Anti-PDL1×EGFR bispecific antibody

Abstract

The present invention provides an anti-PDL1 x EGFR bispecific antibody, and experimental results show that this bispecific antibody can better maintain the activity of anti-EGFR and anti-PD-L1 monoclonal antibodies, and can simultaneously specifically bind to the two targets, PD-L1 and EGFR. More importantly, this bispecific antibody has obvious synergistic effects at the cellular level.

Description

[Technical field]

[0001] The present invention relates to the field of antibodies, more specifically, the present invention discloses an anti-PDL1xEGFR bispecific antibody. [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 major known immune checkpoints (Blank et al., 2005, Cancer Immunotherapy, 54:307-314). PD-1 is expressed on activated T lymphocytes, where it can bind to the ligands PD-L1 (programmed cell death-Ligand 1) and PD-L2 (programmed cell death-Ligand 2) to inhibit the activity of T lymphocytes and the associated in vivo cellular immune response. PD-L2 is mainly expressed on macrophages and dendritic cells, whereas PD-L1 is widely expressed on peripheral cells such as B lymphocytes and T lymphocytes as well as microvascular epithelial cells, and tissue cells of the lung, liver, heart, etc. Numerous studies have shown that the interaction between PD-1 and PD-L1 is not only necessary to maintain the balance of the immune system in vivo, but also the main mechanism and cause of PD-L1-positive tumor cells evading immune surveillance. Blocking the negative regulation of cancer cells on the PD-1 / PD-L1 signaling pathway can activate the immune system and promote tumor-specific cellular immune responses associated with T cells, thus opening the door to a new tumor treatment, tumor immunotherapy.

[0003] PD-1 (encoded by the gene Pdcd1) is a member of the immunoglobulin superfamily that is related to CD28 and CTLA-4. Research results indicate that PD-1 negatively regulates antigen receptor signaling when it binds to its ligands (PD-L1 and / or PD-L2). Currently, the structure of mouse PD-1 and the co-crystal structure of mouse PD-1 with 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 that contain an Ig variable-type (V-type) domain involved in ligand binding and a cytoplasmic tail region involved in binding signaling molecules. The PD-1 cytoplasmic tail region contains two tyrosine-based signaling motifs, ITIM (immunoreceptor tyrosine-based inhibitory effector motif) and ITSM (immunoreceptor tyrosine-based transforming effector motif).

[0004] PD-1 plays an important role in tumor immune evasion mechanisms. Tumor immunotherapy, which uses the body's own immune system to fight cancer, is a groundbreaking tumor treatment, but the tumor microenvironment can protect tumor cells from effective immune destruction, so how to destroy the tumor microenvironment is the focus of antitumor research. Existing research results have identified the role of PD-1 in the tumor microenvironment, and PD-L1 is expressed in many mouse and human tumors (it 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 found expression of PD-1 (on tumor-infiltrating lymphocytes) and / or PD-L1 on tumor cells in many primary human tumors, including lung, liver, ovarian, cervical, skin, colon, glioma, bladder, breast, kidney, esophageal, gastric, oral squamous cell, urothelial and pancreatic cancers, as well as head and neck tumors. This suggests that blocking the PD-1 / PD-L1 interaction can improve the immune activity of tumor-specific T cells and aid the immune system in eliminating tumor cells, making PD-L1 a popular target for the development of tumor immunotherapy drugs.

[0005] EGFR (Epidermal Growth Factor Receptor) is a receptor for epidermal growth factor (EGF) belonging to the ErbB receptor family. EGFR is a transmembrane glycoprotein with a molecular weight of 170KDa belonging to the receptor tyrosine kinase. Under the action of related ligands such as EGF and transforming growth factor α (TGFα), EGFR is activated by converting monomers to dimers, thereby further activating downstream signaling pathways and regulating cell proliferation. Numerous studies have shown that high or abnormal expression of EGFR exists in most tumor tissues, such as glial cell carcinoma, kidney cancer, lung cancer, prostate cancer, pancreatic cancer, and breast cancer. Abnormal EGFR function is associated with inhibition of tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and apoptosis. Its abnormal function is mainly manifested in two aspects: one is excessive abnormal expression in tumor tissues, and the other is continuous activation of EGFR mutants in tumor cells (without the need for ligand stimulation or the formation of an autologous stimulation pathway). In colon cancer patient tissues, the expression rate of EGFR is about 25-77%. Relevant clinical data indicates that the level of EGFR expression is closely related to the malignancy of tumors and the prognosis of tumor patients.

[0006] Erbitux (Cetuximab, IMC-C225) is a human-mouse chimeric monoclonal antibody against EGFR that inhibits EGFR signaling by binding specifically to EGFR and competitively blocking the binding of EGFR to its ligands. Erbitux was approved for marketing in February 2004 and is used in combination with irinotecan to treat EGFR-positive metastatic colorectal cancer that is refractory to irinotecan chemotherapy and in combination with radiation therapy to treat locoregional early stage squamous cell carcinoma of the head and neck (SCCHN).

[0007] Bispecific antibodies have become a new class of therapeutic antibodies that can be used to treat a variety of inflammatory diseases, cancer, and other diseases.

[0008] There is an urgent need in the art to develop bispecific antibodies with superior properties for treating diseases such as cancer. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Blank et al, 2005, Cancer Immunotherapy, 54:307~314 [Non-Patent Document 2] Zhang, X. et al., Immunity, 20:337-347 (2004) [Non-Patent Document 3] Lin et al., Proc. Natl. Acad. Sci. USA, 105:3011-6 (2008). [Non-Patent Document 4] Iwai Y. et al., Proc. Natl. Acad. Sci. USA, 99:12293~12297 (2002) [Non-Patent Document 5] Strome S.E. et al., Cancer Res., 63:6501~6505 (2003) Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention provides an anti-PDL1×EGFR bispecific antibody and applications thereof.

[0011] Therefore, a first object of the present invention is to provide an anti-PDL1 x EGFR bispecific antibody.

[0012] A second object of the present invention is to provide an isolated nucleotide sequence encoding said bispecific antibody.

[0013] A third object of the present invention is to provide an expression vector comprising the above nucleotide.

[0014] A fourth object of the present invention is to provide a host cell containing the expression vector.

[0015] A fifth object of the present invention is to provide a method for preparing said bispecific antibody.

[0016] A sixth object of the present invention is to provide a pharmaceutical composition comprising the bispecific antibody.

