An antibody that binds human PD-L1

Abstract

The present application provides an antibody binding to human PD-L1, and a tetravalent bispecific antibody of anti-PD-1 and PD-L1 constructed based on the antibody binding to human PD-L1. The tetravalent bispecific antibody of the present application does not need to be subjected to Fc modification, does not produce mismatch problems, has a simple preparation method, and has similar or even better biological activity and physicochemical properties than monoclonal antibodies.

Description

[0001] This application is a divisional application of 202180003496.6, filed on April 19, 2021. Technical Field

[0002] This invention relates to the field of antibodies, and more specifically, this invention discloses a quadrivalent bispecific antibody against PD-1 and PD-L1. Background Technology

[0003] Human programmed cell death receptor-1 (PD-1) is a type I membrane protein with 288 amino acids and is one of the known major immune checkpoints (Blank et al., 2005, Cancer Immunotherapy, 54:307-314). PD-1 is expressed on activated T lymphocytes. Its binding to ligands PD-L1 (programmed cell death-ligand 1) and PD-L2 (programmed cell death-ligand 2) can inhibit T lymphocyte activity and related in vivo cellular immune responses. PD-L2 is mainly expressed on macrophages and dendritic cells, while PD-L1 is widely expressed on B and T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, and heart tissue cells. Numerous studies have shown that the interaction between PD-1 and PD-L1 is not only essential for maintaining the balance of the immune system in the body, but also a major mechanism and cause for PD-L1-positive tumor cells to circumvent immune surveillance. By blocking the negative regulation of the PD-1 / PD-L1 signaling pathway by cancer cells, the immune system can be activated, promoting T cell-related tumor-specific cellular immune responses, thus opening the door to a new cancer treatment method—tumor immunotherapy.

[0004] PD-1 (encoded by the gene Pdcd1) is a member of the immunoglobulin superfamily associated with CD28 and CTLA-4. Research shows that PD-1 negatively regulates antigen receptor signal transduction when it binds to its ligands (PD-L1 and / or PD-L2). The structure of mouse PD-1 and the co-crystallization structure of mouse PD-1 and human PD-L1 have been elucidated (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 containing a variable (V-type) Ig domain responsible for ligand binding and a cytoplasmic tail region responsible for binding signal transduction molecules. The PD-1 cytoplasmic tail region contains two tyrosine-based signal transduction motifs: ITIM (immunoreceptor tyrosine inhibition motif) and ITSM (immunoreceptor tyrosine switching motif).

[0005] PD-1 plays a crucial role in the immune evasion mechanisms of tumors. Tumor immunotherapy, which utilizes the body's own immune system to fight cancer, is a groundbreaking cancer treatment method. However, the tumor microenvironment can protect tumor cells from effective immune destruction; therefore, disrupting the tumor microenvironment has become a key focus of anti-tumor research. Existing research has established the role of PD-1 in the tumor microenvironment: PD-L1 is expressed in many mouse and human tumors (and can be induced by IFNγ in most PD-L1-negative tumor cell lines), and is presumed to be an important target 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)). Immunohistochemical evaluation of biopsies has revealed the expression of PD-1 (on tumor-infiltrating lymphocytes) and / or PD-L1 on tumor cells in many primary human tumors. Such cancers 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, pancreatic cancer, and head and neck tumors. Therefore, blocking the interaction between PD-1 and PD-L1 can enhance the immune activity of tumor-specific T cells, helping the immune system to clear tumor cells. Consequently, PD-1 and PD-L1 have become popular targets for developing tumor immunotherapy drugs.

[0006] Bispecific antibodies are antibody molecules that can simultaneously and specifically bind to two antigens or two epitopes. Based on symmetry, bispecific antibodies can be classified into structurally symmetrical and asymmetrical molecules. Based on the number of binding sites, bispecific antibodies can be classified into bivalent, trivalent, tetravalent, and multivalent molecules. Bispecific antibodies are gradually becoming a new class of therapeutic antibodies, which can be used to treat various inflammatory diseases, cancer, and other diseases. Although many new bispecific antibody structures have been reported recently, the main technical challenge in producing bispecific antibodies lies in obtaining correctly paired molecules. Currently existing bispecific antibody forms all suffer from mismatch problems, thus producing one or more mismatch-induced byproducts or aggregates, affecting the yield, purity, and physicochemical stability of the target bispecific antibody, and consequently affecting its safety and efficacy in vivo. Summary of the Invention

[0007] The present invention provides an antibody that binds to human PD-L1, and a tetravalent bispecific antibody against PD-1 and PD-L1 constructed based on the said antibody that binds to human PD-L1.

[0008] Therefore, the first object of the present invention is to provide an antibody or antigen-binding fragment thereof that binds to human PD-L1.

[0009] A second object of the present invention is to provide an isolated nucleotide encoding an antibody or antigen-binding fragment thereof that binds to human PD-L1.

[0010] A third objective of this invention is to provide an expression vector comprising the aforementioned nucleotides.

[0011] A fourth object of the present invention is to provide a host cell comprising the expression vector described above.

[0012] A fifth objective of this invention is to provide a method for preparing the antibody or antigen-binding fragment thereof that binds to human PD-L1.

[0013] A sixth object of the present invention is to provide a pharmaceutical composition comprising the antibody that binds to human PD-L1 or an antigen-binding fragment thereof.

[0014] A seventh object of the present invention is to provide the use of the antibody or antigen-binding fragment thereof that binds to human PD-L1, or the pharmaceutical composition thereof, in the preparation of a medicament for treating diseases of PD-L1 overexpression.

[0015] An eighth object of the present invention is to provide a method for treating diseases with PD-L1 overexpression using the antibody or antigen-binding fragment thereof that binds to human PD-L1 or the pharmaceutical composition thereof.

[0016] The ninth object of the present invention is to provide a quadrivalent bispecific antibody against PD-1 and PD-L1.

[0017] The tenth object of the present invention is to provide an isolated nucleotide encoding the aforementioned tetravalent bispecific antibody.

[0018] The eleventh object of the present invention is to provide an expression vector comprising the aforementioned nucleotides.

[0019] The twelfth object of the present invention is to provide a host cell comprising the expression vector described above.

[0020] The thirteenth objective of this invention is to provide a method for preparing the aforementioned tetravalent bispecific antibody.

[0021] The fourteenth object of the present invention is to provide a pharmaceutical composition comprising the aforementioned tetravalent bispecific antibody.

[0022] The fifteenth object of the present invention is to provide the use of the said tetravalent bispecific antibody or the said pharmaceutical composition in the preparation of a medicament for treating cancer.

[0023] The sixteenth object of the present invention is to provide a method for treating cancer using the aforementioned quadrivalent bispecific antibody or the aforementioned pharmaceutical composition.

[0024] To achieve the above objectives, the present invention provides the following technical solution:

[0025] A first aspect of the present invention provides an antibody or antigen-binding fragment thereof that binds to human PD-L1, comprising:

[0026] (a) Heavy chain complementarity-determining regions H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is shown in SEQ ID NO: 17, the amino acid sequence of H-CDR2 is shown in SEQ ID NO: 18, and the amino acid sequence of H-CDR3 is shown in SEQ ID NO: 19, and

[0027] (b) Light chain complementarity-determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is shown in SEQ ID NO: 20, the amino acid sequence of L-CDR2 is shown in SEQ ID NO: 21, and the amino acid sequence of L-CDR3 is shown in SEQ ID NO: 22.

[0028] According to the present invention, the antibody is a monoclonal antibody or a polyclonal antibody.

[0029] According to the present invention, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0030] According to the present invention, the antigen-binding fragment includes a Fab fragment, an F(ab')2 fragment, an Fv fragment, or a single-chain antibody.

[0031] According to the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment that binds to human PD-L1 is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10.

[0032] According to the present invention, the amino acid sequence of the heavy chain of the antibody binding to human PD-L1 or its antigen-binding fragment is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain is shown in SEQ ID NO: 15.

[0033] A second aspect of the invention provides an isolated nucleotide encoding an antibody or antigen-binding fragment thereof that binds to human PD-L1 as described above.

[0034] According to the present invention, the nucleotide sequence of the heavy chain encoding the antibody or its antigen-binding fragment that binds to human PD-L1 is shown in SEQ ID NO: 14, and the nucleotide sequence encoding the light chain is shown in SEQ ID NO: 16.

[0035] A third aspect of the present invention provides an expression vector containing the nucleotides described above.

[0036] A fourth aspect of the present invention provides a host cell containing the expression vector described above.

[0037] A fifth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof that binds to human PD-L1, characterized in that the method comprises the following steps:

[0038] (a) Under expression conditions, host cells as described above are cultured to express the antibody that binds to human PD-L1 or its antigen-binding fragment;

[0039] (b) Isolate and purify the antibody or antigen-binding fragment thereof that binds to human PD-L1 as described in (a).

[0040] A sixth aspect of the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to human PD-L1 as described above and a pharmaceutically acceptable carrier.

[0041] A seventh aspect of the invention provides the use of the antibody or antigen-binding fragment thereof that binds to human PD-L1, or the pharmaceutical composition described above, in the preparation of a medicament for treating diseases with PD-L1 overexpression.

[0042] According to the present invention, the disease characterized by PD-L1 overexpression is cancer. Preferably, the cancer is selected from the group consisting of: melanoma, renal cell carcinoma, 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 proliferative malignant diseases.

[0043] An eighth aspect of the present invention provides a method for treating a disease of PD-L1 overexpression, comprising administering to a subject in need an antibody that binds to human PD-L1 as described above or an antigen-binding fragment thereof or a pharmaceutical composition as described above.

