A tetravalent bispecific antibody against pd-l1 and egfr

Abstract

The application provides a tetravalent bispecific antibody against PD-L1 and EGFR. The tetravalent bispecific antibody of the application does not need to be subjected to Fc modification, does not produce a mismatch problem, has a simple preparation method, and has similar or even better biological activity and physical and chemical properties than a single antibody.

Description

Technical Field

[0001] This invention relates to the field of antibodies, and more specifically, this invention discloses a tetravalent bispecific antibody against PD-L1 and EGFR. Background Technology

[0002] Human programmed cell death receptor-1 (PD-1) is a type I membrane protein with 288 amino acids and is one of the known major immune checkpoints (Blank et al., 2005, Cancer Immunotherapy, 54:307-314). PD-1 is expressed on activated T lymphocytes. Its binding to ligands PD-L1 (programmed cell death-ligand 1) and PD-L2 (programmed cell death-ligand 2) can inhibit T lymphocyte activity and related in vivo cellular immune responses. PD-L2 is mainly expressed on macrophages and dendritic cells, while PD-L1 is widely expressed on B and T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, and heart tissue cells. Numerous studies have shown that the interaction between PD-1 and PD-L1 is not only essential for maintaining the balance of the immune system in the body, but also a major mechanism and cause for PD-L1-positive tumor cells to circumvent immune surveillance. By blocking the negative regulation of the PD-1 / PD-L1 signaling pathway by cancer cells, the immune system can be activated, promoting T cell-related tumor-specific cellular immune responses, thus opening the door to a new cancer treatment method—tumor immunotherapy.

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

[0004] PD-1 plays a crucial role in the immune evasion mechanisms of tumors. Tumor immunotherapy, which utilizes the body's own immune system to fight cancer, is a groundbreaking cancer treatment method. However, the tumor microenvironment can protect tumor cells from effective immune destruction; therefore, disrupting the tumor microenvironment has become a key focus of anti-tumor research. Existing research has identified the role of PD-1 in the tumor microenvironment: PD-L1 is expressed in many mouse and human tumors (and can be induced by IFN-γ in most PD-L1-negative tumor cell lines), and is presumed to be an important target mediating tumor immune evasion (Iwai Y. et al., Proc. Natl. Acad. Sci. USA 99: 12293-12297 (2002); Strome SE et al., Cancer Res., 63: 6501-6505 (2003)). Immunohistochemical evaluation of biopsies has revealed the expression of PD-1 (on tumor-infiltrating lymphocytes) and / or PD-L1 on tumor cells in many primary human tumors. Such cancers include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma, pancreatic cancer, and head and neck tumors. Therefore, blocking the interaction between PD-1 and PD-L1 can enhance the immune activity of tumor-specific T cells, helping the immune system to clear tumor cells. Consequently, PD-L1 has become a popular target for developing tumor immunotherapy drugs.

[0005] Epidermal growth factor receptor (EGFR) is widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, and other cells. The EGFR signaling pathway plays a crucial role in physiological processes such as cell growth, proliferation, and differentiation. Mutations or abnormal expression of EGFR play a significant role in tumor growth and development. Anti-EGFR monoclonal antibody drugs have functions such as arresting tumor cell cycle progression, accelerating tumor cell apoptosis, inhibiting tumor angiogenesis, inhibiting tumor invasion and metastasis, and enhancing the effects of radiotherapy and chemotherapy. Their mechanisms of action are clear, making them a focus of attention in cancer treatment. Anti-tumor therapy targeting EGFR has become one of the most active areas of cancer research and has achieved significant progress. However, anti-tumor therapy targeting EGFR still has many limitations that need to be addressed.

[0006] Bispecific antibodies are gradually emerging as a new class of therapeutic antibodies, potentially used to treat various inflammatory diseases, cancer, and other conditions. While numerous new bispecific antibody formulations have been reported recently, the main technical challenge in their production lies in obtaining correctly paired molecules. Currently available bispecific antibody formulations all suffer from mismatch issues, leading to one or more mismatch-related byproducts or aggregates. This negatively impacts the yield, purity, and physicochemical stability of the target bispecific antibody, ultimately affecting its safety and efficacy in vivo.

[0007] There is an urgent need in this field to develop bispecific antibodies with excellent efficacy for treating diseases such as cancer. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a quadrivalent bispecific antibody against PD-L1 and EGFR and its applications.

[0009] Therefore, the first objective of this invention is to provide a quadrivalent bispecific antibody against PD-L1 and EGFR.

[0010] A second object of the present invention is to provide an isolated nucleotide encoding the aforementioned tetravalent bispecific antibody.

[0011] A third objective of this invention is to provide an expression vector comprising the aforementioned nucleotides.

[0012] A fourth object of the present invention is to provide a host cell comprising the expression vector described above.

[0013] The fifth object of the present invention is to provide a method for preparing the aforementioned tetravalent bispecific antibody.

[0014] A sixth object of the present invention is to provide a pharmaceutical composition comprising the aforementioned tetravalent bispecific antibody.

[0015] A seventh object of the present invention is to provide the use of the said tetravalent bispecific antibody or the said pharmaceutical composition in the preparation of a medicament for treating cancer.

[0016] An eighth object of the present invention is to provide a method for treating cancer using the aforementioned tetravalent bispecific antibody or the aforementioned pharmaceutical composition. To achieve the above object, the present invention provides the following technical solution:

[0017] A first aspect of the present invention provides a tetravalent bispecific antibody against PD-L1 and EGFR, comprising:

[0018] (a) Two polypeptide chains, wherein the polypeptide chains contain VH-PDL1-CH1-linker-VH-EGFR-CH1-CH2-CH3 or VH-EGFR-CH1-linker-VH-PDL1-CH1-CH2-CH3 from the N-terminus to the C-terminus, wherein VH-PDL1 is a heavy chain variable region binding PD-L1, VH-EGFR is a heavy chain variable region binding EGFR, CH1 is a first domain of a heavy chain constant region, CH2 is a second domain of a heavy chain constant region, and CH3 is a third domain of a heavy chain constant region; and

[0019] (b) Four common light chains, wherein the common light chains contain VL-CL from the N-terminus to the C-terminus, wherein VL is a variable region of the light chain and CL is a constant region of the light chain, wherein VH-PDL1-CH1 and VH-EGFR-CH1 of the polypeptide chain are respectively paired with VL-CL of the common light chain, wherein VH-PDL1 and VL form a PD-L1 antigen binding site, and VH-EGFR and VL form an EGFR binding site;

[0020] Each of the common light chains has an amino acid sequence as shown in SEQ ID NO: 7.

[0021] In another preferred embodiment, the CH1 region of the heavy chain is selected from the CH1 domain of human IgG1 or the CH1 domain of human IgG4.

[0022] In another preferred embodiment, the heavy chain variable region VH-PDL1 combined with PD-L1 is as shown in SEQ ID No:1;

[0023] The heavy chain variable region VH-EGFR that combines with EGFR is shown in SEQ ID No:9.

[0024] In another preferred embodiment, each of the said polypeptide chains has the same amino acid sequence.

