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

Abstract

An antibody that binds to human PD-L1, and a tetravalent bispecific antibody against PD-1 and PD-L1 constructed based on the antibody that binds to human PD-L1. The tetravalent bispecific antibody does not require Fc modification, does not cause mismatching problems, has a simple preparation method, and has similar or even better biological activity and physicochemical properties than monoclonal antibodies.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and more specifically, the present invention discloses a tetravalent bispecific antibody against PD-1 and PD-L1. Background Art

[0002] Human programmed cell death receptor-1 (PD-1) is a type I membrane protein with 288 amino acids and is one of the main known immune checkpoints (Blank et al, 2005, Cancer Immunotherapy, 54: 307-314). PD-1 is expressed in activated T lymphocytes. It binds to its ligands PD-L1 (programmed cell death receptor-ligand 1) and PD-L2 (programmed cell death receptor-ligand 2) to inhibit the activity of T lymphocytes and related cellular immune responses in vivo. PD-L2 is mainly expressed in macrophages and dendritic cells, while PD-L1 is widely expressed in B, T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, heart and other tissue cells. A large number of studies have shown that the interaction between PD-1 and PD-L1 is not only necessary to maintain the balance of the body's immune system, but is also the main mechanism and reason for PD-L1-positive tumor cells to avoid immune surveillance. By blocking the negative regulation of cancer cells on the PD-1 / PD-L1 signaling pathway and activating the immune system, it can promote T cell-related tumor-specific cellular immune responses, thus opening the door to a new tumor treatment method - tumor immunotherapy.

[0003] PD-1 (encoded by the gene Pdcd1) is a member of the immunoglobulin superfamily associated with CD28 and CTLA-4. Research results show that when PD-1 binds to its ligands (PD-L1 and / or PD-L2), it negatively regulates antigen receptor signal transduction. The structure of mouse PD-1 and the co-crystal structure of mouse PD-1 and human PD-L1 have been clarified (Zhang, X. et al., Immunity 20: 337-347 (2004); Lin et al., Proc. Natl. Acad. Sci. USA 105: 3011-6 (2008)). PD-1 and similar family members are type I transmembrane glycoproteins that contain an Ig variable (V-type) domain responsible for ligand binding and a cytoplasmic tail region responsible for binding signal transduction molecules. The cytoplasmic tail of PD-1 contains two tyrosine-based signal transduction motifs, ITIM (immunoreceptor tyrosine-based inhibitory motif) and ITSM (immunoreceptor tyrosine-based switch motif).

[0004] PD-1 plays an important role in the immune escape mechanism of tumors. Tumor immunotherapy, which uses the body's own immune system to resist cancer, is a breakthrough tumor treatment method, but the tumor microenvironment can protect tumor cells from effective immune destruction, so how to break the tumor microenvironment has become the focus of anti-tumor research. Existing research results have determined the role of PD-1 in the tumor microenvironment: PD-L1 is expressed in many mouse and human tumors (and can be induced by IFNγ in most PD-L1-negative tumor cell lines) and is presumed to be an important target for mediating tumor immune escape (Iwai Y. et al., Proc. Natl. Acad. Sci. USA 99: 12293-12297 (2002); Strome SE et al., Cancer Res., 63: 6501-6505 (2003)). By evaluating biopsies with immunohistochemistry, the expression of PD-1 (on tumor-infiltrating lymphocytes) and / or PD-L1 on tumor cells has been found in many primary tumors in humans. Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma and pancreatic cancer, as well as head and neck tumors. It can be seen that blocking the interaction of PD-1 / PD-L1 can enhance the immune activity of tumor-specific T cells and help the immune system to eliminate tumor cells. Therefore, PD-1 and PD-L1 have become popular targets for the development of tumor immunotherapy drugs.

[0005] Bispecific antibodies refer to antibody molecules that can specifically bind to two antigens or two epitopes at the same time. According to symmetry, bispecific antibodies can be divided into structurally symmetrical and asymmetrical molecules. According to the number of binding sites, bispecific antibodies can be divided into divalent, trivalent, tetravalent and multivalent molecules. Bispecific antibodies are gradually becoming a new class of therapeutic antibodies that can be used to treat various inflammatory diseases, cancers and other diseases. Although a large number of new bispecific antibody structures have been reported recently, the main technical difficulty in producing bispecific antibodies is to obtain correctly paired molecules. Currently, all existing bispecific antibody forms have mismatch problems, so one or more by-products or aggregates caused by mismatches will be produced, thereby affecting the yield, purity and physicochemical stability of the target bispecific antibody, and then affecting the safety and effectiveness of the bispecific antibody in vivo. Summary of the invention

[0006] 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 antibody that binds to human PD-L1.

[0007] Therefore, the first object of the present invention is to provide an antibody or an antigen-binding fragment thereof that binds to human PD-L1.

[0008] The second object of the present invention is to provide an isolated nucleotide encoding the antibody or antigen-binding fragment thereof that binds to human PD-L1.

[0009] The third object of the present invention is to provide an expression vector comprising the nucleotide sequence.

[0010] The fourth object of the present invention is to provide a host cell comprising the expression vector.

[0011] The fifth object of the present invention is to provide a method for preparing the antibody or antigen-binding fragment thereof that binds to human PD-L1.

[0012] The sixth object of the present invention is to provide a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof that binds to human PD-L1.

[0013] The 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 in the preparation of a drug for treating a disease in which PD-L1 is overexpressed.

[0014] The eighth object of the present invention is to provide a method for using the antibody or antigen-binding fragment thereof that binds to human PD-L1 or the pharmaceutical composition to treat diseases in which PD-L1 is overexpressed.

[0015] The ninth object of the present invention is to provide a tetravalent bispecific antibody against PD-1 and PD-L1.

[0016] The tenth object of the present invention is to provide an isolated nucleic acid encoding the tetravalent bispecific antibody.

[0017] The eleventh object of the present invention is to provide an expression vector comprising the nucleotide.

[0018] The twelfth object of the present invention is to provide a host cell comprising the expression vector.

[0019] The thirteenth object of the present invention is to provide a method for preparing the tetravalent bispecific antibody.

[0020] The fourteenth object of the present invention is to provide a pharmaceutical composition comprising the tetravalent bispecific antibody.

[0021] The fifteenth object of the present invention is to provide use of the tetravalent bispecific antibody or the pharmaceutical composition in the preparation of a drug for treating cancer.

[0022] The sixteenth object of the present invention is to provide a method for using the tetravalent bispecific antibody or the pharmaceutical composition to treat cancer.

[0023] In order to achieve the above object, the present invention provides the following technical solutions:

[0024] The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof that binds to human PD-L1, comprising:

[0025] (a) heavy chain complementary 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

[0026] (b) light chain complementary 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.

[0027] According to the present invention, the antibody is a monoclonal antibody or a polyclonal antibody.

[0028] According to the present invention, the antibody is a mouse antibody, a chimeric antibody or a humanized antibody.

[0029] According to the present invention, the antigen-binding fragment includes a Fab fragment, a F(ab')2 fragment, a Fv fragment or a single-chain antibody.

[0030] According to the present invention, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds to human PD-L1 is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10.

[0031] According to the present invention, the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof that binds to human PD-L1 is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain is shown in SEQ ID NO: 15.

[0032] The second aspect of the present invention provides an isolated nucleotide encoding the antibody or antigen-binding fragment thereof that binds to human PD-L1 as described above.

[0033] According to the present invention, the nucleotide sequence encoding the heavy chain of the antibody or antigen-binding fragment thereof that binds to human PD-L1 is shown in SEQ ID NO: 14, and the nucleotide sequence encoding the light chain is shown in SEQ ID NO: 16.

[0034] The third aspect of the present invention provides an expression vector, which contains the nucleotides described above.

[0035] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the expression vector described above.

[0036] The 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:

[0037] (a) culturing the host cell as described above under expression conditions to express the antibody or antigen-binding fragment thereof that binds to human PD-L1;

[0038] (b) isolating and purifying the antibody or antigen-binding fragment thereof that binds to human PD-L1 described in (a).

[0039] A sixth aspect of the present invention provides a pharmaceutical composition, comprising the antibody or antigen-binding fragment thereof that binds to human PD-L1 as described above and a pharmaceutically acceptable carrier.

[0040] The seventh aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof that binds to human PD-L1 or the pharmaceutical composition as described above in the preparation of a drug for treating a disease in which PD-L1 is overexpressed.

[0041] According to the present invention, the disease in which PD-L1 is overexpressed is cancer. Preferably, the cancer is selected from the group consisting of melanoma, renal cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases.

[0042] An eighth aspect of the present invention provides a method for treating a disease in which PD-L1 is overexpressed, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof that binds to human PD-L1 as described above, or a pharmaceutical composition as described above.