[0017] A seventh object of the present invention is to provide a use of said bispecific antibody or said pharmaceutical composition in the preparation of a medicament for treating cancer.

[0018] An eighth object of the present invention is to provide a method for treating cancer using said bispecific antibody or said pharmaceutical composition.

[0019] To achieve the above objectives, the present invention provides the following technical solutions: [Means for solving the problem]

[0020] A first aspect of the invention provides an anti-PDL1xEGFR bispecific antibody comprising two polypeptide chains and two light chains selected from the group consisting of: (a) the polypeptide chain comprises, from N-terminus to C-terminus, VL-PDL1-linker1-VH-PDL1-linker2-VH-EGFR-CH1-CH2-CH3 or VH-PDL1-linker1-VL-PDL1-linker2-VH-EGFR-CH1-CH2-CH3 or EGFR-CH1-CH2-CH3-linker2-VL-PDL1-linker1-VH-PDL1 or EGFR-CH1-CH2-CH3-linker2-VH-PDL1-linker1-VL-PDL1; the light chain comprises from N-terminus to C-terminus VL-EGFR-CL; or (b) the polypeptide chain comprises, from N-terminus to C-terminus, VL-EGFR-linker1-VH-EGFR-linker2-VH-PDL1-CH1-CH2-CH3 or VH-EGFR-linker1-VL-EGFR-linker2-VH-PDL1-CH1-CH2-CH3 or PDL1-CH1-CH2-CH3-linker2-VL-EGFR-linker1-VH-EGFR or PDL1-CH1-CH2-CH3-linker2-VH-EGFR-linker1-VL-EGFR; the light chain comprises, from N-terminus to C-terminus, VL-PDL1-CL; wherein VL-PDL1 is a light chain variable region that binds to PD-L1; VH-PDL1 is a heavy chain variable region that binds to PD-L1; the VL-EGFR is a light chain variable region that binds to EGFR; the VH-EGFR is a heavy chain variable region that binds to EGFR; said CH1-CH2-CH3 being a heavy chain constant region and said CL being a light chain constant region; said linker 1 and linker 2 are each independently a flexible peptide linker; the VL-PDL1 and the VH-PDL1 form an antigen-binding site that specifically binds to PD-L1; The VH-EGFR and the VL-EGFR form an antigen-binding site that specifically binds to EGFR.

[0021] In another preferred embodiment, the bispecific antibody has the activity of binding to EGFR and the activity of binding to PD-L1 simultaneously.

[0022] In another preferred embodiment, the flexible peptide linker comprises 6 to 30 amino acids, preferably 10 to 25 amino acids.

[0023] In another preferred embodiment, the flexible peptide linker contains 2 to 6 G4S and / or G3S.

[0024] In another preferred example, the linker 1 is 3 to 5 G4S.

[0025] In another preferred example, the linker 2 is 2 to 4 G4S.

[0026] In another preferred embodiment, the anti-PDL1xEGFR bispecific antibody comprises (a) two polypeptide chains comprising, from N-terminus to C-terminus, VL-PDL1-linker1-VH-PDL1-linker2-VH-EGFR-CH1-CH2-CH3; and (b) two light chains comprising, from N-terminus to C-terminus, VL-EGFR-CL; (a) two polypeptide chains comprising from N-terminus to C-terminus VL-PDL1-linker1-VH-PDL1-linker2-VH-EGFR-CH1-CH2-CH3, VL-PDL1 is a light chain variable region that binds to PD-L1; said linker 1 and linker 2 are each independently a flexible peptide linker; the VH-EGFR is a heavy chain variable region that binds to EGFR; said CH1-CH2-CH3 is a heavy chain constant region, and (b) in two light chains comprising VL-EGFR-CL from the N-terminus to the C-terminus wherein VL-EGFR is a light chain variable region that binds to EGFR, and CL is a light chain constant region; Here, the VL-PDL1 and the VH-PDL1 form an antigen-binding site that specifically binds to PD-L1, and the VH-EGFR and the VL-EGFR form an antigen-binding site that specifically binds to EGFR.

[0027] In another preferred embodiment, the anti-PDL1xEGFR bispecific antibody comprises (a) two polypeptide chains comprising, from N- to C-terminus, VL-PDL1-linker1-VH-PDL1-linker2-VH-EGFR-CH1-CH2-CH3, and (b) two light chains comprising, from N- to C-terminus, VL-EGFR-CL, (a) two polypeptide chains comprising from N-terminus to C-terminus: VL-PDL1-Linker1-VH-PDL1-Linker2-VH-EGFR-CH1-CH2-CH3, wherein the VL-PDL1 is a light chain variable region that binds to PD-L1, the Linker1 has 4 or 5 GGGGS, the VH-PDL1 is a heavy chain variable region that binds to PD-L1, the Linker2 has 3 or 4 GGGGS, the VH-EGFR is a heavy chain variable region that binds to EGFR, and the CH1-CH2-CH3 are heavy chain constant regions; and (b) two light chains comprising, from N-terminus to C-terminus, VL-EGFR-CL, wherein the VL-EGFR is a light chain variable region that binds to EGFR and the CL is a light chain constant region; Here, the VL-PDL1 and the VH-PDL1 form an antigen-binding site that specifically binds to PD-L1, and the VH-EGFR and the VL-EGFR form an antigen-binding site that specifically binds to EGFR.

[0028] According to a preferred embodiment of the present invention, the VL-PDL1 comprises a light chain CDR having an amino acid sequence as shown in SEQ ID NO:1-3, the VH-PDL1 comprises a heavy chain CDR having an amino acid sequence as shown in SEQ ID NO:4-6, the VH-EGFR comprises a heavy chain CDR having an amino acid sequence as shown in SEQ ID NO:7-9, and the VL-EGFR comprises a light chain CDR having an amino acid sequence as shown in SEQ ID NO:10-12.

[0029] According to a preferred embodiment of the present invention, the VL-PDL1 has an amino acid sequence as shown in SEQ ID NO:13, the VH-PDL1 has an amino acid sequence as shown in SEQ ID NO:14, the VH-EGFR has an amino acid sequence as shown in SEQ ID NO:15, and the VL-EGFR has an amino acid sequence as shown in SEQ ID NO:16.