[0025] A second aspect of the present invention provides a tetravalent bispecific antibody against PD-L1 and EGFR, comprising two polypeptide chains and four common light chains, wherein the polypeptide chains have amino acid sequences as shown in SEQ ID NO: 12 or SEQ ID NO: 14, and the common light chains have amino acid sequences as shown in SEQ ID NO: 7.

[0026] A third aspect of the invention provides an isolated nucleotide encoding the tetravalent bispecific antibody.

[0027] In another preferred embodiment, the nucleotides encode the polypeptide chain and the common light chain, wherein the nucleotide sequence encoding the polypeptide chain is as shown in SEQ ID NO: 13 or SEQ ID NO: 15, and the nucleotide sequence encoding the common light chain is as shown in SEQ ID NO: 8.

[0028] A fourth aspect of the present invention provides an expression vector containing the nucleotides described above.

[0029] A fifth aspect of the present invention provides a host cell containing the expression vector described above.

[0030] A sixth aspect of the present invention provides a method for preparing the aforementioned tetravalent bispecific antibody, the method comprising the following steps:

[0031] (a) Under expression conditions, host cells as described above are cultured to express the tetravalent bispecific antibody;

[0032] (b) Isolate and purify the tetravalent bispecific antibody described in (a).

[0033] A seventh aspect of the invention provides a pharmaceutical composition comprising a tetravalent bispecific antibody as described above and a pharmaceutically acceptable carrier. In another preferred embodiment, the pharmaceutical composition further comprises an antitumor agent.

[0034] In another preferred embodiment, the pharmaceutical composition further contains an antitumor agent.

[0035] In another preferred embodiment, the pharmaceutical composition is a unit dosage form.

[0036] In another preferred embodiment, the antitumor agent may be present separately from the bispecific antibody in a separate package, or the antitumor agent may be conjugated to the bispecific antibody.

[0037] In another preferred embodiment, the dosage form of the pharmaceutical composition includes a gastrointestinal dosage form or a parenteral dosage form.

[0038] In another preferred embodiment, the parenteral dosage form includes intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavitary injection.

[0039] The eighth aspect of the invention provides the use of the tetravalent bispecific antibody as described above, or an immunoconjugate thereof, or a pharmaceutical composition as described above, in the preparation of a medicament for treating cancer.

[0040] In another preferred embodiment, the cancer is selected from the group consisting of: melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma and other proliferative malignant diseases.

[0041] A ninth aspect of the present invention provides a method for treating cancer, comprising administering to a subject in need the quadrivalent bispecific antibody as described above, or an immunoconjugate thereof, or a pharmaceutical composition as described above.

[0042] In another preferred embodiment, the cancer is selected from the group consisting of: melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma and other proliferative malignant diseases.

[0043] A tenth aspect of the present invention provides an immunoconjugate comprising:

[0044] (a) a tetravalent bispecific antibody as described in the first aspect of the present invention; and

[0045] (b) The coupling part selected from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

[0046] In another preferred embodiment, the conjugate is partially selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.) capable of producing detectable products.

[0047] In another preferred embodiment, the immunoconjugate includes an antibody-drug conjugate (ADC).

[0048] In another preferred embodiment, the immunoconjugate is used to prepare a pharmaceutical composition for treating tumors.

[0049] Beneficial effects: This invention provides a tetravalent bispecific antibody against PD-L1 and EGFR. The tetravalent bispecific antibody of this invention does not require Fc modification, avoids mismatch problems, has a simple preparation method, and possesses biological activity and physicochemical properties similar to or even superior to monoclonal antibodies. Attached Figure Description

[0050] Figure 1This is a schematic diagram of the structure of the bispecific antibody of the present invention. VH-A represents the heavy chain variable region of Anti-PDL1 or Cetuximab, VH-B represents the heavy chain variable region of Cetuximab or Anti-PDL1, VL represents the light chain variable region of the common light chain, CH1, CH2, and CH3 are the three domains of the heavy chain constant region, CL is the light chain constant region of the common light chain, the line segment between two heavy chains represents a disulfide bond, and the line segment between the heavy chain and the light chain also represents a disulfide bond. The line segment between CH1 and VH-A near the N-terminus of the polypeptide chain represents an artificially designed linker, and the line segment between CH1 and CH2 near the C-terminus of the polypeptide chain represents the antibody's native linker and hinge region (if the heavy chain is the human IgG4 subtype, the hinge region will contain the S228P point mutation, according to EU coding).

[0051] Figure 2 shows the ELISA results of Cetuximab, Anti-PDL1, and their hybrid antibodies; among them, Figure 2A and Figure 2B The microplates were coated with PD-L1-His and EGFR-ECD-hFc, respectively.

[0052] Figure 3 shows the ELISA results for PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1; among them, Figure 3A and Figure 3B The microplates were coated with PD-L1-His and EGFR-ECD-hFc, respectively.

[0053] Figure 4 To evaluate the functional activities of PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 in inhibiting the proliferation of A431 cells.

[0054] Figure 5 shows the HPLC-SEC chromatograms of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1; among them, Figure 5A This represents the HPLC-SEC chromatogram of Anti-PDL1. Figure 5B This is an HPLC-SEC chromatogram of PDL1-Fab-Cetuximab-IgG1.

[0055] Figure 6 shows the NR-CE-SDS and R-CE-SDS spectra of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1; among them... Figure 6A and Figure 6B These represent the NR-CE-SDS and R-CE-SDS spectra of Anti-PDL1, respectively. Figure 6C and Figure 6DThe NR-CE-SDS and R-CE-SDS spectra of PDL1-Fab-Cetuximab-IgG1 are shown respectively.

[0056] Figure 7 shows the results evaluating the ability of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 to enhance MLR; among them, Figure 7A This indicates the results of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 stimulating MLR to secrete IL-2. Figure 7B This indicates the results of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 stimulating MLR to secrete IFN-γ. Detailed Implementation

[0057] Through extensive and in-depth research and numerous screenings, the inventors successfully obtained a tetravalent bispecific antibody against PD-L1 and EGFR. Specifically, the bispecific antibody of this invention possesses a "common light chain," which includes the variable region of the light chain of the anti-PD-L1 antibody and the constant region of the human Kappa chain. The inventors unexpectedly discovered that the hybrid antibody formed by the "common light chain" and the heavy chain of the Cetuximab antibody can effectively bind to EGFR-ECD-hFc. Furthermore, the bispecific antibody of this invention can effectively inhibit the proliferation of human epidermal cancer cells A431 and exhibits similar or even superior biological activity and physicochemical properties to monoclonal antibodies. Based on these findings, this invention was completed.

[0058] The sequence information involved in this invention is summarized in Table 1.

