WO2023165516A1

WO2023165516A1 - Anti-pd-l1 and vegf bispecific antibody and use thereof

Info

Publication number: WO2023165516A1
Application number: PCT/CN2023/079017
Authority: WO
Inventors: 张震; 刘婵娟; 郎国竣; 王立燕; 李文; 李佳星; 苏飞
Original assignee: 三优生物医药(上海)有限公司
Priority date: 2022-03-02
Filing date: 2023-03-01
Publication date: 2023-09-07
Also published as: CN116731188A

Abstract

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

Description

Anti-PD-L1 and VEGF bispecific antibody and its application

technical field

The invention belongs to the field of biomedicine, and in particular relates to a bispecific antibody targeting PD-L1 and VEGF and its preparation method and application.

Background technique

PD-L1 belongs to a type of transmembrane protein on the cell membrane, and is expressed on T cells, B cells and other immune cells and tumor cells. Tasuku Honjo et al. discovered and proved that when PD-L1 on the tumor cell membrane combines with 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 have a killing effect on tumor cells , the immune function of the body is suppressed (Chamoto, K., Al-Habsi, M., & Honjo, T. (2017). Current topics in microbiology and immunology, 410, 75-97). For the first time, Chen Lieping cured about 60% of head and neck cancer mice by combining PD-L1 blocking antibody with T cells (Chen, L et al. (2003). Cancer research, 63 (19), 6501-6505.). At present, 10 anti-PD-1 or PD-L1 monoclonal antibodies have been approved for marketing in China, and the indications are for PD-L1 positive NSCLC, head and neck squamous cell carcinoma and melanoma and other solid tumors (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 a low response rate of anti-PD-1/PD-L1 antibodies, which currently only have an effect on less than 40% of solid tumors, and 30% of patients in the A certain amount of drug resistance appears after treatment.

The most critical cytokine to promote angiogenesis is VEGF-A (also known as VEGF). VEGF activates VEGFR2 (the main receptor tyrosine kinase receptor that mediates angiogenesis) to promote mitosis of vascular endothelial cells and increased vascular permeability, thereby promoting new vessel sprouting. 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 vascular proliferation (Ferrara, N. (2010). Mol Biol Cell 21 (5): 687-690.). The anti-VEGF monoclonal antibody Bevacizumab (Bevacizumab) has good efficacy and target safety in tumor treatment and eye diseases related to abnormal blood vessel proliferation (Pfisterer, J., et al. (2020). Lancet Oncol 21(5):699-709. Bhandari, S., et al. (2020). Ophthalmology 127(5):608-615.).

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 activation and activation, improve tumor microenvironment, and increase anti-PD-1/PD-L1 Drug response rate, further through T cell-mediated tumor killing, restore tumor immune function (Socinski, M.A., et al.(2018).N Engl J Med 378(24):2288-2301.N Engl J Med.2018Jun 14 ; 378(24):2288-2301. N Engl J Med 2020; 382:1894-1905). The latest clinical trial results confirm that anti-VEGF monoclonal antibody combined with anti-PD-L1 monoclonal antibody is more effective than chemical drugs or anti-VEGF monoclonal antibody or anti-PD-L1 monoclonal antibody in the treatment of liver cancer, lung cancer, endometrial cancer and kidney cancer. The superior efficacy of L1 monoclonal antibody has achieved a very significant clinical breakthrough. At present, both anti-PD-L1/VEGF monoclonal antibody combination and anti-PD-1/VEGF bispecific antibody have progressed to clinical phase III, and treatment-related adverse reactions were 91% and 63.4%, respectively. 40% and 19.5%, indicating that compared with monoclonal antibodies, bispecific antibodies have more potential in reducing toxic side effects and dosage, and have better safety; at the same time, anti-PD-L1 has better safety than anti-PD-1 It can better target into the tumor microenvironment, and can improve the toxic and side effects on normal tissues.

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 therapeutic functional and therapeutic behavior requirements of different therapeutic products, there is no theoretical prediction method that can be generally applied to most different desired molecular combinations. Therefore, it is often necessary to develop bispecific antibodies with good biological activity for specific targets and antibody compositions.

Contents of the invention

Anti-PD-L1 and VEGF bispecific antibodies in the prior art are mainly anti-PD-L1 antibody fused to VEGFR1 protein (US20200172623A1, WO2020200210A1) or Mab-VHH anti-PD-L1 and VEGF bispecific antibody (WO2021147829A1), these Bispecific antibodies all contain full-length IgG1 antibodies, which have problems of large molecular weight and druggability. Through in-depth research, on the basis of the developed anti-PD-L1 nanobody and anti-VEGF nanobody, the inventors developed a bispecific nanobody targeting VEGF and PD-L1 at the same time, with a molecular weight of only 107KDa, which has potential With the advantages of strong tumor tissue infiltration and small dosage, it is superior to major competitors in terms of clinical dosage and safety. Further, the in vitro VEGF neutralizing activity of the anti-VEGF nanobody part of the bispecific antibody of the present invention is better than that of the marketed Bevacizumab, and in the animal model of angiogenesis, in the case of equal dose administration, the blood vessels in vivo The production inhibitory activity is also superior to 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 bispecific antibodies of the prior art, the bispecific antibody of the present invention can simultaneously block PD-1/PD-L1 and VEGF/VEGFR signaling pathways, and improve blood vessel and At the same time, by restoring immunity, it has a more significant effect of inhibiting tumors, and has a very broad application prospect.

Therefore, in one aspect, the present invention provides a bispecific antibody specifically binding to PD-L1 and VEGF, said antibody comprising one or two VHH domains specifically binding to PD-L1 (VHH ^PD-L1 ) and one or two VHH domains (VHH ^VEGF ) specifically binding to VEGF, wherein the VHH ^VEGF comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO: 2, and preferably, wherein the VHH ^{PD -L1} comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO:1.

In one embodiment, the VHH ^PD-L1 and VHH ^VEGF domains may be included in a tandem form on the same polypeptide chain (for example, by means of a linker and/or an Fc region in tandem on a polypeptide chain); or Linked to the immunoglobulin Fc region in a Fab-like structure (for example, by means of the immunoglobulin CH1 and CL constant domains to form a Fab-like structure).

In a preferred embodiment, the bispecific antibody comprises a polypeptide chain comprising 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 are The two VHH domains specifically bind 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 from IgG, especially IgG1, and includes 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 linked at the N-terminus to the first VHH domain through an immunoglobulin hinge region sequence. Still preferably, the Fc region polypeptide is connected to the second VHH domain at the C-terminus via a flexible linker peptide, such as (G ₄ S) ₃ (SEQ ID NO: 19). Preferably, the polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 7 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity amino acid sequence.

In another preferred embodiment, the bispecific antibody comprises a polypeptide chain comprising 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 binds 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, said first and second VHH domains are connected in tandem via a linker, eg ( _G4S ) ₃ (SEQ ID NO: 19). Preferably, the Fc region is from IgG, especially IgG1, and includes 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 at the N-terminus Linked to the second VHH domain by an immunoglobulin hinge region sequence. Preferably, the polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 8 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity amino acid sequence.

In yet another preferred embodiment, said 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 second VHH domain and the CL domain in the second polypeptide chain to form a Fab-like structure;

Wherein the first VHH structural domain and the second VHH structural domain specifically bind 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 The first antigen is PD-L1 and the second antigen is VEGF. Preferably, the Fc region is from IgG, especially IgG1, and includes 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 through an immunoglobulin hinge region sequence. Preferably, the first polypeptide chain comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence shown in SEQ ID NO: 9 or 11 Amino acid sequences with % or 99% identity. Preferably, the second polypeptide chain comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence shown in SEQ ID NO: 10 or 12 Amino acid sequences with % or 99% identity.

In another aspect, the present invention also provides nucleic acids encoding the polypeptide chains of the bispecific antibodies of the present invention, vectors and host cells comprising them.

In another aspect, the present invention also provides a pharmaceutical composition comprising the bispecific antibody of the present invention, and its in vitro and in vivo uses, especially in the treatment or diagnosis of diseases.

Description of drawings

Figures 1A-1D show schematic structures of candidate bispecific antibodies. BsAb10 has the format of FIG. 1A; BsAb11 has the format of FIG. 1B; BsAb12 has the format of FIG. 1C; BsAb13 has the format of FIG. 1D.

Figures 2A-2B show gel electrophoresis profiles of candidate bispecific antibodies. The samples in Fig. 2A are BsAb10 and the reference IPI respectively; the samples in Fig. 2B are BsAb11, BsAb12, BsAb13 and the reference IPI respectively.

Figures 3A-3D show the SEC-HPLC monomer detection profiles of candidate bispecific antibodies. Figure 3A is the SEC-HPLC monomer detection spectrum of BsAb10; Figure 3B is the SEC-HPLC monomer detection spectrum of BsAb11; Figure 3C is the SEC-HPLC monomer detection spectrum of BsAb12; Figure 3D is the SEC-HPLC monomer detection spectrum of BsAb13 Atlas.

Figure 4 shows the binding activity of candidate bispecific antibodies to recombinant protein VEGF-His.

Figure 5 shows the binding activity of candidate bispecific antibodies to recombinant protein PD-L1-His.

Figure 6 shows the binding activity of candidate bispecific antibodies to huPD-L1-CHO-K cells.

Figures 7A-7B show the binding activity of candidate bispecific antibodies to both VEGF and PD-L1. Figure 7A shows the activity of candidate bispecific antibodies that first bind to recombinant protein VEGF-Fc and then bind to recombinant protein PD-L1-Fc; Figure 7B shows that candidate bispecific antibodies first bind to recombinant protein PD-L1-Fc and then bind to recombinant protein PD-L1-Fc Binding activity to recombinant protein VEGF-Fc.

Figure 8 shows the detection of the blocking activity of candidate bispecific antibodies against PD-1/PD-L1 by luciferase reporter gene method.

Figure 9 shows the detection of the blocking activity of candidate bispecific antibodies against VEGF/VEGFR2 by luciferase reporter gene method.

Figure 10 shows the inhibitory effect of candidate bispecific antibodies on tumor growth in the COLO205 mouse transplantation model.