[0043] According to the present invention, the disease in which PD-L1 is overexpressed is cancer. Preferably, the cancer is selected from the group consisting of melanoma, renal cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases.

[0044] The 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 the amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO: 31, and the common light chain has the amino acid sequence shown in SEQ ID NO: 15.

[0045] The tenth aspect of the present invention provides an isolated nucleotide encoding the tetravalent bispecific antibody.

[0046] According to a preferred embodiment of the present invention, the nucleotide encodes the polypeptide chain and the common light chain, wherein the nucleotide sequence encoding the polypeptide chain is shown in SEQ ID NO: 30 or SEQ ID NO: 32, and the nucleotide sequence encoding the common light chain is shown in SEQ ID NO: 16.

[0047] The eleventh aspect of the present invention provides an expression vector, which contains the nucleotide described above.

[0048] The twelfth aspect of the present invention provides a host cell, wherein the host cell contains the expression vector described above.

[0049] The thirteenth aspect of the present invention provides a method for preparing the tetravalent bispecific antibody, the method comprising the following steps:

[0050] (a) culturing the host cell as described above under expression conditions, thereby expressing the tetravalent bispecific antibody;

[0051] (b) isolating and purifying the tetravalent bispecific antibody described in (a).

[0052] The fourteenth aspect of the present invention provides a pharmaceutical composition comprising the tetravalent bispecific antibody as described above and a pharmaceutically acceptable carrier.

[0053] The fifteenth aspect of the present invention provides use of the tetravalent bispecific antibody or the pharmaceutical composition as described above in the preparation of a drug for treating cancer.

[0054] According to the present invention, the cancer is selected from the group consisting of: melanoma, renal cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases.

[0055] The sixteenth aspect of the present invention provides a method for treating cancer, comprising administering the tetravalent bispecific antibody as described above or the pharmaceutical composition as described above to a subject in need thereof.

[0056] According to the present invention, the cancer is selected from the group consisting of: melanoma, renal cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases.

[0057] Beneficial effects:

[0058] 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 antibody that binds to human PD-L1. The tetravalent bispecific antibody of the present invention does not require Fc modification, does not cause mismatching problems, has a simple preparation method, and has similar or even better biological activity and physicochemical properties than monoclonal antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 is a schematic diagram of the structure of the bispecific antibody of the present invention, wherein 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 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 an artificially designed linker, and the line segment between CH1 and CH2 near the C-terminus of the polypeptide chain represents the natural linker and hinge region of the antibody (if the heavy chain is of human IgG4 subtype, the hinge region will contain an S228P point mutation according to EU encoding).

[0060] Figure 2 The results of ELISA testing the relative affinity of Anti-PDL1 to PD-L1.

[0061] Figure 3 This is the result of determining the ability of Anti-PDL1 to block the PD-1 / PD-L1 interaction.

[0062] Figure 4A and Figure 4B Results to evaluate the ability of Anti-PDL1 to enhance MLR.

[0063] Figure 5A and Figure 5B ELISA results of Anti-PDL1, 609 and their hybrid antibodies.

[0064] Fig. 6A and Figure 6B ELISA results of PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4.

[0065] FIG. 7A to FIG. 7D The results are to evaluate the ability of 609-Fab-PDL1-IgG4 to enhance MLR.

[0066] Fig. 8A and Figure 8B These are the pharmacokinetic results of 609-Fab-PDL1-IgG4.

[0067] Fig.9A and Fig. 9B This is the HPLC-SEC spectrum of 609-Fab-PDL1-IgG4.

[0068] FIG. 10A to FIG. 10D This is the CE-SDS spectrum of 609-Fab-PDL1-IgG4.

[0069] Fig.11A and Fig. 11B This is the HPLC-IEC spectrum of 609-Fab-PDL1-IgG4.

[0070] Fig. 12A and Fig. 12B This is the DSC spectrum of 609-Fab-PDL1-IgG4.

[0071] Fig.13 This is the molecular weight mass spectrum of 609-Fab-PDL1-IgG4.

[0072] Fig.14 The anti-tumor effect of 609-Fab-PDL1-IgG4 bispecific antibody in mice. DETAILED DESCRIPTION

[0073] The sequence information involved in the present invention is summarized in Table 1.

[0074] Table 1. Sequence information of the antibodies of the present invention

[0075]

[0076]

[0077] In the present invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" are heterotetrameric glycoproteins of about 150,000 daltons with the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a constant region, and the heavy chain constant region is composed of three domains CH1, CH2, and CH3. One end of each light chain has a variable region (VL) and the other end has a constant region, and the light chain constant region includes a domain CL; the constant region of the light chain is paired with the CH1 domain of the heavy chain constant region, and the variable region of the light chain is paired with the variable region of the heavy chain. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC). The heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes κ (Kappa) or λ (Lambda). The heavy chain and light chain of the antibody are covalently linked together by a disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are covalently linked together by an inter-polypeptide disulfide bond formed between the hinge regions. The antibodies of the present invention include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) formed by at least two antibodies, antigen-binding fragments of antibodies, and the like. The antibodies of the present invention include murine antibodies, chimeric antibodies, humanized antibodies, and the like.

[0078] In the present invention, the term "bispecific antibody (bi-antibody)" refers to an antibody molecule that can specifically bind to two antigens (targets) or two epitopes at the same time.

[0079] In the present invention, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a class of substantially homogeneous populations, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may exist. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (usually a mixture of different antibodies directed against different antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture and will not be contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a substantially homogeneous antibody population, and this should not be interpreted as requiring any special method to produce the antibody.

[0080] In the present invention, the term "murine antibody" refers to an antibody derived from rats or mice, preferably mice. The murine antibody of the present invention is obtained by immunizing mice with the extracellular domain of human PD-L1 as an antigen and performing hybridoma cell screening.

[0081] In the present invention, the term "chimeric antibody" refers to an antibody comprising heavy and light chain variable region sequences derived from one species and constant region sequences derived from another species, such as an antibody having mouse heavy and light chain variable regions linked to human constant regions.

[0082] In the present invention, the term "humanized antibody" refers to an antibody whose CDR is derived from a non-human species (preferably a mouse), and the remaining part of the antibody molecule (including the framework region and the constant region) is derived from a human antibody. In addition, the framework region residues may be changed to maintain binding affinity.

[0083] In the present invention, the term "antigen binding fragment" refers to a fragment of an antibody that can specifically bind to a human PD-L1 epitope. Examples of antigen binding fragments of the present invention include Fab fragments, F(ab')2 fragments, Fv fragments, and single-chain antibodies (scFv). The Fab fragment is composed of the VH and CH1 of the heavy chain of the antibody and the VL and CL domains of the light chain. The F(ab')2 fragment is a fragment produced by digesting an antibody with pepsin. The Fv fragment is composed of a dimer of the heavy chain variable region and the light chain variable region of the antibody in a tightly non-covalent association. A single-chain antibody (scFv) is an antibody in which the heavy chain variable region and the light chain variable region of the antibody are connected by a short peptide (linker) of 15 to 20 amino acids.

[0084] In the present invention, the term "Fc" refers to a crystallizable fragment (Fc), which is composed of the CH2 and CH3 domains of an antibody. The Fc segment has no antigen binding activity and is the site where the antibody interacts with effector molecules or cells.

[0085] In the present invention, the term "variable" means that some parts of the variable region in the antibody are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy chain variable region and the light chain variable region. The more conservative part of the variable region is called the framework region (FR). The variable region of the natural heavy chain and light chain each contains four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region 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, Volume I, pp. 647-669 (1991)).

[0086] In the present invention, the terms "anti" and "binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed. -7 M, for example, less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The equilibrium dissociation constant (KD) of M or less binds to the antigen. In the present invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, the binding affinity of the antibody and the antigen is measured in a BIACORE instrument using surface plasmon resonance (Surface Plasmon Resonance, abbreviation SPR) or the relative affinity of the antibody and antigen binding is measured using ELISA.

[0087] In the present invention, the term "valency" refers to the presence of a specified number of antigen binding sites in an antibody molecule. Preferably, the bispecific antibody of the present invention has four antigen binding sites and is tetravalent. In the present invention, the antigen binding site comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0088] In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitope of the present invention is a region of an antigen that is bound by an antibody.

[0089] In the present invention, the term "common light chain" refers to a light chain comprising the same light chain variable region and light chain constant region, which can be paired with the heavy chain of the first antibody that binds to the first antigen to form a binding site that specifically binds to the first antigen, and can also be paired with the heavy chain of the second antibody that binds to the second antigen to form a binding site that specifically binds to the second antigen. Further, 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.

