WO2021244328A1 - Anti-pd-l1 and her2 bispecific antibody - Google Patents

Abstract

Provided in the present invention is an anti-PD-L1 and HER2 bispecific antibody. The bispecific antibody of the present invention is capable of specifically binding with both PD-L1 and HER2 targets, and provides biological activities similar or superior to that of a monoclonal antibody.

Description

A bispecific antibody against PD-L1 and HER2 Technical field

The present invention relates to the field of antibodies. More specifically, the present invention discloses a bispecific antibody against PD-L1 and HER2.

Background technique

Human Programmed Cell Death Receptor-1 (PD-1) is a type I membrane protein with 288 amino acids. It is one of the major known immune checkpoints (Blank et al, 2005, Cancer Immunotherapy) , 54: 307-314). PD-1 is expressed on activated T lymphocytes, and it interacts with the ligand PD-L1 (programmed cell death-Ligand 1) and PD-L2 (programmed cell death receptor- 1). Ligand 2, programmed cell death-Ligand 2) The combination can inhibit the activity of T lymphocytes and related cellular immune responses in the body. PD-L2 is mainly expressed in macrophages and dendritic cells, while PD-L1 is widely expressed in B, T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, heart and other tissue cells. A large number of studies have shown that the interaction of PD-1 and PD-L1 is not only necessary to maintain the balance of the immune system in the body, but also the main mechanism and reason that causes PD-L1 positive tumor cells to evade immune surveillance. By blocking the negative regulation of cancer cells on the PD-1/PD-L1 signaling pathway and activating the immune system, it can promote T cell-related tumor-specific cellular immune responses, thereby opening the door to a new tumor treatment method-- Tumor immunotherapy.

PD-1 (encoded by the gene Pdcd1) is a member of the immunoglobulin superfamily related to CD28 and CTLA-4. Research results show that when PD-1 binds to its ligands (PD-L1 and/or PD-L2), it negatively regulates antigen receptor signal transduction. The structure of mouse PD-1 and the co-crystal structure of mouse PD-1 and human PD-L1 have been clarified (Zhang, X. et al. Immunity 20: 337-347 (2004); Lin et al., Proc. Natl. Acad. Sci. USA 105: 3011-6 (2008)). PD-1 and similar family members are type I transmembrane glycoproteins, which contain an Ig variable (V-type) domain responsible for ligand binding and a cytoplasmic tail region responsible for binding signal transduction molecules. The cytoplasmic tail of PD-1 contains two tyrosine-based signal transduction motifs, ITIM (Immunoreceptor Tyrosine Inhibition Motif) and ITSM (Immune Receptor Tyrosine Switch Motif).

PD-1 plays an important role in the immune evasion mechanism of tumors. Tumor immunotherapy, which uses the body’s own immune system to fight cancer, is a breakthrough tumor treatment method, but the tumor microenvironment can protect tumor cells from effective immune destruction. Therefore, how to break the tumor microenvironment has become an anti-tumor research Focus. Existing research results have determined the role of PD-1 in the tumor microenvironment: PD-L1 is expressed in many mouse and human tumors (and can be induced by IFN-γ in most PD-L1-negative tumor cell lines), It is presumed to be an important target for mediating tumor immune evasion (Iwai Y. et al., Proc. Natl. Acad. Sci. USA 99: 12293-12297 (2002); Strome SE et al., Cancer Res., 63: 6501-6505) (2003)). Through immunohistochemical assessment of biopsies, the expression of PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 on tumor cells has been found in many primary human tumors. Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma and Pancreatic cancer and head and neck tumors. It can be seen that blocking the interaction of PD-1/PD-L1 can improve the immune activity of tumor-specific T cells and help the immune system to clear tumor cells. Therefore, PD-L1 has become a popular target for the development of tumor immunotherapy drugs. .

HER2/neu (human epidermal growth factor receptor 2), also known as erbB2, has tyrosine protein kinase activity and is a member of the human epidermal growth factor receptor family. It is only expressed at low levels in a few normal tissues of adults . However, studies have shown that HER2 is overexpressed in a variety of tumors. For example, there is overexpression in about 30% of breast cancer patients and 16% of gastric cancer patients. Overexpression of HER2 in tumors can significantly promote tumor angiogenesis, The growth of tumors and the enhancement of tumor invasion and metastasis are important indicators of poor prognosis for such patients. Therefore, as early as 1998, Herceptin (Herceptin, Trastuzumab/Trastuzumab, Genentech/Roche), the first monoclonal antibody drug targeting HER2, was approved by the FDA for marketing and used in HER2 overexpressing breast cancer. And the treatment of gastric cancer.

Bispecific antibodies refer to antibody molecules that can specifically bind to two antigens or two epitopes at the same time. According to symmetry, bispecific antibodies can be divided into structurally symmetric and asymmetric molecules. According to the number of binding sites, bispecific antibodies can be divided into bivalent, trivalent, tetravalent and multivalent molecules. Bispecific antibodies are gradually becoming a new class of therapeutic antibodies that can be used to treat various inflammatory diseases, cancers and other diseases.

Summary of the invention

The present invention provides a bispecific antibody against PD-L1 and HER2.

Therefore, the first objective of the present invention is to provide a bispecific antibody against PD-L1 and HER2.

The second object of the present invention is to provide an isolated nucleotide encoding the bispecific antibody.

The third object of the present invention is to provide an expression vector containing the nucleotide.

The fourth object of the present invention is to provide a host cell containing the expression vector.

The fifth object of the present invention is to provide a method for preparing the bispecific antibody.

The sixth object of the present invention is to provide a pharmaceutical composition containing the bispecific antibody.

The seventh object of the present invention is to provide the use of the bispecific antibody or the pharmaceutical composition in the preparation of drugs for the treatment of cancer.

The eighth object of the present invention is to provide a method for treating cancer with the bispecific antibody or the pharmaceutical composition.