[0044] According to the present invention, the disease characterized by PD-L1 overexpression is cancer. Preferably, the cancer is selected from the group consisting of: melanoma, renal cell carcinoma, 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 proliferative malignant diseases.

[0045] A ninth aspect of the present invention provides a tetravalent bispecific antibody against PD-1 and PD-L1, comprising two polypeptide chains and four common light chains, wherein the polypeptide chains have amino acid sequences as shown in SEQ ID NO: 29 or SEQ ID NO: 31, and the common light chains have amino acid sequences as shown in SEQ ID NO: 15.

[0046] A tenth aspect of the present invention provides an isolated nucleotide encoding the tetravalent bispecific antibody.

[0047] According to a preferred embodiment of the present invention, the nucleotides encode the polypeptide chain and the common light chain, wherein the nucleotide sequence encoding the polypeptide chain is as shown in SEQ ID NO: 30 or SEQ ID NO: 32, and the nucleotide sequence encoding the common light chain is as shown in SEQ ID NO: 16.

[0048] The eleventh aspect of the present invention provides an expression vector containing the nucleotides described above.

[0049] A twelfth aspect of the present invention provides a host cell containing the expression vector described above.

[0050] The thirteenth aspect of the present invention provides a method for preparing the aforementioned tetravalent bispecific antibody, the method comprising the following steps:

[0051] (a) Under expression conditions, host cells as described above are cultured to express the tetravalent bispecific antibody;

[0052] (b) Isolate and purify the tetravalent bispecific antibody described in (a).

[0053] The fourteenth aspect of the present invention provides a pharmaceutical composition comprising a tetravalent bispecific antibody as described above and a pharmaceutically acceptable carrier.

[0054] The fifteenth aspect of the invention provides the use of the aforementioned tetravalent bispecific antibody or the pharmaceutical composition described above in the preparation of a medicament for treating cancer.

[0055] 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, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other proliferative malignant diseases.

[0056] The sixteenth aspect of the present invention provides a method for treating cancer, comprising administering to a subject in need a tetravalent bispecific antibody as described above or a pharmaceutical composition as described above.

[0057] 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, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other proliferative malignant diseases.

[0058] Beneficial effects:

[0059] This invention provides an antibody that binds to human PD-L1, and a tetravalent bispecific antibody against PD-1 and PD-L1 constructed based on the aforementioned human PD-L1-binding antibody. The tetravalent bispecific antibody of this invention does not require Fc modification, avoids mismatch issues, has a simple preparation method, and possesses biological activity and physicochemical properties similar to or even superior to monoclonal antibodies. Attached Figure Description

[0060] Figure 1This is a schematic diagram of the structure of the bispecific antibody of the present invention. VH-A represents the heavy chain variable region of Anti-PDL1 or 609, VH-B represents the heavy chain variable region of 609 or Anti-PDL1, VL represents the light chain variable region of the common light chain, CH1, CH2, and CH3 are the three domains of the heavy chain constant region, CL is the light chain constant region of the common light chain, the line segment between two heavy chains represents a disulfide bond, and the line segment between the heavy chain and the light chain also represents a disulfide bond. The line segment between CH1 and VH-A near the N-terminus of the polypeptide chain represents an artificially designed linker, and the line segment between CH1 and CH2 near the C-terminus of the polypeptide chain represents the antibody's native linker and hinge region (if the heavy chain is the human IgG4 subtype, the hinge region will contain the S228P point mutation, according to EU coding).

[0061] Figure 2 Results of ELISA detection of the relative affinity of Anti-PDL1 to PD-L1.

[0062] Figure 3 The results were obtained to determine the ability of Anti-PDL1 to block the interaction between PD-1 and PD-L1.

[0063] Figure 4A and Figure 4B The results were used to evaluate the ability of Anti-PDL1 to enhance MLR.

[0064] Figure 5A and Figure 5B ELISA results for Anti-PDL1 and 609 and their hybrid antibodies.

[0065] Figure 6A and Figure 6B The results are for ELISA of PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4.

[0066] Figures 7A to 7D The results were used to evaluate the ability of 609-Fab-PDL1-IgG4 to enhance MLR.

[0067] Figure 8A and Figure 8B The pharmacokinetic results are for 609-Fab-PDL1-IgG4.

[0068] Figure 9A and Figure 9B The HPLC-SEC chromatogram of 609-Fab-PDL1-IgG4 is shown.

[0069] Figures 10A to 10D The CE-SDS pattern of 609-Fab-PDL1-IgG4.

[0070] Figure 11A and Figure 11B The HPLC-IEC chromatogram of 609-Fab-PDL1-IgG4 is shown.

[0071] Figure 12A and Figure 12B The DSC pattern of 609-Fab-PDL1-IgG4.

[0072] Figure 13 The mass spectrum of 609-Fab-PDL1-IgG4 is shown.

[0073] Figure 14 The antitumor effect of the 609-Fab-PDL1-IgG4 bispecific antibody in mice. Detailed Implementation

[0074] The sequence information involved in this invention is summarized in Table 1.

[0075] Table 1. Sequence information of the antibodies of the present invention

[0076]

[0077]

[0078] In this invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" refer to heterotetraglycoproteins of approximately 150,000 Daltons with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region, which consists 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, which includes a domain CL. The constant region of the light chain pairs with the CH1 domain of the constant region of the heavy chain, and the variable region of the light chain pairs with the variable region of the heavy chain. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). Heavy chain constant regions include IgG1, IgG2, IgG3, and IgG4 subtypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of an antibody are covalently linked by interpeptide disulfide bonds formed between their hinge regions. The antibodies of this invention include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two antibodies, and antigen-binding fragments of antibodies. The antibodies of this invention include murine antibodies, chimeric antibodies, and humanized antibodies.

[0079] In this invention, the term "bispecific antibody (Biantibody)" refers to an antibody molecule that can simultaneously and specifically bind to two antigens (targets) or two epitopes.

[0080] In this invention, the term "monoclonal antibody (MABS)" refers to an antibody obtained from a substantially homogeneous population, meaning that the individual antibodies in this population are identical, except for a few possible naturally occurring mutations. Monoclonal antibodies target a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody formulations (which are typically mixtures of different antibodies targeting different antigenic determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized through hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring any special method to produce the antibody.

[0081] In this invention, the term "mouse antibody" refers to an antibody derived from rats or mice, preferably mice. The mouse antibody of this invention is obtained by immunizing mice with the extracellular domain of human PD-L1 as an antigen and then screening for hybridoma cells.

[0082] In this invention, the term "chimeric antibody" refers to an antibody that comprises heavy and light chain variable region sequences derived from one species and constant region sequences derived from another species, such as a mouse heavy chain variable region and light chain variable region linked to a human constant region.

[0083] In this invention, the term "humanized antibody" refers to an antibody whose CDR is derived from a non-human species (preferably mouse) antibody, and whose residual portions (including the frame region and constant region) are derived from human antibodies. Furthermore, the frame region residues can be modified to maintain binding affinity.

[0084] In this invention, the term "antigen-binding fragment" refers to an antibody fragment capable of specifically binding to the human PD-L1 epitope. Examples of antigen-binding fragments of this invention include Fab fragments, F(ab')2 fragments, Fv fragments, and single-chain antibodies (scFv). The Fab fragment consists of the VH and CH1 domains of the antibody's heavy chain and the VL and CL domains of the light chain. The F(ab')2 fragment is a fragment produced by digesting the antibody with pepsin. The Fv fragment consists of a dimer composed of tightly non-covalently linked variable regions of the antibody's heavy and light chains. A single-chain antibody (scFv) is an antibody formed by linking the variable regions of the antibody's heavy and light chains via a short peptide (linker) of 15-20 amino acids.

[0085] In this invention, the term "Fc" refers to a crystallizable fragment, which is composed of the CH2 and CH3 domains of the antibody. The Fc fragment has no antigen-binding activity and is the site where the antibody interacts with effector molecules or cells.

[0086] In this invention, the term "variable" refers to the fact that certain portions of the variable region in an antibody differ in sequence, resulting in the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the variable regions of the heavy and light chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable regions are called frame regions (FRs). The variable regions of the natural heavy and light chains each contain four FR regions, which are generally β-sheet configurations, linked by three CDRs forming a linking loop, and in some cases may form a partially β-sheet structure. The CDRs in each chain are closely packed together through the FR regions and together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).

[0087] In this invention, the terms "antibody" and "binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. Typically, antibodies bind at a rate of less than approximately 10... -7 M, for example, less than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The antibody binds to the antigen with an equilibrium dissociation constant (KD) of M or smaller. In this invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity between the antibody and the antigen can be determined using surface plasmon resonance (SPR) in a BIACORE instrument or using ELISA to determine the relative affinity of antibody-antigen binding.

[0088] In this invention, the term "valence" refers to the presence of a specified number of antigen-binding sites in an antibody molecule. Preferably, the bispecific antibody of this invention has four antigen-binding sites and is tetravalent. In this invention, the antigen-binding sites comprise a heavy chain variable region (VH) and a light chain variable region (VL).

[0089] In this invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitopes of this invention are regions of an antigen that are bound to antibodies.

[0090] In this invention, the term "common light chain" refers to a light chain containing the same light chain variable region and light chain constant region, which can pair with the heavy chain of a first antibody that binds to a first antigen to form a binding site specifically binding to the first antigen, and can also pair with the heavy chain of a second antibody that binds to a second antigen to form a binding site specifically binding to the second antigen. Furthermore, the light chain variable region of the common light chain forms a first antigen binding site with the heavy chain variable region of the first antibody, and the light chain variable region of the common light chain forms a second antigen binding site with the heavy chain variable region of the second antibody.