[0090] In the present invention, the term "expression vector" can be pTT5, pSECtag series, pCGS3 series, pcDNA series vectors, and other vectors used in mammalian expression systems, etc., and the expression vector includes a fusion DNA sequence connected to appropriate transcription and translation regulatory sequences.

[0091] In the present invention, the term "host cell" refers to a cell suitable for expressing the above-mentioned expression vector, which can be a eukaryotic cell, such as a mammalian or insect host cell culture system can be used for the expression of the fusion protein of the present invention, CHO (Chinese Hamster Ovary), HEK293, COS, BHK and the derivatives of the above cells can be suitable for the present invention.

[0092] In the present invention, the term "pharmaceutical composition" means that the antibody or antigen-binding fragment thereof or bispecific tetravalent antibody binding to human PD-L1 of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to exert the therapeutic effect more stably. These preparations can ensure the conformational integrity of the amino acid core sequence of the antibody or antigen-binding fragment thereof or bispecific tetravalent antibody binding to human PD-L1 disclosed in the present invention, while also protecting the multifunctional groups of the protein to prevent its degradation (including but not limited to aggregation, deamination or oxidation).

[0093] The protein expression and purification methods used in the following examples are as follows: The target gene is constructed into the expression vector pcDNA4, and the constructed expression vector or the combination of expression vectors is transferred into FreeStyle TM HEK293-F Cells (hereinafter referred to as HEK293F, purchased from Thermo Fisher Scientific) were used to express antibodies or recombinant proteins. HEK293F cells were cultured in Free Style 293 Expression Medium (purchased from ThermoFisher Scientific) for 5 days, and the cell supernatant was collected. The antibodies or recombinant proteins were then purified using Protein A affinity chromatography or nickel affinity chromatography.

[0094] The mixed lymphocyte reaction (MLR) method used in the following examples is as follows: Peripheral blood mononuclear cells (PBMC) were separated from human blood using Histopaque (purchased from Sigma), and then the monocytes in the PBMC were separated by the adhesion method, and then the monocytes were induced to differentiate into dendritic cells using IL-4 (25ng / ml) and GM-CSF (25ng / ml). After seven days, the above-induced dendritic cells were collected by digestion. PBMC were separated from the blood of another donor using the above method, and then CD4 was separated from the PBMC using MACS magnets and CD4 MicroBeads (purchased from Miltenyibiotec). + T cells. The induced dendritic cells (10 4 / well) and isolated CD4 + T cells (10 5 / well) were mixed in proportion and inoculated into a 96-well plate, 150 μl per well; a few hours later, 50 μl of gradient diluted antibodies were added to the above 96-well plate; the 96-well plate was placed in a 37°C cell culture incubator and incubated for 3 days. During the above experiment, AIM-V medium (purchased from Thermo Fisher Scientific) was used to culture cells. Then the secretion of IL-2 and IFN-γ was detected according to the standard operating procedure. IL-2 and IFN-γ were detected using double antibody sandwich ELISA (the relevant paired antibodies were purchased from BD Biosciences). OD450 was read using an enzyme reader (SpectraMax 190), and GraphPadPrism6 was used to draw graphs and calculate EC50.

[0095] The physicochemical property detection methods used in the following examples are described as follows:

[0096] HPLC-SEC

[0097] Antibodies are high molecular weight proteins with highly complex secondary and tertiary structures. Due to changes such as post-translational modifications, aggregation and degradation, antibodies are heterogeneous in terms of biochemical and biophysical properties. When bispecific antibodies are analyzed by separation techniques, variants, aggregates and degradation fragments are usually observed, and their presence may impair safety and effectiveness. Aggregates, degradation fragments and incompletely assembled molecules are prone to occur during the production and storage of antibodies. The present invention uses high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) to detect the content of the above impurities in the sample. The molecular weight of the aggregate is greater than that of the monomer, so the retention time of the corresponding peak is shorter; the molecular weight of the degradation fragment or incompletely assembled molecule is less than that of the monomer, so the retention time of the corresponding peak is longer. The chromatograph used for HPLC-SEC was Dionex Ultimate 3000; the mobile phase was prepared as follows: take an appropriate amount of 20 mM sodium dihydrogen phosphate mother solution and adjust the pH to 6.8±0.1 with 20 mM disodium hydrogen phosphate; injection volume: 20 μg; the chromatographic column was TSK G3000SWXL, with specifications of 7.8×300 mm 5 μm; flow rate was 0.5 ml / min, elution time was 30 min; column temperature was 25°C, sample chamber temperature was 10°C; detection wavelength was 214 nm.

[0098] HPLC-IEC

[0099] Many post-translational modifications (such as N-glycosylation, modification of C-terminal lysine residues, cyclization of N-terminal glutamine or glutamic acid, deamidation of asparagine, isomerization of aspartic acid and oxidation of amino acid residues) can directly or indirectly cause changes in the surface charge of antibodies, resulting in the generation of charge heterogeneity. Charge variants can be separated and analyzed based on the charge they carry. Commonly used analytical methods include cation exchange chromatography (CEX) and anion exchange chromatography (AEX). When analyzed by chromatography-based methods, acidic species and basic species are defined based on their retention time relative to the main peak. Acidic species are variants that elute earlier than the main peak of CEX or later than the main peak of AEX, while basic species are variants that elute later than the main peak of CEX or earlier than the main peak of AEX. The peaks corresponding to acidic species and basic species are called acidic peaks and basic peaks, respectively. Charge variants are easily generated during the production and storage of antibodies. High-performance liquid chromatography-ion exchange chromatography (HPLC-IEC) was used to analyze the charge heterogeneity of the samples. The chromatograph used for HPLC-IEC was Dionex Ultimate 3000; mobile phase A: 20mM PB pH 6.3, mobile phase B: 20mM PB + 200mM NaCl pH 6.3, the mixing ratio of the two mobile phases changed over time according to a pre-set program, flow rate 1.0ml / min; chromatographic column: ThermoPropac TM WCX-10; column temperature 30°C, sample chamber temperature 10°C; injection volume: 20 μg; detection wavelength: 214 nm.

[0100] CE-SDS

[0101] The present invention uses CE-SDS (Capillary Electrophoresis-Sodium Dodecyl Sulfate) to analyze the content of degradation fragments or incompletely assembled molecules in the sample. CE is divided into two types: non-reducing and reducing. The sample used for the former does not need to use the reducing agent DTT to destroy the disulfide bonds in the molecule during denaturation, while the sample used for the latter needs to use the reducing agent DTT to destroy the disulfide bonds in the molecule during denaturation. Non-reducing and reducing CE-SDS are respectively recorded as NR-CE-SDS and R-CE-SDS. The capillary electrophoresis instrument used is ProteomeLab TMPA800 plus (Beckman Coulter), equipped with UV214nm detector, capillary model is Bare Fused-Silica Capillary, specification is 30.7cm×50μm, effective length is 20.5cm; other related reagents are purchased from Beckman Coulter. The key parameters of the instrument are set as follows: the temperature of the capillary and sample chamber is 20±2℃, and the separation voltage is 15kV.

[0102] DSC

[0103] Differential Scanning Calorimeter (DSC) mainly reflects the thermal stability of samples by detecting heat changes in biomolecules during a controlled heating or cooling process. Through heating, the unfolding of protein samples absorbs heat, and the supplementary energy required to eliminate the temperature difference in the sample pool is recorded by the equipment. These heat changes will form a peak shape on the spectrum, where the peak temperature corresponding to the unfolding of the protein sample is taken as the melting temperature Tm. Tm is an important indicator of the thermal stability of proteins. The higher the Tm, the better the stability of the protein.

[0104] Molecular weight detection

[0105] Antibodies were treated with PNGase F and endoglycosidase F2 for deglycosylation. UPLC-XEVO G2 Q-TOF LC-MS system (Waters) was used for analysis and identification of sample molecular weights. Mobile phase A was HPLC-grade water containing 0.1% trifluoroacetic acid (TFA). Mobile phase B was acetonitrile containing 0.1% TFA. In the method for detecting intact molecular weight, the chromatographic column used was MassPREPTM Micro Desalting Column (specification 2.1×5 mm). The 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% in 1.5 min; ESI source temperature: 130°C. BiopharmaLynx v1.2 (Waters) was used to control the LC-MS system and collect data, and mass spectrometry signals were deconvoluted using BiopharmaLynx v1.2.