[0030] According to a preferred embodiment of the invention, the polypeptide chain has an amino acid sequence as set forth in SEQ ID NO:17 or SEQ ID NO:18 or SEQ ID NO:23 or SEQ ID NO:24 or SEQ ID NO:25, and the light chain has an amino acid sequence as set forth in SEQ ID NO:19, Or the polypeptide chain has an amino acid sequence as set forth in SEQ ID NO:26 or SEQ ID NO:27 or SEQ ID NO:28 or SEQ ID NO:29, and the light chain has an amino acid sequence as set forth in SEQ ID NO:22.

[0031] According to the invention, the heavy chain constant region comprises an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region and the light chain constant region comprises a κ or λ light chain constant region.

[0032] A second aspect of the invention includes an isolated nucleotide, said nucleotide encoding said bispecific antibody.

[0033] A third aspect of the present invention provides an expression vector, said expression vector comprising a nucleotide sequence as described above.

[0034] A fourth aspect of the invention provides a host cell, said host cell comprising an expression vector as described above.

[0035] A fifth aspect of the invention provides a method for preparing the bispecific antibody, said method comprising the steps of: (a) expressing said bispecific antibody by culturing a host cell as described above under expression conditions; (b) isolating and purifying the bispecific antibody described in (a).

[0036] A sixth aspect of the invention provides a pharmaceutical composition comprising a bispecific antibody as described above and a pharma- ceutically acceptable vector.

[0037] In another preferred embodiment, the pharmaceutical composition includes an antitumor agent.

[0038] In another preferred embodiment, the pharmaceutical composition is in a unit dosage form.

[0039] In another preferred embodiment, the anti-tumor agent can be present in a separate package from the bispecific antibody alone, or the anti-tumor agent can be coupled to the bispecific antibody.

[0040] In another preferred embodiment, the dosage form of the pharmaceutical composition includes a gastrointestinal dosage form or a parenteral dosage form.

[0041] In another preferred embodiment, the parenteral administration form comprises intravenous injection, drip injection, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavity injection.

[0042] A seventh aspect of the invention provides the use of a 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.

[0043] According to the present invention, the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer, squamous cell carcinoma, head and neck cancer, kidney cancer, bladder cancer, ovarian cancer, breast cancer, melanoma, lung cancer, liver cancer, gastric cancer, lymphoma, leukemia, prostate cancer, bone marrow cancer, and other neoplastic malignant diseases.

[0044] An eighth aspect of the invention provides a method for treating cancer comprising administering to a subject in need thereof a bispecific antibody as described above, or an immunoconjugate thereof, or a pharmaceutical composition as described above.

[0045] According to the present invention, the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer, squamous cell carcinoma, head and neck cancer, kidney cancer, bladder cancer, ovarian cancer, breast cancer, melanoma, lung cancer, liver cancer, gastric cancer, lymphoma, leukemia, prostate cancer, bone marrow cancer, and other neoplastic malignant diseases.

[0046] A ninth aspect of the present invention provides an immunoconjugate, said immunoconjugate comprising: (a) a bispecific antibody according to the first aspect of the invention, and (b) a coupling moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0047] In another preferred example, the conjugate moiety is selected from a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biotoxin, a cytokine (e.g., IL-2, etc.).

[0048] In another preferred embodiment, the immunoconjugate comprises an antibody-drug conjugate (ADC).

[0049] In another preferred embodiment, the immunoconjugate is used for the preparation of a pharmaceutical composition for treating a tumor. Effect of the Invention

[0050] Beneficial effects: the present invention provides an anti-PDL1 x EGFR bispecific antibody, and experimental results show that the bispecific antibody can better maintain the activity of anti-EGFR and anti-PD-L1 monoclonal antibodies, and can specifically bind to the two targets of PD-L1 and EGFR simultaneously, with obvious synergistic effect at the cellular level. [Brief description of the drawings]

[0051] [Figure 1] FIG. 1 is a schematic diagram of the structure of the anti-PDL1×EGFR dual antibody molecule of the present invention. [Figure 2A] 2A and 2B show the HPLC detection spectrum and the SDS-PAGE detection result of the anti-PDL1×EGFR dual antibody, in which FIG. 2A is the HPLC detection spectrum and FIG. 2B is the SDS-PAGE detection result. [Figure 2B] 2A and 2B show the HPLC detection spectrum and the SDS-PAGE detection result of the anti-PDL1×EGFR dual antibody, in which FIG. 2A is the HPLC detection spectrum and FIG. 2B is the SDS-PAGE detection result. [Figure 3A] FIG. 3 shows the results of ELISA detection of the binding of anti-PDL1×EGFR dual antibody to PD-L1 and EGFR, respectively, where FIG. 3A shows the results of binding to PD-L1 and FIG. 3B shows the results of binding to EGFR. [Figure 3B] FIG. 3 shows the results of ELISA detection of the binding of anti-PDL1×EGFR dual antibody to PD-L1 and EGFR, respectively, where FIG. 3A shows the results of binding to PD-L1 and FIG. 3B shows the results of binding to EGFR. [Figure 4] The results are shown in the bispecific ELISA detection results of anti-PDL1 x EGFR dual antibody that simultaneously binds to PD-L1 and EGFR. [Figure 5A] 5A and 5B show the results of FACS detection of the binding of anti-PDL1×EGFR dual antibody to A431 and N87-PDL1 cells, respectively, where FIG. 5A shows the results of binding to A431 cells and FIG. 5B shows the results of binding to N87-PDL1 cells. [Figure 5B] 5A and 5B show the results of FACS detection of the binding of anti-PDL1×EGFR dual antibody to A431 and N87-PDL1 cells, respectively, where FIG. 5A shows the results of binding to A431 cells and FIG. 5B shows the results of binding to N87-PDL1 cells. [Figure 6] This shows the results of the activity of anti-PDL1 x EGFR dual antibody in inhibiting the proliferation of A431 cells. [Figure 7] These results are the results of an anti-PDL1 x EGFR dual antibody that blocks the activity of PD1 / PD-L1 on cells. [Figure 8] FIG. 13 is a fitting diagram of affinity detection between an anti-PDL1×EGFR bispecific antibody and an EGFR antigen. [Figure 9] FIG. 1 shows a fitting diagram of affinity detection between anti-PDL1×EGFR bispecific antibody and PD-L1 antigen. [Figure 10]Synergistic effect of anti-PDL1 x EGFR bispecific antibodies at the cellular level. [Figure 11] Antitumor effect of anti-PDL1 x EGFR bispecific antibody in SW48 xenograft tumor model. [Figure 12] B-Anti-tumor effect of anti-PDL1 x EGFR bispecific antibody in hPD-L1 humanized mouse MC38-hPD-L1 xenograft tumor model. [Figure 13] 1 shows the activity results of the in vitro proliferation inhibitory effect of an anti-PDL1 x EGFR dual antibody of a comparative example on A431 cells. [Figure 14A] FIG. 14 shows the activity results of comparative anti-PDL1×EGFR dual antibodies for inhibiting cellular PD1 / PD-L1, where FIG. 14A shows the activity comparison between anti-PDL1×EGFR BsAb rev1, rev2, rev3 and the positive control anti-PD-L1 monoclonal antibody, and FIG. 14B shows the activity comparison between anti-PDL1×EGFR BsAb rev5, rev6, rev7, rev8 and the positive control anti-PD-L1 monoclonal antibody. [Figure 14B] FIG. 14 shows the activity results of comparative anti-PDL1×EGFR dual antibodies for inhibiting cellular PD1 / PD-L1, where FIG. 14A shows the activity comparison between anti-PDL1×EGFR BsAb rev1, rev2, rev3 and the positive control anti-PD-L1 monoclonal antibody, and FIG. 14B shows the activity comparison between anti-PDL1×EGFR BsAb rev5, rev6, rev7, rev8 and the positive control anti-PD-L1 monoclonal antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Through extensive and thorough research and extensive screening, the present inventors have obtained a bispecific antibody consisting of an anti-EGFR antibody and an anti-PD-L1 antibody, and the bispecific antibody of the present invention belongs to a homodimer. The bispecific antibody of the present invention not only maintains the activity of the anti-EGFR antibody and the anti-PDL1 antibody, but can also bind to EGFR and PD-L1 simultaneously, and more importantly, the cell level experiments show that this bispecific antibody can play a synergistic effect in killing tumor cells, and shows a killing activity significantly higher than that of the anti-EGFR antibody and higher than that of the anti-PD-L1 antibody. Therefore, the bispecific antibody of the present invention can be developed as an anti-tumor drug with excellent therapeutic effect. Based on this, the present inventors have completed the present invention.