In order to achieve the above objective, the present invention provides the following technical solutions:

The first aspect of the present invention provides a bispecific antibody against PD-L1 and HER2, comprising two polypeptide chains and two light chains, wherein:

(a) The polypeptide chain includes VH-PDL1-CH1-CH2-CH3-L1-VH-HER2-L2-VL-HER2 or VH-HER2-L2-VL-HER2-L1-VH from N-terminus to C-terminus -PDL1-CH1-CH2-CH3, the light chain includes VL-PDL1-CL from N-terminus to C-terminus; or

(b) The polypeptide chain contains VH-HER2-CH1-CH2-CH3-L1-VH-PDL1-L2-VL-PDL1 or VH-PDL1-L2-VL-PDL1-L1-VH from N-terminus to C-terminus -HER2-CH1-CH2-CH3, the light chain includes VL-HER2-CL from N-terminus to C-terminus;

Wherein, the VH-PDL1 is a heavy chain variable region that binds PD-L1, the VL-PDL1 is a light chain variable region that binds PD-L1, and the VH-HER2 is a heavy chain that binds HER2 The variable region, the VL-HER2 is a light chain variable region that binds to HER2, the L1 and L2 are (G ₄ S)x, and x is 3, 4, 5 or 6, the CH1-CH2 -CH3 is the heavy chain constant region, the CL is the light chain constant region, the VH-PDL1 and the VL-PDL1 form an antigen binding site that specifically binds to PD-L1, the VH-HER2 It forms an antigen binding site that specifically binds to HER2 with the VL-HER2.

According to a preferred embodiment of the present invention, said L1 is (G ₄ S) ₃ and said L2 is (G ₄ S) ₄ .

According to a preferred embodiment of the present invention, the VH-PDL1 includes the amino acid sequence of the heavy chain CDR shown in SEQ ID NO: 1-3, and the VL-PDL1 includes the amino acid sequence of the amino acid sequence shown in SEQ ID NO: 4-6. The light chain CDRs shown, the VH-HER2 includes the amino acid sequence of SEQ ID NO: 7-9 or the heavy chain CDRs of SEQ ID NO: 13-15, and the VL-HER2 includes the amino acid sequence of SEQ ID NO: 10-12 or the light chain CDR shown in SEQ ID NO: 16-18.

According to a preferred embodiment of the present invention, the VH-PDL1 has the amino acid sequence shown in SEQ ID NO: 19, the VL-PDL1 has the amino acid sequence shown in SEQ ID NO: 20, and the VH -HER2 has an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 23, and said VL-HER2 has an amino acid sequence as shown in SEQ ID NO: 22 or SEQ ID NO: 24.

According to a preferred embodiment of the present invention, the polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 31 or SEQ ID NO: 33, and the light chain has an amino acid sequence as shown in SEQ ID NO: 26; Or the polypeptide chain has the amino acid sequence shown in SEQ ID NO: 32 or SEQ ID NO: 34, and the light chain has the amino acid sequence shown in SEQ ID NO: 28; or the polypeptide chain has For example, the amino acid sequence shown in SEQ ID NO: 35 or SEQ ID NO: 36, the light chain has the amino acid sequence shown in SEQ ID NO: 30.

According to a preferred embodiment of the present invention, the polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 34, and the light chain has an amino acid sequence as shown in SEQ ID NO: 28, and the amino acid sequence is The polypeptide chain shown in SEQ ID NO: 34 further includes mutations in the following amino acid residue positions: N76E and/or N213E.

According to a preferred embodiment of the present invention, the polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 37 or SEQ ID NO: 38 or SEQ ID NO: 39, and the light chain has an amino acid sequence as shown in SEQ ID NO: 28 The amino acid sequence shown.

According to the present invention, the heavy chain constant region includes an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, and the light chain constant region includes a kappa or lambda light chain constant region.

The second aspect of the present invention provides an isolated nucleotide, which encodes the bispecific antibody.

The third aspect of the present invention provides an expression vector, which contains the above-mentioned nucleotides.

The fourth aspect of the present invention provides a host cell, which contains the expression vector as described above.

The fifth aspect of the present invention provides a method for preparing the bispecific antibody, the method comprising the following steps:

(a) Culturing the host cell as described above under expression conditions to express the bispecific antibody;

(b) Isolation and purification of the bispecific antibody described in (a).

The sixth aspect of the present invention provides a pharmaceutical composition containing the bispecific antibody as described above and a pharmaceutically acceptable carrier.

The seventh aspect of the present invention provides the use of the above-mentioned bispecific antibody or the above-mentioned pharmaceutical composition in the preparation of a medicament for the treatment of cancer.

According to the present invention, the cancer is selected from the group consisting of: melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer Cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases.

The eighth aspect of the present invention provides a method of treating cancer, comprising administering the bispecific antibody as described above or the pharmaceutical composition as described above to a subject in need.

According to the present invention, the cancer is selected from the group consisting of: melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer Cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases.

Beneficial effects: The present invention provides a bispecific antibody against PD-L1 and HER2. The results of in vitro and in vivo experiments show that it can specifically bind to both PD-L1 and HER2 targets at the same time, which is similar to or even better than that of monoclonal antibodies. Excellent biological activity.

Description of the drawings

Figure 1 shows the result of ELISA to determine the affinity of the bispecific antibody to PDL1-ECD-his.

Figure 2 shows the results of ELISA to determine the affinity of the bispecific antibody to HER2-ECD-his. Figure 2A shows the bispecific antibody combined with M8 and 607, and Figure 2B shows the bispecific antibody combined with M8 and 612.

Figure 3 shows the result of the bispecific antibody competitively inhibiting the binding of PD-1 and PD-L1.

Figure 4 shows the results of determination of the binding activity of bispecific antibodies in inhibiting PD-1 and PD-L1 high-expressing cell lines.

Figure 5 shows the results of measuring the activity of bispecific antibodies in inhibiting the proliferation of BT474 cells.

Figure 6 shows the results of measuring the activity of bispecific antibodies to inhibit the proliferation of N87 cells.

Figure 7 shows the results of measuring the activity of bispecific antibodies in inhibiting the proliferation of BT474 cells.

Figure 8 shows the results of measuring the activity of bispecific antibodies to inhibit the proliferation of N87 cells.

Figure 9 shows the results of the bispecific antibody on the growth inhibition of gastric cancer cell NCI-N87 xenograft tumors.

Figure 10 shows the results of the growth inhibition of MC38-hPD-L1 xenografted tumors by bispecific antibodies.

Figure 11 shows the results of the affinity determination of the bispecific antibody PDL1scFv-607H mutant to PDL1-ECD-his.

Figure 12 shows the results of the affinity determination of the bispecific antibody PDL1scFv-607H mutant to HER2-ECD-his.

Figure 13 shows that the bispecific antibody PDL1scFv-607H mutant inhibits the binding activity of PD-1 and PD-L1 high-expressing cell lines.