[0106] The following examples and experimental examples are provided to further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, insert protein-encoding genes into such vectors and plasmids, or introduce plasmids into host cells. Such methods are well known to those skilled in the art and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

[0107] Example 1 Preparation of humanized anti-human PD-L1 antibody

[0108] Example 1.1 Preparation of PD-1 and PD-L1 recombinant proteins

[0109] The sources of the extracellular region encoding genes of PD-1 and PD-L1 are as described in WO2018 / 137576A1. Using gene recombination technology, polyhistidine coding sequences were connected to the ends of the extracellular region encoding genes of PD-1 and PD-L1, respectively, and then the recombinant genes were cloned into the pcDNA4 expression vector, respectively, and the recombinant proteins were expressed and purified. The resulting recombinant proteins were named PD1-His and PD-L1-His, respectively. Using gene recombination technology, the Fc segment coding sequence of human IgG1 was connected to the ends of the extracellular region encoding genes of PD-1 and PD-L1, respectively, and then the recombinant genes were cloned into the pcDNA4 expression vector, respectively, and the recombinant proteins were expressed and purified. The resulting recombinant proteins were named PD1-ECD-hFc and PD-L1-ECD-hFc, respectively.

[0110] Example 1.2 Preparation of mouse anti-human PD-L1 monoclonal antibody

[0111] The above-mentioned PD-L1-ECD-hFc was used as an antigen to immunize Balb / c mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.). The methods for immunizing mice, titer detection and hybridoma clone screening are described in Example 2 of WO2018 / 137576A1. The ELISA method for screening hybridoma positive clones is as follows: the ELISA plate is coated with the above-mentioned PD-L1-His at a coating concentration of 10 ng / well, and PBST (KH containing 1% bovine serum albumin (BSA)) is used. 2 PO 4 0.2g, Na 2 HPO 4 12H2 O 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20 0.5ml, add pure water to 1L) to seal the ELISA plate. The antibody to be tested was diluted in a gradient manner and then transferred to the above-mentioned ELISA plate coated with the recombinant protein. The plate was incubated at room temperature for half an hour and then washed. An appropriately diluted HRP (Horseradish Peroxidase) labeled goat anti-mouse antibody (Fc-Specific) (purchased from Sigma) was added and incubated at room temperature for half an hour and then washed. 100 μl of a colorimetric solution with TMB (3,3',5,5'-Tetramethylbenzidine) as a substrate was added to each well (substrate colorimetric 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 colorimetric 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; A and B solutions were mixed in equal volumes before use) and incubated at room temperature for 1 to 5 minutes. 50 μl of a stop solution (2M H 2 SO 4 ) to terminate the reaction; and read OD450 using a microplate reader (SpectraMax 190).

[0112] Positive hybridoma clones were selected for expansion culture in 24-well plates and subcloned by limiting dilution. Monoclonal hybridoma cell lines that stably express the target antibody were obtained by the aforementioned method, and these clones were amplified. The aforementioned hybridoma cell lines were cultured with serum-free medium Hybridoma-SFM (purchased from Thermo Fisher Scientific) for 7 days, and then the mouse anti-human PD-L1 monoclonal antibodies were purified from the culture supernatant by Protein A / G affinity chromatography. After purification, several strains of mouse monoclonal antibodies that can bind to human PD-L1 were obtained. The relative affinity of the above-mentioned mouse monoclonal antibodies to human PD-L1 was evaluated by ELISA. Finally, the clone M8 with the highest relative affinity was selected for the next step of development.

[0113] Example 1.3 Determination of mouse anti-PD-L1 monoclonal antibody sequence and humanization

[0114] Step 1: Determination of the variable region sequence of mouse anti-human PD-L1 monoclonal antibody

[0115] 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 the combination 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 sequences were sequenced and analyzed.

[0116] The amino acid sequences of the heavy chain variable region and light chain variable region of the M8 antibody were analyzed, and the antigen complementarity determining regions and framework regions of the heavy chain and light chain of the M8 antibody were determined according to the Kabat rule. The amino acid sequence of the heavy chain CDR of the M8 antibody is 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 sequence of the light chain CDR is L-CDR1: RASQSIGTTIH (SEQ ID NO: 4), L-CDR2: YASESVS (SEQ ID NO: 5) and L-CDR3: QQSNSWPLT (SEQ ID NO: 6).

[0117] Step 2: Humanization of mouse anti-human PD-L1 monoclonal antibody

[0118] exist https: / / www.ncbi.nlm.nih.gov / igblast / , the heavy chain variable region of the mouse M8 antibody was compared with the human IgG germline sequence for homology, IGHV4-59*01 was selected as the heavy chain CDR transplantation template, the heavy chain CDR of the mouse M8 antibody was transplanted into the IGHV4-59*01 framework region, and WGQGTSVTVSS (SEQ ID NO: 7) was added after H-CDR3 as the fourth framework region to obtain the CDR transplanted heavy chain variable region sequence. Similarly, the light chain variable region of the mouse M8 antibody was compared with the human IgG germline sequence for homology, IGKV6-21*01 was selected as the light chain CDR transplantation template, the light chain CDR of the mouse M8 antibody was transplanted into the framework region of IGKV6-21*01, and FGAGTKLEIK (SEQ ID NO: 8) was added after L-CDR3 as the fourth framework region to obtain the CDR transplanted light chain variable region sequence. On the basis of the CDR transplanted variable region, some amino acid sites were mutated. When mutations are made, the amino acid sequence is converted to Kabat code and the position of the site is indicated by the Kabat code.

[0119] Preferably, for the CDR-grafted heavy chain variable region, according to Kabat coding, E at position 6 is mutated to Q, P at position 9 is mutated to G, E at position 16 is mutated to Q, T at position 17 is mutated to S, G at position 27 is mutated to F, I at position 29 is mutated to L, I at position 37 is mutated to V, A at position 61 is mutated to P, A at position 62 is mutated to S, F at position 63 is mutated to L, I at position 64 is mutated to K, V at position 67 is mutated to L, V at position 71 is mutated to R, F at position 78 is mutated to V, L at position 80 is mutated to F, L at position 82 is mutated to I, and V at position 82C is mutated to L. For the CDR-grafted light chain variable region, the Q at position 11 was mutated to L, the E at position 53 was mutated to Q, the V at position 55 was mutated to F, and the L at position 78 was mutated to V.

[0120] The heavy chain variable region and light chain variable region with mutation sites are defined as humanized heavy chain variable region and light chain variable region, respectively (SEQ ID NO: 9 and 10). DNA encoding the humanized heavy chain and light chain variable regions was synthesized by Shanghai Sangon Biotechnology 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 a 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 a full-length humanized light chain gene, named Anti-PDL1-LC (SEQ ID NO: 15 and 16). The Anti-PDL1-HC and Anti-PDL1-LC genes were constructed into pcDNA4 expression vectors, respectively. The obtained heavy chain and light chain expression vectors were transferred into HEK293F cells by PEI transfection method to express the antibody. The antibody was purified by Protein A affinity chromatography, and the obtained antibody was named Anti-PDL1.

[0121] The amino acid sequence of the final Anti-PDL1 antibody heavy chain CDR is 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 sequence of the light chain CDR is L-CDR1: RASQSIGTTIH (SEQ ID NO: 20), L-CDR2: YASQSFS (SEQ ID NO: 21) and L-CDR3: QQSNSWPLT (SEQ ID NO: 22).

[0122] Example 1.4 Preparation of Control Antibody Atezolizumab-IgG1

[0123] The heavy chain variable region and light chain variable region sequences of the positive control antibody Atezolizumab were obtained from WHO Drug Information, Vol 29, No 3, 2015 (SEQ ID NOs: 23 and 24). The DNA encoding the above variable regions was synthesized by Shanghai Sangon Biotechnology 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 a 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 a full-length light chain gene, named Atezolizumab-LC. Atezolizumab-HC and Atezolizumab-LC were constructed into pcDNA4 expression vectors, respectively, and the antibodies were expressed and purified. The resulting antibody was named Atezolizumab-IgG1.

[0124] Example 1.5 ELISA determination of the relative affinity of humanized anti-human PD-L1 antibodies to PD-L1

[0125] The ELISA plate was coated with the above PD-L1-His at a coating concentration of 10 ng / well, and the ELISA plate was blocked with PBST containing 1% BSA. The antibody to be tested was diluted in a gradient manner, and then transferred to the ELISA plate coated with the above recombinant protein, incubated at room temperature for half an hour, and then washed; an appropriately diluted HRP-labeled goat anti-human antibody (Fc-Specific) (purchased from Sigma) was added, incubated at room temperature for half an hour, and then washed; 100 μl of a colorimetric solution with TMB as a substrate was added to each well, and incubated at room temperature for 1 to 5 minutes; 50 μl of a stop solution (2 MH 2 SO 4 ) to terminate the reaction; OD450 was read by microplate reader (SpectraMax 190), and graphing and data analysis were performed using GraphPad Prism6, and EC50 was calculated.