Figure 11 shows the inhibitory effect of candidate bispecific antibodies on tumor growth in the A431 mouse transplantation model.

Detailed description of the invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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 illustrative only and not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the appended claims.

I. Definition

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

As used herein, the term "comprising" or "comprising" means including stated elements, integers or steps, but not excluding any other elements, integers or steps. Where "comprising" is mentioned, the expression also covers "consisting of" unless the context clearly dictates otherwise. For example, when referring to a polypeptide chain comprising a specific amino acid sequence, it is also contemplated that the polypeptide chain consists of the specific amino acid sequence.

As used herein, 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, isoforms such as VEGF121, VEGF165, VEGF189, and VEGF206. In this context, VEGF refers in particular to the isoform VEGF165, for example the human VEGF165 protein described under UniProtKB-P15692 (in particular, the amino acid sequence without amino acids 27-191 of the signal peptide).

VEGF165 is active in angiogenesis, vessel formation, and endothelial cell growth; 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 to the protein Neuropilin-1 (NRP1). Studies have confirmed that blocking the combination of VEGF and the receptor VEGFR has good curative effect and target safety in tumor treatment and eye diseases related to abnormal blood vessel proliferation. Recent studies have also found that the NRP1 protein plays an important role in suppressing the immune response to cancer. Compared with normal mice, mice with NRP1 gene deletion on killer T cells produced better protection against secondary tumors, including relatively "cold" tumors such as melanoma in B16.F10 mice, against PD1 The response to immunotherapy was also more positive (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).

Herein, "antigen-binding specificity for VEGF" refers to a binding site or a binding domain in a molecule that specifically binds to VEGF.

Herein, the term "PD-L1" refers to programmed cell death 1 ligand 1 (Programmed cell death 1 ligand 1). As a ligand for the inhibitory receptor PD-1, this protein regulates T cell activation thresholds and limits T cell effector responses. The PD1/PD-L1-mediated immunosuppressive pathway can be used by tumors to attenuate anti-tumor immunity and evade clearance by the immune system, thereby promoting tumor survival. Blocking this pathway reverses the exhausted T cell phenotype and normalizes antitumor responses, facilitating cancer immunotherapy. The term PD-L1 herein refers especially to human PD-L1 protein, eg human PD-L1 protein under accession number UniProtKB-Q9NZQ7.

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

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

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

Herein, an antibody having "blocking activity" on PD-L1 means that the antibody blocks the binding of PD-L1 to the receptor PD-1, and/or reduces the function of PD-L1/PD-1 signal transmission. Assays that can be used to determine this blocking activity can be FACS assays known in the art; or reporter gene-based signaling pathway blockade assays, such as the reporter gene-based assay described in Example 5. The PD-L1 blocking activity of the antibody to be tested can be determined with reference to the PD-L1/PD-1 binding or PD-L1/PD-1 signaling level in the absence of the antibody and/or in the presence of a positive antibody.

Herein, the "neutralizing activity" of an antibody to VEGF refers to the function of the antibody to block 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 blockade assay, such as the reporter gene-based assay described in Example 6. The neutralizing activity of the antibody to be tested against VEGF can be determined with reference to the VEGF/VEGFR2 signaling level in the absence of the antibody and/or in the presence of a positive antibody.

The term "variable region" or "variable domain" of an antibody refers to the domains of the heavy or light chain of an antibody that participate in the binding of the antibody to an antigen. The variable regions of antibodies can be further subdivided into hypervariable regions (ie, complementarity determining regions (CDRs)) and more conserved regions interspersed between hypervariable regions (ie, framework regions (FRs)). In the case of heavy chain antibodies (also referred to herein as Nanobodies), such as those from Camelidae, the antigen binding site is composed of a single heavy chain variable domain (also referred to herein as a VHH domain). supply. Like the heavy chain variable region VH of an IgG antibody, this 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 carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The "complementarity determining region" of an antibody, used interchangeably herein with "CDR region" or "CDR" or "hypervariable region", is a region within an antibody variable domain (e.g., VHH) that is highly variable in sequence and forms Structurally defined loops ("hypervariable loops") and/or regions containing antigen contact residues ("antigen contact points"). The CDRs are primarily responsible for binding to antigenic epitopes. Numbering sequentially from the N-terminus of the antibody chain, the CDRs located in the VHH variable region domain are called CDR1, CDR2, and CDR3. In a given variable region amino acid sequence, various schemes known in the art can be used to determine its CDR sequence, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat ( Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, USDepartment 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 the 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 Conformations", Journal of Molecular Biology, 406, 228-256 (2011)). Those skilled in the art can readily determine the CDRs of any given antibody variable region amino acid sequence at http://www.abysis.org/abysis/ Sequence range, including the range of CDR regions defined by Kabat, AbM, Chothia, Contact and IMGT schemes and their combined ranges. Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the above ways and combinations thereof.

In the bispecific antibody of the present invention, the antigen-binding site that specifically binds to the antigen is provided by the 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 (herein sometimes referred to as a Nanobody) lacking a light chain, also referred to as a single Variable domain (sVD). Unlike the conventional VH of four-chain immunoglobulins, the VHH domain does not need to be paired with a light chain variable domain to form an antigen-binding site. Such VHH molecules may be derived from antibodies produced in Camelidae species such as llamas, alpacas, dromedaries, llamas and guanacos. In some instances, for therapeutic use of a VHH, it is desirable to reduce its immunogenicity. Thus, preferably, in one embodiment, the invention provides antibodies comprising a humanized VHH domain. In some embodiments according to the invention, in the bispecific antibody according to the invention, the VHH domain from anti-PD-L1 and the VHH domain from anti-VEGF, preferably via an immunoglobulin hinge region or a linker , separately connected to the opposite sides of the Fc region polypeptide of the antibody; or in the form of both in series (for example, from N-terminal to C-terminal, VHH ^PD-L1 -linker-VHH ^VEGF ; or VHH ^VEGF -linked Sub-VHH ^PD-L1 ), linked to the N-terminal side of the Fc region polypeptide of the antibody.

"Fab-like structure" is used herein to refer to, as shown in Figure 1C and Figure 1D, similar to that in a conventional four-chain IgG antibody consisting of a heavy chain variable region and a heavy chain constant region CH1 with complementary light chain variable regions and A structure formed by CL pairing of the light chain constant region, but in which the heavy chain variable region is replaced by a VHH domain and the light chain variable region is replaced by a different VHH domain. In one embodiment according to the present invention, the Fab-like structure comprises the first chain consisting of VHH ^PD-L1 and the CH1 domain of the constant region of the immunoglobulin heavy chain and the CL domain of the constant region of the immunoglobulin light chain consisting of VHH ^VEGF and composed of the second chain. In yet another embodiment according to the present invention, the Fab-like structure comprises a first chain consisting of VHH ^VEGF and the CH1 domain of the constant region of the immunoglobulin heavy chain and a CL structure composed of VHH ^PD-L1 and the constant region of the immunoglobulin light chain The domain consists of the second chain. In still other 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 in a Fab-like structure, preferably by immunoglobulin The hinge region is connected to the N-terminal side of the immunoglobulin Fc region polypeptide. Preferably, the Fab-like structure is linked to the Fc region polypeptide through its CH1 domain.

The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, IgG class immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons consisting of two light chains and two heavy chains that are disulfide bonded. From N-terminus to C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2 and CH3 ). Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL). In an IgG molecule, usually the VH-CH1 of the heavy chain is paired with the VL-CL of the light chain to form a Fab fragment that specifically binds the antigen. Thus, an IgG immunoglobulin essentially consists of two Fab molecules and two dimerized Fc regions linked by the immunoglobulin hinge region. The heavy chains of immunoglobulins can be assigned to one of five classes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), based on the type of their constant region, one of which is These classes 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 assigned to one of two types, called kappa and lambda, based on the amino acid sequence of their constant domains.

"Immunoglobulin heavy chain constant region domain" means a constant region domain from or obtained from or derived from an immunoglobulin heavy chain, including the heavy chain constant regions CH1, CH2 covalently linked sequentially from the N-terminus to the C-terminus , CH3 and optionally heavy chain constant region CH4. In most cases, heavy The chain constant regions CH1 and CH2 are connected by the hinge region of the heavy chain, but they can also be connected by a flexible linking peptide when appropriate. In some preferred embodiments of the present invention, the antibody of the present invention comprises a polypeptide chain consisting of 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 composed of immunoglobulin heavy chain constant regions CH2-CH3. Herein, immunoglobulin constant domains can be selected based on the intended function of the antibody. For example, the constant domain may 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 of human IgG1 constant domain. As an example, the CH1 and Fc fragments of an antibody can both be from IgG1.

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

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 part of the 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, a CH2 domain, a CH3 domain and optionally a CH4 domain. For example, in native antibodies, the immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) of the heavy chain derived from antibodies of the IgG, IgA and IgD classes; and the second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) of the heavy chain of antibodies of the IgE class. Unless otherwise stated herein, amino acid residue numbering in the Fc region or heavy chain constant region is according to, for example, Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, The EU numbering system described in 1991 (also known as the EU Index) is used for numbering. As used herein, the term "Fc domain" or "Fc region" or "Fc polypeptide" does not include the heavy chain variable region VH and the light chain variable region VL and the heavy chain constant region CH1 and the light chain constant region of an immunoglobulin cl.

"Native sequence Fc region" polypeptides encompass naturally occurring Fc region sequences of various immunoglobulins, such as the Fc region sequences of various Ig subtypes and their allotypes (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 carboxy-terminus of the heavy chain. However, the C-terminal terminal lysine (Lys447) of the Fc region may or may not be present. In still other embodiments, the human IgG heavy chain Fc region bears at the N-terminus a hinge sequence or a partial hinge sequence of a native immunoglobulin, for example a sequence from E216 to T225 or a sequence from D221 to T225 according to EU numbering.