Figure 14 shows the determination of the cell proliferation inhibitory activity of the bispecific antibody PDL1scFv-607H mutant on the HER2 high-expressing cell line.

Figure 15 shows the results of a mixed lymphocyte reaction (MLR) experiment measuring that bispecific antibodies promote the secretion of IL-2 by T cells.

Fig. 16 shows the results of the mixed lymphocyte reaction (MLR) experiment measuring that the bispecific antibody promotes the secretion of IFN-γ from T cells.

detailed description

In the present invention, the terms "Antibody (Ab)" and "Immunoglobulin G (Abbreviation IgG)" are heterotetrameric glycoproteins of about 150,000 Daltons with the same structural characteristics, which consist of two The same light chain (L) and two identical heavy chains (H) are composed. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes (isotype) is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. There is a variable region (VH) at one end of each heavy chain, followed by a constant region. The constant region of the heavy chain is composed of three domains, CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end. The light chain constant region includes a structural domain CL; the light chain constant region is paired with the CH1 domain of the heavy chain constant region, and the light chain can be The variable region is paired with the variable region of the heavy chain. Constant regions are not directly involved in the binding of antibodies and antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity) and so on. The heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes kappa (Kappa) or lambda (Lambda). The heavy and light chains of the antibody are covalently linked together by the disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of the antibody are covalently linked together by the inter-polypeptide disulfide formed between the hinge regions. The bonds are linked together covalently.

In the present invention, the term "bispecific antibody (double antibody)" refers to an antibody molecule that can specifically bind to two antigens (targets) or two epitopes at the same time.

In the present invention, the term "monoclonal antibody (monoclonal antibody)" refers to an antibody obtained from a substantially homogeneous population, that is, the single antibodies contained in the population are the same, except for a few naturally occurring mutations that may exist. Monoclonal antibodies are highly specific to a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (usually a mixture of different antibodies directed against different antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by culturing hybridomas without being contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristics of the antibody, which is obtained from a substantially uniform antibody population, which should not be interpreted as requiring any special method to produce the antibody.

In the present invention, the terms "Fab" and "Fc" mean that papain can cleave an antibody into two identical Fab segments and one Fc segment. The Fab segment is composed of the VH and CH1 of the heavy chain of the antibody and the VL and CL domains of the light chain. The Fc segment can be a fragment crystallizable (Fc), which is composed of the CH2 and CH3 domains of the antibody. The Fc segment has no antigen binding activity and is the site where the antibody interacts with effector molecules or cells.

In the present invention, the term "scFv" refers to a single chain antibody (single chain antibody fragment, scFv), which is formed by linking the variable region of the heavy chain of the antibody and the variable region of the light chain through a short peptide (linker) of 15-25 amino acids.

In the present invention, the term "variable" means that certain parts of the variable region of the antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three fragments called the complementarity-determining region (CDR) or hypervariable region in the variable region of the heavy chain and the variable region of the light chain. The more conservative part of the variable region is called the frame region (FR). The variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a β-sheet configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-sheet structure. The CDRs in each chain are closely placed together through the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)).

In the present invention, the terms "anti", "binding", and "specific binding" refer to the non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it is directed against. Generally, the antibody binds to the antigen with an equilibrium dissociation constant (KD) of ^{less than about 10 -7} M, for example, less than about 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^{-11 M or less.} In the present invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, Surface Plasmon Resonance (SPR) is used to measure the binding affinity of an antibody to an antigen in a BIACORE instrument or an ELISA is used to measure the relative binding affinity of an antibody to the antigen.

In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitope of the present invention is a region of an antigen that is bound by an antibody.

In the present invention, the term "expression vector" can be pTT5, pSECtag series, pCGS3 series, pcDNA series vectors, etc., and other vectors used in mammalian expression systems. The expression vector includes those connected with appropriate transcription and translation regulatory sequences. Fusion DNA sequence.

In the present invention, the term "host cell" refers to a cell suitable for expressing the above-mentioned expression vector. It can be a eukaryotic cell. For example, mammalian or insect host cell culture systems can be used for the expression of the fusion protein of the present invention. CHO (Chinese hamster Ovary, Chinese Hamster Ovary), HEK293, COS, BHK and derived cells of the above-mentioned cells are all suitable for the present invention.

In the present invention, the term "pharmaceutical composition" means that the bispecific antibody of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to exert a more stable therapeutic effect. These preparations can ensure that the bispecific antibody disclosed in the present invention The conformational integrity of the amino acid core sequence of the sex antibody, while also protecting the multifunctional groups of the protein from its degradation (including but not limited to aggregation, deamination or oxidation).

The following examples and experimental examples are to further illustrate the present invention, and should not be construed as limiting the present invention. The examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art, and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd edition, Cold spring Harbor Laboratory Press.

Example 1 Construction of anti-PD-L1 and HER2 bispecific antibodies

The general structural formula of the bispecific antibody of the present invention is as follows:

1) Ab-L1-scFv;

2) scFv-L1-Ab;

Wherein Ab is an antibody, which contains two heavy chains and two light chains;

The scFv is a single-chain antibody, which is formed by connecting the variable region of the heavy chain and the variable region of the light chain of another antibody through the linking sequence L2, and is connected to the N-terminus or C of the two heavy chains of the Ab through the linking sequence L1. End

L1 and L2 are the connection sequence (G ₄ S)x, and x can be 3, 4, 5, or 6.

The present invention uses anti-human PD-L1 antibody M8 (sequence derived from PCT/CN2020/090442), anti-human HER2 antibody 607 (prepared according to trastuzumab sequence, sequence derived from US5821337) and anti-human HER2 antibody 612 (sequence source) The bispecific antibody was constructed by the combination of WO2020/025013A1), and the specific structure is shown in Table 1.

Table 1. The structure of the bispecific antibody of the present invention

name	Polypeptide chain (N-terminal to C-terminal)	Light chain
PDL1H-607scFv	PDL1-HC-(G 4 S) 3 -607-VH-(G 4 S) 4 -607-VL	PDL1-LC
607H-PDL1scFv	607-HC-(G 4 S) 3 -PDL1-VH-(G 4 S) 4 -PDL1-VL	607-LC
607scFv-PDL1H	607-VH-(G 4 S) 4 -607-VL-(G 4 S) 3 -PDL1-HC	PDL1-LC
PDL1scFv-607H	PDL1-VH-(G 4 S) 4 -PDL1-VL-(G 4 S) 3 -607-HC	607-LC
PDL1scFv-612H	PDL1-VH-(G 4 S) 4 -PDL1-VL-(G 4 S) 3 -612-HC	612-LC
612H-PDL1scFv	612-HC-(G 4 S) 3 -PDL1-VH-(G 4 S) 4 -PDL1-VL	612-LC

Among them, PDL1-HC represents the heavy chain of M8, PDL1-LC represents the light chain of M8, 607-VH represents the variable region of the heavy chain of 607, and 607-VL represents the variable region of the light chain of 607, and the rest are similar.