[0126] like Figure 2 As shown in the figure, both Anti-PDL1 and Atezolizumab-IgG1 can effectively bind to PD-L1-His, with EC50 of 0.1018nM and 0.09351nM, respectively, and their apparent affinities are comparable. The isotype control antibody is a human IgG1 antibody that does not bind to human PD-L1.

[0127] Example 1.6 Determination of the ability of humanized anti-human PD-L1 antibodies to block PD-1 / PD-L1 interactions

[0128] PD-L1-ECD-hFc was biotinylated using Biotin N-hydroxysuccinimide ester (purchased from Sigma, catalog number / specification: H1759-100MG). 2 CO 3 and 2.93 g NaHCO 3 Dissolve in 1L pure water) to dilute human PD-1-ECD-hFc to 2μg / ml, add to 96-well ELISA plate with a dispenser, 100μl / well, incubate at room temperature for 4h; wash once with PBST, block with PBST containing 1% BSA, 200μl / well, incubate at room temperature for 2h; discard the blocking solution, pat dry, and store at 4℃ for use. 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 graded diluted with the above protein solution; the mixed solution of the diluted antibody and biotinylated PD-L1-ECD-hFc was transferred to the ELISA plate coated with human PD1-ECD-hFc and incubated at room temperature for 1 hour; the plate was washed 3 times with PBST; Streptavidin-HRP (purchased from BD Biosciences) diluted 1:1000 with 1% BSA in PBST solution was added and incubated at room temperature for 45 minutes; the plate was washed 3 times with PBST; the color developing solution (TMB substrate solution) was added, 100 μl / well, and incubated at room temperature for 1 to 5 minutes; the stop solution (2M H 2 SO 4 ) to terminate the color development reaction, 50 μl / well; read the OD450 value with an enzyme reader; use GraphPad Prism6 to organize and analyze the data and draw graphs to calculate IC50.

[0129] like Figure 3 As shown in the figure, both Anti-PDL1 and Atezolizumab-IgG1 can effectively block the interaction between PD-1 and PD-L1, with IC50 of 1.366nM and 1.471nM, respectively, and their blocking abilities are comparable. The isotype control antibody is a human IgG1 antibody that does not bind to human PD-L1.

[0130] Example 1.7 Determination of the functional activity of humanized anti-human PD-L1 antibodies using mixed lymphocyte reaction

[0131] like Figure 4AAs shown in Figure 2, both Anti-PDL1 and Atezolizumab-IgG1 can effectively stimulate MLR to secrete IL-2, with EC50 values ​​of 0.306 nM and 0.29 nM, respectively. Figure 4B As shown, both Anti-PDL1 and Atezolizumab-IgG1 can effectively stimulate MLR to secrete IFN-γ, with EC50 of 0.1464nM and 0.1294nM, respectively. The isotype control antibody is a human IgG1 antibody that does not bind to human PD-L1.

[0132] Example 2 Construction of tetravalent bispecific antibodies against PD-1 and PD-L1

[0133] Example 2.1 Sequence

[0134] mAb1-25-Hu (hereinafter referred to as 609) is a humanized anti-human PD-1 monoclonal antibody, whose heavy chain variable region and light chain variable region sequences are from WO2018 / 137576A1. The humanized heavy chain variable region and light chain variable region (SEQ ID NO: 25 and 26) are respectively connected to the human IgG4 (S228P) heavy chain constant region (SEQ ID NO: 27) and the Kappa light chain constant region (SEQ ID NO: 12), and finally the complete heavy chain and light chain amino acid sequences of the humanized mAb1-25-Hu monoclonal antibody (609) are obtained.

[0135] Anti-PDL1 is a humanized monoclonal antibody against human PD-L1, and its sequence is shown in Example 1.3.

[0136] Example 2.2 Selection of common light chain

[0137] The amino acid sequences of the light chain variable region of Anti-PDL1 and 609 were compared and analyzed using BLAST (Basic Local Alignment Search Tool). The results showed that 74% of the amino acids were exactly the same (Identities) and 86% of the amino acids were similar in nature (Positives).

[0138] The heavy chain and light chain genes of Anti-PDL1 were named Anti-PDL1-HC and Anti-PDL1-LC, respectively, and the heavy chain and light chain genes of 609 were named 609-HC and 609-LC, respectively. They were constructed into pcDNA4 expression vectors, and the heavy chain and light chain expression vectors were combined as follows: Anti-PDL1-HC+Anti-PDL1-LC, 609-HC+609-LC, Anti-PDL1-HC+609-LC and 609-HC+Anti-PDL1-LC. The antibodies were expressed and purified, and the obtained antibodies were named Anti-PDL1, 609, Anti-PDL1-HC+609-LC and 609-HC+Anti-PDL1-LC, respectively.

[0139] The ELISA plate was coated with the above-mentioned PD1-ECD-hFc and PD-L1-ECD-hFc, respectively, and the coating concentration was 10 ng / well. The ELISA plate was blocked with PBST containing 1% BSA. The antibody to be tested was diluted in a gradient manner and then transferred to the ELISA plate coated with the above-mentioned recombinant protein. The plate was washed after incubation at room temperature for half an hour; an appropriately diluted HRP-labeled goat anti-human antibody (Fab-specific) (purchased from Sigma) was added, and the plate was washed after incubation at room temperature for half an hour; 100 μl of the colorimetric solution with TMB as the substrate was added to each well, and incubated at room temperature for 1 to 5 minutes; 50 μl of the stop solution (2M H 2 SO 4 ) to terminate the reaction; OD450 was read by microplate reader (SpectraMax 190), and graphing and data analysis were performed using GraphPad Prism6, and EC50 was calculated.

[0140] like Figure 5A As shown in the figure, 609 and 609-HC+Anti-PDL1-LC can effectively bind to PD1-ECD-hFc, with EC50 of 0.2001nM and 0.2435nM respectively; while Anti-PDL1 and Anti-PDL1-HC+609-LC cannot bind to PD1-ECD-hFc. Figure 5B As shown, Anti-PDL1 can effectively bind to PD-L1-ECD-hFc with an EC50 of 0.1246 nM, while 609, Anti-PDL1-HC+609-LC and 609-HC+Anti-PDL1-LC cannot effectively bind to PD1-ECD-hFc. Anti-PDL1-LC (SEQ ID NOs: 15 and 16) was selected as the common light chain to construct a bispecific antibody.

[0141] Example 2.3 Construction of bispecific antibodies

[0142] The heavy chain variable region of Anti-PDL1 was connected to the CH1 domain of human IgG4, and then connected to the heavy chain variable region of 609 through an artificial linker (the linker used here is three tandem GGGGS, SEQ ID NO: 28), and finally connected to the heavy chain constant region of human IgG4 (CH1+CH2+CH3, the hinge region contains S228P mutation). 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 NOs: 29 and 30). Similarly, the heavy chain variable region of 609 was connected to the CH1 domain of human IgG4, and then connected to the heavy chain variable region of Anti-PDL1 through an artificial linker (the linker used here is three tandem GGGGS, SEQ ID NO: 28), and finally connected to the heavy chain constant region of human IgG4 (CH1+CH2+CH3, hinge region containing S228P mutation). 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 NOs: 31 and 32).

[0143] The above sequences were constructed into pcDNA4 expression vectors, respectively. The PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4 expression vectors were combined with the Anti-PDL1-LC expression vector, respectively, and the antibodies were expressed and purified. The obtained antibodies were named PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4, respectively (for the sake of simplicity, only the name of the heavy chain is taken as the name of the antibody here).

[0144] Example 2.4 ELISA determination of relative affinity

[0145] The ELISA detection method was as described in Example 1.3.

[0146] like Fig. 6A As shown, 609-HC+Anti-PDL1-LC, PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4 can effectively bind to PD1-His, and the EC50 values ​​are 0.3821nM, 5.308nM and 0.4213nM, respectively. Figure 6BAs shown, Anti-PDL1, PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4 can effectively bind to PD-L1-His, and the EC50 are 0.1204nM, 0.1400nM and 0.1350nM, respectively. Among them, the isotype control antibody is a human IgG4 antibody that binds neither PD-1 nor PD-L1. The above results show that PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4 can bind to both PD-1 and PD-L1, which indicates that they are bispecific antibodies.

