HK40066552B - ANTI-PD-l AND PD-Ll TETRAVALENT BISPECIFIC ANTBODY - Google Patents

Description

A tetravalent bispecific antibody against PD-1 and PD-L1

Technical Field

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

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.

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).

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, so disrupting the tumor microenvironment has become a key focus of anti-tumor research. Existing research has identified 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 has been presumed to be an important target mediating tumor immune evasion (Iwai Y. et al., Proc. Natl. Acad. Sci. U.S.A. 99: 12293-12297 (2002); Strome S.E. 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. These tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial 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 the development of tumor immunotherapy drugs.

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

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.

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

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.

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

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

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.

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.

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.

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.

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

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

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

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

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

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

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.

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.

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

A first aspect of the present invention provides 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.

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

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

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.

According to the present invention, the amino acid sequence of the heavy chain variable region of the antibody binding to human PD-L1 or its antigen-binding fragment 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.

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.

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.

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.

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

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

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:

(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;

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

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.

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.

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.

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.

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.

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.

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

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.

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

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

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

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

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

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

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.

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.

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.

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.

Beneficial effects:

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

Figure 1 is a schematic diagram of the structure of the bispecific antibody of the present invention. In this diagram, 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 the two heavy chains represents a disulfide bond, 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 the 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).

Figure 2 shows the results of ELISA detection of the relative affinity of Anti-PDL1 to PD-L1.

Figure 3 shows the results of measuring the ability of Anti-PDL1 to block the interaction between PD-1 and PD-L1.

Figures 4A and 4B show the results of evaluating the ability of Anti-PDL1 to enhance MLR.

Figures 5A and 5B show the ELISA results of Anti-PDL1 and 609 and their hybrid antibodies.

Figures 6A and 6B show the ELISA results for PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4.

Figures 7A to 7D show the results of evaluating the ability of 609-Fab-PDL1-IgG4 to enhance MLR.

Figures 8A and 8B show the pharmacokinetic results of 609-Fab-PDL1-IgG4.

Figures 9A and 9B are HPLC-SEC chromatograms of 609-Fab-PDL1-IgG4.

Figures 10A to 10D show the CE-SDS patterns of 609-Fab-PDL1-IgG4.

Figures 11A and 11B are HPLC-IEC chromatograms of 609-Fab-PDL1-IgG4.

Figures 12A and 12B show the DSC patterns of 609-Fab-PDL1-IgG4.

Figure 13 shows the molecular weight mass spectrum of 609-Fab-PDL1-IgG4.

Figure 14 shows the antitumor effect of the 609-Fab-PDL1-IgG4 bispecific antibody in mice.

Detailed Implementation

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

Table 1. Sequence information of the antibodies of the present invention

In this invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" refer to isotetraglycoproteins 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, the constant region of 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, the constant region of 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.

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.

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.

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.

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.

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.

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.

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

In 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 partial 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)).

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 to the antigen with an equilibrium dissociation constant ⁽ KD) of less than about ^10⁻⁷ M, for example, less than about 10⁻⁸ M, ^10⁻⁹ M, ^10⁻¹⁰ M, ^10⁻¹¹ M, or even 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 an ELISA to determine the relative affinity of antibody-antigen binding.

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).

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.

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.

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.

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.

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).

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

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 with 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 ⁺ T cells were isolated from the PBMCs using a MACS magnet and CD4 MicroBeads (from Miltenyibiotec). Induced dendritic cells ( ^10⁴ /well) and isolated CD4 ⁺ T cells ( ^10⁵ /well) were mixed in a specific ratio and seeded into 96-well plates at 150 μl per well. Several hours later, 50 μl of serially diluted antibody was added to each well. The 96-well plates were 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 corresponding paired antibodies were purchased from BD Biosciences). OD450 was read using a SpectraMax 190 microplate reader, and graphs were plotted using GraphPadPrism6 to calculate EC50.

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

HPLC-SEC

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.

HPLC-IEC

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 was performed using a Dionex Ultimate 3000 chromatograph; 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 ^™ WCX-10; the column temperature was 30 °C, the sample chamber temperature was 10 °C; the injection volume was 20 μg; and the detection wavelength was 214 nm.

CE-SDS

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 instrument used was a ProteomeLab ^™ PA800 plus (Beckman Coulter), equipped with a UV 214nm detector. The capillary model was Bare Fused-Silica Capillary, with dimensions of 30.7cm × 50μm and an effective length of 20.5cm. Other relevant reagents were purchased from Beckman Coulter. The key instrument parameters were set as follows: capillary and sample chamber temperature 20±2℃, separation voltage 15kV.

DSC

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.

Molecular weight detection

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 °C; 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 °C. BiopharmaLynx v1.2 (Waters) was used to control the LC-MS system and collect data; mass spectrometry signals were deconvolved using BiopharmaLynx v1.2.