[0091] In this invention, the term "expression vector" can refer to pTT5, pSECtag series, pCGS3 series, pcDNA series vectors, and other vectors used in mammalian expression systems. The expression vector includes a fusion DNA sequence linked with suitable transcription and translation regulatory sequences.

[0092] In this invention, the term "host cell" refers to a cell suitable for expressing the above-mentioned expression vector. It can be a eukaryotic cell, such as a mammalian or insect host cell culture system, which can be used for the expression of the fusion protein of this invention. CHO (Chinese Hamster Ovary), HEK293, COS, BHK, and derived cells of the above cells can all be used in this invention.

[0093] In this invention, the term "pharmaceutical composition" refers to the antibody or antigen-binding fragment of the human PD-L1 antibody or bispecific tetravalent antibody of this invention, which can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical formulation composition to exert a more stable therapeutic effect. These formulations can ensure the conformational integrity of the amino acid core sequence of the human PD-L1 antibody or antigen-binding fragment of the human PD-L1 antibody or bispecific tetravalent antibody disclosed in this invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation).

[0094] The protein expression and purification methods used in the following examples are described below: The target gene was constructed into the expression vector pcDNA4, and the constructed expression vector or a combination of expression vectors was transformed into FreeStyle using PEI (Polyethylenimine). TM To express antibodies or recombinant proteins, HEK293F cells (hereinafter referred to as HEK293F, purchased from Thermo Fisher Scientific) were cultured in Free Style 293 Expression Medium (purchased from Thermo Fisher Scientific) for 5 days. The cell supernatant was then collected and purified by Protein A affinity chromatography or nickel affinity chromatography.

[0095] The Mixed Lymphocyte Reaction (MLR) method used in the following examples is described below: Peripheral Blood Mononuclear Cells (PBMCs) were isolated from human blood using Histopaque (from Sigma). The PBMCs were then separated using an adherent method, and their differentiation into dendritic cells was induced using IL-4 (25 ng / ml) and GM-CSF (25 ng / ml). Seven days later, the induced dendritic cells were digested and collected. PBMCs were then isolated from the blood of another donor using the same method, and CD4+ was isolated from the PBMCs using a MACS magnet and CD4 MicroBeads (from Miltenyibiotec). + T cells. Induced dendritic cells (10 4 / hole) and separated CD4 + T cells (10) 5 After mixing the antibody in the specified proportions, 150 μl was seeded into each well of a 96-well plate. Several hours later, 50 μl of serially diluted antibody was added to each well. The 96-well plate was then incubated at 37°C for 3 days. AIM-V medium (Thermo Fisher Scientific) was used to culture the cells during the above experiments. The secretion of IL-2 and IFN-γ was then detected according to standard operating procedures. IL-2 and IFN-γ were detected using a double-antibody sandwich ELISA (the relevant paired antibodies were purchased from BD Biosciences). OD450 was read using a SpectraMax 190 microplate reader, and EC50 was calculated using a GraphPad Prism6.

[0096] The physicochemical property detection methods used in the following examples are described below:

[0097] HPLC-SEC

[0098] Antibodies are high-molecular-weight proteins with highly complex secondary and tertiary structures. Due to post-translational modifications, aggregation, and degradation, antibodies are heterogeneous in their biochemical and biophysical properties. When analyzing bispecific antibodies using separation techniques, variants, aggregates, and degradation fragments are commonly observed, and their presence may compromise safety and efficacy. Aggregates, degradation fragments, and incompletely assembled molecules are prone to occur during antibody production and storage. This invention uses high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) to detect the content of these impurities in samples. Aggregates have a larger molecular weight than monomers, resulting in shorter retention times for their corresponding peaks; degradation fragments or incompletely assembled molecules have smaller molecular weights than monomers, resulting in longer retention times for their corresponding peaks. The HPLC-SEC instrument used was a Dionex Ultimate 3000; the mobile phase was prepared as follows: take an appropriate amount of 20mM sodium dihydrogen phosphate stock solution and adjust the pH to 6.8±0.1 with 20mM disodium hydrogen phosphate; injection volume: 20μg; the column was a TSK G3000SWXL, with dimensions of 7.8×300mm 5μm; the flow rate was 0.5ml / min, the elution time was 30min; the column temperature was 25℃, the sample chamber temperature was 10℃; and the detection wavelength was 214nm.

[0099] HPLC-IEC

[0100] Many post-translational modifications (such as N-glycosylation, C-terminal lysine residue modification, N-terminal glutamine or glutamate cyclization, asparagine deamidation, aspartic acid isomerization, and amino acid residue oxidation) can directly or indirectly cause changes in the surface charge of antibodies, leading to charge heterogeneity. Charge variants can be separated and analyzed based on their charge, with commonly used analytical methods including cation exchange chromatography (CEX) and anion exchange chromatography (AEX). When analyzed using chromatographic methods, acidic and basic species are defined based on their retention times relative to the main peak. Acidic species are variants eluted from the main peak earlier than the CEX peak or later than the AEX peak, while basic species are variants eluted from the main peak later than the CEX peak or earlier than the AEX peak. The peaks corresponding to acidic and basic species are called acidic peaks and basic peaks, respectively. Charge variants are easily generated during antibody production and storage. High-performance liquid chromatography-ion exchange chromatography (HPLC-IEC) was used to analyze the charge heterogeneity of the samples. The HPLC-IEC instrument used was a Dionex Ultimate 3000; mobile phase A: 20 mM PB pH 6.3, mobile phase B: 20 ​​mM PB + 200 mM NaCl pH 6.3, the mixing ratio of the two mobile phases was changed over time according to a pre-set program, the flow rate was 1.0 mL / min; the column was a ThermoPropac. TM WCX-10; column temperature 30℃, sample chamber temperature 10℃; injection volume: 20μg; detection wavelength: 214nm.

[0101] CE-SDS

[0102] This invention uses CE-SDS (Capillary Electrophoresis-Sodium Dodecyl Sulfate) to analyze the content of degraded fragments or incompletely assembled molecules in samples. CE is divided into two types: non-reducing and reducing. Samples used for the former do not require the use of the reducing agent DTT to break the disulfide bonds within the molecules during denaturation, while samples used for the latter require the use of the reducing agent DTT to break the disulfide bonds within the molecules during denaturation. Non-reducing and reducing CE-SDS are denoted as NR-CE-SDS and R-CE-SDS, respectively. The capillary electrophoresis apparatus used is ProteomeLab. TMThe PA800 plus (Beckman Coulter) instrument is equipped with a UV 214nm detector and a Bare Fused-Silica Capillary measuring 30.7cm × 50μm with an effective length of 20.5cm. Other relevant reagents were purchased from Beckman Coulter. Key instrument parameters were set as follows: capillary and sample chamber temperature 20±2℃, separation voltage 15kV.

[0103] DSC

[0104] Differential scanning calorimetry (DSC) primarily reflects the thermal stability of a sample by detecting changes in heat within biomolecules during controlled heating or cooling processes. Upon heating, the unfolding of a protein sample absorbs heat, and the additional energy required to eliminate the temperature difference in the sample cell is recorded by the device. These heat changes form a peak on the chromatogram, with the peak temperature corresponding to the unfolding of the protein sample being taken as the melting temperature (Tm). Tm is an important indicator of protein thermal stability; the higher the Tm, the better the protein's stability.

[0105] Molecular weight detection

[0106] Antibodies were treated with PNGase F and endoglucosidase F2 for deglycosylation. A UPLC-XEVO G2 Q-TOF liquid chromatography-mass spectrometry (LC-MS) system (Waters) was used for sample molecular weight analysis and identification. Mobile phase A was HPLC-grade water containing 0.1% trifluoroacetic acid (TFA). Mobile phase B was acetonitrile containing 0.1% TFA. For the method of determining whole molecular weight, a MassPREP™ Micro Desalting Column (2.1 × 5 mm) was used. Key parameters were set as follows: column temperature: 80℃; mobile phase flow rate: 0.2 mL / min; mobile phase gradient: mobile phase B increased from 5% to 90% over 1.5 min; ESI source temperature: 130℃. BiopharmaLynx v1.2 (Waters) was used to control the LC-MS system and collect data; mass spectrometry signals were deconvolved using BiopharmaLynx v1.2.

[0107] The following examples and experimental cases are further illustrative of the present invention and should not be construed as limiting the invention. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) *Molecular Cloning: A Laboratory Manual*, 2nd edition, Cold Spring Harbor Laboratory Press.

[0108] Example 1: Preparation of humanized anti-human PD-L1 antibody

[0109] Example 1.1 Preparation of recombinant PD-1 and PD-L1 proteins

[0110] The extracellular coding genes for PD-1 and PD-L1 were derived as described in WO2018 / 137576A1. Using recombination technology, polyhistidine coding sequences were ligated to the ends of the extracellular coding genes for PD-1 and PD-L1, respectively. The recombinant genes were then cloned into pcDNA4 expression vectors, expressed, and purified. The resulting recombinant proteins were named PD1-His and PD-L1-His, respectively. Using recombination technology, the Fc coding sequence of human IgG1 was ligated to the ends of the extracellular coding genes for PD-1 and PD-L1, respectively. The recombinant genes were then cloned into pcDNA4 expression vectors, expressed, and purified. The resulting recombinant proteins were named PD1-ECD-hFc and PD-L1-ECD-hFc, respectively.