[0147] Example 2.5 Evaluation of the ability to enhance MLR

[0148] The results of A and C are from the same MLR experiment, and the results of B and D are from another independent MLR experiment, in which the isotype control antibody is a human IgG4 antibody that binds neither PD-1 nor PD-L1. Fig. 7A and 7B As shown, Anti-PDL1, 609-HC+Anti-PDL1-LC and 609-Fab-PDL1-IgG4 can effectively stimulate MLR to secrete IL-2. Fig. 7A The EC50 values ​​were 0.306nM, 0.5384nM and 0.1023nM, respectively. Figure 7B The EC50 values ​​of 1, 2 and 3 were 0.1016 nM, 0.6819 nM and 0.1259 nM respectively. Figure 7C and 7D As shown, Anti-PDL1, 609-HC+Anti-PDL1-LC and 609-Fab-PDL1-IgG4 can effectively stimulate MLR to secrete IFN-γ. Figure 7C The EC50 values ​​were 0.5119nM, 1.21nM and 0.1675nM, Fig.7D The EC50 values ​​of 1,2-dihydropyridine-1,4 ...2,4-dihydropyridine-3,4-dihydropyridine-4,4-dihydropyridine-5,4-dihydropyridine-6,4-dihydropyridine-7,4-dihydropyridine-8,4-dihydropyridine-9,4-dihydropyridine-1,4-dihydropyridine- Fig. 7A and 7B It was shown that at the same concentration, 609-Fab-PDL1-IgG4 stimulated MLR to secrete more IL-2 than monoclonal antibody Anti-PDL1 or 609-HC+Anti-PDL1-LC.

[0149] Example 2.6 Biacore affinity determination

[0150] Here, the affinity between the above antibodies and PD-1 or PD-L1 was detected by Biacore 8K (GE healthcare). On Biacore 8K, various antibodies were captured using a chip coupled with ProteinA / G, and then the recombinant protein PD1-His or PD-L1-His was injected to obtain the binding-dissociation curve, and the next cycle was repeated after elution with 6M guanidine hydrochloride regeneration buffer; the data was analyzed using Biacore 8K Evaluation Software. The results are shown in Table 2.

[0151] Table 2-1. Kinetic parameters of binding and dissociation to PD-1 and equilibrium dissociation constant

[0152]

[0153] Table 2-2. Kinetic parameters of binding and dissociation to PD-L1 and equilibrium dissociation constant

[0154]

[0155] The experimental results show that the binding constant (Kon) and dissociation constant (Koff) of 609-Fab-PDL1-IgG4 and 609-HC+Anti-PDL1-LC to PD-1 are very similar, and the equilibrium dissociation constant (KD) is basically the same, KD is 2.57E-08 and 3.49E-08 respectively. The binding constant (Kon) and dissociation constant (Koff) of 609-Fab-PDL1-IgG4 and 609-HC+Anti-PDL1-LC to PD-L1 are also very similar, and the equilibrium dissociation constant (KD) is basically the same, KD is 6.08E-10 and 8.43E-10 respectively. The equilibrium dissociation constant (KD) is inversely proportional to the affinity.

[0156] Example 2.7 Pharmacokinetic study

[0157] In this example, SD (Sprague-Dawley) rats (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were used to study the pharmacokinetics of 609-Fab-PDL1-IgG4. Each group had five rats, weighing about 200 g, and each rat was intravenously injected (IV) with a dose of 1 mg of the antibody; blood was collected from the eye sockets at a specific time after administration, and the blood was centrifuged to obtain serum after natural coagulation.

[0158] The method for measuring the concentration of the target antibody in the serum is as follows: PD1-His and PD-L1-His are coated on the ELISA plate at a coating concentration of 20 ng / well and 10 ng / well, respectively, and then the ELISA plate is blocked with PBST containing 1% bovine serum albumin. The appropriately diluted rat serum is transferred to the above-mentioned ELISA plates coated with PD1-His and PD-L1-His, respectively; after 1 hour of room temperature incubation, the plate is washed, and then HRP-labeled goat anti-human (Fc-Specific) antibody (purchased from Sigma; the antibody has been cross-adsorbed by species and does not recognize rat antibodies) is added; after half an hour of room temperature incubation, the plate is washed, and 100 μl of color development solution with TMB as the substrate is added to each well, and incubated at room temperature for 1 to 5 minutes; 50 μl of stop solution (2M H 2 SO 4 ) to terminate the reaction; read OD450 with a microplate reader and convert OD450 into antibody serum concentration using a standard curve; use GraphPad Prism6 for data analysis and plotting; and use Phoenix software to calculate the half-life of the antibody drug in rats.

[0159] according to Fig. 8A The half-life of 609-Fab-PDL1-IgG4 was calculated to be 365 hours (15.2 days). Figure 8B The half-life of 609-Fab-PDL1-IgG4 was calculated to be 446 hours (18.6 days), and the half-life of monoclonal antibody Anti-PDL1 was 361 hours (15.0 days). The above results show that 609-Fab-PDL1-IgG4 has similar pharmacokinetic properties to Anti-PDL1 monoclonal antibody.

[0160] Example 2.8 Characterization of physicochemical properties

[0161] Example 2.8.1 HPLC-SEC

[0162] Fig.9A The HPLC-SEC spectrum of 609 monoclonal antibody shows that there are three obvious peaks, namely Peak1, Peak2 and Peak3, accounting for 0.7%, 0.3% and 99.0% respectively. The retention time of Peak1 and Peak2 is shorter than that of the main peak Peak3, indicating that Peak1 and Peak2 may be produced by aggregates, and the sum of the two accounts for 1.0%; there are no peaks that may represent degradation fragments or incomplete assembly molecules in the figure. Fig. 9BThe HPLC-SEC spectrum of 609-Fab-PDL1-IgG4 shows that there are three obvious peaks, namely 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 produced by aggregates; the retention time of Peak3 is longer than that of the main peak Peak2, indicating that Peak3 may be produced by degradation fragments or incomplete assembly molecules.

[0163] Example 2.8.2 CE-SDS

[0164] Fig. 10A and 10B Respectively represent the NR-CE-SDS and R-CE-SDS spectra of 609 monoclonal antibody, Fig. 10C and Fig. 10D They represent the NR-CE-SDS and R-CE-SDS spectra of 609-Fab-PDL1-IgG4, respectively. The main NR-CE-SDS peak Peak 8 of 609 accounts for 98.92%, and the main NR-CE-SDS peak Peak 9 of 609-Fab-PDL1-IgG4 accounts for 97.70%. The main R-CE-SDS peaks Peak 4 (corresponding to the light chain) and Peak 8 (corresponding to the heavy chain) of 609 account for 32.03% and 66.99%, respectively, and the peak area ratio between the two is 1:2.09; the main R-CE-SDS peaks Peak 3 (corresponding to the light chain) and Peak 9 (corresponding to the heavy chain) of 609-Fab-PDL1-IgG4 account for 38.73% and 58.78%, respectively, and the peak area ratio between the two is 2:3.03. In NR-CE-SDS, the main peak proportions of 609 mAb and 609-Fab-PDL1-IgG4 were very similar; in R-CE-SDS, the peak area ratios of the light chain and heavy chain of 609 mAb and 609-Fab-PDL1-IgG4 were in line with expectations.

[0165] Example 2.8.3 HPLC-IEC

[0166] Fig.11A and 11B The HPLC-IEC spectra of 609 and 609-Fab-PDL1-IgG4 are shown respectively, and their main peaks account for 82.95% and 92.70%, respectively. The results show that the charge heterogeneity of 609-Fab-PDL1-IgG4 is better than that of 609 monoclonal antibody.

[0167] Example 2.8.4 DSC

[0168] Fig. 12A and 12BThe DSC spectra of 609 and 609-Fab-PDL1-IgG4 are shown respectively. TmOnset indicates the temperature at which the protein begins to unfold or denature, and Tm corresponds to the peak temperature. The TmOnset and Tm of 609 are 63.68°C and 72.36°C, respectively, and the TmOnset and Tm of 609-Fab-PDL1-IgG4 are 64.22°C and 76.25°C, respectively. The above results show that the thermal stability of 609 and 609-Fab-PDL1-IgG4 is very similar.

[0169] Example 2.8.5 Molecular weight detection

[0170] Each molecule of 609-Fab-PDL1-IgG4 contains two long heavy chains and four light chains. Its expected molecular weight (Expected Molecular Weight) is 238099 Da, taking into account the modification of the C-terminal lysine residue of the heavy chain. Fig.13 As shown, the actual measured molecular weight is 238100Da, which is only 1Da different from the theoretical molecular weight. The above results show that the molecular structure of 609-Fab-PDL1-IgG4 is in line with expectations.