[0059] Table 1. Sequence information of the antibodies of the present invention

[0060] SEQ ID NO: sequence name 1 The amino acid sequence of the heavy chain variable region of Anti-PDL1 2 The amino acid sequence of the light chain variable region of Anti-PDL1 3 Amino acid sequence of the constant region of human IgG1 heavy chain 4 The amino acid sequence of the heavy chain of Anti-PDL1 5 Nucleotide sequence of the heavy chain of Anti-PDL1 6 Amino acid sequence of the constant region of the human Kappa light chain 7 The amino acid sequence of the light chain of Anti-PDL1 8 The nucleotide sequence of the light chain of Anti-PDL1 9 The amino acid sequence of the heavy chain variable region of Cetuximab 10 The amino acid sequence of the light chain variable region of Cetuximab 11 Connector (GGGGSGGGGSGGGGS) 12 The amino acid sequence of PDL1-Fab-Cetuximab-IgG1 13 The nucleotide sequence of PDL1-Fab-Cetuximab-IgG1 14 The amino acid sequence of Cetuximab-Fab-PDL1-IgG1 15 Nucleotide sequence of Cetuximab-Fab-PDL1-IgG1

[0061] the term

[0062] In this invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" refer to heterotetraglycoproteins with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region, which consists of three domains: CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the light chain constant region including a domain CL; the light chain constant region pairs with the CH1 domain of the heavy chain constant region, and the light chain variable region pairs with the heavy chain variable region. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). Heavy chain constant regions include IgG1, IgG2, IgG3, and IgG4 isotypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of an antibody are covalently linked by interpeptide disulfide bonds formed between their hinge regions.

[0063] In this invention, the term "bispecific antibody (or bispecific antibody)" refers to an antibody molecule that can simultaneously and specifically bind to two antigens (targets) or two epitopes. Based on symmetry, bispecific antibodies can be classified into structurally symmetrical and asymmetrical molecules. Based on the number of binding sites, bispecific antibodies can be classified into bivalent, trivalent, tetravalent, and multivalent molecules.

[0064] In this invention, the term "monoclonal antibody (MABS)" refers to an antibody obtained from a substantially homogeneous population, meaning that the individual antibodies in this population are identical, except for a few possible naturally occurring mutations. Monoclonal antibodies target a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody formulations (which are typically mixtures of different antibodies targeting different antigenic determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized through hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring any special method to produce the antibody.

[0065] In this invention, the term "humanized" refers to a antibody whose CDR is derived from a non-human species (preferably mouse) antibody, and whose residual portions (including the frame region and constant region) are derived from human antibodies. Furthermore, the frame region residues can be modified to maintain binding affinity.

[0066] In this invention, the terms "Fab" and "Fc" refer to the ability of papain to cleave an antibody into two identical Fab fragments and one Fc fragment. The Fab fragment consists of the VH and CH1 domains of the antibody's heavy chain and the VL and CL domains of its light chain. The Fc fragment, or crystallizable fragment, consists of the antibody's CH2 and CH3 domains. The Fc fragment lacks antigen-binding activity and is the site of interaction between the antibody and effector molecules or cells.

[0067] In this invention, the term "variable" refers to the fact that certain portions of the variable region in an antibody differ in sequence, resulting in the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the variable regions of the heavy and light chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable regions are called frame regions (FRs). The variable regions of the natural heavy and light chains each contain four FR regions, which are generally β-sheet configurations, linked by three CDRs forming a linking loop, and in some cases may form a partial β-sheet structure. The CDRs in each chain are closely packed together through the FR regions and together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).

[0068] As used herein, the term "frame region" (FR) refers to the amino acid sequence inserted between CDRs, specifically those portions of the variable regions of the light and heavy chains of immunoglobulins that are relatively conserved among different immunoglobulins within a single species. Each immunoglobulin light and heavy chain has four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain can be represented as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, wherein the derivative of the human antibody FR is substantially identical to the naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0069] Knowing the amino acid sequence of the CDR, those skilled in the art can easily determine the frame regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0070] As used herein, the term “human frame region” is a frame region that is substantially identical (approximately 85% or more, specifically 90%, 95%, 97%, 99%, or 100%) to the frame region of a naturally occurring human antibody.

[0071] As used herein, the term "connector" or "linker" refers to one or more amino acid residues inserted into the immunoglobulin domain that provide sufficient mobility for both the light and heavy chain domains to fold into an exchangeable dual variable region immunoglobulin.

[0072] In one specific embodiment, the linker of the present invention links the heavy chain variable region of Anti-PDL1 to the CH1 domain of human IgG4, and then links the heavy chain variable region of Cetuximab through a linker (preferably an artificial linker, the linker used here being three GGGGS in series, SEQ ID NO: 11).

[0073] Suitable examples of linkers include monoglycine (Gly) or serine (Ser) residues, and the identification and sequence of amino acid residues in the linker can vary depending on the type of secondary structural element that needs to be achieved in the linker.

[0074] Bispecific antibodies

[0075] The bispecific antibody of the present invention is a tetravalent bispecific antibody against PD-L1 and EGFR, comprising an anti-PD-L1 antibody portion and an anti-EGFR antibody portion.

[0076] Preferably, the sequence of the anti-PD-L1 antibody of the present invention is as described in patent application PCT / CN2020 / 090442. Those skilled in the art can also modify or alter the anti-PD-L1 antibody of the present invention using techniques well known in the art, such as adding, deleting, and / or substituting one or more amino acid residues, thereby further increasing the affinity or structural stability of anti-PD-L1, and obtaining the modified or altered results by conventional assay methods.

[0077] In this invention, the terms "antibody," "binding," and "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. Typically, antibodies bind at a rate of less than approximately 10... -7 M, for example, less than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The antibody binds to the antigen with an equilibrium dissociation constant (KD) of M or smaller. In this invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity between the antibody and the antigen can be determined using surface plasmon resonance (SPR) in a BIACORE instrument or using ELISA to determine the relative affinity of antibody-antigen binding.

[0078] In this invention, the term "valence" refers to the presence of a specified number of antigen-binding sites in an antibody molecule. Preferably, the bispecific antibody of this invention has four antigen-binding sites and is tetravalent. In this invention, the antigen-binding sites comprise a heavy chain variable region (VH) and a light chain variable region (VL).

[0079] In this invention, the term "epitope" refers to a polypeptide determinant cluster that specifically binds to an antibody. The epitope of this invention is a region of an antigen that is bound by an antibody. In this invention, the term "common light chain" refers to a light chain containing the same light chain variable region and light chain constant region, which can pair with the heavy chain of a first antibody binding to a first antigen to form a binding site specifically binding to the first antigen, and can also pair with the heavy chain of a second antibody binding to a second antigen to form a binding site specifically binding to the second antigen. Further, the light chain variable region of the common light chain and the heavy chain variable region of the first antibody form a first antigen binding site, and the light chain variable region of the common light chain and the heavy chain variable region of the second antibody form a second antigen binding site.

[0080] The bispecific antibody of the present invention can be used alone or in combination or conjugated with a detectable marker (for diagnostic purposes), a therapeutic agent, or any combination of the above substances.