[0111] Example 1.2 Preparation of mouse-derived anti-human PD-L1 monoclonal antibody

[0112] Balb / c mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were immunized using the above-mentioned PD-L1-ECD-hFc as the antigen. The methods for immunizing mice, titer detection, and hybridoma clone screening were as described in Example 2 of WO2018 / 137576A1. The method for screening hybridoma-positive clones by ELISA was as follows: the above-mentioned PD-L1-His was used to coat the ELISA plate at a coating concentration of 10 ng / well, and the ELISA plate was blocked with PBST containing 1% bovine serum albumin (BSA) (KH2PO4 0.2 g, Na2HPO4·12H2O 2.9 g, NaCl 8.0 g, KCl 0.2 g, Tween-20 0.5 ml, and pure water to 1 L). The antibody to be tested was serially diluted and then transferred to the ELISA plate coated with the recombinant protein. After incubation at room temperature for half an hour, the plate was washed. Appropriately diluted HRP (Horseradish Peroxidase)-labeled goat anti-mouse antibody (Fc-Specific) (purchased from Sigma) was added, and after incubation at room temperature for half an hour, the plate was washed. 100 μl of chromogenic solution with TMB (3,3',5,5'-Tetramethylbenzidine) as the substrate was added to each well (Substrate A: 13.6 g sodium acetate trihydrate, 1.6 g citric acid monohydrate, 0.3 ml 30% hydrogen peroxide, 500 ml pure water; Substrate B: 0.2 g disodium ethylenediaminetetraacetate, 0.95 g citric acid monohydrate, 50 ml glycerol, 0.15 g TMB dissolved in 3 ml DMSO, 500 ml pure water; solutions A and B should be mixed in equal volumes before use). The plate was incubated at room temperature for 1–5 min. 50 μl of stop solution (2M) was added. The reaction was terminated with H2SO4; the OD450 was read using an ELISA reader (SpectraMax 190).

[0113] Positive hybridoma clones were selected and expanded in 24-well plates, then subcloned using limiting dilution. Monoclonal hybridoma cell lines stably expressing the target antibody were obtained using the aforementioned method, and these clones were amplified. The hybridoma cell lines were cultured for 7 days in serum-free Hybridoma-SFM (Thermo Fisher Scientific), and then mouse-derived anti-human PD-L1 monoclonal antibodies were purified from the culture supernatant using Protein A / G affinity chromatography. Several mouse monoclonal antibodies capable of binding to human PD-L1 were obtained after purification. The relative affinity of these mouse monoclonal antibodies for human PD-L1 was assessed using ELISA. Finally, clone M8, with the highest relative affinity, was selected for further development.

[0114] Example 1.3 Determination of the sequence and humanization of mouse anti-PD-L1 monoclonal antibody

[0115] Step 1: Determination of the variable region sequence of the murine anti-human PD-L1 monoclonal antibody

[0116] Total RNA was extracted from the M8 hybridoma monoclonal cell line using Trizol. The mRNA was reverse transcribed into cDNA using a reverse transcription kit. The light chain variable region and heavy chain variable region genes of M8 were amplified by PCR using a combination of primers reported in the literature (Antibody Engineering, Volume 1, Edited by Roland Kontermann and Stefan Dübel, the sequence of the combination primers is from page 323). The PCR products were then cloned into the pMD18-T vector, and the variable region gene sequence was sequenced and analyzed.

[0117] The amino acid sequences of the variable regions of the heavy and light chains of the M8 antibody were analyzed, and the antigen complementarity-determining regions and framework regions of the heavy and light chains of the M8 antibody were determined according to Kabat rules. The amino acid sequences of the heavy chain CDRs of the M8 antibody are H-CDR1: SYGVH (SEQ ID NO: 1), H-CDR2: LIWSGGGTDYNAAFIS (SEQ ID NO: 2), and H-CDR3: QLGLRAMDY (SEQ ID NO: 3), and the amino acid sequences of the light chain CDRs are L-CDR1: RASQSIGTTIH (SEQ ID NO: 4), L-CDR2: YASESVS (SEQ ID NO: 5), and L-CDR3: QQSNSWPLT (SEQ ID NO: 6).

[0118] Step 2: Humanization of mouse-derived anti-human PD-L1 monoclonal antibody

[0119] exist https: / / www.ncbi.nlm.nih.gov / igblast / Homology comparison was performed between the heavy chain variable region of the murine M8 antibody and the germline sequence of human IgG. IGHV4-59*01 was selected as the heavy chain CDR transplantation template. The heavy chain CDR of the murine M8 antibody was transplanted into the IGHV4-59*01 backbone region, and WGQGTSVTVSS (SEQ ID NO: 7) was added after H-CDR3 as the fourth frame region to obtain the CDR transplanted heavy chain variable region sequence. Similarly, homology comparison was performed between the light chain variable region of the murine M8 antibody and the germline sequence of human IgG. IGKV6-21*01 was selected as the light chain CDR transplantation template. The light chain CDR of the murine M8 antibody was transplanted into the IGKV6-21*01 backbone region, and FGAGTKLEIK (SEQ ID NO: 8) was added after L-CDR3 as the fourth frame region to obtain the CDR transplanted light chain variable region sequence. Based on the CDR transplanted variable regions, mutations were performed at some amino acid sites. When mutations are performed, the amino acid sequence is encoded using Kabat codes, and the location of the site is indicated by the Kabat code.

[0120] Preferably, for the variable region of the CDR transplanted heavy chain, according to the Kabat encoding, the 6th position E is mutated to Q, the 9th position P is mutated to G, the 16th position E is mutated to Q, the 17th position T is mutated to S, the 27th position G is mutated to F, the 29th position I is mutated to L, the 37th position I is mutated to V, the 61st position A is mutated to P, the 62nd position A is mutated to S, the 63rd position F is mutated to L, the 64th position I is mutated to K, the 67th position V is mutated to L, the 71st position V is mutated to R, the 78th position F is mutated to V, the 80th position L is mutated to F, the 82nd position L is mutated to I, and the 82nd position V is mutated to L. For CDR transplantation of the light chain variable region, the Q at position 11 is mutated to L, the E at position 53 is mutated to Q, the V at position 55 is mutated to F, and the L at position 78 is mutated to V.

[0121] The aforementioned variable regions of the heavy and light chains with mutation sites are defined as humanized heavy and light chain variable regions (SEQ ID NO: 9 and 10), respectively. DNA encoding these humanized heavy and light chain variable regions was synthesized by Shanghai Sangon Biotech Co., Ltd. The synthesized humanized heavy chain variable region was linked to the human IgG1 heavy chain constant region (SEQ ID NO: 11) to obtain the full-length humanized heavy chain gene, named Anti-PDL1-HC (SEQ ID NO: 13 and 14); the humanized light chain variable region was linked to the human Kappa chain constant region (SEQ ID NO: 12) to obtain the full-length humanized light chain gene, named Anti-PDL1-LC (SEQ ID NO: 15 and 16). The Anti-PDL1-HC and Anti-PDL1-LC genes were constructed into pcDNA4 expression vectors, respectively. The resulting heavy and light chain expression vectors were transfected into HEK293F cells using PEI transfection to express the antibody. The antibody was purified using Protein A affinity chromatography and named Anti-PDL1.

[0122] The final amino acid sequences of the Anti-PDL1 antibody heavy chain CDR are H-CDR1: SYGVH (SEQ ID NO: 17), H-CDR2: LIWSGGGTDYNPSLKS (SEQ ID NO: 18) and H-CDR3: QLGLRAMDY (SEQ ID NO: 19), and the amino acid sequences of the light chain CDR are L-CDR1: RASQSIGTTIH (SEQ ID NO: 20), L-CDR2: YASQSFS (SEQ ID NO: 21) and L-CDR3: QQSNSWPLT (SEQ ID NO: 22).

[0123] Example 1.4 Preparation of control antibody Atezolizumab-IgG1

[0124] The heavy chain and light chain variable region sequences (SEQ ID NO: 23 and 24) of the positive control antibody Atezolizumab were obtained from *WHO Drug Information, Vol. 29, No. 3, 2015*. DNA encoding these variable regions was synthesized by Shanghai Sangon Biotech Co., Ltd. The heavy chain variable region of Atezolizumab (Atezolizumab-VH) was linked to the human IgG1 heavy chain constant region (SEQ ID NO: 11) to obtain the full-length heavy chain gene, named Atezolizumab-HC; the light chain variable region of Atezolizumab (Atezolizumab-VL) was linked to the human Kappa light chain constant region (SEQ ID NO: 12) to obtain the full-length light chain gene, named Atezolizumab-LC. Atezolizumab-HC and Atezolizumab-LC were constructed into pcDNA4 expression vectors, expressed, and purified to obtain the antibody, named Atezolizumab-IgG1.

[0125] Example 1.5 ELISA determination of the relative affinity of humanized anti-human PD-L1 antibody for PD-L1

[0126] The ELISA plate was coated with PD-L1-His at a concentration of 10 ng / well, and blocked with PBST containing 1% BSA. The antibody to be tested was serially diluted and transferred to the ELISA plate coated with the recombinant protein. After incubation at room temperature for half an hour, the plate was washed. Appropriately diluted HRP-labeled goat anti-human antibody (Fc-Specific) (purchased from Sigma) was added, and after incubation at room temperature for half an hour, the plate was washed. 100 μl of chromogenic solution with TMB as substrate was added to each well, and the plate was incubated at room temperature for 1–5 min. The reaction was terminated by adding 50 μl of stop solution (2M H2SO4). OD450 was read using a SpectraMax 190 microplate reader, and graphing and data analysis were performed using GraphPad Prism6, with EC50 calculated.

[0127] like Figure 2 As shown, both Anti-PDL1 and Atezolizumab-IgG1 can effectively bind to PD-L1-His, with EC50 values ​​of 0.1018 nM and 0.09351 nM, respectively, indicating comparable apparent affinity. The isotype control antibody is a human IgG1 antibody that does not bind to human PD-L1.