The term "variant Fc region" or "Fc region variant" polypeptide herein, used interchangeably herein, refers to an Fc region polypeptide comprising a modification relative to a native sequence Fc region polypeptide. Fc region variant polypeptides of the invention are defined by the amino acid modifications that make them up. Thus, for example, L234A is an Fc region variant with alanine substituted for leucine at position 234 relative to the parental polypeptide, where numbering is according to the EU index. Modifications can be additions, deletions or substitutions. Substitutions can include naturally occurring amino acids and non-naturally occurring amino acids. Variants may contain unnatural amino acids. The purpose of the modification may be to alter the binding of the Fc region to its receptor and the effector functions elicited thereby.

The term "effector functions" refers to those biological activities attributable 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 (eg, B-cell receptors), and B-cell activation. Depending on the intended use of the antibody, the antibody of the invention may have altered effector functions relative to an antibody with a wild-type Fc region, such as reduced or eliminated ADCC activity and the like.

As used herein, the term "flexible linker" or "linker" or "linker" is used interchangeably to refer to a short amino acid sequence consisting of amino acids such as glycine (G) and/or serine ( S) and/or threonine residues (T), or from the hinge region of an immunoglobulin. In one embodiment, the connecting peptide is 5-50 amino acids in length, eg, 10, 15, 20, 25, 30 amino acids in length. In one embodiment, the connecting peptide comprises the amino acid sequence (G ₄ S) _n (SEQ ID NO: 20), wherein n is an integer equal to or greater than 1, for example, n is 2, 3, 4, 5, 6 or 7 an integer of . In one embodiment, the connecting peptide comprises the amino acid sequence TS(G ₄ S) _n (SEQ ID NO: 21), wherein n is an integer equal to or greater than 1, for example, n is 2, 3, 4, 5, 6 or Integer of 7. In yet another embodiment, the connecting peptide is a hinge region from an immunoglobulin, for example comprising a hinge region amino acid sequence of "CPPC", for example, the amino acid sequence "EPKSCDKTHTCPPCP" (SEQ ID NO: 22) or "EPKSSDKTHTCPPCP" (SEQ ID NO:23). The connecting peptide that can be used to link the various domains in the antibody of the present invention can also be, for example but not limited to, the following amino acid sequence: GGG (SEQ ID NO: 24); DGGGS (SEQ ID NO: 25); TGEKP (SEQ ID NO :26); GGRR (SEQ ID NO:27); EGKSSGSGSESKVD (SEQ ID NO:28); KESGSVSSEQLAQFRSLD (SEQ ID NO:29); GGRRGGGS (SEQ ID NO:30); LRQRDGERP (SEQ ID NO:31); (SEQ ID NO:32) and GSTSGSGKPGSGEGSTKG (SEQ ID NO:33). Alternatively, suitable flexible linker peptides can be rationally designed using computer programs to model the three-dimensional structures of proteins and peptides, or by phage display methods.

The term "chimeric antibody" is an antibody in which (a) the constant region, or part thereof, has been altered, substituted or exchanged such that the antigen binding site corresponds to a constant region of a different or altered class, effector function and/or species or linking entirely different molecules (e.g., enzymes, toxins, hormones, growth factors, drugs) etc. that confer new properties on the chimeric antibody; Variable regions are altered, substituted or swapped.

A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (eg, an alpaca heavy chain antibody), while being less immunogenic when administered to humans as a therapeutic. This can be achieved, for example, by retaining the non-human antigen binding sites and replacing the remainder of the antibodies with their human counterparts (ie, the constant regions and the parts of the variable regions that do not participate in binding are those of human antibodies).

"Percent identity (%)" of an amino acid sequence refers to after aligning a candidate sequence with the specific amino acid sequence shown in this specification and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without taking into account any When conservative substitutions are taken as part of sequence identity, the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular amino acid sequence shown in this specification. In some embodiments, the invention contemplates variants of the antibodies of the invention that have a substantial degree of identity, for example at least 80%, 85% identity, relative to the antibodies and sequences thereof specifically disclosed herein. %, 90%, 95%, 97%, 98%, or 99% or higher. Such variants may contain conservative modifications.

With respect to polypeptide sequences, "conservative modifications" include substitutions, deletions or additions to a polypeptide sequence that result in the substitution of an amino acid for 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 polymorphic variants, interspecies homologues and alleles of the invention. The following 8 groups contain amino acids that are mutually conservative substitutions: 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 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine Acid (C), Methionine (M) (See eg, Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modification" is used to refer to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence.

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 the primary transformed cell and progeny derived therefrom. A host cell is any type of cellular system that can be used to produce antibodies of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells. Host cells include cultured cells as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. Expression vectors contain 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) that incorporate recombinant polynucleotides. virus and adeno-associated virus).

The terms "individual" or "subject" are used interchangeably to refer to a mammal. 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 rodents). mouse). In particular, an individual is a human being.

The term "treatment" refers to clinical intervention intended to alter the natural course of disease in the individual being treated. Desirable therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or to slow the progression of a disease. In the context of tumor or cancer treatment, "treatment" encompasses anti-tumor biological effects that can be induced by human intervention (e.g., administration of drugs such as antibodies of the invention), including, but not limited to, for example, reduction in tumor volume, tumor Decreased cell number, decreased tumor cell proliferation, or decreased tumor cell survival.

The terms "cancer" and "tumor" are used interchangeably to refer to or describe a physiological disorder in mammals that is often characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, solid tumors, and liquid tumors.

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

I. Bispecific antibodies of the invention

In one aspect, the present invention provides a VHH domain-based bispecific antibody specifically binding to PD-L1 and VEGF, said antibody comprising a VHH domain specifically binding to PD-L1 (also abbreviated herein as VHH ^PD-L1 ) and a VHH domain specifically binding to VEGF (also abbreviated herein as VHH ^VEGF ).

In some specific embodiments, in the bispecific antibody of the present invention, the VHH ^PD-L1 and VHH ^VEGF domains may be in a tandem form contained on the same polypeptide chain (for example, by means of a linker and/or or Fc regions in series on one polypeptide chain); or can form a Fab-like structure (for example, by means of immunoglobulin CH1 and CL constant domains to form a Fab-like structure).

In some specific embodiments, the bispecific antibody according to the invention further comprises an Fc region. In the bispecific antibody according to the present invention, said VHH ^PD-L1 and VHH ^VEGF domains may be in a tandem form comprising both VHH ^PD-L1 and VHH ^VEGF domains (for example, as shown in Figure 1A and Figure 1B ), or in a Fab-like structure comprising both VHH ^PD-L1 and VHH ^VEGF domains (eg, as shown in Figure 1C and Figure 1D ), linked to the Fc region. Since antibody polypeptide chains comprising the Fc region can associate through dimerization of the Fc region, in some embodiments, the bispecific antibody according to the present invention can be a tetravalent antibody that specifically binds PD-L1 and VEGF. Two-chain antibody, or can be specific Quadrivalent 4-chain antibody that heterosexually binds PD-L1 and VEGF.

The components of the bispecific antibody of the present invention and the bispecific antibody of the present invention composed of them are described in detail below. Those skilled in the art can understand that unless the context clearly indicates otherwise, any combination of any technical features of these components is within the scope of the present invention. And, those skilled in the art will understand that unless the context clearly indicates otherwise, the antibodies of the invention (including antibodies in any form, such as diabodies and tetrabodies) may comprise any such combination of features.

VHH ^VEGF domain and VHH ^PD-L1 domain according to the invention

In the bispecific antibody 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 provided by the VHH ^PD-L1 according to the present invention; and The antigen binding site that specifically binds VEGF is provided by the VHH ^VEGF according to the invention.

A VHH ^PD-L1 domain according to the invention comprises a VHH domain from an anti-PD-L1 Nanobody. Preferably, the VHH comprises CDR1, CDR2 and CDR3 sequences having the variable region shown in SEQ ID NO:1. The CDR sequence range of the variable region SEQ ID NO: 1 amino acid sequence 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. Those skilled in the art can easily know the CDR sequences defined by these definitions through http://www.abysis.org/abysis/. In a preferred embodiment, the VHH ^PD-L1 domain according to the invention comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence defined according to AbM in the variable region having SEQ ID NO:1.

In yet another preferred embodiment, the VHH ^PD-L1 domain according to the invention comprises

(i) CDR1 comprising or consisting of SEQ ID NO:13;

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

(iii) CDR3 comprising or consisting of SEQ ID NO:15.

In one embodiment, the VHH ^PD-L1 domain comprises the amino acid sequence shown in SEQ ID NO:1. In yet another embodiment, the VHH ^PD-L1 domain comprises at least 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 1 and retains the ability to specifically bind PD-L1 amino acid sequence. In yet another preferred embodiment, the VHH ^PD-L1 domain comprises one or more (preferably 1-10, more preferably 1-5) amino acid additions compared to SEQ ID NO: 1, Amino acid sequences that are deleted and/or substituted (eg, conservatively substituted) and retain the ability to specifically bind PD-L1. Preferably, said amino acid additions, deletions and/or substitutions do not occur in the CDR regions. Most preferably, in the bispecific antibody according to the present invention, the VHH ^PD-L1 domain according to the present invention comprises the amino acid sequence of SEQ ID NO: 1, or consists of the amino acid sequence shown in SEQ ID NO: 1.

A VHH ^VEGF domain according to the invention comprises a VHH domain from an anti-VEGF Nanobody. Preferably, the VHH comprises CDR1, CDR2 and CDR3 sequences of the variable region shown in SEQ ID NO:2. The CDR sequence range of the variable region SEQ ID NO: 2 amino acid sequence 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. Those skilled in the art can easily know the CDR sequences defined by these definitions through http://www.abysis.org/abysis/. In a preferred embodiment, the VHH ^VEGF domain according to the invention comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence defined according to AbM in the variable region having SEQ ID NO:2.

In yet another preferred embodiment, the VHH ^VEGF domain according to the invention comprises

(i) CDR1 comprising or consisting of SEQ ID NO: 16;

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

(iii) CDR3 comprising or consisting of SEQ ID NO:18.