[0081] Nucleic acid encoding and expression vector

[0082] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0083] In this invention, the term "expression vector" refers to a vector carrying an expression cassette for expressing a specific target protein or other substance, such as plasmids, viral vectors (e.g., adenovirus, retrovirus), bacteriophages, yeast plasmids, or other vectors. Representative examples include, but are not limited to, pTT5, pSECtag series, pCGS3 series, pcDNA series vectors, and other vectors used in mammalian expression systems. The expression vector includes a fusion DNA sequence linked to suitable transcriptional and translational regulatory sequences.

[0084] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0085] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0086] In this invention, the term "host cell" refers to a cell suitable for expressing the above-mentioned expression vector. It can be a eukaryotic cell, such as a mammalian or insect host cell culture system, which can be used for the expression of the fusion protein of this invention. CHO (Chinese Hamster Ovary), HEK293, COS, BHK, and derived cells of the above cells can all be used in this invention.

[0087] Pharmaceutical Compositions and Applications

[0088] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the above-described antibody or its active fragment or fusion protein, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavitary injection.

[0089] In this invention, the term "pharmaceutical composition" refers to the fact that the tetravalent bispecific antibody of this invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical formulation composition, thereby exerting its therapeutic effect more stably. These formulations can ensure the conformational integrity of the amino acid core sequence of the antibody that binds to human PD-L1 disclosed in this invention or its antigen-binding fragment or the tetravalent bispecific antibody, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation).

[0090] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described tetravalent bispecific antibody (or its conjugates) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the tetravalent bispecific antibody of the present invention can also be used with other therapeutic agents.

[0091] When using a pharmaceutical composition, a safe and effective amount of the tetravalent bispecific antibody or its immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0092] The protein expression and purification methods used in the following examples are described below: The target gene was constructed into the expression vector pcDNA4, and the constructed expression vector or a combination of expression vectors was transformed into FreeStyle using PEI (Polyethylenimine). TM To express antibodies or recombinant proteins, HEK293F cells (hereinafter referred to as HEK293F, purchased from Thermo Fisher Scientific) were cultured in Free Style 293 Expression Medium (purchased from Thermo Fisher Scientific) for 5 days. The cell supernatant was then collected and purified by Protein A affinity chromatography or nickel affinity chromatography.

[0093] The physicochemical property detection methods used in the following examples are described below:

[0094] HPLC-SEC

[0095] Antibodies are high-molecular-weight proteins with highly complex secondary and tertiary structures. Due to post-translational modifications, aggregation, and degradation, antibodies are heterogeneous in their biochemical and biophysical properties. When analyzing bispecific antibodies using separation techniques, variants, aggregates, and degradation fragments are commonly observed, and their presence may compromise safety and efficacy. Aggregates, degradation fragments, and incompletely assembled molecules are prone to occur during antibody production and storage. This invention uses high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) to detect the content of these impurities in samples. Aggregates have a larger molecular weight than monomers, resulting in shorter retention times for their corresponding peaks; degradation fragments or incompletely assembled molecules have smaller molecular weights than monomers, resulting in longer retention times for their corresponding peaks. The HPLC-SEC instrument used was a Dionex Ultimate 3000; the mobile phase was prepared as follows: take an appropriate amount of 20mM sodium dihydrogen phosphate stock solution and adjust the pH to 6.8±0.1 with 20mM disodium hydrogen phosphate; injection volume: 20μg; the column was a TSK G3000SWXL, with dimensions of 7.8×300mm 5μm; the flow rate was 0.5ml / min, the elution time was 30min; the column temperature was 25℃, the sample chamber temperature was 10℃; and the detection wavelength was 214nm.

[0096] CE-SDS

[0097] This invention uses CE-SDS (Capillary Electrophoresis-Sodium Dodecyl Sulfate) to analyze the content of degraded fragments or incompletely assembled molecules in samples. CE is divided into two types: non-reducing and reducing. Samples used for the former do not require the use of the reducing agent DTT to break the disulfide bonds within the molecules during denaturation, while samples used for the latter require the use of the reducing agent DTT to break the disulfide bonds within the molecules during denaturation. Non-reducing and reducing CE-SDS are denoted as NR-CE-SDS and R-CE-SDS, respectively. The capillary electrophoresis apparatus used is ProteomeLab. TM The PA800 plus (Beckman Coulter) instrument is equipped with a UV 214nm detector and a Bare Fused-Silica Capillary measuring 30.7cm × 50μm with an effective length of 20.5cm. Other relevant reagents were purchased from Beckman Coulter. Key instrument parameters were set as follows: capillary and sample chamber temperature 20±2℃, separation voltage 15kV.

[0098] The following examples and experimental cases are further illustrative of the invention and should not be construed as limiting the invention. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) *Molecular Cloning: A Laboratory Manual*, 2nd edition, Cold Spring Harbor Laboratory Press. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0099] Example 1: Construction of a bispecific antibody against PD-L1 and EGFR

[0100] Example 1.1 Sequence

[0101] Anti-PDL1 is a humanized monoclonal antibody against human PD-L1. Its heavy chain variable region and light chain variable region sequences (SEQ ID NO: 1 and 2) are derived from PCT / CN2020 / 090442. The synthesized humanized heavy chain variable region was linked to the human IgG1 heavy chain constant region (SEQ ID NO: 3) to obtain the full-length humanized heavy chain gene, named Anti-PDL1-HC (SEQ ID NO: 4 and 5); the humanized light chain variable region was linked to the human Kappa chain constant region (SEQ ID NO: 6) to obtain the full-length humanized light chain gene, named Anti-PDL1-LC (SEQ ID NO: 7 and 8).

[0102] The heavy chain and light chain variable region sequences (SEQ ID NO: 9 and 10) of the Cetuximab antibody were obtained from publicly available literature (Magdelaine-Beuzelin C, Kaas Q, Wehbi V, et al. Structure–function relationships of the variable domains of monoclonal antibodies approved for cancer treatment[J]. Critical reviews in oncology / hematology, 2007, 64(3):210-225.). DNA encoding the heavy chain variable region (Cetuximab-VH) and light chain variable region (Cetuximab-VL) was synthesized by Shanghai Sangon Biotech Co., Ltd. Cetuximab-VH and Cetuximab-VL were linked to the human IgG1 heavy chain constant region (SEQ ID NO: 3) and the human Kappa light chain constant region (SEQ ID NO: 6), respectively, to construct the full-length heavy chain and light chain genes of the Cetuximab antibody, named Cetuximab-HC and Cetuximab-LC, respectively.

[0103] The amino acid sequence of the heavy chain variable region of Anti-PDL1 (SEQ ID NO: 1)

[0104]

[0105] The amino acid sequence of the light chain variable region of Anti-PDL1 (SEQ ID NO: 2)

[0106]

[0107] Amino acid sequence of the constant region of the human IgG1 heavy chain (SEQ ID NO: 3)

[0108]

[0109]

[0110] The amino acid sequence of the heavy chain of Anti-PDL1 (SEQ ID NO: 4)

[0111]

[0112] Amino acid sequence of the constant region of the human Kappa light chain (SEQ ID NO: 6)

[0113]

[0114] The amino acid sequence of the light chain of Anti-PDL1 (SEQ ID NO: 7)

[0115]

[0116] The amino acid sequence of the heavy chain variable region of Cetuximab (SEQ ID NO: 9)

[0117]

[0118] The amino acid sequence of the light chain variable region of Cetuximab (SEQ ID NO: 10)

[0119]

[0120] Example 1.2 Selection of Common Light Chain

[0121] Comparative analysis of the amino acid sequences of the Anti-PDL1 light chain variable region and the Cetuximab light chain variable region using BLAST showed that 75% of the amino acids were identical (identities), and 89% of the amino acids had similar properties (positives).