[0128] Example 1.6 Determination of the ability of humanized anti-human PD-L1 antibody to block PD-1 / PD-L1 interaction

[0129] Biotinylated PD-L1-ECD-hFc was labeled using Biotin N-hydroxysuccinimide ester (Sigma, catalog number / specification: H1759-100MG). Human PD-1-ECD-hFc was diluted to 2 μg / ml with sodium carbonate buffer (1.59 g Na2CO3 and 2.93 g NaHCO3 dissolved in 1 L of pure water), and added to 96-well ELISA plates at 100 μl / well using a multipipeline. The plates were incubated at room temperature for 4 h. After washing once with PBST, the plates were blocked with PBST containing 1% BSA at 200 μl / well and incubated at room temperature for 2 h. The blocking solution was discarded, the plates were patted dry, and stored at 4°C. In a 96-well plate, biotinylated PD-L1-ECD-hFc was diluted to 500 ng / ml with PBST solution containing 1% BSA. Anti-human PD-L1 antibody was serially diluted with the above protein solution. The diluted antibody and biotinylated PD-L1-ECD-hFc mixture was transferred to the ELISA plate coated with human PD-L1-ECD-hFc and incubated at room temperature for 1 hour. The plate was washed three times with PBST. Streptavidin-HRP (purchased from BD Biosciences) diluted 1:1000 with PBST solution containing 1% BSA was added and incubated at room temperature for 45 min. The plate was washed three times with PBST. 100 μl of chromogenic reagent (TMB substrate solution) was added to each well and incubated at room temperature for 1–5 min. 50 μl of stop solution (2M H2SO4) was added to terminate the chromogenic reaction. The OD450 value was read using a microplate reader. Data were processed, analyzed, and plotted using GraphPad Prism6, and the IC50 was calculated.

[0130] like Figure 3 As shown, both Anti-PDL1 and Atezolizumab-IgG1 effectively blocked the interaction between PD-1 and PD-L1, with IC50 values ​​of 1.366 nM and 1.471 nM, respectively, indicating comparable blocking abilities. The isotype control antibody was a human IgG1 antibody that does not bind to human PD-L1.

[0131] Example 1.7 Determination of the functional activity of humanized anti-human PD-L1 antibody using mixed lymphocyte reaction

[0132] like Figure 4A As shown, both Anti-PDL1 and Atezolizumab-IgG1 can effectively stimulate MLR secretion of IL-2, with EC50 values ​​of 0.306 nM and 0.29 nM, respectively. Figure 4BAs shown, both Anti-PDL1 and Atezolizumab-IgG1 effectively stimulated MLR secretion of IFN-γ, with EC50 values ​​of 0.1464 nM and 0.1294 nM, respectively. The isotype control antibody was a human IgG1 antibody that does not bind to human PD-L1.

[0133] Example 2: Construction of a tetravalent bispecific antibody against PD-1 and PD-L1

[0134] Example 2.1 Sequence

[0135] mAb1-25-Hu (hereinafter referred to as 609) is a humanized anti-human PD-1 monoclonal antibody. Its heavy chain variable region and light chain variable region sequences are derived from WO2018 / 137576A1. The humanized heavy chain variable region and light chain variable region (SEQ ID NO: 25 and 26) are linked to the heavy chain constant region (SEQ ID NO: 27) of human IgG4 (S228P) and the light chain constant region (SEQ ID NO: 12) of Kappa, respectively, and finally the complete heavy chain and light chain amino acid sequences of the humanized mAb1-25-Hu monoclonal antibody (609) are obtained.

[0136] Anti-PDL1 is a humanized monoclonal antibody against human PD-L1, and its sequence is shown in Example 1.3.

[0137] Example 2.2 Selection of Common Light Chain

[0138] Comparative analysis of the amino acid sequences of the Anti-PDL1 light chain variable region and the 609 light chain variable region using BLAST (Basic Local Alignment Search Tool) showed that 74% of the amino acids were identical (Identities), and 86% of the amino acids had similar properties (Positives).

[0139] The heavy and light chain genes of Anti-PDL1 were named Anti-PDL1-HC and Anti-PDL1-LC, respectively, and the heavy and light chain genes of 609 were named 609-HC and 609-LC, respectively. They were constructed into pcDNA4 expression vectors, and the above heavy and light chain expression vectors were combined in the following ways: Anti-PDL1-HC+Anti-PDL1-LC, 609-HC+609-LC, Anti-PDL1-HC+609-LC, and 609-HC+Anti-PDL1-LC. Antibodies were expressed and purified, and the resulting antibodies were named Anti-PDL1, 609, Anti-PDL1-HC+609-LC, and 609-HC+Anti-PDL1-LC, respectively.

[0140] The ELISA plates were coated with PD1-ECD-hFc and PD-L1-ECD-hFc at a concentration of 10 ng / well. The plates were blocked with PBST containing 1% BSA. The antibodies to be tested were serially diluted and transferred to the ELISA plates coated with the recombinant proteins. After incubation at room temperature for half an hour, the plates were washed. Appropriately diluted HRP-labeled goat anti-human antibody (Fab-specific, purchased from Sigma) was added, and the plates were incubated at room temperature for half an hour, followed by washing. 100 μl of TMB-based chromogenic solution was added to each well, and the plates were incubated at room temperature for 1–5 min. The reaction was terminated by adding 50 μl of stop solution (2M H2SO4). OD450 was read using a SpectraMax 190 microplate reader, and graphing and data analysis were performed using a GraphPad Prism6, with EC50 calculated.

[0141] like Figure 5A As shown, 609 and 609-HC+Anti-PDL1-LC can effectively bind PD1-ECD-hFc, with EC50 values ​​of 0.2001 nM and 0.2435 nM, respectively; while Anti-PDL1 and Anti-PDL1-HC+609-LC cannot bind PD1-ECD-hFc. Figure 5B As shown, Anti-PDL1 can effectively bind to PD-L1-ECD-hFc with an EC50 of 0.1246 nM, while 609, Anti-PDL1-HC+609-LC, and 609-HC+Anti-PDL1-LC cannot effectively bind to PD1-ECD-hFc. Therefore, Anti-PDL1-LC (SEQ ID NO: 15 and 16) was selected as the common light chain for constructing the bispecific antibody.

[0142] Example 2.3 Construction of bispecific antibodies

[0143] The heavy chain variable region of Anti-PDL1 was linked to the CH1 domain of human IgG4, and then the heavy chain variable region of 609 was linked through an artificial linker (the linker used here is three tandem GGGGS, SEQ ID NO: 28). Finally, the heavy chain constant region of human IgG4 (CH1+CH2+CH3, the hinge region contains the S228P mutation) was linked. The long heavy chain gene containing two heavy chain variable regions and two CH1 domains constructed by this procedure was named PDL1-Fab-609-IgG4 (SEQ ID NO: 29 and 30). Similarly, the heavy chain variable region of 609 was linked to the CH1 domain of human IgG4, and then the heavy chain variable region of Anti-PDL1 was linked through an artificial linker (the linker used here is three tandem GGGGS, SEQ ID NO: 28). Finally, the heavy chain constant region of human IgG4 (CH1+CH2+CH3, the hinge region contains the S228P mutation) was linked. The long heavy chain gene containing two heavy chain variable regions and two CH1 domains constructed by this procedure was named 609-Fab-PDL1-IgG4 (SEQ ID NO: 31 and 32).

[0144] The above sequences were constructed into the pcDNA4 expression vector. The PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4 expression vectors were combined with the Anti-PDL1-LC expression vector, respectively, to express and purify the antibodies. The resulting antibodies were named PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4, respectively (for simplicity, only the name of the heavy chain is used as the name of the antibody here).

[0145] Example 2.4 ELISA determination of relative affinity

[0146] The ELISA detection method is as described in Example 1.3.

[0147] like Figure 6A As shown, 609-HC+Anti-PDL1-LC, PDL1-Fab-609-IgG4, and 609-Fab-PDL1-IgG4 can all effectively bind to PD1-His, with EC50 values ​​of 0.3821 nM, 5.308 nM, and 0.4213 nM, respectively. Figure 6BAs shown, Anti-PDL1, PDL1-Fab-609-IgG4, and 609-Fab-PDL1-IgG4 can all effectively bind to PD-L1-His, with EC50 values ​​of 0.1204 nM, 0.1400 nM, and 0.1350 nM, respectively. The isotype control antibody is a human IgG4 antibody that does not bind to either PD-1 or PD-L1. These results indicate that PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4 can bind to both PD-1 and PD-L1, demonstrating that they are bispecific antibodies.

[0148] Example 2.5: Evaluating the ability to enhance MLR

[0149] Results A and C came from the same MLR experiment, while results B and D came from a separate MLR experiment. The isotype control antibody in this study was a human IgG4 antibody that does not bind to either PD-1 or PD-L1. Figure 7A and 7B As shown in Figure 7A, Anti-PDL1, 609-HC+Anti-PDL1-LC, and 609-Fab-PDL1-IgG4 can all effectively stimulate MLR secretion of IL-2, with EC50 values ​​of 0.306 nM, 0.5384 nM, and 0.1023 nM, respectively. Figure 7B The EC50 values ​​are 0.1016 nM, 0.6819 nM, and 0.1259 nM, respectively. Additionally, as... Figure 7C and 7D As shown, Anti-PDL1, 609-HC+Anti-PDL1-LC, and 609-Fab-PDL1-IgG4 can all effectively stimulate MLR to secrete IFN-γ. Figure 7C The EC50 values ​​were 0.5119 nM, 1.21 nM, and 0.1675 nM, respectively. Figure 7D The EC50 values ​​were 0.1464 nM, 1.29 nM, and 0.05491 nM, respectively. Furthermore, Figure 7A and 7B The results showed that, at the same concentration, 609-Fab-PDL1-IgG4 stimulated MLR to secrete more IL-2 compared to monoclonal antibody Anti-PDL1 or 609-HC+Anti-PDL1-LC.