[0053] term In the present invention, the terms "antibody (abbreviated as Ab)" and "immunoglobulin G (abbreviated as IgG)" refer to a heterotetrameric glycoprotein with identical structural characteristics consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has intrachain disulfide bonds arranged in a regular sense. At one end of each heavy chain is a variable region (VH) followed by a constant region, the heavy chain constant region consisting of three domains CH1, CH2, and CH3. At one end of each light chain is a variable region (VL) and at the other end is a constant region, the light chain constant region contains one domain CL, the light chain constant region is paired with the CH1 domain of the heavy chain constant region, and the light chain variable region is paired with the heavy chain variable region. Although the constant regions are not directly involved in binding between the antibody and the antigen, they exhibit various effector functions, such as being involved in antibody-dependent cell-mediated cytotoxicity (ADCC). The heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes, and the light chain constant region includes κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently bound by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of an antibody are covalently bound by disulfide bonds between polypeptides formed in the hinge region.

[0054] In the present invention, the term "bispecific antibody (or double antibody)" refers to an antibody molecule capable of specifically binding to two antigens (targets) or two epitopes simultaneously. Based on symmetry, bispecific antibodies can be divided into structurally symmetric and asymmetric molecules. Depending on the number of binding sites, bispecific antibodies can be divided into bivalent, trivalent, tetravalent and multivalent molecules.

[0055] In the present invention, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies within the population are identical except for a small number of naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. And unlike traditional polyclonal antibody preparations (which are usually mixtures of different antibodies bearing different antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, an advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring a particular method of antibody production.

[0056] In the present invention, the terms "Fab" and "Fc" refer to the fact that papain can cleave an antibody into two identical Fab segments and one Fc segment. The Fab segment consists of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain of an antibody. The Fc segment is a fragment crystallizable (Fc) that consists of the CH2 and CH3 domains of an antibody. The Fc segment does not have antigen binding activity and is the site where the antibody interacts with effector molecules or cells.

[0057] In the present invention, the term "scFv" refers to a single chain antibody fragment (scFv) formed by linking the heavy chain variable region and the light chain variable region of an antibody via a linker generally consisting of 15 to 25 amino acids.

[0058] In the present invention, the term "variable" refers to the difference in sequence of certain parts of the variable region of an antibody, which forms a variety of specific binding and specificity for a particular antigen. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy and light chain variable regions. The more conserved parts of the variable regions are called framework regions (FRs). Natural heavy and light chain variable regions each contain four FR regions, which are mostly in a β-folded configuration connected by three CDRs that form a connecting ring, and can sometimes form a partial β-folded structure. The CDRs of each chain are closely juxtaposed by 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)).

[0059] As used herein, the term "framework region" (FR) refers to the amino acid sequences inserted between the CDRs, i.e., the portions of the light and heavy chain variable regions of an immunoglobulin that are relatively conserved among different immunoglobulins of a single species. The light and heavy chains of an immunoglobulin have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Correspondingly, the light chain variable domain can be designated (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L), and the heavy chain variable domain can be represented 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, and the derivative of said human antibody FR is substantially identical to a naturally occurring human antibody FR, i.e., the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0060] A person skilled in the art knowing the amino acid sequences of the CDRs can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0061] As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, particularly 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.

[0062] As used herein, the term "linker" refers to one or more amino acid groups inserted into an immunoglobulin domain to provide sufficient flexibility for the light and heavy chain domains to fold into an exchanged dual variable region immunoglobulin. In the present invention, preferred linkers refer to linker 1 and linker 2, where linker 1 is used to connect the VH and VL of a single chain antibody (scFv) and linker 2 is used to connect the scFv to the heavy chain of another antibody.

[0063] Examples of suitable linkers include monoglycine (Gly), or serine (Ser) residues, and the label and sequence of amino acid residues in the linker will vary depending on the type of secondary structural element that needs to be achieved in the linker.

[0064] bispecific antibody The bispecific antibody of the present invention is an anti-PDL1 x EGFR bispecific antibody comprising an anti-PDL1 antibody portion and an anti-EGFR antibody portion.