[0122] The gene sequences of Cetuximab-HC and Cetuximab-LC were constructed into pcDNA4 expression vectors, respectively. The expression vectors of Anti-PDL1-HC, Anti-PDL1-LC, Cetuximab-HC, and Cetuximab-LC were combined in the following ways: Anti-PDL1-HC+Anti-PDL1-LC, Cetuximab-HC+Cetuximab-LC, Anti-PDL1-HC+Cetuximab-LC, and Cetuximab-HC+Anti-PDL1-LC. Antibodies were expressed and purified, and the resulting antibodies were named Anti-PDL1, Cetuximab, Anti-PDL1-HC+Cetuximab-LC, and Cetuximab-HC+Anti-PDL1-LC, respectively.

[0123] The extracellular region coding gene of PD-L1 was obtained as described in WO2018 / 137576A1. Using recombination technology, a polyhistidine coding sequence was ligated to the end of the extracellular region coding gene of PD-L1. The recombinant gene was then cloned into the pcDNA4 expression vector, expressed, and purified. The resulting recombinant protein was named PD-L1-His. Using recombination technology, the Fc coding sequence of human IgG1 was ligated to the end of the extracellular region coding gene of human EGFR (sequence from NCBI, Accession: NP_005219). The recombinant gene was then cloned into the pcDNA4 expression vector, expressed, and purified. The resulting recombinant protein was named EGFR-ECD-hFc. ELISA plates were coated with EGFR-ECD-hFc and PD-L1-His at concentrations of 40 ng / well and 10 ng / well, respectively. The ELISA plate was blocked with PBST containing 1% bovine serum albumin (KH2PO4 0.2g, Na2HPO4·12H2O 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20 0.5ml, and pure water to 1L). The antibody to be tested was serially diluted and then transferred to the ELISA plate coated with the recombinant protein. After incubation at room temperature for half an hour, the plate was washed. Appropriately diluted HRP (Horseradish Peroxidase)-labeled goat anti-human antibody (Fab-specific, purchased from Sigma) was added, and after incubation at room temperature for half an hour, the plate was washed. 100 μl of TMB-based chromogenic buffer was added to each well (Substrate A: 13.6 g sodium acetate trihydrate, 1.6 g citric acid monohydrate, 0.3 ml 30% hydrogen peroxide, 500 ml pure water; Substrate B: 0.2 g disodium EDTA, 0.95 g citric acid monohydrate, 50 ml glycerol, 0.15 g TMB dissolved in 3 ml DMSO, 500 ml pure water; equal volumes of solutions A and B were mixed before use). The plate was incubated at room temperature for 1–5 min. 50 μl of stop solution (2 M H2SO4) was added to terminate the reaction. The plate was then readjusted using a SpectraMax ELISA reader. 190) Read OD450, use GraphPad Prism6 for plotting and data analysis, and calculate EC50.

[0124] like Figure 2A As shown, Anti-PDL1 can effectively bind PD-L1-His, with an EC50 of 0.0924 nM; while Cetuximab, Anti-PDL1-HC+Cetuximab-LC, and Cetuximab-HC+Anti-PDL1-LC cannot bind PD-L1-His. Figure 2BAs shown, both Cetuximab and Cetuximab-HC+Anti-PDL1-LC effectively bind to EGFR-ECD-hFc, with EC50 values ​​of 0.2096 nM and 0.2484 nM, respectively, while Anti-PDL1 and Anti-PDL1-HC+Cetuximab-LC do not effectively bind to EGFR-ECD-hFc. Therefore, Anti-PDL1-LC (SEQ ID NO: 7 and 8) was selected as the common light chain for constructing the bispecific antibody.

[0125] Example 1.3 Construction of bispecific antibodies

[0126] The heavy chain variable region of Anti-PDL1 was linked to the CH1 domain of human IgG4, and then the heavy chain variable region of Cetuximab was linked through an artificial linker (the linker used here is three tandem GGGGS, SEQ ID NO: 11). Finally, the heavy chain constant region (CH1+CH2+CH3) of human IgG1 was linked. The long heavy chain gene containing two heavy chain variable regions and two CH1 domains constructed by this procedure was named PDL1-Fab-Cetuximab-IgG1 (SEQ ID NO: 12 and 13). Similarly, the heavy chain variable region of Cetuximab was linked to the CH1 domain of human IgG4, and then the heavy chain variable region of Anti-PDL1 was linked through an artificial linker (the linker used here is three tandem GGGGS, SEQ ID NO: 11). Finally, the heavy chain constant region (CH1+CH2+CH3) of human IgG1 was linked. The long heavy chain gene containing two heavy chain variable regions and two CH1 domains constructed by this procedure was named Cetuximab-Fab-PDL1-IgG1 (SEQ ID NO: 14 and 15).

[0127] The above sequences were constructed into the pcDNA4 expression vector. The PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 expression vectors were combined with the Anti-PDL1-LC expression vector to express and purify the antibodies. The resulting antibodies were named PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1, respectively (for simplicity, only the name of the heavy chain is used as the name of the antibody here).

[0128] The amino acid sequence of PDL1-Fab-Cetuximab-IgG1 is shown below (SEQ ID NO: 12):

[0129]

[0130] The amino acid sequence of Cetuximab-Fab-PDL1-IgG1 is shown below (SEQ ID NO: 14):

[0131]

[0132] Example 2: ELISA determination of relative affinity

[0133] The ELISA detection method is as described in Example 1.2.

[0134] like Figure 3A As shown, Anti-PDL1, PDL1-Fab-Cetuximab-IgG1, and Cetuximab-Fab-PDL1-IgG1 can all effectively bind to PD-L1-His, with EC50 values ​​of 0.2177 nM, 0.2003 nM, and 0.3356 nM, respectively. Figure 3B As shown, Cetuximab-HC+Anti-PDL1-LC, PDL1-Fab-Cetuximab-IgG1, and Cetuximab-Fab-PDL1-IgG1 can all effectively bind to EGFR-ECD-hFc, with EC50 values ​​of 0.2253 nM, 0.2388 nM, and 0.1852 nM, respectively. These results indicate that PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 can bind to both PD-L1 and EGFR, suggesting they are bispecific antibodies.

[0135] Example 3: Evaluation of the functional activity in inhibiting A431 cell proliferation

[0136] A431( CRL-1555 TM A431 is a human epidermal cancer cell line that overexpresses wild-type EGFR. Anti-EGFR antibodies can inhibit the proliferation of A431 cells both in vitro and in vivo.