[0150] Example 2.6 Biacore determination of affinity

[0151] The affinity between the aforementioned antibodies and PD-1 or PD-L1 was detected using a Biacore 8K (GE Healthcare) microarray. Various antibodies were captured on the Biacore 8K using a chip coupled with Protein A / G. Recombinant proteins PD1-His or PD-L1-His were then injected, and binding-dissociation curves were obtained. The mixture was eluted with 6M guanidine hydrochloride regeneration buffer, and the cycle was repeated. The data were analyzed using Biacore 8K Evaluation Software. The results are shown in Table 2.

[0152] Table 2-1. Binding and dissociation kinetic parameters and equilibrium dissociation constants of PD-1

[0153]

[0154] Table 2-2. Binding and dissociation kinetic parameters and equilibrium dissociation constants of PD-L1

[0155]

[0156] Experimental results showed that the binding constants (Kon) and dissociation constants (Koff) of 609-Fab-PDL1-IgG4 and 609-HC+Anti-PDL1-LC for PD-1 were very similar, and their equilibrium dissociation constants (KD) were also essentially the same, at 2.57E-08 and 3.49E-08, respectively. Similarly, the binding constants (Kon) and dissociation constants (Koff) of 609-Fab-PDL1-IgG4 and 609-HC+Anti-PDL1-LC for PD-L1 were also very similar, and their equilibrium dissociation constants (KD) were also essentially the same, at 6.08E-10 and 8.43E-10, respectively. The equilibrium dissociation constant (KD) is inversely proportional to the affinity.

[0157] Example 2.7 Pharmacokinetic Study

[0158] This study used SD (Sprague-Dawley) rats (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) to conduct a pharmacokinetic study of 609-Fab-PDL1-IgG4. Five rats, weighing approximately 200g, were used in each group. Each rat was intravenously injected with 1mg of the antibody. Blood samples were collected from the orbital sinus at specific time points after administration, and the blood was centrifuged after natural clotting to obtain serum.

[0159] The method for measuring the concentration of the target antibody in serum is as follows: ELISA plates are coated with PD1-His and PD-L1-His at concentrations of 20 ng / well and 10 ng / well, respectively, and then the ELISA plates are blocked with PBST containing 1% bovine serum albumin. Appropriately diluted rat serum was transferred to ELISA plates coated with PD1-His and PD-L1-His, respectively. After incubation at room temperature for 1 hour, the plates were washed, and HRP-labeled goat anti-human (Fc-Specific) antibody (purchased from Sigma; this antibody underwent species cross-adsorption treatment and does not recognize rat antibodies) was added. After incubation at room temperature for half an hour, the plates were washed, and 100 μl of chromogenic solution with TMB as substrate was added to each well. The plates were incubated at room temperature for 1–5 min. The reaction was terminated by adding 50 μl of stop solution (2M H2SO4). The OD450 was read using an ELISA reader, and the OD450 was converted into antibody serum concentration using a standard curve. Data analysis and plotting were performed using GraphPad Prism6. The half-life of the antibody drug in rats was calculated using Phoenix software.

[0160] according to Figure 8A The half-life of 609-Fab-PDL1-IgG4 was calculated to be 365 hours (15.2 days). According to... Figure 8B The half-life of 609-Fab-PDL1-IgG4 was calculated to be 446 hours (18.6 days), while the half-life of the monoclonal antibody Anti-PDL1 was 361 hours (15.0 days). These results indicate that 609-Fab-PDL1-IgG4 has pharmacokinetic properties similar to those of the Anti-PDL1 monoclonal antibody.

[0161] Example 2.8 Characterization of physicochemical properties

[0162] Example 2.8.1 HPLC-SEC

[0163] Figure 9A The HPLC-SEC chromatogram of monoclonal antibody 609 shows three distinct peaks: Peak1, Peak2, and Peak3, accounting for 0.7%, 0.3%, and 99.0%, respectively. The retention times of Peak1 and Peak2 are shorter than that of the main peak, Peak3, suggesting that Peak1 and Peak2 may be generated by aggregates. Their combined percentage is 1.0%. No peaks representing degradation fragments or incompletely assembled molecules are present in the chromatogram. Figure 9BThe HPLC-SEC chromatogram of 609-Fab-PDL1-IgG4 shows three distinct peaks: Peak1, Peak2, and Peak3, accounting for 0.2%, 99.5%, and 0.3%, respectively. Peak1 has a shorter retention time than the main peak Peak2, suggesting that Peak1 may be generated by aggregates. Peak3 has a longer retention time than Peak2, suggesting that Peak3 may be generated by degradation fragments or incompletely assembled molecules.

[0164] Example 2.8.2 CE-SDS

[0165] Figure 10A and 10B These represent the NR-CE-SDS and R-CE-SDS spectra of the 609 monoclonal antibody, respectively. Figure 10C and Figure 10D These figures represent the NR-CE-SDS and R-CE-SDS spectra of 609-Fab-PDL1-IgG4, respectively. The main NR-CE-SDS peak of 609, Peak8, accounts for 98.92%, while the main NR-CE-SDS peak of 609-Fab-PDL1-IgG4, Peak9, accounts for 97.70%. In the R-CE-SDS of 609, the main peaks Peak4 (corresponding to the light chain) and Peak8 (corresponding to the heavy chain) account for 32.03% and 66.99%, respectively, with a peak area ratio of 1:2.09. In the R-CE-SDS of 609-Fab-PDL1-IgG4, the main peaks Peak3 (corresponding to the light chain) and Peak9 (corresponding to the heavy chain) account for 38.73% and 58.78%, respectively, with a peak area ratio of 2:3.03. In NR-CE-SDS, the peak proportions of 609 monoclonal antibody and 609-Fab-PDL1-IgG4 were very similar; in R-CE-SDS, the peak area ratios of the light and heavy chains of 609 monoclonal antibody and 609-Fab-PDL1-IgG4 were as expected.

[0166] Example 2.8.3 HPLC-IEC

[0167] Figure 11A and 11B The HPLC-IEC spectra of 609 and 609-Fab-PDL1-IgG4 are shown, respectively. Their main peaks account for 82.95% and 92.70%, respectively, indicating that the charge heterogeneity of 609-Fab-PDL1-IgG4 is better than that of 609 monoclonal antibody.

[0168] Example 2.8.4 DSC

[0169] Figure 12A and 12BThe DSC spectra of 609 and 609-Fab-PDL1-IgG4 are shown below. TmOnset represents the temperature at which the protein begins to unfold or denature, and Tm corresponds to the peak temperature. The TmOnset and Tm of 609 are 63.68℃ and 72.36℃, respectively, while those of 609-Fab-PDL1-IgG4 are 64.22℃ and 76.25℃, respectively. These results indicate that the thermal stability of 609 and 609-Fab-PDL1-IgG4 is very similar.

[0170] Example 2.8.5 Molecular weight determination

[0171] Each molecule of 609-Fab-PDL1-IgG4 contains two long heavy chains and four light chains. Its expected molecular weight, calculated to account for the modification of the C-terminal lysine residue of the heavy chain, is 238099 Da. Figure 13 As shown, the actual measured molecular weight was 238,100 Da, differing from the theoretical molecular weight by only 1 Da. These results indicate that the molecular structure of 609-Fab-PDL1-IgG4 is as expected.

[0172] Example 3: Antitumor effect of anti-PD-1 and PD-L1 bispecific antibodies in mice.

[0173] Human peripheral blood mononuclear cells (PBMCs) were used to reconstruct the human immune system in NSG mice, and a subcutaneous xenograft model of human lung cancer NCI-H292 was established in these mice. This mouse model simultaneously possesses T cells expressing human PD-1 and human tumor cells expressing human PD-L1, thus it can be used to evaluate the in vivo antitumor activity of anti-PD-1 and anti-PD-L1 bispecific antibodies. The specific implementation steps are as follows: Human non-small cell lung cancer NCI-H292 cells were collected from in vitro culture (…). CRL-1848 TM The cell suspension concentration was adjusted to 1×10⁻⁶. 8 / ml, mixed with an equal volume of Matrigel (purchased from BD Biosciences, catalog number: 356234). PBMCs (purchased from Allcells, catalog number: PB005-C) were resuspended in PBS and the PBMC suspension concentration was adjusted to 1×10⁻⁶. 7 / ml. The mixed tumor cell suspension and PBMC suspension were mixed in equal volumes. Under aseptic conditions, 200 μl of the cell mixture was subcutaneously injected into the right upper back of M-NSG mice (purchased from the Shanghai Southern Model Organism Research Center). On the same day, the mice inoculated with the mixed cells were randomly divided into groups of 10 mice each according to their body weight. The drug treatments for each group were as follows: control group, injected with saline; Opdivo group, injected with 10 mg / kg of the anti-PD-1 positive control antibody Opdivo (manufactured by Bristol Myers Squibb); Tecentriq group, injected with 10 mg / kg of the anti-PD-L1 positive control antibody Tecentriq (manufactured by Roche); 609-Fab-PDL1-IgG4 group, injected with 16 mg / kg of 609-Fab-PDL1-IgG4. Considering the difference in molecular weight between bispecific antibodies and monoclonal antibodies, the drug dosages in this experiment were set according to equimolar amounts. Subsequently, medication was administered according to the pre-designed protocol, twice a week for a total of eight doses, with tumor volume measured twice weekly. Finally, the growth curves of each group of tumors over time are shown below. Figure 14 As shown.