The related sequence information of the bispecific antibody constructed in the present invention is shown in Table 2, where the CDR is encoded according to the Kabat rule.

Table 2. Sequence information of the antibody of the present invention

SEQ ID NO:	Sequence name
1	The amino acid sequence of H-CDR1 of the heavy chain complementarity determining region of anti-PD-L1 antibody
2	The amino acid sequence of H-CDR2 of the heavy chain complementarity determining region of anti-PD-L1 antibody
3	The amino acid sequence of H-CDR3 of the heavy chain complementarity determining region of anti-PD-L1 antibody
4	The amino acid sequence of L-CDR1 of the light chain complementarity determining region of anti-PD-L1 antibody
5	The amino acid sequence of L-CDR2 of the light chain complementarity determining region of anti-PD-L1 antibody
6	The amino acid sequence of L-CDR3 of the light chain complementarity determining region of anti-PD-L1 antibody
7	The amino acid sequence of the H-CDR1 of the heavy chain complementarity determining region of 607
8	The amino acid sequence of the H-CDR2 of the heavy chain complementarity determining region of 607
9	The amino acid sequence of the H-CDR3 of the heavy chain complementarity determining region of 607
10	The amino acid sequence of L-CDR1 of the light chain complementarity determining region of 607
11	The amino acid sequence of L-CDR2 of the light chain complementarity determining region of 607
12	The amino acid sequence of L-CDR3 of the light chain complementarity determining region of 607
13	The amino acid sequence of the H-CDR1 of the heavy chain complementarity determining region of 612
14	The amino acid sequence of the H-CDR2 of the heavy chain complementarity determining region of 612
15	The amino acid sequence of the H-CDR3 of the heavy chain complementarity determining region of 612
16	The amino acid sequence of L-CDR1 of the light chain complementarity determining region of 612
17	The amino acid sequence of L-CDR2 of the light chain complementarity determining region of 612
18	The amino acid sequence of L-CDR3 of the light chain complementarity determining region of 612
19	Amino acid sequence of heavy chain variable region of anti-PD-L1 antibody

20	Amino acid sequence of light chain variable region of anti-PD-L1 antibody
twenty one	Amino acid sequence of the variable region of the heavy chain of 607
twenty two	Amino acid sequence of the light chain variable region of 607
twenty three	Amino acid sequence of the variable region of the heavy chain of 612
twenty four	Amino acid sequence of the light chain variable region of 612
25	Amino acid sequence of heavy chain of anti-PD-L1 antibody
26	Amino acid sequence of light chain of anti-PD-L1 antibody
27	Amino acid sequence of the heavy chain of 607
28	Amino acid sequence of the light chain of 607
29	Amino acid sequence of the heavy chain of 612
30	Amino acid sequence of the light chain of 612
31	Amino acid sequence of the polypeptide chain of PDL1H-607scFv
32	Amino acid sequence of the polypeptide chain of 607H-PDL1scFv
33	The amino acid sequence of the polypeptide chain of 607scFv-PDL1H
34	Amino acid sequence of the polypeptide chain of PDL1scFv-607H
35	Amino acid sequence of the polypeptide chain of PDL1scFv-612H
36	Amino acid sequence of the polypeptide chain of 612H-PDL1scFv

Through gene synthesis and conventional molecular cloning methods, the pTT5 expression vector (purchased from NRC Biotechnology Research Institute) was constructed through the two restriction sites of EcoRI and HindIII to obtain the heavy chain of each bispecific antibody and its corresponding monoclonal antibody And light chain expression vector, the above heavy chain sequence expression vector and its corresponding light chain expression vector were co-transfected into HEK293F cells (purchased from Thermo Fisher, catalog number A14527), expressed and purified to obtain each bispecific antibody PDL1H-607scFv, 607H-PDL1scFv, 607scFv-PDL1H, PDL1scFv-607H, PDL1scFv-612H and 612H-PDL1scFv, and the corresponding monoclonal antibodies M8, 607, 612.

Example 2 Determination of the affinity of bispecific antibodies targeting HER2 and PD-L1 by ELISA

PDL1-ECD-his (NCBI registration number is NP_054862.1) and HER2-ECD-his protein (NCBI registration number is NP_004439.2) are prepared as follows: according to the sequence provided by NCBI, the PD-L1 and HER2 extracellular domain genes are synthesized and A signal peptide sequence was added to the N-terminus, and a 6×His tag was added to the C-terminus. The two restriction sites of EcoRI and HindIII were respectively constructed into pTT5 expression vector, transfected into HEK-293F cells for expression and purified.

Dilute PDL1-ECD-his or HER2-ECD-his protein and coat 96-well ELISA plate respectively, 0.05μg/well, coat overnight at 4℃, wash 3 times with PBST (PBS containing 0.05% Tween20), and prepare 2 with PBS PDL1H-607scFv, 607H-PDL1scFv, 607scFv-PDL1H, PDL1scFv-607H, PDL1scFv-612H, 612H-PDL1scFv and 1% BSA prepared with PBST were blocked for 2h at room temperature. M8 was diluted to different concentrations and added to the ELISA wells, 100μL/well, 3 replicate wells for each concentration, incubated for 1h at room temperature, washed with PBST 3 times, and diluted the secondary antibody (HRP-anti Human IgG Fc, purchased from Millipore, catalog number AP101P), add to ELISA wells, 100μL/well, incubate at room temperature for 1h, wash 3 times with PBST, add TMB color developing solution, 100μL/well, develop to the expected color, use 2M H ₂ The _{color reaction was terminated by SO 4} , 50 μL/well, the reaction solution was shaken uniformly and the OD450nm was measured in a microplate reader, the data was analyzed, and the EC _{50 was} calculated.