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.

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

Example 1.1 Preparation of recombinant PD-1 and PD-L1 proteins

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.

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

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 chromogenic solution A: 13.6 g sodium acetate trihydrate, 1.6 g citric acid monohydrate, 0.3 ml 30% hydrogen peroxide, 500 ml pure water; Substrate chromogenic solution 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 (2 M _H₂SO₄ ) was added. ₄ ) Terminate the reaction; read the OD450 using an ELISA reader (SpectraMax 190).

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 the murine anti-human PD-L1 monoclonal antibody was purified from the culture supernatant using Protein A/G affinity chromatography. Several murine monoclonal antibodies capable of binding to human PD-L1 were obtained after purification. The relative affinity of these murine monoclonal antibodies for human PD-L1 was assessed using ELISA. Finally, clone M8, with the highest relative affinity, was selected for further development.

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

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

Total RNA was extracted from the M8 hybridoma monoclonal cell line using Trizol, and 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.

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).

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

At https://www.ncbi.nlm.nih.gov/igblast/ , the homology of the heavy chain variable region of the murine M8 antibody with the germline sequence of human IgG was compared. 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, the homology of the light chain variable region of the murine M8 antibody with the germline sequence of human IgG was compared. 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 backbone region of IGKV6-21*01, 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 region, mutations are performed on some amino acid sites. During mutation, the amino acid sequence is Kabat encoded, and the site position is indicated by the Kabat code.

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.

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 the pcDNA4 expression vector, 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.

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).

Example 1.4 Preparation of control antibody Atezolizumab-IgG1

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.

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

The ELISA plate was coated with the above-mentioned 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 then transferred to the ELISA plate coated with the above-mentioned 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. 50 μl of stop solution ( _2MH2SO4 ) was added to stop the reaction. The _OD450 was read using an ELISA reader (SpectraMax 190), and the data was plotted and analyzed using GraphPad Prism6, and the EC50 was calculated.

As shown in Figure 2, 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.

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

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 an 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 minutes. 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 minutes. 50 μl of stop solution (2 M _H₂SO₄ ) was added to terminate the chromogenic reaction. The OD450 value was read using a microplate reader. The ELISA plate was _then printed using a GraphPad microplate reader. Prism6 is used for data processing, analysis, and graphing to calculate IC50.

As shown in Figure 3, 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.

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

As shown in Figure 4A, both Anti-PDL1 and Atezolizumab-IgG1 effectively stimulated MLR secretion of IL-2, with EC50 values of 0.306 nM and 0.29 nM, respectively. As shown in Figure 4B, 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.

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

Example 2.1 Sequence

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.

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

Example 2.2 Selection of Common Light Chain

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).

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.

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 (2 M _{H₂SO₄ )} . OD450 was read using a SpectraMax 190 microplate reader, and graphing and data analysis were performed using GraphPad Prism 6, with EC50 calculated.

As shown in Figure 5A, 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. As shown in Figure 5B, Anti-PDL1 can effectively bind 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 PD1-ECD-hFc. Therefore, Anti-PDL1-LC (SEQ ID NO: 15 and 16) were selected as the common light chain for constructing the bispecific antibody.

Example 2.3 Construction of bispecific antibodies

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).

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).

Example 2.4 ELISA determination of relative affinity

The ELISA detection method is as described in Example 1.3.

As shown in Figure 6A, 609-HC+Anti-PDL1-LC, PDL1-Fab-609-IgG4, and 609-Fab-PDL1-IgG4 can all effectively bind PD1-His, with EC50 values of 0.3821 nM, 5.308 nM, and 0.4213 nM, respectively. As shown in Figure 6B, Anti-PDL1, PDL1-Fab-609-IgG4, and 609-Fab-PDL1-IgG4 can all effectively bind 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 show that PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4 can bind to both PD-1 and PD-L1, indicating that they are bispecific antibodies.

Example 2.5: Evaluating the ability to enhance MLR

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

Example 2.6 Biacore determination of affinity

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 samples were 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.

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

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

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 basically equivalent, 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 basically equivalent, at 6.08E-10 and 8.43E-10, respectively. The equilibrium dissociation constant (KD) is inversely proportional to the affinity.

Example 2.7 Pharmacokinetic Study

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.

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 did 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.

The half-life of 609-Fab-PDL1-IgG4 was calculated to be 365 hours (15.2 days) according to Figure 8A. The half-life of 609-Fab-PDL1-IgG4 was calculated to be 446 hours (18.6 days) according to Figure 8B, 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.