In one embodiment, the VHH ^VEGF domain comprises the amino acid sequence shown in SEQ ID NO:2. In yet another embodiment, the VHH ^VEGF 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 VEGF. In yet another preferred embodiment, the VHH ^VEGF domain comprises one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and and/or amino acid sequences that are substituted (eg, conservatively substituted) and retain the ability to specifically bind VEGF. Preferably, said amino acid additions, deletions and/or substitutions do not occur in the CDR regions. Most preferably, in the bispecific antibody according to the present invention, the VHH ^VEGF domain according to the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO:2.

immunoglobulin constant domain

According to the bispecific antibody of the present invention, in addition to the aforementioned VHH ^VEGF domain and VHH ^PD-L1 domain, in some embodiments, it may also include an immunoglobulin constant domain, for example, the immunoglobulin constant regions CH2 and CH3 Constituent Fc region, and/or CH1 and CL constant domains in a Fab-like structure.

In some embodiments of the invention, therefore, the invention provides a bispecific antibody comprising an Fc region polypeptide chain consisting of immunoglobulin heavy chain constant regions CH2-CH3. In some other embodiments of the present invention, the present invention provides a bispecific antibody comprising an Fc region and further comprising an immunoglobulin CH1 domain and a CL domain, preferably, wherein the CH1 domain passes through an immunoglobulin hinge region connects the Fc region polypeptide, 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 comprised in the bispecific antibody according to the 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), May be, independently of each other, an immunoglobulin constant domain from human IgG, eg, a constant domain of human IgGl, IgG2, IgG3 or IgG4, preferably a constant domain of human IgGl. Furthermore, the immunoglobulin constant domains comprised in the bispecific antibodies according to the invention, including the constant regions CH2 and CH3 and CH1 and CL (if present), may independently of each other be native sequence constant domains (for example, Human native sequence constant domain) or an amino acid sequence variant thereof, preferably a native sequence constant domain.

In a preferred embodiment, the Fc region comprised in the bispecific antibody according to the invention is an Fc region sequence from human IgG1. In a more preferred embodiment, the Fc region has the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence at least 90%, 95% or 99% identical thereto, or no more than Amino acid sequence with 1-10 (preferably 1-5) amino acid residues altered.

In some specific embodiments, the bispecific antibody according to the present invention may comprise two polypeptide chains having an Fc region, for example, the di-chain antibody and the tetra-chain antibody of the present invention as shown in FIG. 1 , wherein the two polypeptide chains The Fc region of is capable of pairing and dimerization. In such an embodiment, both the first Fc region polypeptide and the second Fc region polypeptide constituting the dimerized Fc region of the antibody may be Fc domains from IgG, such as human IgG1, IgG2, IgG3 or IgG4, preferably human IgG1 and may independently of each other be a native sequence Fc region and a variant Fc region as defined herein, provided that said first Fc region polypeptide and said second Fc region polypeptide can pair and dimerize. 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 at least 90%, 95% or 99% identical thereto, or identical thereto An amino acid sequence that has no more than 1-10 (preferably 1-5) amino acid residue changes.

In yet another embodiment, the antibody according to the present invention (for example, 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 structure The domain is preferably linked C-terminally to the first VHH domain and the light chain constant region CL domain is linked C-terminally to the second VHH domain. In such bispecific antibodies, the heavy chain constant regions CH1 and CL constituting the antibody may be constant domains from IgG, such as human IgG1, IgG2, IgG3 or IgG4, preferably 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 bispecific antibodies, the CH1 and CL domains may independently of each other be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof, provided that the CH1 structure is included Domain and the antibody polypeptide chain of the first VHH domain connected at its N-terminus, and the antibody polypeptide chain comprising said CL domain and the second VHH domain connected at its N-terminus can be paired with each other to form a Fab according to the invention like structure. Preferably, the CH1 and CL domains are native sequence constant domains, especially human IgG1 native sequence CH1 domains and CL domains. 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.

Linker

In the bispecific antibody according to the present invention, linkers can be used to connect antibody components. For example, a VHH domain according to the present invention may be linked to the N-terminal or C-terminal of the Fc region via a linker, or may be linked to another VHH domain according to the present invention via a linker. Linkers that can be used in the antibodies of the present invention are not particularly limited. Those skilled in the art can easily determine the available linker sequences according to the components to be linked and the position of the link.

In some embodiments, in the bispecific antibody according to the invention, the VHH domains according to the invention are in tandem form (eg, as shown in Figures 1A and 1B ) or in a Fab-like structure (eg, as shown in Figure 1C and 1D), connected to the Fc region polypeptide chain through a linker. Those skilled in the art can select different linkers according to the connection position of the VHH, that is, the connection at the N-terminal or C-terminal of the Fc region.

In one embodiment, the polypeptide chains of the Fc region are connected at the N-terminus to a single VHH domain or two VHH domains in tandem, or a Fab-like structure via a linker, wherein the linker preferably comprises (one or two) cysteine Amino acid residues, which are capable of forming (one or two) disulfide bonds when the two antibody polypeptide chains comprising the Fc region pair 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 FIG. 1 . Therefore, in a preferred embodiment, the Fc region polypeptide chain contained in the bispecific antibody of the present invention has a linker sequence comprising "CPPC" at the N-terminus. In yet another preferred embodiment, the Fc region polypeptide chain contained in the bispecific antibody of the present invention has a hinge region derived from an immunoglobulin at the N-terminus, for example, the amino acid sequence of the hinge region comprising "CPPC", for example, amino acid The sequence "EPKSCDKTHTCPPCP" (SEQ ID NO:22) or "EPKSSDKTHTCPPCP" (SEQ ID NO:23); more preferably, the amino acid sequence "EPKSCDKTHTCPPCP" (SEQ ID NO:22).

In another embodiment, the Fc region polypeptide chain is connected to the VHH domain at the C-terminus by a linker, wherein the linker is preferably a flexible connecting peptide of 5-50 amino acids, preferably comprising glycine (G) and/or serine (S) and/or a linking peptide of a threonine residue (T). In one embodiment, the linker is 5-50 amino acids in length, eg, 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 (G ₄ S) _n , wherein n is an integer equal to or greater than 1, eg, n is an integer of 2, 3, 4, 5, 6 or 7. In a preferred embodiment, the linker consists of the amino acid sequence (G ₄ S) ₃ .

In still other embodiments, in the bispecific antibody according to the invention, the VHH ^VEGF domain and the VHH ^PD-L1 domain according to the invention form a tandem VHH domain via a linker. In the bispecific antibody according to the invention having such tandem VHH domains, preferably Optionally, the first and second domains are connected in series through a linker, wherein the linker is a flexible connecting peptide of 5-50 amino acids. In one embodiment, the linker is 5-50 amino acids in length, eg, 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 is a linker peptide comprising glycine (G) and/or serine (S) and/or threonine residues (T). In yet another embodiment, the linker comprises the amino acid sequence (G ₄ S) _n (SEQ ID NO: 20), wherein n is an integer equal to or greater than 1, for example, n is 2, 3, 4, 5, 6 or Integer of 7. In a preferred embodiment, the linker consists of the amino acid sequence (G ₄ S) ₃ (SEQ ID NO: 19).

Bispecific antibody embodiments of the invention

In one aspect, the present invention provides a bispecific antibody specifically binding to PD-L1 and VEGF, wherein the antibody comprises a VHH domain specifically binding to a first antigen and a VHH domain specifically binding 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 one embodiment, the VHH domain specifically binding to VEGF comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO:2. In another embodiment, the VHH domain specifically binding to PD-L1 comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO:1.

In another embodiment, the VHH domain specifically binding to VEGF comprises the amino acid sequence shown in SEQ ID NO: 2, or has at least 80%, 85%, 90%, 95%, or An amino acid sequence with 99% identity, or an amino acid sequence with one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and/or substitutions 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.

In yet another embodiment, the VHH domain specifically binding to PD-L1 comprises the amino acid sequence shown in SEQ ID NO: 1, or has at least 80%, 85%, 90%, 95% with SEQ ID NO: 1 Amino acid sequence with % or 99% identity, or with one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and/or substitutions compared to SEQ ID NO: 1 Amino acid sequence, 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.

In some embodiments, the bispecific antibody according to the present invention further comprises a linker comprising 10-20 amino acids in length, preferably comprising the amino acid sequence (G ₄ S) ₃ (SEQ ID NO: 19) . In some further embodiments, the bispecific antibody according to the present invention further comprises an Fc region of an immunoglobulin, wherein the Fc region is an IgG Fc region, for example, the Fc domain of human IgG1, IgG2, IgG3 or IgG4, preferably human The Fc domain of IgG1, more preferably the Fc region carries an immunoglobulin hinge region sequence at the N-terminus.

In some preferred embodiments, the bispecific antibody according to the invention comprises a polypeptide chain comprising VHH ^PD-L1 and VHH ^VEGF domains connected in series by means of an Fc region and a linker. Preferably, the bispecific antibody comprises a polypeptide chain comprising 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 The two VHH domains specifically bind 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 bispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 7 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof or amino acid sequences with 99% identity.

In other preferred embodiments, the bispecific antibody according to the invention comprises a polypeptide chain comprising VHH ^PD-L1 and VHH ^VEGF domains connected in series by means of a linker. Preferably, the bispecific antibody comprises a polypeptide chain comprising from the N-terminus to the C-terminus Containing 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 antigens 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 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof or amino acid sequences with 99% identity.

In further preferred embodiments, the bispecific antibody according to the 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 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 the same as the second VHH in the second polypeptide chain domain and CL domain pair to form a Fab-like structure; wherein the first VHH domain and the second VHH domain specifically bind a first antigen and a second antigen, respectively, wherein the first and second antigens are different and independent of each other is selected from PD-L1 and VEGF, preferably, the first antigen is PD-L1 and the second antigen is VEGF. In such embodiments, preferably, the bispecific antibody comprises first and second polypeptide chains, wherein the first antigen is PD-L1 and the second antigen is VEGF, wherein the first polypeptide chain comprises SEQ The amino acid sequence shown in 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 two polypeptide chains comprise an amino acid sequence shown in SEQ ID NO: 10 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith amino acid sequence.