The experimental results are shown in Figure 1 and Figure 2, respectively. The affinities (EC ₅₀ ) of each bispecific antibody to PDL1-ECD-his and HER2-ECD-his are shown in Table 3 and Table 4, respectively. It can be seen that each bispecific antibody The affinity of the antibody to PDL1-ECD-his is comparable to that of M8, both at the same level, and PDL1scFv-607H is slightly better. And the affinity to HER2-ECD-his, in the combination of 607 and M8 bispecific antibody, 607scFv-PDL1H and PDL1H-607scFv are better, and the affinity is equivalent to 607 monoclonal antibody, PDL1scFv-607H and 607H-PDL1scFv are Relatively weak; in the bispecific antibody combination of 612 and M8, the affinity of PDL1scFv-612H and 612H-PDL1scFv to HER2-ECD-his is similar, and both are slightly weaker than that of 612 monoclonal antibody.

_{Table 3. EC 50 of} each bispecific antibody to PDL1-ECD-his

_{Table 4. EC 50 of} each bispecific antibody to HER2-ECD-his

Example 3 Determination of the binding activity of bispecific antibodies to competitively inhibit PD-1 and PD-L1

Dilute the PDL1-ECD-Fc protein (the preparation method is the same as above, but replace the C-terminal label with adult Fc sequence) and coat it on a 96-well ELISA plate, 0.2μg/well, overnight at 4°C, PBST (PBS containing 0.05% Tween20 ) Wash 3 times, prepare 2% BSA with PBS, 200μL/well, block at room temperature for 2h, wash 2 times with PBST, use 500ng/ml biotin-labeled PD1-Fc (ie PD1- Fc-biotin, NCBI registration number is NP_005009.2, according to the registration information on NCBI to obtain the extracellular domain gene of PD-1, the cloning and preparation method is the same as PDL1-ECD-Fc) solution PDL1H-607scFv, 607H-PDL1scFv, 607scFv-PDL1H , PDL1scFv-607H, PDL1scFv-612H, 612H-PDL1scFv and M8 were diluted to different concentrations and added to the ELISA wells, 100μL/well, 3 replicate wells for each concentration, incubated at room temperature for 1h, washed with PBST 3 times, prepared with PBST Dilute the secondary antibody (HRP-labeled streptavidin, purchased from Sigma, Catalog No. S4672-5MG) with 1% BSA according to the appropriate ratio, add it to the ELISA well, 100μL/well, incubate at room temperature for 1h, wash with PBST 3 times and add TMB chromogenic solution, 100μL/ Well, develop the color to the expected color, _{stop the color reaction with 2M H 2} SO ₄ , 50 μL/well, make each reaction solution oscillate uniformly and measure OD450nm in a microplate reader, analyze the data, and calculate EC ₅₀ .

The experimental results are shown in Figure 3. Each bispecific antibody competitively inhibits the binding activity (IC ₅₀ ) of PD-1 and PD-L1 as shown in Table 5. It can be seen that each bispecific antibody competitively inhibits PD-1 and PD-L1. L1 activity is comparable.

_{Table 5. IC 50 of} each bispecific antibody competitively inhibiting the binding of PD-1 and PD-L1

Example 4 Bispecific antibodies inhibit the binding activity of PD-1 and PD-L1 high-expressing cell lines

The PD-1 high-expressing cell line PD-1 Effector Cells and the PD-L1 high-expressing cell line PD-L1 aAPC/CHO-K1 Cells were purchased from Promega (Cat. No.: J1252).

1) PDL1-aAPC/CHO-K1 cells in the logarithmic growth phase were trypsinized, centrifuged, counted, and resuspended in 10% FBS Ham/F12 medium (purchased from Thermo Fisher, Catalog No. 11765054), according to 100μL/ Pour 40,000 cells per well, inoculate them into a 96-well plate with a transparent bottom on a white plate, and grow overnight at 37°C _{in a CO 2 cell incubator.}

2) Prepare M8, 607H-PDL1scFv, PDL1scFv-607H, PDL1H-607scFv, 607scFv-PDL1H, PDL1scFv-612H, 612H with the highest concentration of 200nM RPMI1640 medium (purchased from Thermo Fisher, article number 61870036) containing 1% FBS -PDL1scFv, and diluted in a 3-fold gradient (the highest working concentration is 100nM), and at the same time dilute PD-1 Effector Cells with 1% FBS in RPMI1640 medium to 1.25×10 ⁶ cells/mL.

3) Discard the above-mentioned 96-well plate with PDL1-aAPC/CHO-K1 cells, add 40 μL of the diluted PD-1 Effector Cell suspension and 40 μL of the diluted antibody, tap the well plate and shake well, ℃ Cell incubator continued to culture for 6h.

4) Add Bio-Glo Luciferase Assay Reagent (purchased from Promega, catalog number: G7941) that has been restored to room temperature according to 80 μL/well, and incubate at room temperature for 10 minutes.

5) Measure the luminous intensity in a multifunctional microplate reader, and analyze the data through GraphPad Prism 6.

_{The experimental results are shown in Figure 4. The IC 50 of} each bispecific antibody inhibiting the binding activity of PD-1 and PD-L1 high-expressing cell lines is shown in Table 6. It can be seen that PDL1H-607scFv and 607scFv-PDL1H have relatively better activities. PDL1scFv-612H and PDL1scFv-607H followed.

_{Table 6. IC 50 of} each bispecific antibody inhibiting the binding of PD-1 and PD-L1 high-expressing cell lines

Example 5 Determination of cell proliferation inhibitory activity of bispecific antibodies against HER2 high-expressing cell lines

1) BT474 and N87 cells in the logarithmic growth phase were trypsinized, counted, and resuspended in a complete medium, namely RPMI1640 medium (purchased from Thermo Fisher, catalog number 61870036) containing 10% FBS, and 150μL/well , Were inoculated into 96-well cell culture plates, breast cancer cells BT474 (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu143) 10,000 cells per well, and gastric cancer cells N87 (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP- 534) Pour 8000 cells in each well and incubate overnight at 37°C _{in a CO 2 cell incubator.}

2) Dilute the antibody to be tested in a 3-fold gradient with complete medium, each group is serially diluted 9 gradients, the highest concentration of each antibody is 400nM (final working concentration is 100nM), add 50μL of the above antibody to a 96-well cell culture plate, each well The final volume is 200 μL, the non-administered group is set as a negative control, and the culture is continued for 5 days in a 37 ℃ cell incubator, and 2 replicate wells are made in parallel for each concentration.

3) Discard the cell culture supernatant, add 100 μL/well of CCK8 reaction solution (diluted with complete medium at a ratio of 1:10, purchased from Dojindo, catalog number CK04), and incubate at 37°C.