Example 2.8 Characterization of physicochemical properties

Example 2.8.1 HPLC-SEC

Figure 9A shows the HPLC-SEC chromatogram of the 609 monoclonal antibody, with 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, indicating that Peak1 and Peak2 may be generated by aggregates; their combined percentage is 1.0%. No peaks representing degradation fragments or incompletely assembled molecules appear in the figure. Figure 9B shows the HPLC-SEC chromatogram of 609-Fab-PDL1-IgG4, with three distinct peaks: Peak1, Peak2, and Peak3, accounting for 0.2%, 99.5%, and 0.3%, respectively. The retention time of Peak1 is shorter than that of the main peak, Peak2, indicating that Peak1 may be generated by aggregates; the retention time of Peak3 is longer than that of the main peak, Peak2, indicating that Peak3 may be generated by degradation fragments or incompletely assembled molecules.

Example 2.8.2 CE-SDS

Figures 10A and 10B show the NR-CE-SDS and R-CE-SDS spectra of the 609 monoclonal antibody, respectively, while Figures 10C and 10D show the NR-CE-SDS and R-CE-SDS spectra of the 609-Fab-PDL1-IgG4 antibody, respectively. The dominant NR-CE-SDS peak, Peak8, accounts for 98.92% of the NR-CE-SDS spectrum for 609, while the dominant NR-CE-SDS peak, Peak9, accounts for 97.70% of the NR-CE-SDS spectrum for 609-Fab-PDL1-IgG4. In the R-CE-SDS of 609, Peak4 (corresponding to the light chain) and Peak8 (corresponding to the heavy chain) accounted 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, Peak3 (corresponding to the light chain) and Peak9 (corresponding to the heavy chain) accounted 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 both 609 monoclonal antibody and 609-Fab-PDL1-IgG4 were as expected.

Example 2.8.3 HPLC-IEC

Figures 11A and 11B show the HPLC-IEC spectra of 609 and 609-Fab-PDL1-IgG4, respectively, with their main peaks accounting for 82.95% and 92.70%, respectively. The results indicate that the charge heterogeneity of 609-Fab-PDL1-IgG4 is better than that of 609 monoclonal antibody.

Example 2.8.4 DSC

Figures 12A and 12B show the DSC spectra of 609 and 609-Fab-PDL1-IgG4, respectively. TmOnset represents the temperature at which the protein begins to unfold or denature, and Tm corresponds to the peak temperature. The TmOnset and Tm for 609 are 63.68℃ and 72.36℃, respectively, while those for 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.

Example 2.8.5 Molecular weight determination

Each molecule of 609-Fab-PDL1-IgG4 contains two long heavy chains and four light chains. Its theoretical molecular weight (Expected Molecular Weight), calculated to account for the modification of the C-terminal lysine residue of the heavy chain, is 238099 Da. As shown in Figure 13, the actual measured molecular weight is 238100 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.

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

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-PDL-1 bispecific antibodies. The specific implementation steps are as follows: Human non-small cell lung cancer NCI-H292 cells (CRL-1848 ^™ ) cultured in vitro were collected, and the cell suspension concentration was adjusted to 1× ^10⁸ /ml. The cells were then mixed with an equal volume of Matrigel (purchased from BD Biosciences, catalog number: 356234). Purchased PBMCs (purchased from Allcells, catalog number: PB005-C) were resuscitated in vitro, and the PBMC cells were resuspended in PBS, adjusting the PBMC suspension concentration to 1× ^10⁷ /ml. The mixed tumor cell suspension and PBMC suspension were then mixed with an equal volume. Under aseptic conditions, 200 μl of a mixed cell suspension 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 dosage was set at equimolar amounts in this experiment. Subsequently, the drugs were administered according to the designed protocol, twice a week for a total of 8 weeks, and tumor volume was measured twice a week. Finally, the tumor growth curves of each group over time are shown in Figure 14.

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> A tetravalent bispecific antibody against PD-1 and PD-L1

<130> SH363-21P450348

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<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> 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> 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 human IgG1 heavy chain constant region

<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 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> Nucleotide sequence of the heavy chain of Anti-PDL1

<400> 14

caggtccagc tgcagcagtc aggagggggc ctggtgaagc catcacagag cctgtccctg 60

acctgcacag tctctgggtt cagtctgact tcatacggag tgcactgggt ccgacagccc 120

cctggaaagg gactggagtg gatcggcctg atttggtctg gcgggggaac agactataac 180

cccagcctga aatcccggct gaccatctct agagatacca gtaagaatca agtgagcttt 240

aaaattagct ccctgacagc cgctgacact gcagtgtact attgtgcaag gcagctggga 300

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 ccaggacac 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> 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> Amino acid sequence of H-CDR2, the heavy chain complementarity-determining region 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> 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> 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> 21

<211> 7

<212> PRT

<213> Artificial

<220>

<223> Amino acid sequence of the light chain complementarity-determining region L-CDR2 of Anti-PDL1