Two-chain bispecific antibody embodiments of the invention

In some embodiments of the bispecific antibody of the invention having VHH ^PD-L1 and VHH ^VEGF domains arranged in tandem, the antibody may comprise two polypeptide chains comprising an Fc region, wherein the two polypeptide chains Dimers can be formed by dimerizing association of the Fc region. As will be appreciated by those skilled in the art, the two polypeptide chains that associate to form a dimer need not be identical. For example, asymmetric modifications can be introduced in one or both polypeptide chains (eg, in the Fc region of the polypeptide chains) without affecting the desired target activity. However, in some aspects, preferably, the two polypeptide chains are identical. For example, as shown in Figures 1A and 1B.

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

Each polypeptide chain comprises a first VHH domain, an Fc region, a linker, and a second VHH domain from N-terminus to C-terminus (as shown in Figure 1A); or 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 in Figure 1B);

The Fc region polypeptides of the two polypeptide chains are paired to form a dimerized Fc region, wherein the first VHH domain and the second VHH domain specifically bind the first antigen and the second antigen respectively, wherein the 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.

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

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

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 the first antigen and the second antigen respectively, wherein the first antigen and the second antigen The two antigens 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 from IgG, especially IgG1, and includes 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 linked at the N-terminus to the first VHH domain through an immunoglobulin hinge region sequence. Still preferably, the Fc region polypeptide is connected to the second VHH domain at the C-terminus through a flexible linking peptide with a length of 10-20 amino acids, such as (G ₄ S) ₃ . Preferably, the first VHH domain is a VHH ^PD-L1 domain according to the present invention, preferably, comprising the CDR1-3 sequence of the variable region shown in SEQ ID NO: 1, more preferably comprising SEQ ID NOs: 13- 15 CDR1-3 sequence; most preferably comprising the variable region sequence shown in SEQ ID NO: 1; and the second VHH domain is a VHH ^VEGF domain according to the present invention, preferably comprising SEQ ID NO: 2 The CDR1-3 sequence of the variable region, more preferably comprising the CDR1-3 sequence of SEQ ID NOs: 16-18; most preferably comprising 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 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences of 98% or 99% identity. Most preferably, the two polypeptide chains are identical and each comprise the amino acid sequence of SEQ ID NO:7.

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

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

Wherein the Fc region polypeptides of the two polypeptides pair and form a dimerized Fc region, wherein the first VHH domain and the second VHH domain specifically bind the first antigen and the second antigen respectively, wherein the first antigen and The second antigens 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 (G ₄ S) ₃ (SEQ ID NO: 19) _. Still more preferably, the Fc region is from IgG, especially IgG1, and includes 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 linked at the N-terminus to a tandem VHH domain through an immunoglobulin hinge region sequence. Preferably, the first VHH domain is a VHH ^PD-L1 domain according to the present invention, preferably, comprising the CDR1-3 sequence of the variable region shown in SEQ ID NO: 1, more preferably comprising SEQ ID NOs: 13- 15 CDR1-3 sequence; most preferably comprising the variable region sequence shown in SEQ ID NO: 1; and the second VHH domain is a VHH ^VEGF domain according to the present invention, preferably comprising SEQ ID NO: 2 The CDR1-3 sequence of the variable region, more preferably comprising the CDR1-3 sequence of SEQ ID NOs: 16-18; most preferably comprising 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 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences of 98% or 99% identity. Most preferably, the two polypeptide chains are identical and each comprise the amino acid sequence of SEQ ID NO:8.

Four-chain bispecific antibody embodiments of the invention

In some embodiments of the bispecific antibody of the invention having VHH ^PD-L1 and VHH ^VEGF domains comprised in a Fab-like structure, said antibody may comprise two first polypeptide chains comprising an Fc region and two The second polypeptide chain, wherein the two first polypeptide chains can form a dimer through dimerization association of the Fc region. As understood by those skilled in the art, the two first polypeptide chains comprised in the bispecific antibody need not be identical; and similarly, the two second polypeptide chains need not be identical either. For example, in one or both first polypeptide chains and/or introduce asymmetric modifications into the second polypeptide chain 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.

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

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;

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

wherein the Fc region polypeptides in the two first polypeptide chains pair and form a dimerized Fc region, and

wherein 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 respectively to form two Fab-like structures;

Wherein the first VHH structural domain and the second VHH structural domain specifically bind 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. For example, as shown in 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 from IgG, especially IgG1, and includes 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 through an immunoglobulin hinge region sequence. Preferably, the first VHH domain is a VHH ^PD-L1 domain according to the present invention, preferably, comprising the CDR1-3 sequence of the variable region shown in SEQ ID NO: 1, more preferably comprising SEQ ID NOs: 13- 15 CDR1-3 sequence; most preferably comprising the variable region sequence shown in SEQ ID NO: 1; and the second VHH domain is a VHH ^VEGF domain according to the present invention, preferably comprising SEQ ID NO: 2 The CDR1-3 sequence of the variable region, more preferably comprising the CDR1-3 sequence of SEQ ID NOs: 16-18; most preferably comprising the variable region sequence shown in SEQ ID NO:2.

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:

The two first polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO: 9 or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences; and

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

Most preferably, the two first polypeptide chains are identical, respectively comprising the amino acid sequence of SEQ ID NO:9; and the two second polypeptide chains are identical, respectively comprising the amino acid sequence of SEQ ID NO:10.

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:

The two first polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO: 11 or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences; and

The two second polypeptide chains respectively comprise the amino acid sequence shown in SEQ ID NO: 12 and have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% % identity amino acid sequence.

Most preferably, the two first polypeptide chains are identical, respectively comprising the amino acid sequence of SEQ ID NO: 11; and the two second polypeptide chains are identical, respectively comprising the amino acid sequence of SEQ ID NO: 12.

In the above-described embodiments of the four-chain antibody according to the invention, the invention also contemplates an 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 linked by the CL domain of the Fab-like structure through an immunoglobulin hinge region.

II. Production and Purification of Antibodies of the Invention

In yet another aspect, the invention provides methods for producing antibodies of the invention. To produce the antibodies of the invention, the polypeptide chains of the antibodies of the invention can be obtained, eg, by solid-state peptide synthesis (eg, Merrifield solid-phase synthesis) or recombinant production, and assembled under suitable conditions.

For recombinant production, polynucleotides encoding any polypeptide chain and/or multiple polypeptide chains of the antibody can be isolated and inserted into one or more vectors for further cloning and/or expression in host cells. The polynucleotides can be readily isolated and sequenced using conventional methods. In one embodiment, polynucleotides encoding one or more polypeptide chains of an antibody of the invention are provided. In yet another embodiment, the invention provides a vector, preferably an expression vector, comprising one or more polynucleotides of the invention. Accordingly, in one embodiment, the invention provides a method for producing an antibody of the invention, said method comprising: culturing a host cell comprising a polypeptide chain encoding said antibody under conditions suitable for expressing said polypeptide chain; and The antibody is produced by assembling the polypeptide chains under conditions suitable for assembly of the polypeptide chains into the antibody.

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 phage, or yeast artificial chromosomes (YACs).

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. Various techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.

In one embodiment, the invention also provides host cells comprising one or more polynucleotides of the invention. In some embodiments, host cells comprising an expression vector of the invention are provided. As used herein, the term "host cell" refers to any kind of cellular system that can be engineered to produce the antibodies of the invention. Host cells suitable for replicating and supporting expression of the antibodies of the invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors as needed, and large numbers of cells containing the vector can be grown for inoculation of large-scale fermenters to obtain sufficient quantities of the antibodies of the invention for clinical use. 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 and the like. 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, NSO, P3X63 and Sp2/0, etc. For a review of mammalian host cell lines suitable for protein production see, eg, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (ed. B.K.C. Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO, HEK293 or NSO cell.

Antibodies prepared by the methods described herein can be purified by known art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. After purification, the antibodies of the invention can be determined by any of a variety of well known assays. The well-known analytical methods include 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.

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

III. Pharmaceutical Compositions, Drug Combinations and Kits

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

In another aspect, the invention also provides pharmaceutical combinations comprising an antibody described herein and more than one therapeutic agent.

Therapeutic agents suitable for use in the pharmaceutical compositions and drug combinations of the invention may be therapeutic agents selected from any of the following categories (i)-(iv): (i) agents that enhance antigen presentation (e.g., tumor antigen presentation) Drugs; (ii) drugs that enhance effector cell responses (eg, B cell and/or T cell activation and/or mobilization); (iii) drugs that reduce immunosuppression; (iv) drugs that have tumor suppressive effects.

The compositions of the invention can be in a variety of forms. These forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. Commonly preferred compositions are in the form of injectable solutions or infusible solutions. The preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal (i.p.), 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, intraperitoneal or subcutaneous injection.

The phrases "parenteral administration" and "parenteral administration" as used herein 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.

Therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage. Compositions can be formulated as solutions, microemulsions, dispersions, liposomes or in lyophilized form. Sterile injectable solutions can be prepared by incorporating the active compound (ie, antibody) in the required amount in an appropriate solvent, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and other ingredients. Coating agents such as lecithin and the like can be used. In the case of a dispersion, proper fluidity of the solution can be maintained by using a surfactant. Prolonged absorption of the injectable compositions can be brought about by including in the composition substances which delay absorption, for example, monostearate salts and gelatin.

The pharmaceutical composition of the present invention may contain "therapeutically effective amount" or "prophylactically effective amount" of the antibody of the present invention. A "therapeutically effective amount" refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired therapeutic result. can be based on a variety of factors such as disease status, individual age, A therapeutically effective amount for such changes as sex and weight. A therapeutically effective amount is one in which any noxious or detrimental effects are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g. tumor growth rate) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60% and still more relative to an untreated subject. Preferably at least about 80%. The ability of the antibodies of the invention to inhibit a measurable parameter (eg, tumor volume) can be evaluated in animal model systems predictive of efficacy in human tumors.

A "prophylactically effective amount" refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired prophylactic result. Typically, the prophylactically effective amount is less than the therapeutically effective amount because the prophylactic dose is used in the subject before or at an earlier stage of the disease.

Kits comprising the antibodies described herein are also within the scope of the invention. A kit may comprise one or more other elements including, for example: instructions for use; other reagents, such as labels or reagents for conjugation; a pharmaceutically acceptable carrier; and devices or other materials for administration to a subject.