[0065] Preferably, the sequences of the anti-PD-L1 antibodies of the invention are as set forth in patent application PCT / CN2020 / 090442, and those skilled in the art can modify or transform the anti-PD-L1 antibodies of the invention by techniques known in the art, such as adding, deleting, and / or substituting one or several amino acid residues, to further increase the affinity or structural stability of anti-PD-L1, and obtain modified or altered results according to conventional measurement methods.

[0066] In the present invention, "conservative variants of a bispecific antibody of the invention" refers to a polypeptide in which up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids having the same or similar properties compared to the amino acid sequence of a bispecific antibody of the invention. These conservative variant polypeptides are preferably produced by amino acid replacement according to Table A.

[0067] [Table 1]

[0068] In the present 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 corresponding antigen. Typically, an antibody binds to a specific antigen at a concentration of about 10 -7 M, for example, about 10 -8 Less than M, 10 -9 Less than M, 10 -10 Less than M, 10 -11It binds to an antigen with an equilibrium dissociation constant (KD) of less than or equal to M. In the present invention, the term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction and is used to describe 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, Surface Plasmon Resonance (SPR) is used to measure the binding affinity between the antibody and the antigen on a BIACORE instrument, or ELISA is used to measure the relative affinity of antibody-antigen binding.

[0069] In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. An epitope of the present invention is a region of an antigen to which an antibody binds.

[0070] The bispecific antibodies of the present invention can be used alone or can be conjugated or coupled to a detectable marker (for diagnostic purposes), a therapeutic agent, or a combination of any of these substances.

[0071] Encoding nucleic acids and expression vectors The present invention further provides a polynucleotide molecule encoding the above antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA or artificial DNA. The DNA can be single-stranded or double-stranded. The DNA can be coding strand or non-coding strand.

[0072] In the present invention, the term "expression vector" refers to a vector carrying an expression cassette for expressing a specific target protein or other substance, such as a plasmid, a viral vector (e.g., adenovirus, retrovirus), a phage, a yeast plasmid, or other vector. Representative examples include, but are not limited to, pTT5, pSECtag series, pCGS3 series, pcDNA series vectors, and other vectors used in mammalian expression systems. An expression vector includes a bound DNA sequence linked to appropriate transcriptional and translational regulatory sequences.

[0073] Once the relevant sequence is obtained, recombinant methods can be used to obtain the relevant sequence in large quantities, typically by cloning it into a vector, then transforming it into a cell, and then isolating the relevant sequence from the host cell which has been propagated by conventional methods.

[0074] The present invention further relates to vectors containing said appropriate DNA sequences and appropriate promoter or control sequences, which can be used to transform appropriate host cells so as to express the protein.

[0075] In the present invention, the term "host cell" refers to a cell suitable for expressing the above-mentioned expression vector, which may be a eukaryotic cell such as a mammalian or insect host cell culture system that can be used for expressing the fusion protein of the present invention, and CHO (Chinese Hamster Ovary), HEK293, COS, BHK and derived cells of the above-mentioned cells can be applied to the present invention.

[0076] Pharmaceutical Compositions and Applications The present invention further provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above-mentioned antibody or an active fragment thereof or a fusion protein thereof, and a pharma- ceutically acceptable vector. In general, these substances can be formulated in a non-toxic, inert, pharma- ceutically acceptable aqueous vector medium, where the pH is usually about 5-8, preferably about 6-8, and the pH value varies depending on the nature of the formulated substance and the disease to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal), intracranial injection, or intracavity injection.

[0077] In the present invention, the term "pharmaceutical composition" refers to the formation of a pharmaceutical formulation composition of the bispecific antibody of the present invention together with a pharma- ceutical acceptable vector, which can exert a more stable therapeutic effect, and these formulations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in the present invention, and at the same time, protect the multiple functional groups of the protein from degradation (including, but not limited to, condensation, deamination, or oxidation).

[0078] The pharmaceutical composition of the present invention comprises 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 bispecific antibody of the present invention (or a conjugate thereof) and a pharma- ceutically acceptable vector or excipient. Such vectors include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The drug formulation should be consistent with the method of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection by conventional methods, for example, using saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be prepared under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 times daily. μgThe bispecific antibody of the present invention can be used in combination with other therapeutic agents.

[0079] When using a pharmaceutical composition, a safe and effective amount of the bispecific antibody or immunoconjugate thereof is administered to a mammal, where the safe and effective amount is typically at least about 10 μg / kg body weight, in most cases not exceeding about 50 mg / kg body weight, preferably the dosage is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dosage must take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0080] The description of the experimental materials used in the following examples is as follows:

[0081] pcDNA TM 3.4 TOPO( Registered trademark) Vector: Purchased from Thermo Fisher, catalog number A14697; CHO cells: purchased from Thermo Fisher, catalog number A29133; 293E cells: purchased from NRC biotechnology Research Institute; PD-1 / PD-L1 Blockade Bioassay, Propagation model: purchased from Promega, catalog number J1252; Human gastric cancer cell line NCI-N87: purchased from the American Type Culture Collection (ATCC); Human epidermal carcinoma cell line A431: purchased from ATCC. SD rats: purchased from Shanghai Lingcheng Bioscience and Technology Co., Ltd. SW48 cells: purchased from Shanghai Institute of Biochemistry, Chinese Academy of Sciences; MC38 cells: purchased from Guangzhou Geneo Biotechnology Co., Ltd. B-hPD-L1 humanized mice: purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.

[0082] Descriptions of experimental reagents used in the following examples are as follows:

[0083] Anti-PD-L1 monoclonal antibody: prepared according to the sequence in PCT / CN2020 / 090442; Anti-EGFR monoclonal antibody: Prepared according to the sequence of cetuximab, HRP-conjugated sheep anti-human Fc antibody: purchased from Sigma, catalog number A0170; HRP-labeled mouse anti-human Fab antibody: purchased from Sigma, catalog number A0293; HRP-labeled anti-6×His antibody: purchased from Abcam, catalog number ab178563; Goat anti-human IgG-FITC: purchased from Sigma, catalog number F4143; PBS: purchased from Sangon Bioengineering (Shanghai) Co., Ltd., catalog number B548117; PBST: PBS + 0.05% Tween 20, BSA: purchased from Sangon Bioengineering (Shanghai) Co., Ltd., catalog number A60332; FBS: purchased from Gibco, catalog number 10099; TMB: purchased from BD Company, catalog number 555214; RPMI 1640 medium: purchased from Gibco, catalog number 22400-089; Bio-Glo Luciferase Assay System: purchased from Promega, catalog number G7940; Cell Counting Kit-8: purchased from Dojindo, catalog number CK04. DMEM medium: purchased from Cellgro, catalog number 10-013-CV.