4) After the color of the cell culture well develops to the expected depth, measure its absorbance at a wavelength of 450nm in a multifunctional microplate reader, and analyze the data through GraphPad Prism 6.

5) Calculate cell survival rate and growth inhibition rate according to the following formula:

Survival rate=(OD administration-OD blank)/(OD control-OD blank)×100%. Growth inhibition rate = 1-survival rate.

The experimental results are shown in Fig. 5, Fig. 6, Fig. 7 and Fig. 8. The IC _{50 of} each bispecific antibody on the proliferation inhibition of HER2 high-expressing cell lines BT474 and N87 are shown in Table 7, Table 8, Table 9, and Table 10, respectively. As shown, it can be seen that for the combination of 607 and M8 double antibodies, PDL1scFv-607H and 607H-PDL1scFv have better inhibitory activity on the proliferation of BT474 cells; the activity of PDL1H-607scFv on the proliferation of N87 cells is the weakest, although PDL1scFv-607H, _{The IC 50 of} 607H-PDL1scFv is comparable, but PDL1scFv-607H has the strongest maximum killing effect on N87 cells at high concentrations. For the combination of 612 and M8 double antibodies, PDL1scFv-612H and 612H-PDL1scFv have the same inhibitory activity on cell proliferation.

_{Table 7. The IC 50 of} bispecific antibodies inhibiting the proliferation of BT474 cells

_{Table 8. The IC 50 of} bispecific antibodies inhibiting the proliferation of N87 cells

_{Table 9. The IC 50 of} bispecific antibodies inhibiting the proliferation of BT474 cells

_{Table 10. The IC 50 of} bispecific antibodies inhibiting the proliferation of N87 cells

Example 6 In vivo drug efficacy study based on gastric cancer cell NCI-N87 xenograft tumor

The gastric cancer cells NCI-N87 (purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) in the logarithmic growth phase were digested by trypsin, collected by centrifugation and resuspended in RPMI1640 serum-free medium (purchased from Thermo Fisher, catalog number 61870036) to adjust the cells Concentration, mix the cell suspension with an equal volume of Matrigel so that the final cell concentration is 8*10 ⁷ /mL. Aseptically, inoculate 100 μL of cell suspension into BALB/c nude mice (female, weighing 18-20 g, purchased from Beijing Weitong Lihua Biotechnology Co., Ltd.) subcutaneously on the right side of the rib. After the tumor grows to 100-200 mm ³ , the animals are randomly divided into 5 groups, each with 8 animals. Including solvent control group (Control), monoclonal antibody control group 607, and test sample group PDL1scFv-607H. The administration was started on the day of grouping. The dose of 607 was 20 mg/kg and the dose of PDL1scFv-607H was 27 mg/kg. The dose was intraperitoneally administered twice a week for 7 consecutive times. Measure the tumor diameter twice a week, record, and calculate the tumor volume and tumor growth inhibition rate according to the following formula.

Tumor volume (tumor volume, TV) is calculated as follows: TV = 1/2 × length × width ^2.

Tumor growth inhibition rate (TGI)=(1-experimental group tumor volume/solvent control group tumor volume)×100%.

The experimental results are shown in Figure 9. Both bispecific antibodies PDL1scFv-607H and monoclonal antibody 607 can effectively inhibit the growth of gastric cancer cell NCI-N87 xenograft tumors, and PDL1scFv-607H inhibits tumor growth on the 25th day after administration The rate is 57%, which is significantly better than 40% of the monoclonal antibody reference 607.

Example 7 In vivo pharmacodynamic study of MC38-hPD-L1 colon cancer animal model based on B-hPD-L1 humanized mice

Collect MC38-hPD-L1 colon cancer cells cultured in vitro, and resuspend them with PBS to a concentration of 5*10 ⁵ /0.1mL, and inoculate them into the right subcutaneously of B-hPD-L1 humanized mice at a rate of 0.1 mL/mouse. When the average tumor volume reaches about 138mm ³ , select mice with appropriate individual tumor volume into the experimental group and assign them to 3 experimental groups, namely the solvent control group (Control), the monoclonal antibody control group M8 and the test sample group PDL1scFv-607H, 8 mice per group. On the day of grouping, the administration was started by intraperitoneal injection, once every two days, for 8 consecutive administrations. The dosage of M8 was 20 mg/kg and the dosage of PDL1scFv-607H was 27.5 mg/kg. Measure the tumor diameter twice a week, record, and calculate the tumor volume and tumor growth inhibition rate according to the following formula.

Tumor volume (tumor volume, TV) is calculated as follows: TV = 1/2 × length × width ^2.

Tumor growth inhibition rate (TGI)=(1-experimental group tumor volume/solvent control group tumor volume)×100%.

In this experiment, B-hPD-L1 humanized mice and MC38-hPD-L1 colon cancer cells were both provided by Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.

The experimental results are shown in Figure 10. The bispecific antibody PDL1scFv-607H and the monoclonal antibody M8 can effectively inhibit the growth of MC38-hPD-L1 transplanted tumors, and the tumor growth inhibition rate of PDL1scFv-607H on the 23rd day after administration 74%, the tumor growth inhibition rate of M8 is 89%. Although the bispecific antibody PDL1scFv-607H had a slightly lower tumor growth inhibition rate than the monoclonal antibody reference M8 at the end of this experiment, there was no significant difference between the two and it was not statistically significant.

Example 8 Preparation of mutants of bispecific antibody PDL1scFv-607H

According to the applicant's previous research results (refer to Chinese patent application 202110085824.3), the specific asparagine (N) mutation on the scFv fragment can effectively improve the physicochemical properties of the bispecific antibody. Therefore, the present invention uses conventional molecular cloning technology to perform single-point or combined mutations of asparagine (N) on the scFv fragment of PDL1scFv-607H into glutamic acid (E), extract each mutant plasmid and associate it with the corresponding light chain Paired transfection HEK293F cell expression and purification to obtain the bispecific antibody PDL1scFv-607H mutant samples. Excluding the sites that are not expressed, or that have a greater impact on antibody activity after mutation, and where the physical and chemical properties become worse after mutation, it is finally determined that the 76th N and 213th N on the polypeptide chain of PDL1scFv-607H are mutated to E After that, the physical and chemical properties of the samples were significantly improved. The above mutants were named PDL1scFv-607H-VHN76E, PDL1scFv-607H-VLN76E and PDL1scFv-607H-VHN76E-VLN76E, and the corresponding polypeptide chain sequences are as SEQ ID NO: 37. SEQ ID NO: 38 and SEQ ID NO: 39 are shown.