[0137] This embodiment evaluates the functional activity of the above-mentioned antibody in inhibiting the proliferation of A431 cells. The method is as follows: A431 cells in the logarithmic growth phase were washed twice with DMEM and centrifuged at 1000 rpm for 5 min; the cells were resuspended at an appropriate density with DMEM containing 1% fetal bovine serum (fetal bovine serum and DMEM were purchased from Gibco), and seeded into 96-well plates. 4150 μl / well; then serially dilute the above antibody in DMEM containing 1% fetal bovine serum; add 50 μl / well to the above-mentioned 96-well plate inoculated with A431 cells; incubate at 37°C and 5% CO2 for 3 days; after 3 days, add 20 μl of CCK-8 (purchased from Dojindo) solution to each well and continue incubation in the incubator for 4 hours; read OD450 using a microplate reader; perform data analysis, plot and calculate IC50 using GraphPad Prism6.

[0138] like Figure 4 As shown, Cetuximab, Cetuximab-HC+Anti-PDL1-LC, PDL1-Fab-Cetuximab-IgG1, and Cetuximab-Fab-PDL1-IgG1 all effectively inhibited the proliferation of A431 cells, with IC50 values ​​of 0.6322 nM, 0.5629 nM, 0.7094 nM, and 0.9597 nM, respectively.

[0139] Example 4: Biacore determination of affinity

[0140] The affinity between the aforementioned antibodies and PD-L1 or EGFR was detected using a Biacore 8K (GE Healthcare) microarray. Various antibodies were captured on the Biacore 8K using a chip coupled with Protein A / G. Recombinant proteins PD-L1-His (self-made) or EGFR-His (His-tagged EGFR recombinant protein, purchased from Beijing Sinocare) were then injected, and binding-dissociation curves were obtained. The samples were eluted with 6M guanidine hydrochloride regeneration buffer, and the cycle was repeated. The data were analyzed using Biacore 8K Evaluation Software. The results are shown in Table 2.

[0141] Table 2-1. Binding and dissociation kinetic parameters and equilibrium dissociation constants of PD-L1

[0142]

[0143] Table 2-2. Binding and dissociation kinetic parameters and equilibrium dissociation constants of EGFR

[0144]

[0145] Table 2-1 shows that the binding constant (Kon) and dissociation constant (Koff) of Anti-PDL1, PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 to PD-L1 are very similar, and their equilibrium dissociation constants (KD) are also basically the same, with KD of 9.66E-10, 6.46E-10 and 7.79E-10, respectively. Table 2-2 shows that the binding constants (Kon) and dissociation constants (Koff) of Cetuximab, Cetuximab-HC+Anti-PDL1-LC, and Cetuximab-Fab-PDL1-IgG1 for EGFR are very similar, and their equilibrium dissociation constants (KD) are also basically the same, at 6.14E-10, 9.46E-10, and 9.57E-10, respectively. Compared with the first three, the equilibrium dissociation constant (KD) of PDL1-Fab-Cetuximab-IgG1 for EGFR is slightly larger, at 14.2E-10. The equilibrium dissociation constant (KD) is inversely proportional to the affinity.

[0146] Example 5 Characterization of physicochemical properties

[0147] 5.1 HPLC-SEC

[0148] Figure 5A The HPLC-SEC chromatogram of Anti-PDL1 shows two distinct peaks, Peak1 and Peak2, accounting for 0.2% and 99.8% (main peaks), respectively. Figure 5B The HPLC-SEC chromatogram of PDL1-Fab-Cetuximab-IgG1 shows three distinct peaks: Peak1, Peak2, and Peak3, accounting for 0.3%, 99.5% (main peak), and 0.2%, respectively. The main peak percentages of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 are similar.

[0149] 5.2 CE-SDS

[0150] Figure 6A and Figure 6B These represent the NR-CE-SDS and R-CE-SDS spectra of Anti-PDL1, respectively. Figure 6C and Figure 6DThe figures represent the NR-CE-SDS and R-CE-SDS spectra of PDL1-Fab-Cetuximab-IgG1, respectively. For Anti-PDL1, the main NR-CE-SDS peak, Peak8, accounts for 98.11%, while for PDL1-Fab-Cetuximab-IgG1, Peak9 accounts for 97.14%. For Anti-PDL1, the main R-CE-SDS peaks, Peak5 (corresponding to the light chain) and Peak10 (corresponding to the heavy chain), account for 32.62% and 63.55% respectively, with a peak area ratio of 1:1.95 and a combined peak area percentage of 96.17%. For PDL1-Fab-Cetuximab-IgG1, the main R-CE-SDS peaks, Peak4 (corresponding to the light chain) and Peak11 (corresponding to the heavy chain), account for 38.27% and 57.37% respectively, with a peak area ratio of 2:3.0 and a combined peak area percentage of 95.64%. In NR-CE-SDS, the peak proportions of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 are very similar; in R-CE-SDS, the peak area ratios of the light and heavy chains of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 are as expected, and the sum of their peak proportions is very similar.

[0151] Example 6: Evaluating the ability to enhance MLR

[0152] The Mixed Lymphocyte Reaction (MLR) method used in this embodiment is described as follows: Peripheral Blood Mononuclear Cells (PBMCs) were isolated from human blood using Histopaque (purchased from Sigma). The PBMCs were then separated using an adherent method, and monocytes were induced to differentiate into dendritic cells using IL-4 (25 ng / ml) and GM-CSF (25 ng / ml). Seven days later, the induced dendritic cells were digested and collected. PBMCs were then isolated from the blood of another donor using the same method, and CD4+ was isolated from the PBMCs using a MACS magnet and CD4 MicroBeads (purchased from Miltenyi Biotec). + T cells. Induced dendritic cells (10 4 / hole) and separated CD4 + T cells (10) 5After mixing the antibody in the specified proportions, 150 μl was seeded into each well of a 96-well plate. Several hours later, 50 μl of serially diluted antibody was added to each well. The 96-well plate was then incubated at 37°C for 3 days. AIM-V medium (purchased from Thermo Fisher Scientific) was used to culture the cells in the above experiments. The secretion of IL-2 and IFN-γ was then detected according to standard operating procedures. The secretion of IL-2 and IFN-γ was detected using a double-antibody sandwich ELISA (the relevant paired antibodies were purchased from BD Biosciences). OD450 was read using a SpectraMax 190 microplate reader, and graphs were plotted using GraphPad Prism 6 to calculate EC50.

[0153] Results A and B both come from the same MLR experimental sample. For example... Figure 7A As shown, both Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 can effectively stimulate MLR secretion of IL-2, with EC50 values ​​of 0.1578 nM and 0.409 nM, respectively. Additionally, as... Figure 7B As shown, both Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 effectively stimulated MLR secretion of IFN-γ, with EC50 values ​​of 0.1369 nM and 0.08084 nM, respectively. These results indicate that Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 have comparable functional activities. The isotype control antibody was a human IgG1 antibody unrelated to the target.

