a second VHH domain, wherein the first VHH domain and the second VHH domain specifically bind to the first antigen and the second antigen, respectively, Wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF.

22. The bispecific antibody according to claim 21, wherein The first antigen is PD-L1 and the second antigen is VEGF.

23. The bispecific antibody according to claim 1, wherein the bispecific antibody comprises a polypeptide chain comprising, from N-terminus to C-terminus, a first VHH domain, a linker, a second VHH domain and an Fc region, wherein the first VHH domain and the second VHH domain specifically bind to the first antigen and the second antigen, respectively, Wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF.

24. The bispecific antibody according to claim 23, wherein The first antigen is PD-L1 and the second antigen is VEGF.

25. The bispecific antibody according to claim 1, wherein the bispecific antibody comprises a first and a second polypeptide chain, wherein: The first polypeptide chain comprises a first VHH domain, a CH1 domain and an Fc region from the N-terminus to the C-terminus; the second polypeptide chain comprises a second VHH domain and a CL domain from the N-terminus to the C-terminus; wherein the first VHH domain and the CH1 domain in the first polypeptide chain are paired with the first VHH domain and the CL domain in the second polypeptide chain to form a Fab-like structure; wherein the first VHH domain and the second VHH domain specifically bind to the first antigen and the second antigen, respectively, Wherein the first antigen and the second antigen are different from each other and are independently selected from PD-L1 and VEGF.

26. The bispecific antibody according to claim 25, wherein The first antigen is PD-L1 and the second antigen is VEGF.

27. The bispecific antibody according to claim 1, wherein the bispecific antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:7; or comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:

8.

28. The bispecific antibody according to claim 1, wherein the bispecific antibody comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:7; or comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:

8.

29. The bispecific antibody according to claim 1, wherein the bispecific antibody comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:7; or comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:

8.

30. The bispecific antibody according to claim 27, wherein the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO:

7.

31. The bispecific antibody according to claim 27, wherein the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO:

8.

32. The bispecific antibody according to claim 1, wherein the bispecific antibody comprises a first and a second polypeptide chain, wherein: The first polypeptide chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:9; and The second polypeptide chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:

10.

33. The bispecific antibody according to claim 32, wherein the first polypeptide chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:9; and the second polypeptide chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:

10.

34. The bispecific antibody according to claim 32, wherein the first polypeptide chain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:9; and the second polypeptide chain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:

10.

35. The bispecific antibody according to claim 32, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:9; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:

10.

36. A polynucleotide encoding the bispecific antibody of any one of claims 1-35.

37. A vector comprising the polynucleotide of claim 36.

38. The vector according to claim 37, which is an expression vector.

39. A host cell comprising the polynucleotide of claim 36 or the vector of claim 37 or 38, wherein: The host cell is a mammalian cell.

40. A method for producing the bispecific antibody of any one of claims 1 to 35, the method comprising: Cultivating a host cell containing a protein encoding the polypeptide chain of the antibody under conditions suitable for expressing the polypeptide chain of the antibody; and assembling the polypeptide chains under conditions suitable for the assembly of the polypeptide chains into the antibody to produce the antibody.

41. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-35 and a pharmaceutically acceptable carrier.

42. Use of the bispecific antibody according to any one of claims 1 to 35 for non-diagnostic purposes in vitro - binds to human PD-L1 and human VEGF; or - Neutralizes VEGF activity.

43. Use of the bispecific antibody according to any one of claims 1 to 35 for preparing a composition for any of the following uses: - binds to human PD-L1 and human VEGF; or - Neutralizes VEGF activity.

44. Use according to claim 43, wherein the composition is used as a diagnostic tool.

45. Use of the bispecific antibody according to any one of claims 1 to 35 or the pharmaceutical composition according to claim 41 in the preparation of a medicament for treating a disease in an individual, wherein the disease is a PD-L1 positive solid tumor or a hematological tumor, The PD-L1 positive solid tumor or blood tumor is selected from head and neck squamous cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, urothelial carcinoma, gastric cancer, colon cancer, ovarian cancer, breast cancer, lung cancer, cervical cancer, glioblastoma, pancreatic cancer, prostate cancer, esophageal cancer, lymphoma and liver cancer.

46. ​​The use according to claim 45, wherein the PD-L1 positive solid tumor or blood tumor is non-small cell lung cancer or colorectal cancer.

47. The use according to claim 45, wherein the PD-L1 positive solid tumor is a microsatellite unstable solid tumor.

48. The use according to claim 45, wherein the individual is a human.

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