[0171] Example 3 Anti-tumor effect of anti-PD-1 and PD-L1 bispecific antibodies in mice

[0172] Human peripheral blood mononuclear cells (PBMCs) were used to reconstruct the human immune system in NSG mice, and a subcutaneous transplant tumor model of human lung cancer NCI-H292 was established in this mouse. This mouse model contains both T cells expressing human PD-1 and human tumor cells expressing human PD-L1, so it can be used to evaluate the in vivo anti-tumor activity of anti-PD-1 and PDL-1 bispecific antibodies. The specific implementation steps are as follows: Collect in vitro cultured human non-small cell lung cancer NCI-H292 cells ( CRL-1848 TM ), and the cell suspension concentration was adjusted to 1×10 8 / ml, and mixed with Matrigel (purchased from BD Biosciences, catalog number: 356234) in equal volumes. PBMC purchased in vitro (purchased from Allcells, catalog number: PB005-C) were resuspended in PBS and the concentration of PBMC suspension was adjusted to 1×10 7 / ml. The mixed tumor cell suspension and PBMC suspension were mixed in equal volumes. Under sterile conditions, 200 μl of the mixed cell suspension was inoculated subcutaneously on the right upper back of M-NSG mice (purchased from Shanghai Southern Model Organism Research Center). The mice inoculated with mixed cells were randomly divided into groups according to body weight on the same day, with 10 mice in each group. The drug treatment of each group of mice is as follows: control group, injected with normal saline; Opdivo group, injected with 10 mg / kg of anti-PD-1 positive control antibody Opdivo (produced by Bristol-Myers Squibb); Tecentriq group, injected with 10 mg / kg of anti-PD-L1 positive control antibody Tecentriq (produced by Roche Pharmaceuticals); 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 dose in this experiment was set according to the amount of equal substances. Subsequently, the drug was administered according to the above-designed scheme, twice a week, for a total of 8 times, and the tumor volume was measured twice a week. Finally, the growth curves of each group of tumors over time were measured as follows Fig.14 shown.