[0174] The results showed that at the end of the experiment on day 30, the tumor inhibition rates of Opdivo, Tecentriq, and 609-Fab-PDL1-IgG4 were 50.5%, 84.4%, and 96.0%, respectively (tumor inhibition rate = (average volume of control group - average volume of experimental group) / average volume of control group × 100%). Compared with Opdivo and Tecentriq, 609-Fab-PDL1-IgG4 was more effective in inhibiting tumor growth. SEQUENCE LISTING <110> 3SBio (Shanghai) Co., Ltd. <120> An antibody that binds to human PD-L1 <130> BJ3501-21P450590CN <160> 32 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Mus musculus <400> 1 Ser Tyr Gly Val His 1 5 <210> 2 <211> 16 <212> PRT <213> Mus musculus <400> 2 Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Ala Ala Phe Ile Ser 1 5 10 15 <210> 3 <211> 9 <212> PRT <213> Mus musculus <400> 3 Gln Leu Gly Leu Arg Ala Met Asp Tyr 1 5 <210> 4 <211> 11 <212> PRT <213> Mus musculus <400> 4 Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Mus musculus <400> 5 Tyr Ala Ser Glu Ser Val Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Mus musculus <400> 6 Gln Gln Ser Asn Ser Trp Pro Leu Thr 1 5 <210> 7 <211> 11 <212> PRT <213> Artificial <220> <223> The fourth frame region of the heavy chain <400> 7 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5 10 <210> 8 <211> 10 <212> PRT <213> Artificial <220> <223> Light Chain Fourth Frame Area <400> 8 Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 9 <211> 117 <212> PRT <213> Artificial <220> <223> The amino acid sequence of the heavy chain variable region of Anti-PDL1 <400> 9 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 10 <211> 107 <212> PRT <213> Artificial <220> <223> The amino acid sequence of the light chain variable region of Anti-PDL1 <400> 10 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 11 <211> 330 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the constant region of human IgG1 heavy chain <400> 11 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 12 <211> 107 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the constant region of the human Kappa light chain <400> 12 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 13 <211> 447 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain of Anti-PDL1 <400> 13 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 14 <211> 1341 <212> DNA <213> Artificial <220> <223> Anti‑PDL1 antibodies have been released <400> 14 60. caggtccagc tgcagcagtc aggagggggc ctggtgaagc catcacagag cctgtccctg acctgcacag tctctgggtt cagtctgact tcatacggag tgcactgggt ccgacagccc 120 cctggaaagg gactggagtg gatcggcctg atttggtctg gcggggac agactataac cccagcctga aatcccggct gaccatctct aggatacca gtaagaatca agtgagcttt aaaattagct ccctgacagc cgctgacact gcagtgtact attgtgcaag gcagctggga ctgcgagcta tggattactg gggacagggc acttccgtga ccgtctctag tgcgagcacc 360 aagggacctt ccgtgtttcc cctcgccccc agctccaaaa gcaccagcgg cggaacagct 420 gctctcggct gtctcgtcaa ggattacttc cccgagcccg tgaccgtgag ctggaacagc 480 ggagccctga caagcggcgt ccacaccttc cctgctgtcc tacagtcctc cggactgtac 540 agcctgagca gcgtggtgac agtccctagc agctccctgg gcacccagac atatatttgc 600 aacgtgaatc acaagcccag caacaccaag gtcgataaga aggtggagcc taagtcctgc 660 gacaagaccc acacatgtcc cccctgtccc gctcctgaac tgctgggagg cccttccgtg 720 ttcctgttcc cccctaagcc caaggacacc ctgatgattt ccaggacacc cgaggtgacc 780 tgtgtggtgg tggacgtcag ccacgaggac cccgaggtga aattcaactg gtacgtcgat 840 ggcgtggagg tgcacaacgc taagaccaag cccagggagg agcagtacaa ttccacctac 900 agggtggtgt ccgtgctgac cgtcctccat caggactggc tgaacggcaa agagtataag 960 tgcaaggtga gcaacaaggc cctccctgct cccatcgaga agaccatcag caaagccaag 1020 ggccagccca gggaacctca agtctatacc ctgcctccca gcagggagga gatgaccaag 1080 aaccaagtga gcctcacatg cctcgtcaag ggcttctatc cttccgatat tgccgtcgag 1140 tgggagtcca acggacagcc cgagaacaac tacaagacaa caccccccgt gctcgattcc 1200 gatggcagct tcttcctgta ctccaagctg accgtggaca agtccagatg gcaacaaggc 1260 aacgtcttca gttgcagcgt catgcatgag gccctccaca accactacac ccagaagagc 1320 ctctccctga gccctggaaa g 1341 <210> 15 <211> 214 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the light chain of Anti-PDL1 <400> 15 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 16 <211> 642 <212> DNA <213> Artificial <220> <223> Nucleotide sequence of the light chain of Anti-PDL1 <400> 16 gaaatcgtgc tgacacagag ccctgacttt ctgtccgtga cacccaagga gaaagtcact 60 atcacctgcc gggctagcca gtccatcgga accacaattc actggtacca gcagaagccc 120 gaccagagcc ctaagctgct gattaaatat gcctctcaga gtttctcagg cgtgccatcc 180 agatttagcg gctccgggtc tggaactgac ttcacactga ctatcaactc tgtcgaggca 240 gaagatgccg ctacctacta ttgtcagcag agtaattcat ggcccctgac ctttggcgcc 300 gggacaaagc tggaaattaa aagaaccgtc gccgctccca gcgtcttcat cttccccccc 360 agcgatgagc agctgaagag cggaaccgcc agcgtggtgt gcctgctgaa caacttctac 420 cccagggagg ccaaggtgca atggaaggtg gacaacgccc tacagagcgg caactcccag 480 gagagcgtga ccgagcagga cagcaaggat agcacctaca gcctgagcag caccctcacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccatcagggc 600 ctgagcagcc ctgtgaccaa gagcttcaac aggggcgagt gc 642 <210> 17 <211> 5 <212> PRT <213> Artificial <220> <223> The amino acid sequence of the heavy chain complementarity-determining region H-CDR1 of Anti-PDL1 <400> 17 Ser Tyr Gly Val His 1 5 <210> 18 <211> 16 <212> PRT <213> Artificial <220> <223> The amino acid sequence of the heavy chain complementarity-determining region H-CDR2 of Anti-PDL1. <400> 18 Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 19 <211> 9 <212> PRT <213> Artificial <220> <223> The amino acid sequence of the heavy chain complementarity-determining region H-CDR3 of Anti-PDL1. <400> 19 Gln Leu Gly Leu Arg Ala Met Asp Tyr 1 5 <210> 20 <211> 11 <212> PRT <213> Artificial <220> <223> The amino acid sequence of the light chain complementarity-determining region L-CDR1 of Anti-PDL1 <400> 20 Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His 1 5 10 <210> twenty one <211> 7 <212> PRT <213> Artificial <220> <223> The amino acid sequence of the L-CDR2 light chain complementarity-determining region of Anti-PDL1. <400> twenty one Tyr Ala Ser Gln Ser Phe Ser 1 5 <210> twenty two <211> 9 <212> PRT <213> Artificial <220> <223> The amino acid sequence of the light chain complementarity-determining region L-CDR3 of Anti-PDL1 <400> twenty two Gln Gln Ser Asn Ser Trp Pro Leu Thr 1 5 <210> twenty three <211> 118 <212> PRT <213> Artificial <220> <223> The amino acid sequence of the heavy chain variable region of Atezolizumab <400> twenty three Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> twenty four <211> 107 <212> PRT <213> Artificial <220> <223> The amino acid sequence of the light chain variable region of Atezolizumab <400> twenty four Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 25 <211> 117 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain variable region of mAb1-25-Hu(609) <400> 25 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 26 <211> 107 <212> PRT <213> Artificial <220> <223> The amino acid sequence of the light chain variable region of mAb1-25-Hu(609) <400> 26 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Phe 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro His 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 27 <211> 327 <212> PRT <213> Artificial <220> <223> The amino acid sequence of the constant region of the IgG4 (S228P) heavy chain <400> 27 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 28 <211> 15 <212> PRT <213> Artificial <220> <223> Linker (GGGGSGGGGSGGGGS) <400> 28 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 29 <211> 674 <212> PRT <213> Artificial <220>[[ID=4*]] <223> Amino acid sequence of PDL1-Fab-609-IgG4 <400> 29 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly 225 230 235 240 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 245 250 255 Phe Ala Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly 260 265 270 Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr 275 280 285 Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 290 295 300 Ala Lys Asn Ser His Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 305 310 315 320 Thr Ala Val Tyr Phe Cys Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val 325 330 335 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 340 345 350 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 355 360 365 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 370 375 380 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 385 390 395 400 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 405 410 415 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 420 425 430 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 435 440 445 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 450 455 460 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 465 470 475 480 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 485 490 495 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 500 505 510 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 515 520 525 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 530 535 540 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 545 550 555 560 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 565 570 575 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 580 585 590 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 595 600 605 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 610 615 620 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 625 630 635 640 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 645 650 655 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 660 665 670 Gly Lys <210> 30 <211> 2022 <212> DNA <213> Artificial <220> <223> PDL1‐Fab‐609‐IgG4 inhibitory antibodies <400> 30 60. caggtccagc tgcagcagtc aggagggggc ctggtgaagc catcacagag cctgtccctg acctgcacag tctctgggtt cagtctgact tcatacggag tgcactgggt ccgacagccc 120 cctggaaagg gactggagtg gatcggcctg atttggtctg gcggggac agactataac cccagcctga aatcccggct gaccatctct aggatacca gtaagaatca agtgagcttt aaaattagct ccctgacagc cgctgacact gcagtgtact attgtgcaag gcagctggga ctgcgagcta tggattactg gggacagggc acttccgtga ccgtctctag tgcaagtacc 360 aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420 gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480 ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540 agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600 aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660 ggcggaggtg gcagcggagg tggcgggagt gaggtcaagc tggtggaaag cggcggcggc 720 ctggtgcagc ctggaggatc cctgcggctg agctgcgctg cctccggctt cgctttcagc 780 tcctatgaca tgtcctgggt gaggcaggcc cctggaaaga ggctggagtg ggtggctacc 840 atctccggag gcggaaggta cacctactac cccgacacag tgaagggaag gttcaccatc 900 agccgggata acgccaaaaa cagccactat ctccagatga actccctgag ggccgaagat 960 acagccgtgt atttctgtgc ctccccctac ggaggctatt ttgacgtgtg gggacagggc 1020 accctggtga ccgtctcctc cgcaagtacc aagggaccta gtgttttccc tcttgcacct 1080 tgctccaggt caacatcaga gtccacagct gctcttggat gtctcgttaa ggactacttc 1140 ccagagccag ttaccgtatc ctggaactcc ggagctttga caagcggcgt tcatacattc 1200 ccagctgtgt tgcagagttc tgggttgtac agtttgagct cagtggtgac cgtgccttca 1260 tcttctttgg gcactaagac ctacacctgc aacgtggatc acaagccaag caacaccaag 1320 gtggataaga gggtggagtc caagtacggc ccaccatgtc ctccatgtcc agcccctgaa 1380 tttttgggcg ggccttctgt ctttctgttt cctcctaaac ctaaagatac cctgatgatc 1440 agccgcacac ccgaagtcac ttgtgtggtc gtggatgtgt ctcaggaaga tcccgaagtg 1500 cagtttaact ggtatgtcga tggcgtggaa gtgcataatg ccaaaactaa gccccgcgaa 1560 gaacagttca acagcactta tcgggtcgtg tctgtgctca cagtcctcca tcaggattgg 1620 ctgaatggga aagaatataa gtgcaaggtg agcaataagg gcctccccag cagcatcgag 1680 aagactatta gcaaagccaa agggcagcca cgggaacccc aggtgtacac tctgcccccc 1740 tctcaggagg agatgactaa aaatcaggtc tctctgactt gtctggtgaa agggttttat 1800 cccagcgaca ttgccgtgga gtgggagtct aatggccagc ccgagaataa ttataagaca 1860 actccccccg tcctggactc tgacggcagc tttttcctgt attctcggct gacagtggac 1920 aaaagtcgct ggcaggaggg caatgtcttt agttgcagtg tcatgcatga ggccctgcac 1980 aatcactata cacagaaaag cctgtctctg agtctgggca aa 2022 <210> 31 <211> 674 <212> PRT <213> Artificial <220> <223> Amino acid sequence of 609-Fab-PDL1-IgG4 <400> 31 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Gly Gly 225 230 235 240 Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Ser Gly 245 250 255 Phe Ser Leu Thr Ser Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly 260 265 270 Lys Gly Leu Glu Trp Ile Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp 275 280 285 Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser 290 295 300 Lys Asn Gln Val Ser Phe Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr 305 310 315 320 Ala Val Tyr Tyr Cys Ala Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr 325 330 335 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly 340 345 350 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 355 360 365 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 370 375 380 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 385 390 395 400 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 405 410 415 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 420 425 430 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 435 440 445 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 450 455 460 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 465 470 475 480 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 485 490 495 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 500 505 510 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 515 520 525 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 530 535 540 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 545 550 555 560 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 565 570 575 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 580 585 590 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 595,600,605 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 610 615 620 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 625,630,635,640 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 645,650,655 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 660 665 670 Gly Lys <210> 32 <211> 2022 <212> DNA <213> Artificial <220> <223> 609‐Fab‐PDL1‐IgG4 <400> 32 gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60 agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120 cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180 cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240 ctccagatga actccctgag ggccgagat acagccgtgt atttctgtgc ctccccctac ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360 aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420 gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480 ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540 agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600 aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca ggcggaggtg gcagcggagg tggcgggagt caggtccagc tgcagcagtc aggaggggc 720 ctggtgaagc catcacagag cctgtccctg acctgcacag tctctgggtt cagtctgact 780 tcatacggag tgcactgggt ccgacagccc cctggaaagg gactggagtg gatcggcctg 840 atttggtctg gcgggggac agactatac cccagcctga aatcccggct gaccatctct 960. sightseeing sightseeing aaaattagct ccctgacagc cgctgacact gcagtgtact attgtgcaag gcagctggga ctgcgagcta tggattactg gggacagggc 1020 acttccgtga ccgtctctag tgcaagtacc aagggaccta gtgttttcc tcttgcacct 1080 tgctccaggt caacatcaga gtccacagct gctcttggat gtctcgttaa ggactacttc 1140 ccagagccag ttaccgtatc ctggaactcc ggagctttga caagcggcgt tcatacattc 1200 ccagctgtgt tgcagagttc tgggttgtac agtttgagct cagtggtgac cgtgccttca 1260 tcttctttgg gcactaagac ctacacctgc aacgtggatc acaagccaag caacaccaag 1320 gtggataaga gggtggagtc caagtacggc ccaccatgtc ctccatgtcc agcccctgaa 1380 tttttgggcg ggccttctgt ctttctgttt cctcctaaac ctaaagatac cctgatgatc 1440 agccgcacac ccgaagtcac ttgtgtggtc gtggatgtgt ctcaggaaga tcccgaagtg 1500 cagtttaact ggtatgtcga tggcgtggaa gtgcataatg ccaaaactaa gccccgcgaa 1560 gaacagttca acagcactta tcgggtcgtg tctgtgctca cagtcctcca tcaggattgg 1620 ctgaatggga aagaatataa gtgcaaggtg agcaataagg gcctccccag cagcatcgag 1680 aagactatta gcaaagccaa agggcagcca cgggaacccc aggtgtacac tctgcccccc 1740 tctcaggagg agatgactaa aaatcaggtc tctctgactt gtctggtgaa agggttttat 1800 cccagcgaca ttgccgtgga gtgggagtct aatggccagc ccgagaataa ttataagaca 1860 actccccccg tcctggactc tgacggcagc tttttcctgt attctcggct gacagtggac 1920 aaaagtcgct ggcaggaggg caatgtcttt agttgcagtg tcatgcatga ggccctgcac 1980 aatcactata cacagaaaag cctgtctctg agtctgggca aa 2022