[0154] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> 3SBio (Shanghai) Co., Ltd. <120> A tetravalent bispecific antibody against PD-L1 and EGFR <130> P2021-1248 <150> 2020104876208 <151> 2020-06-02 <160> 15 <170> PatentIn version 3.5 <210> 1 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the heavy chain variable region of Anti-PDL1 <400> 1 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> 2 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the light chain variable region of Anti-PDL1 <400> 2 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> 3 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the constant region of human IgG1 heavy chain <400> 3 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> 4 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the heavy chain of Anti-PDL1 <400> 4 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> 5 <211> 1341 <212> DNA <213> Artificial Sequence <220> <223> Anti‑PDL1 antibodies have been released <400> 5 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 cctcgcccc 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> 6 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the constant region of human Kappa light chain <400> 6 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> 7 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the light chain of Anti-PDL1 <400> 7 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> 8 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of the light chain of Anti-PDL1 <400> 8 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 ccagggagg 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> 9 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Cetuximab is the most commonly used drug <400> 9 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the light chain variable region of Cetuximab <400> 10 Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 11 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 11 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 12 <211> 679 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of PDL1-Fab-Cetuximab-IgG1 <400> 12 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 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly 225 230 235 240 Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly 245 250 255 Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg Gln Ser Pro Gly 260 265 270 Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp 275 280 285 Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser 290 295 300 Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr 305 310 315 320 Ala Ile Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe 325 330 335 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr 340 345 350 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 355 360 365 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 370 375 380 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 385 390 395 400 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 405 410 415 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 420 425 430 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 435 440 445 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 450 455 460 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 465 470 475 480 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 485 490 495 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 500 505 510 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 515 520 525 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 530 535 540 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 545 550 555 560 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 565 570 575 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 580 585 590 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 595 600 605 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 610 615 620 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 625 630 635 640 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 645 650 655 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 660 665 670 Leu Ser Leu Ser Pro Gly Lys 675 <210> 13 <211> 2037 <212> DNA <213> Artificial Sequence <220> <223> PDL1‐Fab‐Cetuximab‐IgG1 <400> 13 caggtccagc tgcagcagtc aggagggggc ctggtgaagc catcacagag cctgtccctg 60 acctgcacag tctctgggtt cagtctgact tcatacggag tgcactgggt ccgacagcc 120 cctggaaagg gactggagtg gatcggcctg atttggtctg gcgggggaac agactataac 180 ccagcctga aatcccggct gaccatctct agagatacca gtaagaatca agtgagcttt 240 aaaattagct ccctgacagc cgctgacact gcagtgtact attgtgcaag gcagctggga 300 ctgcgagcta tggattactg gggacagggc acttccgtga ccgtctctag tgcaagtacc 360 aagggaccta gtgttttccc tcttgcacct tgctccaggt caacatcaga gtccacagct 420 gctcttggat gtctcgttaa ggactacttc ccagagccag ttaccgtatc ctggaactcc 480 ggagctttga caagcggcgt tcatacattc ccagctgtgt tgcagagttc tgggttgtac 540 agtttgagct cagtggtgac cgtgccttca tcttctttgg gcactaagac ctacacctgc 600 aacgtggatc acaagccaag caacaccaag gtggataaga gggtgggtgg aggcggttca 660 ggcggaggtg gcagcggagg tggcgggagt caggtgcagc tgaagcagtc cggacctggc 720 ctggtgcagc cttcccagtc cctgtccatc acctgcaccg tgtccggctt ttccctgacc 780 aactacggcg tgcactgggt gaggcagtcc cctggcaagg gcctggaatg gctgggcgtg 840 atctggtccg gcggcaacac cgactacaac acccccttca cctcccggct gtccatcaac 900 aaggacaaca gcaagtccca ggtgttcttc aagatgaact ccctgcagag caacgacacc 960 gccatctact actgcgccag agccctgacc tattacgact acgagttcgc ctactggggc 1020 cagggcacac tggtgaccgt gtccgccgcg agcaccaagg gaccttccgt gtttcccctc 1080 gcccccagct ccaaaagcac cagcggcgga acagctgctc tcggctgtct cgtcaaggat 1140 tacttccccg agcccgtgac cgtgagctgg aacagcggag ccctgacaag cggcgtccac 1200 accttccctg ctgtcctaca gtcctccgga ctgtacagcc tgagcagcgt ggtgacagtc 1260 cctagcagct ccctgggcac ccagacatat atttgcacg tgaatcaca gcccagcaac 1320 accaaggtcg ataagaggt ggagcctaag tcctgcgaca agacccacac atgtcccccc 1380 tgtcccgctc ctgactgct gggaggccct tccgtgttcc tgttcccccc taagcccaag 1440 gandaccctga tgatttccag gandacccgag gtgacctgtg tggtggtgga cgtcagccac 1500 gaggaccccg aggtgaatt caacgtac gtcgatggcg tggaggtgca caacgctaag 1560 accaagcccca gggaggagca gtacaatcc acctacaggg tggtgtccgt gctgaccgtc 1620 ctccatcagg actggctgaa cggcaagag tataagtgca aggtgagcaa caggccctc 1680 cctgctccca tcgagagac catcagcaaa gccaagggcc agcccaggga acccaagtc 1740 tataccctgc ctcccagcag ggaggagat accagaacc aagtgagcct cacatgccctc 1800 gtcaagggct tctatccttc cgatattgcc gtcgagtggg agtccaacgg acagcccgag 1860 aacaactaca agacacacc cccgtgctc gattccgatg gcagctctt cctgtactcc 1920 aagctgaccg tggacaagtc cagatggcaa caaggcaacg tcttcagttg cagcgtcatg 1980 catgaggccc tccacaacca ctacacccag aagagcctct ccctgagccc tggaaag 2037 <210> 14 <211> 679 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Cetuximab‑Fab‑PDL1‑IgG1 <400> 14 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser Gly Gly 210 215 220 Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly 225 230 235 240 Gly Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val 245 250 255 Ser Gly Phe Ser Leu Thr Ser Tyr Gly Val His Trp Val Arg Gln Pro 260 265 270 Pro Gly Lys Gly Leu Glu Trp Ile Gly Leu Ile Trp Ser Gly Gly Gly 275 280 285 Thr Asp Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Arg Asp 290 295 300 Thr Ser Lys Asn Gln Val Ser Phe Lys Ile Ser Ser Leu Thr Ala Ala 305 310 315 320 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Gly Leu Arg Ala Met 325 330 335 Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr 340 345 350 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 355 360 365 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 370 375 380 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 385 390 395 400 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 405 410 415 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 420 425 430 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 435 440 445 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 450 455 460 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 465 470 475 480 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 485 490 495 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 500 505 510 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 515 520 525 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 530 535 540 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 545 550 555 560 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 565,570,575 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 580,585,590 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 595,600,605 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 610 615 620 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 625,630,635,640 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 645,650,655 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 660 665 670 Leu Ser Leu Ser Pro Gly Lys 675 <210> 15 <211> 2037 <212> DNA <213> Artificial Sequence <220> <223> Cetuximab‑Fab‑PDL1‑IgG1 <400> 15 caggtgcagc tgaagcagc cggacctggc ctggtgcagc cttcccagtc cctgtccatc 60 acctgcaccg tgtccggctt ttccctgacc aactacggcg tgcactgggt gaggcagtcc 120 cctggcaagg gcctggaatg gctgggcgtg atctggtccg gcggcaacac cgactacaac 180 acccccttca cctcccggct gtccatcaac aaggacaaca gcaagtccca ggtgttcttc 240 aagatgaact ccctgcagag caacgacacc gccatctact actgcgccag agccctgacc 300 tattacgact acgagttcgc ctactggggc cagggcacac tggtgaccgt gtccgccgca 360 agtaccaagg gacctagtgt tttccctctt gcaccttgct ccaggtcaac atcagagtcc 420 acagctgctc ttggatgtct cgttaaggac tacttcccag agccagttac cgtatcctgg 480 aactccggag ctttgacaag cggcgttcat acattcccag ctgtgttgca gagttctggg 540 ttgtacagtt tgagctcagt ggtgaccgtg ccttcatctt ctttgggcac taagacctac 600 acctgcaacg tggatcacaa gccaagcaac accaaggtgg ataagagggt gggtggaggc 660 ggttcaggcg gaggtggcag cggaggtggc gggagtcagg tccagctgca gcagtcagga 720 gggggcctgg tgaagccatc acagagcctg tccctgacct gcacagtctc tgggttcagt 780 ctgacttcat acggagtgca ctgggtccga cagccccctg gaaagggact ggagtggatc 840 ggcctgattt ggtctggcgg gggaacagac tataacccca gcctgaaatc ccggctgacc 900 atctctagag ataccagtaa gaatcaagtg agctttaaaa ttagctccct gacagccgct 960 gacactgcag tgtactattg tgcaaggcag ctgggactgc gagctatgga ttactgggga 1020 cagggcactt ccgtgaccgt ctctagtgcg agcaccaagg gaccttccgt gtttcccctc 1080 gcccccagct ccaaaagcac cagcggcgga acagctgctc tcggctgtct cgtcaaggat 1140 tacttccccg agcccgtgac cgtgagctgg aacagcggag ccctgacaag cggcgtccac 1200 accttccctg ctgtcctaca gtcctccgga ctgtacagcc tgagcagcgt ggtgacagtc 1260 cctagcagct ccctgggcac ccagacatat atttgcaacg tgaatcacaa gcccagcaac 1320 accaaggtcg ataagaaggt ggagcctaag tcctgcgaca agacccacac atgtcccccc 1380 tgtcccgctc ctgaactgct gggaggccct tccgtgttcc tgttcccccc taagcccaag 1440 gacaccctga tgatttccag gacacccgag gtgacctgtg tggtggtgga cgtcagccac 1500 gaggaccccg aggtgaatt caacgtac gtcgatggcg tggaggtgca caacgctaag 1560 accaagcccca gggaggagca gtacaatcc acctacaggg tggtgtccgt gctgaccgtc 1620 ctccatcagg actggctgaa cggcaagag tataagtgca aggtgagcaa caggccctc 1680 cctgctccca tcgagagac catcagcaaa gccaagggcc agcccaggga acccaagtc 1740 tataccctgc ctcccagcag ggaggagat accagaacc aagtgagcct cacatgccctc 1800 gtcaagggct tctatccttc cgatattgcc gtcgagtggg agtccaacgg acagcccgag 1860 aacaactaca agacacacc cccgtgctc gattccgatg gcagctctt cctgtactcc 1920 aagctgaccg tggacaagtc cagatggcaa caggcaacg tctcagttg cagcgtcatg 1980 catgaggcccc tccaacca ctacacccag agagcctct ccctgagcccc tggaag 2037