IV. Uses and methods of the molecules of the invention

In one aspect, the present invention provides in vivo and in vitro uses and methods of application of the antibodies of the present invention.

In some embodiments, the uses and methods of the invention involve the in vivo and/or in vitro application of the antibodies of the invention to:

- binds human PD-L1 and human VEGF; or

- Block the human PD-L1/PD-1 signaling pathway; or

- neutralizes VEGF activity; or

- inhibition of tumor cell growth,

Or for the preparation of medicines for any of the above purposes.

In some embodiments, the antibody of the present invention or a pharmaceutical composition comprising the antibody of the present invention is used as a drug for treating and/or preventing a disease or as a diagnostic tool for a disease in an individual, preferably, the individual is a mammal, more preferably Preferably a human.

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 can be PD-L1 positive solid tumors or blood cancers, 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 Carcinoma, esophageal cancer, lymphoma, liver cancer, microsatellite unstable solid tumors. Preferably, said cancer is colorectal cancer, liver cancer, ovarian cancer, breast cancer, lung cancer.

In one aspect, the invention provides in vitro or in vivo diagnostic methods for detecting the presence of relevant antigens in biological samples such as serum, semen or urine or tissue biopsy samples (eg, from hyperproliferative or cancerous lesions). The diagnostic method comprises: (i) contacting a 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 detected Complex formation between the antibody and the sample (and optionally, a control sample) is performed. Formation of the complex indicates the presence of the relevant antigen and may indicate suitability or need for treatment and/or prophylaxis as described herein.

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

The following examples are described to aid in the understanding of the present invention. The examples are not intended and should not be construed in any way as limiting the scope of the invention.

Example

Embodiment 1 raw material preparation

1.1 Antigen preparation

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 General Biosystems (Anhui) Co., Ltd. for the target fragment gene. Each target fragment was amplified by PCR, and a His tag or hIgG1Fc (shown in the amino acid sequence SEQ ID NO: 3) was introduced at the C-terminus by primers, and then respectively constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) by homologous recombination . The constructed recombinant protein expression vectors were respectively transformed into Escherichia coli DH5α, cultured overnight at 37°C, and then plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids and For eukaryotic expression.

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 passed through the Expi293 transient expression system (ThermoFisher, A14635) expression, see Expi293 ^TM Expression System Kit Manual for the transient method. After 5-7 days of transfection, the cell expression supernatant was centrifuged at a high speed of 15000 g for 10 min. The resulting 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 filter concentration tube (Millipore, UFC901096) was replaced with PBS buffer; the resulting Fc-tagged recombinant protein expression supernatant was filtered through a 0.22 μm filter membrane, and then purified by Protein A/G affinity chromatography column affinity method. The target protein was eluted with 100mM glycine salt (pH3.0), then concentrated and replaced, and then frozen at -80°C after being identified by SDS-PAGE and qualified for activity identification.

1.2 Preparation of control antibody

In this example, anti-human VEGF antibody P30-10-26 (VHH ^VEGF -hIgG1Fc, VHH ^VEGF amino acid sequence shown in SEQ ID NO: 2) is derived from patent application CN202110995278.7; anti-human PD-L1 antibody D21-4 (VHH ^PD-L1 -hIgG1Fc, VHH ^PD-L1 amino acid sequence shown in SEQ ID NO: 1) is derived from the patent application PCT/CN2020/125301; the anti-VEGF control antibody Bevacizumab (Bevacizumab, abbreviated as Beva) sequence is derived from Drug Bank (Drug Bank No: DB00112); anti-PD-L1 control antibody Atezolizumab (Ate) sequence is derived from Drug Bank (Drug Bank No: DB11595); anti-PD-L1 and VEGF control bispecific The anti-antibody W3256 sequence is derived from WO2021147829A1; the anti-PD-1 and VEGF control bispecific antibody AK112 sequence is derived from the VP101 sequence in US20210340239A1. After the above amino acid sequences were converted into gene sequences, General Biotechnology Co., Ltd. carried out gene synthesis of the target fragment. Each target fragment was amplified by PCR, and then constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) by homologous recombination.

The antibodies were all expressed using the transient system (ExpiCHO). For the transient method, see ExpiCHO ^™ Expression System Kit User Guide. The expressed cell suspension was subjected to high-speed centrifugation and the supernatant was taken, and the obtained supernatant was filtered through a 0.22 μm filter membrane, and then purified by using a Protein A/G affinity chromatography column affinity method. After purification, the target protein was eluted with 100 mM glycine salt (pH 3.0), then concentrated and replaced, and then frozen at -80°C after being identified by SDS-PAGE and qualified for activity identification.

1.3 Cell line construction

1.3.1 Construction of huPD-L1-CHO-K cell line

The CHO-K cells (Thermo, A1461801) were passaged to 5×10 ⁶ cells/mL, and the next day, the constructed cells containing full-length human PD-L1 Plasmid introduction of (UniProtKB-Q9NZQ7) gene sequence in CHO-K cells. The cells after electroporation were transferred to CD-CHO medium (Gibco, 10743029), and placed in a cell culture incubator at 37°C for 48 hours. Then, 2000 cells/well were plated into a 96-well plate, and MSX (Millipore, GSS-1015-F) and GS supplement (Sigma, 58672C) were added to a final concentration of 30 μM for pressurized screening. Single-cell clones grown in 96-well plates were picked and expanded to obtain huPD-L1-CHO-K cell lines through FACS identification.

1.3.2 Construction of HEK293-VEGFR2-NFAT cell line

The pGL4.30 plasmid (promega, #E8481) containing the NF-AT-re nucleic acid sequence was electroporated into HEK293 cells ( CRL-1573 ^TM ) to obtain HEK293-NFAT cell line. The HEK293-NFAT cells were passaged to 2× ¹⁰⁵ cells/mL, and the next day, the electroporation kit (Invitrogen, MPK10096) and electroporation instrument (Invitrogen, MP922947) were used to transform the constructed human VEGFR2 gene sequence (NCBI Gene ID: 3791) plasmid into HEK293-NFAT cells. The electroporated cells were transferred to DMEM medium (Gibco, 12634010), and placed in a 37°C cell culture incubator for 48 hours. Then, 1000 cells/well were plated in a 96-well plate, and puromycin at a final concentration of 2 μg/mL was added for pressure selection. The single-cell clones grown in the 96-well plate were picked, and after expanded culture, the HEK293-VEGFR2-NFAT cell line was obtained by adding a luciferase catalytic substrate to detect the fluorescent signal and identify it.

1.3.3 Construction of Jurkat-PD-1-NFAT cell line

The pGL4.30 plasmid (promega, #E8481) containing the NF-AT-re nucleic acid sequence was electroporated into Jurkat cells ( TIB-152) to obtain Jurkat-NFAT cell line. On this basis, stably transform the full-length expression gene sequence of PD-1 (NCBI Gene ID: 5133), the method is consistent with 1.3.2, add puromycin and 0.5 μg/mL hygromycin B solution at a final concentration of 2 μg/mL Monoclonal cell lines were screened, and Jurkat-PD-1-NFAT cell lines were obtained by FACS identification after expansion.

1.3.4 Construction of CHO-PD-L1-CD3L cell line

The scFv sequence of OKT-3 (Drug Bank No: DB00075) was electrotransfected into CHO cells to obtain CHO-CD3L stably transfected cell line, on this basis, the full-length expression gene sequence of PD-L1 was stably transfected (NCBI Gene ID: 29126), The method was consistent with 1.3.2. The final concentration of 30 mM methionine iminosulfone and 8 μg/mL puromycin were added to screen monoclonal cell lines, and the CHO-PD-L1-CD3L cell line was obtained by FACS identification after expansion.

Example 2 Construction of anti-PD-L1 and VEGF bispecific antibody

This example describes the construction of exemplary anti-PD-L1 and VEGF bispecific antibody (BsAb) structures and expression vectors. Four constructs were designed and constructed: the amino acid sequence of VHH ^PD-L1 is from D21-4, and its variable region amino acid sequence is shown in SEQ ID NO:1; the VHH ^VEGF amino acid sequence is from P30-10-26, and its variable region The amino acid sequence is shown in SEQ ID NO: 2; the amino acid sequence of the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 19). Exemplary BsAb constructs are shown in Table 1 and the corresponding amino acid sequences are provided in Table 2.

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

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

Construct BsAb12: Contains two identical first polypeptide chains, including anti-PD-L1 VHH domain from N-terminal to C-terminal, IgG1 heavy Chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, IgG1 heavy chain Fc); contains two identical second polypeptide chains, from N-terminal to C-terminal contains anti-VEGF VHH domain, antibody Kappa light chain CL domain. BsAb12 has the format of Figure 1C.

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

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

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

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

Example 3 Expression, purification, and analysis of physicochemical properties of anti-PD-L1 and VEGF bispecific antibody

3.1 Expression and purification of anti-PD-L1 and VEGF bispecific antibody

The construct in Example 2 was expressed through the ExpiCHO transient expression system (Thermo Fisher, A29133), and the specific method was as follows: on the day of transfection, confirm that the cell density was about 7×10 ⁶ to 1×10 ⁷ cells/mL, and the cell survival At this point, adjust the cells to a final concentration of 6×10 ⁶ cells/mL with fresh ExpiCHO expression medium pre-warmed at 37°C. Dilute the target plasmid with 4°C pre-cooled OptiPRO ^TM SFM (add 1 μg of plasmid to 1 mL of the medium), and at the same time, dilute ExpiFectamine ^TM CHO with OptiPRO ^TM SFM, then mix the two in equal volumes and gently blow and mix to prepare Form ExpiFectamine ^TM CHO/plasmid DNA mixture, incubate at room temperature for 1-5min, slowly add to the prepared cell suspension and shake gently at the same time, and finally place in a cell culture shaker at 37°C, 8% CO ₂ conditions under cultivation. 18-22 hours after transfection, add ExpiCHO ^™ Enhancer and ExpiCHO ^™ Feed to the culture medium, and place the shaker flask in a shaker at 32°C and 5% CO ₂ to continue culturing. On day 5 after transfection, the same volume of ExpiCHO ^TM Feed was added, and the cell suspension was mixed gently while adding slowly. After 7-15 days of transfection, the cell culture supernatant expressing the target protein was centrifuged at a high speed of 15000g for 10min, and the resulting supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), and then purified with 100mM sodium acetate (pH3.0 ) to elute the target protein, then neutralize it with 1M Tris-HCl, and finally replace the obtained protein into PBS buffer through an ultrafiltration concentrator tube (Millipore, UFC901096).