[0084] The description of the experimental equipment used in the following examples is as follows:

[0085] PCR equipment: purchased from BioRad, catalog number C1000 Touch Thermal Cycler; HiTrap MabSelectSuRe column: purchased from GE, catalog number 11-0034-95; Beckman Coulter CytoFLEX flow cytometer: purchased from Beckman Company. SpectraMax i3x microplate reader: purchased from Molecular Devices, SpectraMaxM5 microplate reader: purchased from Molecular Devices.

[0086] The following examples and experimental examples are for further illustrating the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of conventional methods, such as those methods of constructing vectors and plasmids, those methods of inserting protein-encoding genes into such vectors and plasmids, or those methods of introducing plasmids into host cells. Such methods are well known to those skilled in the art and are described in many publications, such as Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press. For experimental methods for which no specific conditions are given in the following examples, conventional conditions or conditions suggested by the manufacturer are usually followed. Unless otherwise stated, percentages and parts are percentages and parts by weight.

[0087] Example 1. Construction of anti-PDL1 x EGFR dual antibody molecule The present invention relates to an anti-human PD-L1 monoclonal antibody M8 (sequence from PCT / CN2020 / 090442) scFv (VL-linker1-VH, linker1 is 4 or 5 GGGGS) connected in tandem to the N-terminus of the heavy chain of an anti-human EGFR monoclonal antibody (prepared according to the sequence of cetuximab, the sequence from 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) via linker 2 (3 or 4 GGGGS) to construct an anti-PDL1 x EGFR bispecific antibody. Here, the bibody in which linker 1 is 4 GGGGS and linker 2 is 3 GGGGS is named anti-PDL1×EGFR BsAb1, and the bibody in which linker 1 is 5 GGGGS and linker 2 is 3 GGGGS is named anti-PDL1×EGFR BsAb2. The structure is as shown in FIG.

[0088] The present invention uses several other conjugation methods to construct anti-PDL1 x EGFR dual antibody molecules, as shown below.

[0089] anti-PDL1×EGFR BsAb rev1: anti-human PD-L1 monoclonal antibody scFv (VL-linker1-VH from N-terminus to C-terminus, linker1 is 4 GGGGS) is used to tandemly construct the VL of the scFv to the C-terminus of the heavy chain of anti-human EGFR monoclonal antibody via linker2 (3 GGGGS); Anti-PDL1×EGFR BsAb rev2: Using an anti-human PD-L1 monoclonal antibody scFv (VH-linker1-VL from N-terminus to C-terminus, where linker1 is 4 GGGGS), the VH of the scFv is tandemly constructed via linker2 (3 GGGGS) to the heavy chain C-terminus of an anti-human EGFR monoclonal antibody; Anti-PDL1×EGFR BsAb rev3: Using an anti-human PD-L1 monoclonal antibody scFv (VH-linker1-VL from N-terminus to C-terminus, where linker1 is 4 GGGGS), the VL of the scFv is tandemly constructed with the N-terminus of the heavy chain of an anti-human EGFR monoclonal antibody via linker2 (3 GGGGS); anti-PDL1×EGFR BsAb rev5: anti-human EGFR monoclonal antibody scFv (VL-linker1-VH from N-terminus to C-terminus, linker1 is 4 GGGGS) is used to tandemly construct the VL of the scFv to the C-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody via linker2 (3 GGGGS); anti-PDL1×EGFR BsAb rev6: anti-human EGFR monoclonal antibody scFv (VH-linker1-VL from N-terminus to C-terminus, linker1 is 4 GGGGS) is used to tandemly construct the VH of the scFv to the C-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody via linker2 (3 GGGGS); anti-PDL1×EGFR BsAb rev7: anti-human EGFR monoclonal antibody scFv (VH-linker1-VL from N-terminus to C-terminus, linker1 is 4 GGGGS) is used to construct the VL of the scFv in tandem with the N-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody via linker2 (3 GGGGS); Anti-PDL1×EGFR BsAb rev8: Using an scFv of an anti-human EGFR monoclonal antibody (VL-linker1-VH from N-terminus to C-terminus, where linker1 has 4 GGGGS residues), the VH of the scFv is constructed in tandem with the N-terminus of the heavy chain of an anti-human PD-L1 monoclonal antibody via linker2 (which has 3 GGGGS residues).

[0090] By gene synthesis and conventional molecular cloning methods, the bispecific antibody and its corresponding monoclonal antibody heavy and light chain expression vectors were obtained, whose corresponding amino acid sequences are as shown in Table 1, where the CDRs are encoded according to the Kabat rules.

[0091] [Table 2]

[0092] TIFF0007656635000003.tif254163

[0093] TIFF0007656635000004.tif254158

[0094] TIFF0007656635000005.tif254162

[0095] TIFF0007656635000006.tif174170

[0096] Example 2. Expression and purification of anti-PDL1 x EGFR dual antibodies The heavy and light chain DNA fragments of the anti-PDL1 x EGFR dual antibody were subcloned into the pcDNA3.4 vector, respectively, and the recombinant plasmids were extracted and co-transfected into CHO cells and / or 293E cells. After culturing the cells for 5 to 7 days, the culture medium was centrifuged at high speed and suction filtered through a micro-multiwell membrane. The sample was then loaded onto a HiTrap MabSelectSuRe column, and the protein was eluted with an eluent containing 100 mM citric acid, pH 3.5, and dialyzed into PBS, pH 7.4.

[0097] The purified protein was detected by HPLC, and the HPLC-SEC detection spectrum of the anti-PDL1×EGFR double antibody was shown in Figure 2 A. The monomer purity of the two double antibodies, anti-PDL1×EGFR BsAb1 and anti-PDL1×EGFR BsAb2, reached more than 92%, where the monomer purity of anti-PDL1×EGFR BsAb2 was slightly higher than that of anti-PDL1×EGFR BsAb1.

[0098] The results of SDS-PAGE detection are shown in Figure 2B, where lanes 1 and 2 are non-reduced and reduced SDS-PAGE of anti-PDL1 x EGFR double antibody, and lanes 3 and 4 are non-reduced and reduced SDS-PAGE of anti-EGFR monoclonal antibody. The theoretical molecular weight of the double antibody is 198KD.