After each mutant of PDL1scFv-607H was purified by one-step proteinA, the ultra-high performance liquid chromatography (UPLC) detection results are shown in Table 11.

Table 11. UPLC detection results of each mutant of PDL1scFv-607H after one-step proteinA purification

Example 9 Affinity determination of the affinity of the bispecific antibody PDL1scFv-607H mutant to PD-L1 and HER2

Refer to Example 2 for the experimental method.

The experimental results are shown in Figure 11 and Figure 12, respectively. The affinity (EC ₅₀ ) of each bispecific antibody to PDL1-ECD-his and HER2-ECD-his is shown in Table 12 and Table 13, respectively. The affinity of PDL1-ECD-his is comparable to that of PDL1scFv-607H and M8, and there is no obvious attenuation. The affinity of each mutant to HER2-ECD-his is comparable to that of PDL1scFv-607H, and slightly weaker than that of monoclonal antibody 607.

_{Table 12. EC 50 of} PDL1scFv-607H mutants binding to PDL1-ECD-his

_{Table 13. EC 50 of} each mutant of PDL1scFv-607H binding to HER2-ECD-his

Example 10 The bispecific antibody PDL1scFv-607H mutant inhibits the binding activity of PD-1 and PD-L1 high-expressing cell lines

Refer to Example 4 for the experimental method.

The experimental results are shown in Figure 13. The binding activity of each mutant bispecific antibody of PDL1scFv-607H in inhibiting PD-1 and PD-L1 high-expressing cell lines is equivalent to that of PDL1scFv-607H and M8. After no mutation, the activity decreases, and their respective ICs ₅₀ see Table 14 for details.

_{Table 14. The IC 50 of} each mutant of PDL1scFv-607H inhibiting the combination of PD-1 and PD-L1 high-expressing cell lines

Example 11 Determination of the proliferation inhibitory activity of the bispecific antibody PDL1scFv-607H mutant on the cell line with high expression of HER2

Refer to Example 5 for the experimental method.

The experimental results are shown in Figure 14. The inhibitory effect of each mutant bispecific antibody of PDL1scFv-607H on the proliferation of the HER2 high-expressing cell line BT474 is the same as that of PDL1scFv-607H, and their respective IC _50s are shown in Table 15.

_{Table 15. The IC 50 of} each mutant of PDL1scFv-607H in inhibiting the proliferation of BT474 cells

Example 12 Mixed lymphocyte reaction (MLR) experiment to determine the activity of bispecific antibodies

The experimental steps are as follows:

1. Induction of dendritic cells

1) Take an appropriate amount of PBMC (peripheral blood mononuclear cells, purchased from Shanghai Cycla Biotechnology Co., Ltd.) and appropriate volume of CD14MicroBeads (purchased from Miltenyi Biotec, catalog number 130-050-201) and mix them in a refrigerator at 4°C. Leave it for 15 minutes; then use the MACS magnetic cell sorting device to separate the CD14+ monocytes according to the instructions provided by the manufacturer.

2) Resuspend CD14+ monocytes with 10% FBS-containing RPMI1640 medium (purchased from Thermo Fisher, article number 61870036) to a concentration of 5×10 ⁵ cells/mL, and add 50ng/mL GM-CSF (purchased from R&D, Product number 215-GM-050) and IL-4 (purchased from R&D, product number: 204-IL-050) for induction.

3) On the fourth day, RPMI1640 medium containing 10% FBS, 50ng/mL GM-CSF and 50ng/mL IL-4 was half-changed for the above cells.

4) On the seventh day, the DC cells were induced to mature by adding 1 μg/ml LPS (purchased from sigma, item number L6529-1MG) for 24 hours.

2. Isolation and administration of T cells

1) Use the Pan T cell Isolation Kit (purchased from Miltenyi Biotec, catalog number 130-096-535) to separate T cells from PBMC according to the instructions, and use PBS cells preheated at 37°C once to count the T cells for use.

2) Collect the above-mentioned induced maturation DC cells (mDC), wash 3 times with PBS containing 2% FBS, and count them for later use.

3) Dilute the antibody to be tested with RPMI1640 medium containing 10% FBS in a 3-fold gradient, with the highest final concentration of 300 nM.

4) Take the above diluted antibody and add 50μL/well to a 96-well U-shaped bottom culture plate, and mix mDC and T cells at a concentration ratio of ^{2×10 5} /mL and 1×10 ^{6 /mL.} And take 100 μL of cell mixture and add it to the above 96-well U-shaped bottom culture plate.

5) Incubate in a 37°C, 5% CO ₂ incubator for 3 days, collect 50 μL of cell culture supernatant for IL-2 detection; culture for 5 days, collect 120 μL of cell culture supernatant for IFN-γ detection.

3. Detection of IL-2 and IFN-γ secretion levels

1) Dilute mouse-anti-human IL-2 protein (purchased from BD Pharmingen, catalog number 555051) to 2.5μg/mL and mouse-anti-human IFN-γ (purchased from BD Pharmingen, catalog number 551221) with ELISA coating solution The protein was diluted to 4μg/mL, respectively coated on the ELISA plate, 100μL/well, placed in a wet box, 4℃, coated for 16h.

2) Wash the ELISA plate three times with PBST to remove unbound antigen, and pat dry the ELISA plate on absorbent paper to remove excess liquid, then use 2% BSA prepared with PBS, 200 μL/well, and block at room temperature for 1-2 hours.

3) Preparation of IL-2/IFN-γ standard products: Prepare standard products with RPMI1640 medium, make the initial concentration of IL-2 40ng/ml, and make the initial concentration of IFN-γ 400ng/ml, respectively, carry out 2-fold dilutions, each A total of 12 gradients were diluted for each standard. Add 100μl of standard diluent to each well, and set up 2 replicate wells for each concentration.

4) Wash once with PBST to remove excess blocking solution, and pat dry the ELISA plate to remove excess liquid. Dilute the cell supernatant for 5 days with 1% BSA prepared by PBST and add it to mouse-anti-human. IFN-γ coated plate: After mixing 45 μL of 3d cell supernatant and 65 μL of 5d cell supernatant, add 100 μL of the mixture to the mouse-anti-human IL-2 coated plate, and incubate at room temperature for 1 hour.