[0173] The results showed that at the end of the 30th day of the experiment, 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 the control group - average volume of the experimental group) / average volume of the control group × 100%). Compared with Opdivo and Tecentriq, 609-Fab-PDL1-IgG4 can inhibit tumor growth more effectively. SEQUENCE LISTING <110> Sunshine Guojian Pharmaceutical (Shanghai) Co., Ltd. <120> A tetravalent bispecific antibody against PD-1 and PD-L1 <130> SH363-21P450348 <160> 32 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Mus musculus <400> 1 Ser Tyr Gly Val His 1 5 <210> 2 <211> 16 <212> PRT <213> Mus musculus <400> 2 Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Ala Ala Phe Ile Ser 1 5 10 15 <210> 3 <211> 9 <212> PRT <213> Mus musculus <400> 3 Gln Leu Gly Leu Arg Ala Met Asp Tyr 1 5 <210> 4 <211> 11 <212> PRT <213> Mus musculus <400> 4 Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Mus musculus <400> 5 Tyr Ala Ser Glu Ser Val Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Mus musculus <400> 6 Gln Gln Ser Asn Ser Trp Pro Leu Thr 1 5 <210> 7 <211> 11 <212> PRT <213> Artificial <220> <223> Heavy chain fourth framework region <400> 7 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5 10 <210> 8 <211> 10 <212> PRT <213> Artificial <220> <223> Light chain fourth framework region <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 human kappa light chain constant region <400> 12 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 13 <211> 447 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain of Anti-PDL1 <400> 13 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 14 <211> 1341 <212> DNA <213> Artificial <220> <223> Anti-PDL1 antibody inhibitor antibody <400> 14 60. caggtccagc tgcagcagtc aggagggggc ctggtgaagc catcacagag cctgtccctg acctgcacag tctctgggtt cagtctgact tcatacggag tgcactgggt ccgacagccc 120 cctggaaagg gactggagtg gatcggcctg atttggtctg gcggggac agactataac cccagcctga aatcccggct gaccatctct aggatacca gtaagaatca agtgagcttt aaaattagct ccctgacagc cgctgacact gcagtgtact attgtgcaag gcagctggga ctgcgagcta tggattactg gggacagggc acttccgtga ccgtctctag tgcgagcacc 360 aagggacctt ccgtgtttcc cctcgccccc agctccaaaa gcaccagcgg cggaacagct 420 gctctcggct gtctcgtcaa ggattacttc cccgagcccg tgaccgtgag ctggaacagc 480 ggagccctga caagcggcgt ccacaccttc cctgctgtcc tacagtcctc cggactgtac 540 agcctgagca gcgtggtgac agtccctagc agctccctgg gcacccagac atatatttgc 600 aacgtgaatc acaagcccag caacaccaag gtcgataaga aggtggagcc taagtcctgc 660 gacaagaccc acacatgtcc cccctgtccc gctcctgaac tgctgggagg cccttccgtg 720 ttcctgttcc cccctaagcc caaggacacc ctgatgattt ccaggacacc cgaggtgacc 780 tgtgtggtgg tggacgtcag ccacgaggac cccgaggtga aattcaactg gtacgtcgat 840 ggcgtggagg tgcacaacgc taagaccaag cccagggagg agcagtacaa ttccacctac 900 agggtggtgt ccgtgctgac cgtcctccat caggactggc tgaacggcaa agagtataag 960 tgcaaggtga gcaacaaggc cctccctgct cccatcgaga agaccatcag caaagccaag 1020 ggccagccca gggaacctca agtctatacc ctgcctccca gcagggagga gatgaccaag 1080 aaccaagtga gcctcacatg cctcgtcaag ggcttctatc cttccgatat tgccgtcgag 1140 tgggagtcca acggacagcc cgagaacaac tacaagacaa caccccccgt gctcgattcc 1200 gatggcagct tcttcctgta ctccaagctg accgtggaca agtccagatg gcaacaaggc 1260 aacgtcttca gttgcagcgt catgcatgag gccctccaca accactacac ccagaagagc 1320 ctctccctga gccctggaaa g 1341 <210> 15 <211> 214 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the light chain of Anti-PDL1 <400> 15 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 16 <211> 642 <212> DNA <213> Artificial <220> <223> Nucleotide sequence of the light chain of Anti-PDL1 <400> 16 gaaatcgtgc tgacacagag ccctgacttt ctgtccgtga cacccaagga gaaagtcact 60 atcacctgcc gggctagcca gtccatcgga accacaattc actggtacca gcagaagccc 120 gaccagagcc ctaagctgct gattaaatat gcctctcaga gtttctcagg cgtgccatcc 180 agatttagcg gctccgggtc tggaactgac ttcacactga ctatcaactc tgtcgaggca 240 gaagatgccg ctacctacta ttgtcagcag agtaattcat ggcccctgac ctttggcgcc 300 gggacaaagc tggaaattaa aagaaccgtc gccgctccca gcgtcttcat cttccccccc 360 agcgatgagc agctgaagag cggaaccgcc agcgtggtgt gcctgctgaa caacttctac 420 cccagggagg ccaaggtgca atggaaggtg gacaacgccc tacagagcgg caactcccag 480 gagagcgtga ccgagcagga cagcaaggat agcacctaca gcctgagcag caccctcacc 540 ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccatcagggc 600 ctgagcagcc ctgtgaccaa gagcttcaac aggggcgagt gc 642 <210> 17 <211> 5 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain complementary 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 the heavy chain complementary determining region H-CDR2 of Anti-PDL1 <400> 18 Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 19 <211> 9 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain complementary 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 complementary determining region L-CDR1 of Anti-PDL1 <400> 20 Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His 1 5 10 <210> twenty one <211> 7 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the light chain complementary determining region L-CDR2 of Anti-PDL1 <400> twenty one Tyr Ala Ser Gln Ser Phe Ser 1 5 <210> twenty two <211> 9 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the light chain complementary determining region L-CDR3 of Anti-PDL1 <400> twenty two Gln Gln Ser Asn Ser Trp Pro Leu Thr 1 5 <210> twenty three <211> 118 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain variable region of atezolizumab <400> twenty three Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> twenty four <211> 107 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the light chain variable region of atezolizumab <400> twenty four Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 25 <211> 117 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain variable region of mAb1-25-Hu(609) <400> 25 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 26 <211> 107 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the light chain variable region of mAb1-25-Hu (609) <400> 26 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Phe 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro His 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 27 <211> 327 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain constant region of IgG4 (S228P) <400> 27 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 28 <211> 15 <212> PRT <213> Artificial <220> <223> Linker (GGGGSGGGGSGGGGS) <400> 28 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 29 <211> 674 <212> PRT <213> Artificial <220> <223> Amino acid sequence of PDL1-Fab-609-IgG4 <400> 29 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe 65 70 75 80 Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly 225 230 235 240 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 245 250 255 Phe Ala Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly 260 265 270 Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr 275 280 285 Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 290 295 300 Ala Lys Asn Ser His Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 305 310 315 320 Thr Ala Val Tyr Phe Cys Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val 325 330 335 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 340 345 350 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 355 360 365 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 370 375 380 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 385 390 395 400 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 405 410 415 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 420 425 430 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 435 440 445 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 450 455 460 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 465 470 475 480 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 485 490 495 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 500 505 510 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 515 520 525 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 530 535 540 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 545 550 555 560 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 565 570 575 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 580 585 590 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 595 600 605 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 610 615 620 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 625 630 635 640 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 645 650 655 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 660 665 670 Gly Lys <210> 30 <211> 2022 <212> DNA <213> Artificial <220> <223> PDL1-Fab-609-IgG4 inhibitor scaffold <400> 30 60. caggtccagc tgcagcagtc aggagggggc ctggtgaagc catcacagag cctgtccctg acctgcacag tctctgggtt cagtctgact tcatacggag tgcactgggt ccgacagccc 120 cctggaaagg gactggagtg gatcggcctg atttggtctg gcggggac agactataac cccagcctga aatcccggct gaccatctct aggatacca gtaagaatca agtgagcttt aaaattagct ccctgacagc cgctgacact gcagtgtact attgtgcaag gcagctggga ctgcgagcta tggattactg gggacagggc acttccgtga ccgtctctag tgcaagtacc 360 aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420 gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480 ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540 agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600 aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660 ggcggaggtg gcagcggagg tggcgggagt gaggtcaagc tggtggaaag cggcggcggc 720 ctggtgcagc ctggaggatc cctgcggctg agctgcgctg cctccggctt cgctttcagc 780 tcctatgaca tgtcctgggt gaggcaggcc cctggaaaga ggctggagtg ggtggctacc 840 atctccggag gcggaaggta cacctactac cccgacacag tgaagggaag gttcaccatc 900 agccgggata acgccaaaaa cagccactat ctccagatga actccctgag ggccgaagat 960 acagccgtgt atttctgtgc ctccccctac ggaggctatt ttgacgtgtg gggacagggc 1020 accctggtga ccgtctcctc cgcaagtacc aagggaccta gtgttttccc tcttgcacct 1080 tgctccaggt caacatcaga gtccacagct gctcttggat gtctcgttaa ggactacttc 1140 ccagagccag ttaccgtatc ctggaactcc ggagctttga caagcggcgt tcatacattc 1200 ccagctgtgt tgcagagttc tgggttgtac agtttgagct cagtggtgac cgtgccttca 1260 tcttctttgg gcactaagac ctacacctgc aacgtggatc acaagccaag caacaccaag 1320 gtggataaga gggtggagtc caagtacggc ccaccatgtc ctccatgtcc agcccctgaa 1380 tttttgggcg ggccttctgt ctttctgttt cctcctaaac ctaaagatac cctgatgatc 1440 agccgcacac ccgaagtcac ttgtgtggtc gtggatgtgt ctcaggaaga tcccgaagtg 1500 cagtttaact ggtatgtcga tggcgtggaa gtgcataatg ccaaaactaa gccccgcgaa 1560 gaacagttca acagcactta tcgggtcgtg tctgtgctca cagtcctcca tcaggattgg 1620 ctgaatggga aagaatataa gtgcaaggtg agcaataagg gcctccccag cagcatcgag 1680 aagactatta gcaaagccaa agggcagcca cgggaacccc aggtgtacac tctgcccccc 1740 tctcaggagg agatgactaa aaatcaggtc tctctgactt gtctggtgaa agggttttat 1800 cccagcgaca ttgccgtgga gtgggagtct aatggccagc ccgagaataa ttataagaca 1860 actccccccg tcctggactc tgacggcagc tttttcctgt attctcggct gacagtggac 1920 aaaagtcgct ggcaggaggg caatgtcttt agttgcagtg tcatgcatga ggccctgcac 1980 aatcactata cacagaaaag cctgtctctg agtctgggca aa 2022 <210> 31 <211> 674 <212> PRT <213> Artificial <220> <223> Amino acid sequence of 609-Fab-PDL1-IgG4 <400> 31 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Gly Gly 225 230 235 240 Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Ser Gly 245 250 255 Phe Ser Leu Thr Ser Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly 260 265 270 Lys Gly Leu Glu Trp Ile Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp 275 280 285 Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser 290 295 300 Lys Asn Gln Val Ser Phe Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr 305 310 315 320 Ala Val Tyr Tyr Cys Ala Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr 325 330 335 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly 340 345 350 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 355 360 365 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 370 375 380 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 385 390 395 400 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 405 410 415 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 420 425 430 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 435 440 445 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 450 455 460 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 465 470 475 480 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 485 490 495 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 500 505 510 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 515 520 525 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 530 535 540 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 545 550 555 560 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 565 570 575 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 580 585 590 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 595,600,605 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 610 615 620 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 625,630,635,640 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 645,650,655 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 660 665 670 Gly Lys <210> 32 <211> 2022 <212> DNA <213> Artificial <220> <223> 609-Fab-PDL1-IgG4 <400> 32 gaggtcaagc tggtggaaag cggcggcggc ctggtgcagc ctggaggatc cctgcggctg 60 agctgcgctg cctccggctt cgctttcagc tcctatgaca tgtcctgggt gaggcaggcc 120 cctggaaaga ggctggagtg ggtggctacc atctccggag gcggaaggta cacctactac 180 cccgacacag tgaagggaag gttcaccatc agccgggata acgccaaaaa cagccactat 240 ctccagatga actccctgag ggccgagat acagccgtgt atttctgtgc ctccccctac ggaggctatt ttgacgtgtg gggacagggc accctggtga ccgtctcctc cgcaagtacc 360 aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420 gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480 ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540 agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600 aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca ggcggaggtg gcagcggagg tggcgggagt caggtccagc tgcagcagtc aggaggggc 720 ctggtgaagc catcacagag cctgtccctg acctgcacag tctctgggtt cagtctgact 780 tcatacggag tgcactgggt ccgacagccc cctggaaagg gactggagtg gatcggcctg 840 atttggtctg gcgggggac agactatac cccagcctga aatcccggct gaccatctct 960. sightseeing sightseeing aaaattagct ccctgacagc cgctgacact gcagtgtact attgtgcaag gcagctggga ctgcgagcta tggattactg gggacagggc 1020 acttccgtga ccgtctctag tgcaagtacc aagggaccta gtgttttcc tcttgcacct 1080 tgctccaggt caacatcaga gtccacagct gctcttggat gtctcgttaa ggactacttc 1140 ccagagccag ttaccgtatc ctggaactcc ggagctttga caagcggcgt tcatacattc 1200 ccagctgtgt tgcagagttc tgggttgtac agtttgagct cagtggtgac cgtgccttca 1260 tcttctttgg gcactaagac ctacacctgc aacgtggatc acaagccaag caacaccaag 1320 gtggataaga gggtggagtc caagtacggc ccaccatgtc ctccatgtcc agcccctgaa 1380 tttttgggcg ggccttctgt ctttctgttt cctcctaaac ctaaagatac cctgatgatc 1440 agccgcacac ccgaagtcac ttgtgtggtc gtggatgtgt ctcaggaaga tcccgaagtg 1500 cagtttaact ggtatgtcga tggcgtggaa gtgcataatg ccaaaactaa gccccgcgaa 1560 gaacagttca acagcactta tcgggtcgtg tctgtgctca cagtcctcca tcaggattgg 1620 ctgaatggga aagaatataa gtgcaaggtg agcaataagg gcctccccag cagcatcgag 1680 aagactatta gcaaagccaa agggcagcca cgggaacccc aggtgtacac tctgcccccc 1740 tctcaggagg agatgactaa aaatcaggtc tctctgactt gtctggtgaa agggttttat 1800 cccagcgaca ttgccgtgga gtgggagtct aatggccagc ccgagaataa ttataagaca 1860 actccccccg tcctggactc tgacggcagc tttttcctgt attctcggct gacagtggac 1920 aaaagtcgct ggcaggaggg caatgtcttt agttgcagtg tcatgcatga ggccctgcac 1980 aatcactata cacagaaaag cctgtctctg agtctgggca aa 2022

Claims

1. 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 shown in SEQ ID NO: 29 or SEQ ID NO: 31, and the amino acid sequence of the common light chain is shown in SEQ ID NO:

15.

2. An isolated polynucleotide, characterized in that The polynucleotide encodes the tetravalent bispecific antibody according to claim 1.

3. The polynucleotide according to 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 according to claim 2 or 3.

5. A host cell, characterized in that The host cell contains the expression vector as claimed in claim 4.

6. The method for preparing a tetravalent bispecific antibody according to claim 1, characterized in that: The method comprises the following steps: (a) culturing the host cell of claim 5 under expression conditions, thereby expressing the tetravalent bispecific antibody; (b) isolating and purifying the tetravalent bispecific antibody described in (a).

7. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the tetravalent bispecific antibody according to 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 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, lymphoma and other neoplastic malignancies.

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