<400> 21

Tyr Ala Ser Gln Ser Phe Ser

1 5

<210> 22

<211> 9

<212> PRT

<213> Artificial

<220>

<223> Amino acid sequence of the light chain complementarity-determining region L-CDR3 of Anti-PDL1

<400> 22

Gln Gln Ser Asn Ser Trp Pro Leu Thr

1 5

<210> 23

<211> 118

<212> PRT

<213> Artificial

<220>

<223> Amino acid sequence of the heavy chain variable region of Atezolizumab

<400> 23

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 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> 24

<211> 107

<212> PRT

<213> Artificial

<220>

<223> Amino acid sequence of the light chain variable region of Atezolizumab

<400> 24

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 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>

The amino acid sequence of the light chain variable region of <223>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> 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> Connector (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>

<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 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> Nucleotide sequence of PDL1-Fab-609-IgG4

<400> 30

caggtccagc tgcagcagtc aggagggggc ctggtgaagc catcacagag cctgtccctg 60

acctgcacag tctctgggtt cagtctgact tcatacggag tgcactgggt ccgacagccc 120

cctggaaagg gactggagtg gatcggcctg atttggtctg gcgggggaac agactataac 180

cccagcctga aatcccggct gaccatctct agagatacca gtaagaatca agtgagcttt 240

aaaattagct ccctgacagc cgctgacact gcagtgtact attgtgcaag gcagctggga 300

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 tggcggggagt 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 aagaataa gtgcaaggtg agcaataagg gcctccccag cagcatcgag 1680

aagactatta gcaaagccaa agggcagcca cgggaacccc aggtgtacac tctgcccccc 1740

tctcaggagg agatgactaa aaatcaggtc tctctgactt gtctggtgaa agggttttat 1800

cccagcgaca ttgccgtgga gtggggagtct 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 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 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 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> Nucleotide sequence of 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 ggccgaagat acagccgtgt atttctgtgc ctccccctac 300

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 660

ggcggaggtg gcagcggagg tggcggggagt caggtccagc tgcagcagtc aggagggggc 720

ctggtgaagc catcacagag cctgtccctg acctgcacag tctctgggtt cagtctgact 780

tcatacggag tgcactgggt ccgacagccc cctggaaagg gactggagtg gatcggcctg 840

atttggtctg gcgggggaac agactataac cccagcctga aatcccggct gaccatctct 900

agagatacca gtaagaatca agtgagcttt aaaattagct ccctgacagc cgctgacact 960

gcagtgtact attgtgcaag gcagctggga ctgcgagcta tggattactg gggacagggc 1020

acttccgtga ccgtctctag tgcaagtacc 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 aagaataa gtgcaaggtg agcaataagg gcctccccag cagcatcgag 1680

aagactatta gcaaagccaa agggcagcca cgggaacccc aggtgtacac tctgcccccc 1740

tctcaggagg agatgactaa aaatcaggtc tctctgactt gtctggtgaa agggttttat 1800

cccagcgaca ttgccgtgga gtggggagtct 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 (8)

1. A tetravalent bispecific antibody against PD-1 and PD-L1, characterized in that it comprises two polypeptide chains and four common light chains, wherein the amino acid sequence of the polypeptide chains is as shown in SEQ ID NO: 29 or SEQ ID NO: 31, and the amino acid sequence of the common light chains is as shown in SEQ ID NO: 15. 2. An isolated polynucleotide, characterized in that the polynucleotide encodes the tetravalent bispecific antibody as described in claim 1. 3. The polynucleotide of claim 2, wherein the polynucleotide encodes the polypeptide chain and the common light chain, wherein the polynucleotide sequence encoding the polypeptide chain is shown in SEQ ID NO: 30 or SEQ ID NO: 32, and the polynucleotide sequence encoding the common light chain is shown in SEQ ID NO: 16. 4. An expression vector, characterized in that the expression vector contains the polynucleotide as described in claim 2 or 3. 5. A host cell, characterized in that the host cell contains the expression vector as described in claim 4. 6. The method for preparing the tetravalent bispecific antibody as described in claim 1, characterized in that the method comprises the following steps: (a) Under expression conditions, host cells as described in claim 5 are cultured to express the tetravalent bispecific antibody; (b) Isolate and purify the tetravalent bispecific antibody described in (a). 7. A pharmaceutical composition, characterized in that the pharmaceutical composition contains the tetravalent bispecific antibody as described in claim 1 and a pharmaceutically acceptable carrier. 8. Use of the tetravalent bispecific antibody of claim 1 or the pharmaceutical composition of claim 7 in the preparation of a medicament for treating cancer, wherein 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, lymphoma and other neoplastic malignant tumors.