5) Wash off unbound or non-specifically bound primary antibodies, and use PBST (antibody dilution) containing 1% BSA to combine biotin-mouse-anti-human IL-2 (purchased from BD Pharmingen, catalog number 555040) and biotin- Mouse-anti-human IFN-γ (purchased from BD Pharmingen, catalog number 554550) secondary antibodies were diluted 1000 times, added to the ELISA plate, 100μL/well, and incubated for 1h at room temperature. After washing with PBST three times, Streptavidin-HRP (purchased from BD Pharmingen, catalog number 554066) was diluted 5000 times with antibody diluent, added to an ELISA plate, 100 μL/well, and incubated for 1 h at room temperature.

6) Wash five times with PBST, pat dry the ELISA plate on absorbent paper, remove excess liquid, add TMB color developing solution, 100μL/well, develop the color to the appropriate shade, add 2M H2SO4, 50μL/well to stop Develop the color and measure its absorbance at 450nm wavelength in a multifunctional microplate reader to analyze the data.

The experimental results are shown in Figure 15 and Figure 16, respectively. It can be seen that the mutant PDL1scFv-607H-VHN76E can significantly promote the secretion of IL-2 and IFN-γ of T cells, and has an excellent effect on the promotion of IL-2 secretion at high concentrations. For PDL1scFv-607H, their respective EC _50s are shown in Tables 16 and 17, respectively.

_{Table 16. EC 50 of} PDL1scFv-607H and mutants promoting IL2 secretion

_{Table 17. EC 50 of} PDL1scFv-607H and mutants promoting IFN-γ secretion

Claims (17)

1. The bispecific antibody against PD-L1 and HER2 is characterized by comprising two polypeptide chains and two light chains, wherein:

   (a) The polypeptide chain includes VH-PDL1-CH1-CH2-CH3-L1-VH-HER2-L2-VL-HER2 or VH-HER2-L2-VL-HER2-L1-VH from N-terminus to C-terminus -PDL1-CH1-CH2-CH3, the light chain includes VL-PDL1-CL from N-terminus to C-terminus; or

   (b) The polypeptide chain contains VH-HER2-CH1-CH2-CH3-L1-VH-PDL1-L2-VL-PDL1 or VH-PDL1-L2-VL-PDL1-L1-VH from N-terminus to C-terminus -HER2-CH1-CH2-CH3, the light chain includes VL-HER2-CL from N-terminus to C-terminus;

   Wherein, the VH-PDL1 is a heavy chain variable region that binds PD-L1, the VL-PDL1 is a light chain variable region that binds PD-L1, and the VH-HER2 is a heavy chain that binds HER2 The variable region, the VL-HER2 is a light chain variable region that binds to HER2, the L1 and L2 are (G ₄ S)x, and x is 3, 4, 5 or 6, the CH1-CH2 -CH3 is the heavy chain constant region, the CL is the light chain constant region, the VH-PDL1 and the VL-PDL1 form an antigen binding site that specifically binds to PD-L1, the VH-HER2 It forms an antigen binding site that specifically binds to HER2 with the VL-HER2.
2. The bispecific antibody of claim 1, wherein said L1 is (G ₄ S) ₃ and said L2 is (G ₄ S) ₄ .
3. The bispecific antibody of claim 1, wherein the VH-PDL1 comprises the heavy chain CDR shown in the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The VL-PDL1 includes an amino acid sequence such as the light chain CDR shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and the VH-HER2 includes an amino acid sequence such as SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 or heavy chain CDRs as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, and said VL-HER2 includes an amino acid sequence as SEQ ID NO : 10, SEQ ID NO: 11 and SEQ ID NO: 12 or the light chain CDR shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
4. The bispecific antibody of claim 3, wherein the VH-PDL1 has the amino acid sequence shown in SEQ ID NO: 19, and the VL-PDL1 has the amino acid sequence shown in SEQ ID NO: 20 The VH-HER2 has the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 23, and the VL-HER2 has the amino acid sequence shown in SEQ ID NO: 22 or SEQ ID NO: 24 The amino acid sequence.
5. The bispecific antibody of claim 4, wherein the polypeptide chain has the amino acid sequence shown in SEQ ID NO: 31 or SEQ ID NO: 33, and the light chain has the amino acid sequence shown in SEQ ID NO: : The amino acid sequence shown in 26; or the polypeptide chain has the amino acid sequence shown in SEQ ID NO: 32 or SEQ ID NO: 34, and the light chain has the amino acid sequence shown in SEQ ID NO: 28 Or the polypeptide chain has the amino acid sequence shown in SEQ ID NO: 35 or SEQ ID NO: 36, and the light chain has the amino acid sequence shown in SEQ ID NO: 30.
6. The bispecific antibody of claim 5, wherein the polypeptide chain has the amino acid sequence shown in SEQ ID NO: 34, and the light chain has the amino acid sequence shown in SEQ ID NO: 28 The polypeptide chain shown in SEQ ID NO: 34 further includes mutations in the following amino acid residue positions: N76E and/or N213E.
7. The bispecific antibody of claim 6, wherein the polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 37 or SEQ ID NO: 38 or SEQ ID NO: 39, and the light The chain has an amino acid sequence as shown in SEQ ID NO: 28.
8. The bispecific antibody of claim 1, wherein the heavy chain constant region comprises an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, and the light chain constant region comprises a kappa or lambda light chain constant region. Area.
9. An isolated nucleotide, characterized in that the nucleotide encodes the bispecific antibody according to any one of claims 1-8.
10. An expression vector, characterized in that the expression vector contains the nucleotide according to claim 9.
11. A host cell, characterized in that the host cell contains the expression vector according to claim 9.
12. The method for preparing a bispecific antibody according to any one of claims 1-8, wherein the method comprises the following steps:

    (a) Culturing the host cell according to claim 11 under expression conditions, thereby expressing the bispecific antibody;

    (b) Isolation and purification of the bispecific antibody described in (a).
13. A pharmaceutical composition, characterized in that it contains the bispecific antibody according to any one of claims 1-8 and a pharmaceutically acceptable carrier.
14. Use of the bispecific antibody according to any one of claims 1-8 or the pharmaceutical composition according to claim 13 in the preparation of a medicament for the treatment of cancer.
15. The use according to claim 14, wherein the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous Cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases.
16. A method for treating cancer, which is characterized by comprising administering the bispecific antibody according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 13 to a subject in need.
17. The method of claim 16, wherein the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous Cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases.

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