[0099] Example 3. Enzyme-linked immunosorbent assay (ELISA) to detect the affinity of anti-PDL1 x EGFR dual antibodies to antigens 3.1.Affinity detection by PD-L1 antigen To detect the affinity of anti-PDL1×EGFR dual antibody and PD-L1 antigen, PDL1-ECD-His protein (PD-L1 extracellular domain gene was synthesized according to the sequence provided by NCBI (NCBI accession number is NP_054862.1)) in PBS buffer at pH 7.4, and a signal peptide sequence was added to its N-terminus and a 6×His tag was added to its C-terminus. The gene was then constructed into an expression vector with EcoRI and HindIII restriction sites, and transfected into HEK-293E cells. ELISA plate (obtained by incubating at 4℃ overnight) was diluted to 1000ng / ml, then 100μl / well was added to the plate, incubated at 4℃ overnight, the next day, the plate was washed twice with PBST, each well was blocked by adding PBST+1%BSA, blocked at 37℃ for 1 hour, the plate was washed twice with PBST, and the detection antibody diluted in a gradient of PBS+1%BSA was added, the anti-PD-L1 monoclonal antibody was used as a positive control, the initial concentration was 100nM, and 12 gradients were serially diluted 3-fold. Incubated at 37℃ for 1 hour, the plate was washed twice with PBST, the secondary antibody HRP-labeled goat anti-human Fc antibody was added, further incubated at 37℃ for 40 minutes, the plate was washed three times with PBST, and tapped dry, 100μl of TMB was added to each well, and placed in the dark at room temperature (20±5℃) for 5 minutes, and 50μl of 2M H 2 SO 4 The substrate reaction was stopped by adding stop solution, and the OD value at 450 nm was read using a microplate reader. The data was analyzed and plotted using GraphPad Prism, and the EC 50 Calculate.

[0100] The experimental results are shown in Figure 3A. The EC 50 are all 0.11 nM and the affinities of the three are similar.

[0101] 3.2.Affinity detection using EGFR antigen To detect the affinity of anti-PDL1 × EGFR double antibody and EGFR antigen, EGFR-ECD-hFc protein (extracllel domain gene was synthesized according to the sequence provided by UniProt (serial number P00533) in pH 7.4 PBS buffer, and a signal peptide sequence was added to its N-terminus and an hFc tag was added to its C-terminus. The gene was constructed into an expression vector with EcoRI and HindIII restriction sites, respectively, and transfected into HEK-293E cells for expression and analysis. Dilute the IgG antibody (obtained by purification and purification) to 1000ng / ml, then add 100μl / well to the ELISA plate, incubate overnight at 4℃, wash the plate twice with PBST the next day, add PBST+1%BSA to each well to block, block at 37℃ for 1 hour, wash the plate twice with PBST, then add the detection antibody diluted with a gradient of PBS+1%BSA, anti-EGFR monoclonal antibody is used as a positive control, the initial concentration is 100nM, and 12 gradients are serially diluted 3-fold. Incubate at 37℃ for 1 hour, wash the plate twice with PBST, add the secondary antibody HRP-anti-Fab antibody, further incubate at 37℃ for 40 minutes, wash the plate three times with PBST, and tap dry, add 100μl of TMB to each well, place in the dark at room temperature (20±5℃) for 5 minutes, add 50μl of 2M H 2 SO 4 The substrate reaction was stopped by adding stop solution, and the OD value at 450 nm was read using a microplate reader. The data was analyzed and plotted using GraphPad Prism 6, and the EC 50 Calculate.

[0102] The experimental results are shown in FIG. 3B. The EC 50 is 0.15 nM, and the EC 50 is 0.15 nM, and the EC 50 is 0.21 nM and the affinities of the three are similar.

[0103] Example 4. Bispecific ELISA to detect the ability of anti-PDL1 x EGFR dual antibodies to simultaneously bind two antigens To detect the ability of anti-PDL1 x EGFR dual antibody to simultaneously bind to EGFR antigen and PD-L1 antigen, dilute PDL1-ECD-hFc protein (replace the C-terminus of PDL1-ECD-His protein with hFc tag) to 1 μg / ml in PBS buffer at pH 7.4, then add 100 μl / well to ELISA plate, incubate overnight at 4℃, wash plate twice with PBST the next day, add PBST+1% BSA to each well to block, block at 37℃ for 1 hour, wash plate twice with PBST, then add detection antibody diluted with PBS+1% BSA gradient, initial concentration is 300 nM, and 12 gradients are serially diluted 3-fold. Incubate at 37°C for 1 hour, wash the plate twice with PBST, add 1 μg / ml EGFR-ECD-His antigen (the C-terminus of EGFR-ECD-hFc protein is replaced with 6×His tag) diluted with PBS at pH 7.4, and add 100 μl / well to the ELISA plate. Incubate at 37°C for 1 hour, wash the plate twice with PBST, add secondary antibody HRP-anti-His, further incubate at 37°C for 40 minutes, wash the plate three times with PBST, and tap dry, add 100 μl of TMB to each well, place in the dark at room temperature (20±5°C) for 5 minutes, and add 50 μl of 2M H 2 SO 4 The substrate reaction was stopped by adding stop solution, and the OD value at 450 nm was read using a microplate reader. The data was analyzed and plotted using GraphPad Prism, and the EC 50 Calculate.

[0104] The experimental results are shown in FIG. 4. The EC 50 are 0.66 nM and 0.63 nM, respectively, and the monoclonal antibody is not capable of binding to both antigens simultaneously.

[0105] Example 5. FACS detection of binding affinity of anti-PDL1 x EGFR dual antibodies to target cells In this experiment, human epidermal carcinoma cells A431, which highly express EGFR on the cell surface, were used as target cells. They were washed three times with PBS containing 0.5% BSA, centrifuged at 300g for 5 min each time, and the supernatant was discarded. The cells were resuspended in PBS with 0.5% BSA at a cell density of 1 × 10 6 cells / mL and 100 μL / well are added to a 96-well plate. Anti-PDL1×EGFR dual antibody and positive control anti-EGFR monoclonal antibody are diluted to 600 nM, serially diluted with 11 gradients, 100 μL / well are added to a 96-well plate and mixed evenly with A431 cells. Incubate at 4°C for 1 hour. Wash the cells twice with PBS to remove unbound detection antibodies. Add 100 μL / well of goat anti-human IgG-FITC to the dish and incubate at 4°C for 30 minutes. Centrifuge at 300g for 5 minutes and wash the cells twice with PBS to remove unbound secondary antibodies. Finally, resuspend the cells in 200 μl of PBS and measure the binding affinity of the dual antibodies to the cells by a Beckman Coulter CytoFLEX flow cytometer. The obtained data are fitted and analyzed by GraphPad Prism7 software.