3.2 Concentration determination of anti-PD-L1 and VEGF bispecific antibody

The concentration of the purified bispecific antibody in Example 3.1 was measured with a verified ultra-micro spectrophotometer (Hangzhou Aosheng Instrument Co., Ltd., Nano-300), and the measured A280 value was divided by the theoretical extinction coefficient of the antibody. The value is used as the antibody concentration value for follow-up research. After passing the quality inspection, it is aliquoted and stored at -80°C.

3.3 SDS-PAGE identification of anti-PD-L1 and VEGF bispecific antibody

Non-reducing solution preparation: 1 μg of candidate bispecific antibody and reference product IPI (the IPI is the abbreviation of Ipilimumab (Ipilimumab), prepared by the method in Example 3.1) was added to 5×SDS loading buffer and 40mM Iodoacetamide was heated in a dry bath at 75°C for 10 minutes, cooled to room temperature, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was taken. Preparation of reducing solution: Add 2 μg of candidate bispecific antibody and reference IPI to 5×SDS loading buffer and 5 mM DTT, heat in a dry bath at 100°C for 10 min, cool to room temperature, centrifuge at 12,000 rpm for 5 min, and take the supernatant. Add the supernatant to Bis-tris 4-15% gradient gel (purchased from GenScript), electrophoresis at a constant voltage of 110V, stop running when the Coomassie brilliant blue migrates to the bottom of the gel, take out the gel slice and place it in the Coomassie brilliant blue staining solution Incubate for 1-2 hours, discard the staining solution, add destaining solution, replace the destaining solution 2-3 times as needed, decolorize until the gel background is transparent, and store in deionized water. After decolorization, it was scanned with EPSON V550 color scanner, and the purity of reduced and non-reduced bands was calculated by ImageJ according to the peak area normalization method.

The results are shown in Figures 2A-2B: the bands of the candidate bispecific antibody and the reference product IPI reduced gel and non-reduced gel all conformed to the expected size, and the purity of the reduced gel was greater than 95%.

3.4 SEC-HPLC monomer purity identification of anti-PD-L1 and VEGF bispecific antibody

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

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

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

Example 4 Affinity activity analysis of anti-PD-L1 and VEGF bispecific antibody

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

Coat the recombinant protein VEGF-His on a 96-well ELISA plate, overnight at 4°C. On the next day, the well plate was washed 3 times with PBST and blocked with 5% skimmed milk for 2 h. After the plate was washed 3 times with PBST, different concentrations of candidate bispecific antibody, control antibody bevacizumab and P30-10- 26 for 1 h. Afterwards, after washing with PBST for 3 times, the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab79225) was added and incubated for 1 h. After incubation, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added to develop the color. According to the color development results, 2M HCl was added to terminate the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Using PRISM ^TM (GraphPad Software, San Diego, CA) analyzed data and calculated _EC50 values.

The results of the ELISA binding assay are shown in Figure 4 and Table 4, the bispecific antibodies BsAb11, BsAb12 and BsAb13 exhibited better binding ability to VEGF than the control antibody bevacizumab, and comparable to the control antibody P30-10-26 .

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

Coat the recombinant protein PD-L1-His on a 96-well ELISA plate overnight at 4°C. The next day, the well plate was washed 3 times with PBST and blocked with 5% skimmed milk for 2 h. After the plate was washed 3 times with PBST, different concentrations of candidate bispecific antibody and control antibody D21-4 were added and incubated for 1 h. Afterwards, after washing with PBST for 3 times, the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab79225) was added and incubated for 1 h. After incubation, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added to develop the color. According to the color development results, 2M HCl was added to terminate the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Data were analyzed using PRISM ^™ (GraphPad Software, San Diego, CA), and _EC50 values were calculated.

The results of the ELISA binding assay are shown in Figure 5 and Table 4, and the candidate bispecific antibodies all showed comparable ability to bind to PD-L1 as the control antibody D21-4.

4.3 Detection of the binding ability of candidate bispecific antibodies to huPD-L1-CHO-K based on FACS method

Collect the huPD-L1-CHO-K cells in the exponential growth phase, centrifuge at 300g to remove the supernatant, resuspend the cells in FACS buffer (PBS containing 1% BSA), count and adjust the density of the cell suspension to 2×10 ⁶ individual/mL. Subsequently, huPD-L1-CHO-K cells were added to a 96-well round bottom plate at 100 μL per well, and centrifuged at 300 g to remove the supernatant. Different concentrations of candidate bispecific antibody and control antibody D21-4 dilutions were added to the corresponding wells, the cells were resuspended and incubated at 4°C for 30 min. After the incubated cell mixture was washed 3 times, a PE-labeled anti-human-IgG-Fc flow antibody (Abcam, 98596) was added, resuspended and incubated at 4°C for 30 min. After the incubated cell mixture was washed three times, 200 μL of FACS buffer was added to resuspend the cells, and the cells were detected and analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102). Data were analyzed using PRISM ^™ (GraphPad Software, San Diego, CA), and _EC50 values were calculated.

FACS binding assay results are shown in Figure 6 and Table 4, bispecific antibodies BsAb10 and BsAb12 showed better binding ability to PD-L1 expressed on cells than control antibody D21-4.

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

Coat the recombinant protein VEGF-Fc on a 96-well ELISA plate, overnight at 4°C. On the next day, the well plate was washed 3 times with PBST and then blocked with 5% skim milk for 2 hours. After the plate was washed 3 times with PBST, different concentrations of candidate bispecific antibodies were added and incubated for 1 hour. After that, add recombinant protein PD-L1-His after washing with PBST for 3 times, wash the plate with PBST for 3 times, add Anti-6×His-HRP (Proteintech, HRP-660050), incubate at room temperature for 50min, and then wash the plate with PBST Twelve times, add TMB (SurModics, TMBS-1000-01) for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Data were analyzed using PRISM ^™ (GraphPad Software, San Diego, CA), and _EC50 values were calculated.

The recombinant protein PD-L1-Fc was coated on a 96-well ELISA plate overnight at 4°C. On the next day, the well plate was washed 3 times with PBST and then blocked with 5% skim milk for 2 hours. After the plate was washed 3 times with PBST, different concentrations of candidate bispecific antibodies were added and incubated for 1 hour. Afterwards, wash 3 times with PBST, add recombinant protein VEGF-His, wash the plate 3 times with PBST, add Anti-6×His-HRP (Proteintech, HRP-660050), incubate at room temperature for 50 min, after completion, wash the plate with PBST for 12 The second time, add TMB (SurModics, TMBS-1000-01) for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). use PRISM ^™ (GraphPad Software, San Diego, CA) analyzed data and calculated _EC50 values.

The results of the ELISA binding assay are shown in Figures 7A-7B, the bispecific antibody BsAb10 showed the ability to simultaneously bind to PD-L1 and VEGF.

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

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

In this example, the luciferase reporter gene system was used to detect the activity of candidate anti-PD-L1 and VEGF bispecific antibodies in blocking the PD-1/PD-L1 signaling pathway, and the combination of PD-1 and PD-L1 can block the downstream signal of CD3 Transduced to inhibit luciferase expression system, when adding anti-PD-L1 and VEGF bispecific antibody, the blocking effect is reversed, luciferase expression, stimulated with antibodies with different concentration gradients, will have antibody concentration-dependent A specific fluorescence readout curve allows the evaluation of the blocking activity of the antibody. In this example, the luciferase reporter gene system was used 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:

Add 2× ¹⁰⁴ /well CHO-PD-L1-CD3L cells and 1× ¹⁰⁵ /well Jurkat-PD-1-NFAT cells in a volume of 50 μL in a 96-well permeable cell culture plate with white edges. Add 50 μL of serially diluted candidate bispecific antibody and control antibody D21-4, and incubate in a 37°C incubator for 6h. Add 50 μL Bright-Lite (vazyme, product number: DD1204-03) to each well, incubate in the dark for 10 min, and detect the fluorescent signal.

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

Example 6 Detection of VEGF neutralizing activity of anti-PD-L1 and VEGF bispecific antibody based on reporter gene method

In this example, in order to determine the function of the candidate bispecific antibody neutralizing the VEGF-VEGFR2 signaling pathway, the HEK293-VEGFR2-NFAT cell line was used (the VEGF recombinant protein was added to the cell line culture system to activate the cell line through the VEGF-VEGFR2 signaling axis). Transcription and expression of the internal NFAT luciferase reporter gene, adding the catalytic substrate of luciferase to generate a fluorescent signal) as a material to detect the binding of the candidate bispecific antibody to VEGF-VEGFR2 to block the expression of the downstream NFAT luciferase reporter gene ability. The specific implementation is as follows:

Adjust the HEK293-VEGFR2-NFAT cell line to 4×10 ⁵ cells/mL, add 100 μL per well to a new 96-well cell culture plate, and place it in a 37°C cell culture incubator. At the same time, the candidate bispecific antibody and control antibody P30-10-26 were serially diluted in DMEM medium, 60 ng/mL VEGF-Fc was added, mixed and incubated at room temperature for 30 min. Subsequently, the co-incubated serially diluted antibody and VEGF-Fc mixture was added to a 96-well cell culture plate, and cultured in a 37° C. incubator for 18 hours. After the incubation, 30 μL of luciferase substrate Bright-Lite (Vazyme, DD1204-03) was added to each well, and the fluorescence value of the 96-well plate was detected after shaking for 2 minutes.

The results are shown in Figure 9 and Table 5, the neutralization effect of bispecific antibody BsAb10 on VEGF was significantly better than that of control antibody P30-10-26, and the neutralization effect of bispecific antibody BsAb11, BsAb12 and BsAb13 on VEGF was comparable to that of control antibody P30- 10-26 is equivalent.