Claims

1. An antibody or antigen-binding fragment thereof that binds to human PD-L1, comprising: (a) Heavy chain complementarity-determining regions H-CDR1, H-CDR2, and H-CDR3, wherein the amino acid sequence of H-CDR1 is shown in SEQ ID NO: 17, the amino acid sequence of H-CDR2 is shown in SEQ ID NO: 18, and the amino acid sequence of H-CDR3 is shown in SEQ ID NO: 19, and (b) Light chain complementarity-determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the amino acid sequence of L-CDR1 is shown in SEQ ID NO: 20, the amino acid sequence of L-CDR2 is shown in SEQ ID NO: 21, and the amino acid sequence of L-CDR3 is shown in SEQ ID NO:

22.

2. The antibody that binds to human PD-L1 or its antigen-binding fragment according to claim 1, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

3. The antibody or antigen-binding fragment of human PD-L1 according to claim 2, wherein the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

4. The antibody binding to human PD-L1 according to claim 1, or its antigen-binding fragment, wherein the antigen-binding fragment includes a Fab fragment, an F(ab')2 fragment, an Fv fragment, or a single-chain antibody.

5. The antibody or antigen-binding fragment of human PD-L1 according to any one of claims 1-4, wherein the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment of human PD-L1 is as shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

10.

6. The antibody or antigen-binding fragment of human PD-L1 according to claim 1, wherein the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment of human PD-L1 is as shown in SEQ ID NO: 13, and the amino acid sequence of the light chain is as shown in SEQ ID NO:

15.

7. An isolated polynucleotide, said polynucleotide encoding an antibody or antigen-binding fragment thereof that binds to human PD-L1 as described in any one of claims 1-6.

8. The isolated polynucleotide according to claim 7, wherein the polynucleotide sequence encoding the heavy chain of the antibody or antigen-binding fragment thereof that binds to human PD-L1 is shown in SEQ ID NO: 14, and the polynucleotide sequence encoding the light chain is shown in SEQ ID NO:

16.

9. An expression vector comprising the isolated polynucleotide as described in any one of claims 7-8.

10. A host cell comprising the expression vector of claim 9.

11. A method for preparing an antibody or antigen-binding fragment of human PD-L1, characterized in that, The method includes the following steps: (a) Under expression conditions, host cells as described in claim 10 are cultured to express the antibody that binds to human PD-L1 or its antigen-binding fragment; (b) Isolate and purify the antibody or antigen-binding fragment thereof that binds to human PD-L1 as described in (a).

12. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof for binding human PD-L1 as described in any one of claims 1-6 and a pharmaceutically acceptable carrier.

13. Use of the antibody binding to human PD-L1 as claimed in claim 1 or its antigen-binding fragment, or the pharmaceutical composition as claimed in claim 12, in the preparation of a medicament for treating diseases with PD-L1 overexpression; The disease in which PD-L1 is overexpressed is cancer; the cancer is selected from the group consisting of the following: 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, and lymphoma.

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