Claims

1. A tetravalent bispecific antibody against PD-L1 and EGFR, characterized in that, Include: (a) Two polypeptide chains, wherein the polypeptide chains contain VH-PDL1-CH1-linker-VH-EGFR-CH1-CH2-CH3 or VH-EGFR-CH1-linker-VH-PDL1-CH1-CH2-CH3 from the N-terminus to the C-terminus, wherein VH-PDL1 is a heavy chain variable region binding PD-L1, VH-EGFR is a heavy chain variable region binding EGFR, CH1 is a first domain of a heavy chain constant region, CH2 is a second domain of a heavy chain constant region, and CH3 is a third domain of a heavy chain constant region; and (b) Four common light chains, wherein the common light chains contain VL-CL from the N-terminus to the C-terminus, wherein VL is a variable region of the light chain and CL is a constant region of the light chain, wherein VH-PDL1-CH1 and VH-EGFR-CH1 of the polypeptide chain are respectively paired with VL-CL of the common light chain, wherein VH-PDL1 and VL form a PD-L1 antigen binding site, and VH-EGFR and VL form an EGFR binding site; Each of the common light chains has an amino acid sequence as shown in SEQ ID NO: 7; The heavy chain variable region VH-PDL1 that binds PD-L1 is shown in SEQ ID No: 1; the heavy chain variable region VH-EGFR that binds EGFR is shown in SEQ ID No:

9.

2. The tetravalent bispecific antibody as described in claim 1, characterized in that, The CH1 region of the heavy chain is selected from the CH1 domain of human IgG1 or the CH1 domain of human IgG4.

3. The tetravalent bispecific antibody as described in claim 1, characterized in that, Each of the aforementioned polypeptide chains has the same amino acid sequence.

4. A tetravalent bispecific antibody against PD-L1 and EGFR, characterized in that, It comprises two polypeptide chains and four common light chains, wherein the polypeptide chains have amino acid sequences as shown in SEQ ID NO: 12 or SEQ ID NO: 14, and the common light chains have amino acid sequences as shown in SEQ ID NO:

7.

5. An isolated nucleotide, characterized in that, The nucleotides encode the tetravalent bispecific antibody as described in any one of claims 1-4.

6. The nucleotide as claimed in claim 5, characterized in that, The nucleotides encode the polypeptide chain and the common light chain, wherein the nucleotide sequence encoding the polypeptide chain is shown in SEQ ID NO: 13 or SEQ ID NO: 15, and the nucleotide sequence encoding the common light chain is shown in SEQ ID NO:

8.

7. An expression carrier, characterized in that, The expression vector contains the nucleotides as described in claim 5 or 6.

8. A host cell, characterized in that, The host cell contains the expression vector as described in claim 7.

9. The method for preparing the tetravalent bispecific antibody according to any one of claims 1-4, characterized in that, The method includes the following steps: (a) Under expression conditions, host cells as described in claim 8 are cultured to express the tetravalent bispecific antibody; (b) Isolate and purify the tetravalent bispecific antibody described in (a).

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains a tetravalent bispecific antibody as described in any one of claims 1-4 and a pharmaceutically acceptable carrier.

11. Use of the tetravalent bispecific antibody as described in any one of claims 1-4 or the pharmaceutical composition as described in claim 10 in the preparation of a medicament for treating cancer, wherein the cancer is selected from the group consisting of: melanoma, renal cell carcinoma, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, and glioma.

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