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

Example 7 In vivo tumor suppression experiment of anti-PD-L1 and VEGF bispecific antibody (COLO205 mouse tumor-bearing model)

6-8 weeks old female NOG mice (purchased from Shanghai Weitong Lihua, strain: NOD.Cg-Prkdc ^scid Il2rg ^tm1Sug /JicCrl) were used, and the experimental mice were kept in an independent ventilated box with constant temperature and humidity, and the temperature of the feeding room was 21 -24°C, humidity 30-53%. The huPD-L1-COLO205 cells (a stably transfected cell line overexpressing human PD-L1 obtained by stably transfecting PD-L1 (Gene ID: 29126) into COLO205 cells (purchased from Beina Biotech, catalog number: BNCC338682)) were 3×10 ⁶ mice/mouse were subcutaneously injected into the right back (day 0), and then randomly divided into groups (6 mice per 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 has a high-dose group, in which the bispecific antibody BsAb10 administration group and AK112 The administration group also set up a middle and low dose group. On the second day after tumor bearing, PBMC (C2106025) was injected through the tail vein, and each mouse was injected with 3×10 ⁶ PBMC cells. The first administration was performed 2 hours later, twice a week, intraperitoneal injection (ip) medicine for a total of 3 weeks.

Observe and record the tumor length (mm) and width (mm) at any time, calculate its tumor volume (V), the calculation method is V=(length×width ² )/2, tumor inhibition rate TGI (%)=(1-administration The average tumor volume of the group/the average tumor volume of the PBS-treated group)×100%. The tumor suppression results are shown in Figure 10 and Table 6. It can be seen from the results that all drug groups exhibited tumor growth inhibition relative to the PBS treatment group; the Atezolizumab+Bevacizumab combination group was superior to the Atezolizumab single drug group and Bevacizumab single drug group, proving that in this model, anti-PD-L1 and anti-VEGF can present a combined drug effect; at a high dose of 1.87 μM, all mice in the bispecific antibody BsAb10 administration group showed a trend of complete tumor remission on the 13th day, and the tumor inhibition rate was 89.75%, which was significant It was better than the control antibody W3256 administration group (the tumor inhibition rate was 80.74%), the control antibody AK112 administration group (the tumor inhibition rate was 54.79%) and the combination administration group of Atezolizumab+Bevacizumab (the tumor inhibition rate was 76.52%); At the medium dose of 0.47μM, all mice in the bispecific antibody BsAb10-administered group showed a trend of complete tumor remission on the 13th day, and the tumor inhibition rate was 83.68%, which was significantly better than that of the control antibody AK112-administered group (inhibition The tumor rate was 35.69%); at a low dose of 0.094 μM, the bispecific antibody BsAb10 showed a 36.26% tumor inhibitory effect on the 23rd day, which was significantly better than the control bispecific antibody AK112 (the tumor inhibitory rate was 14.25%).

The tumor inhibition rate TGI (%) of different administration groups of table 6

Example 8 In vivo tumor suppression experiment of anti-PD-L1 and VEGF bispecific antibody (A431 mouse tumor-bearing model)

6-8 weeks old female NSG mice (purchased from Shanghai Nanfang Model Organisms, strain: M-NSG) were used, and the experimental mice were kept in an independent ventilation box with constant temperature and humidity. %. A431 cells (human epidermal cancer cells) were subcutaneously injected into the right back of each mouse at 5×10 ⁶ cells (day 0), and then randomly divided into groups (8 mice per group): PBS treatment group respectively , D21-4 low-dose administration group (0.37mpk (mg/kg)), P30-10-26 low-dose administration group (0.37mpk), bispecific antibody BsAb10 high-dose administration group (2mpk), bispecific Antibody BsAb10 low-dose administration group (0.5mpk), D21-4 and P30-10-26 high-dose combination treatment group (1.48+1.48mpk), D21-4 and P30-10-26 low-dose combination treatment group (0.37 +0.37mpk). On the second day after bearing the tumor, inject PBMC (C2106025) through the tail vein, inject 5×10 ⁶ PBMC cells per mouse, and give the first administration 2 hours later, twice a week, intraperitoneal injection (ip) medicine for a total of 5 weeks.

Observe and record the tumor length (mm) and width (mm) at any time, calculate its tumor volume (V), the calculation method is V=(length×width ² )/2, tumor inhibition rate TGI (%)=(1-administration The average tumor volume of the group/the average tumor volume of the PBS-treated group)×100%. The tumor suppression results are shown in Figure 11 and Table 7. As can be seen from the results: all administration groups exhibited tumor growth inhibition relative to the PBS treatment group; BsAb10 (tumor inhibition rate of 75.94%) at high doses compared to D21-4 and P30-10-26 combined treatment group ( Inhibition rate of 66.1%) showed better antitumor efficacy; BsAb10 (inhibition rate of 43.14%) at low doses compared with D21-4 and P30-10-26 combination group (inhibition rate of 36.06 %) showed better antitumor efficacy.

The tumor inhibition rate TGI (%) of different administration groups of table 7

Sequence Listing Overview

Accompanying this application is a Sequence Listing containing a number of nucleic acid and amino acid sequences. The table below provides an overview of the sequences included.

Claims

A bispecific antibody specifically binding to PD-L1 and VEGF, wherein the antibody comprises a VHH domain specifically binding to a first antigen and a VHH domain specifically binding to a second antigen, wherein the first antigen and the second the antigens are different from each other and are independently selected from PD-L1 and VEGF;

Wherein, the VHH structural domain specifically binding to VEGF comprises the CDR1-3 sequence of the VHH structural domain shown in SEQ ID NO:2;

And preferably, wherein the VHH domain specifically binding to PD-L1 comprises the CDR1-3 sequence of the VHH domain shown in SEQ ID NO:1.
The bispecific antibody of claim 1, wherein the VHH domain specifically binding to VEGF comprises the amino acid sequence shown in SEQ ID NO: 2, or has at least 80%, 85%, 90%, 95% with SEQ ID NO: 2 Amino acid sequence with % or 99% identity, or with one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and/or substitutions compared to SEQ ID NO: 2 amino acid sequence,

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.
The bispecific antibody according to any one of claims 1-2, wherein the VHH domain specifically binding to PD-L1 comprises the amino acid sequence shown in SEQ ID NO: 1, or has at least 80%, An amino acid sequence that is 85%, 90%, 95% or 99% identical, or has one or more (preferably 1-10, more preferably 1-5) amino acid additions compared to SEQ ID NO: 1 , deleted and/or substituted amino acid sequences,

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.
The bispecific antibody according to any one of claims 1-3, wherein said bispecific antibody comprises a linker comprising 10-20 amino acids in length, preferably comprising an amino acid sequence (G 4 S) 3 .
The bispecific antibody according to any one of claims 1-4, wherein the bispecific antibody comprises an Fc region of an immunoglobulin, wherein the Fc region is an IgG Fc region, for example, the Fc of human IgG1, IgG2, IgG3 or IgG4 Region, preferably the Fc region of human IgG1, more preferably the Fc region has an immunoglobulin hinge region sequence at the N-terminus.
The bispecific antibody according to any one of claims 1-5, wherein said bispecific antibody comprises a polypeptide chain comprising 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 the first antigen and the second antigen, respectively,

Wherein said first antigen and 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.
The bispecific antibody according to any one of claims 1-5, wherein said bispecific antibody comprises a polypeptide chain comprising 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 the first antigen and the second antigen, respectively,

Wherein said first antigen and 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.
The bispecific antibody according to any one of claims 1-5, wherein said bispecific antibody comprises a first and a second polypeptide chain, wherein said first polypeptide chain comprises a first polypeptide chain from N-terminus to C-terminus VHH domain, CH1 domain and Fc region; the second polypeptide chain comprises a second VHH domain and a CL domain from N-terminal to C-terminal;

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 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.
The bispecific antibody according to any one of claims 1-8, wherein said bispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 7 or has at least 90%, 91%, 92%, 93%, 94% thereof , 95%, 96%, 97%, 98% or 99% identical amino acid sequences; or

Comprising 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.
The bispecific antibody of any one of claims 1-8, wherein said bispecific antibody comprises a first and a second polypeptide chain, wherein:

The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 9 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto a specific amino acid sequence; and

The second polypeptide chain comprises an amino acid sequence shown in SEQ ID NO: 10 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith amino acid sequence.
A polynucleotide encoding the bispecific antibody of any one of claims 1-10.
A vector, preferably an expression vector, comprising the polynucleotide of claim 11.
A host cell comprising the polynucleotide of claim 11 or the vector of claim 12, for example, the host cell is a mammalian cell.
A method for producing the bispecific antibody of any one of claims 1-10, said method comprising:

culturing a host cell comprising a polypeptide chain encoding said antibody under conditions suitable for expressing said polypeptide chain; and allowing assembly of said polypeptide chain into said antibody to produce said antibody under conditions suitable for assembly of said polypeptide chain into said antibody.
A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1-10 and a pharmaceutically acceptable carrier.
Use of the bispecific antibody according to any one of claims 1-10 or the pharmaceutical composition of claim 15, for in vivo or in vitro

- binds human PD-L1 and human VEGF; or

- Block the human PD-L1/PD-1 signaling pathway; or

- neutralizes VEGF activity; or

- inhibition of tumor cell growth,

Or for the preparation of medicines for any of the above purposes.
The use of claim 16, wherein the bispecific antibody or pharmaceutical composition is used as a drug for treating and/or preventing diseases in an individual or as a diagnostic tool for a disease, preferably said individual is a mammal, more preferably a human.
The use according to claim 17, wherein the disease is PD-L1 positive cancer, preferably PD-L1 positive solid tumor or blood tumor, such as head and neck squamous cell carcinoma, melanoma, renal cell carcinoma, non-small cell lung cancer, bladder cancer, urinary tract cancer Skin cancer, gastric cancer, colon cancer, colorectal cancer, ovarian cancer, breast cancer, lung cancer, cervical cancer, glioblastoma cancer, pancreatic cancer, prostate cancer, esophageal cancer, lymphoma, liver cancer, microsatellite unstable solid tumors .