[0106] The experimental results are shown in FIG. 5A. The EC values ​​of the three anti-EGFR monoclonal antibodies, anti-PDL1×EGFR BsAb1 and anti-PDL1×EGFR BsAb2 were 50 are all 1.56 nM and the affinities of the three are similar.

[0107] N87-PDL1 is a stable cell line constructed in our laboratory by transfecting PD-L1 into NCI-N87 using lentiviral transfection method. After logarithmic growth phase N87-PDL1 is digested with trypsin, it is washed three times with PBS containing 0.5% BSA, centrifuged at 300g for 5 min each time, and the supernatant is discarded. The cells are resuspended in PBS with 0.5% BSA, the cell density is 1×106 cells / mL, and 100μL / well is added to a 96-well plate. Anti-PDL1×EGFR dual antibody and positive control anti-PD-L1 monoclonal antibody are diluted to 120nM, serially diluted with 11 gradients, and 100μL / well is added to a 96-well plate and mixed evenly with N87-PDL1 cells. The remaining method is the same as above.

[0108] The experimental results are shown in Figure 5B. The EC 50 was 0.11 nM, and the EC 50 The EC of anti-PDL1×EGFR BsAb2 was 0.16 nM. 50 is 0.22 nM and the affinities of the three are similar.

[0109] Example 6. Inhibitory effect of anti-PDL1 x EGFR dual antibody on A431 cell proliferation in vitro Human epidermal carcinoma cell line A431 expresses EGFR at high levels on its surface, and anti-EGFR antibodies can effectively inhibit the proliferation of A431 cells in vitro. A431 cells in logarithmic growth phase were cultured at 5 × 10 4 100 μL / mL and inoculated into a 96-well culture plate at 100 μL / well. After 24 h of adherent growth, add different concentrations of the detecting antibody diluted in RPMI 1640 medium with 1% FBS. The initial working concentration of the detecting antibody is 50 nM, and a total of 10 different concentrations are serially diluted 3-fold, with duplicate wells set up for each concentration. Incubate at 37 °C in 5% CO. 2After culturing the cells for 72 hours under the conditions, 20 μl / well of CCK-8 color development solution was added and incubated at room temperature for 1.5 hours. The plate was read on a SpectraMax M5 microplate reader using 450 nm as the detection wavelength and 650 nm as the reference wavelength. The data was analyzed and plotted in GraphPad Prism, and the IC was calculated. 50 Calculate.

[0110] The experimental results are shown in Figure 6. The IC of anti-PDL1×EGFR BsAb1, anti-PDL1×EGFR BsAb2, and anti-EGFR monoclonal antibodies inhibiting A431 proliferation. 50 are 6.63 nM, 7.17 nM, and 5.23 nM, respectively. The three inhibitory activities are comparable.

[0111] Seven bispecific antibodies, including anti-PDL1×EGFR BsAb rev1, rev2, rev3, rev5, rev6, rev7 and rev8, were tested for their inhibitory effect on the proliferation of A431 cells in vitro, with the results shown in Figure 13. The activities of the three bispecific antibodies, anti-PDL1×EGFR BsAb rev2, rev7 and rev8, were significantly worse than the positive control anti-EGFR monoclonal antibody, anti-PDL1×EGFR rev5 was slightly weaker than the positive control, and the activity of anti-PDL1×EGFR rev3 was not significantly different from the positive control anti-EGFR monoclonal antibody.

[0112] Furthermore, the expression level of anti-PDL1×EGFR rev6 is low.

[0113] Example 7. Anti-PDL1 x EGFR dual antibody blocks the activity of PD1 / PD-L1 on cells This experiment uses Promega's PD-1 / PD-L1 Blockade Bioassay, Propagation model and methods.

[0114] PD-L1 aAPC / CHO-K1 in the logarithmic growth phase were digested into single cells with trypsin and then transferred to a white-bottom clear 96-well plate, 100 μL / well, 40,000 cells / well, 37℃, 5% CO 2 Place in a dish and incubate overnight. Take anti-PDL1 x EGFR dual antibody and anti-PD-L1 monoclonal antibody and dilute them to a 2x working solution concentration, the initial concentration is 24 nM, and serially dilute 3x. The density is 1.4-2 x 10 6 Take PD1 effector cells with cell viability of 95% or higher and trypsinize them at 1.25 × 10 cells / mL. 6 Digest into a single cell suspension of 1000 cells / mL. Take the PD-L1 aAPC / CHO-K1 cells plated the day before, discard the supernatant, add 40μl of serially diluted dual antibody / PD-L1 monoclonal antibody working solution, and add an equal volume of PD1 effector cells. Incubate at 37℃, 5% CO 2 Place plate in a dark place and incubate for 6 hours. Add 80μl of Bio-Glo detection reagent to each well, incubate at room temperature for 10 minutes, and read luminescence on a Spectramax i3. Seal the bottom of the plate with a light-tight membrane before reading the plate. Analyze and plot data in GraphPad Prism and obtain IC values. 50 Calculate.

[0115] The experimental results are shown in Figure 7. The IC of anti-PDL1×EGFR BsAb1, anti-PDL1×EGFR BsAb2, and anti-PD-L1 monoclonal antibody 50 are 1.06 nM, 1.21 nM, and 0.78 nM, respectively, and the three effects are substantial.

[0116] Seven bispecific antibodies, including anti-PDL1×EGFR BsAb rev1, rev2, rev3, rev5, rev6, rev7, and rev8, blocked the activity of PD1 / PD-L1 signaling pathway on cells, and the results are shown in Figure 14. Four bispecific antibodies, anti-PDL1×EGFR BsAb rev1, rev2, rev3, rev7, were significantly worse than the positive control anti-PD-L1 monoclonal antibody (Figure 14A), while the activity of anti-PDL1×EGFR BsAb rev5, rev6, and rev8 was not significantly different from the positive control anti-PD-L1 monoclonal antibody (Figure 14B).

[0117] Based on the experimental results of Examples 6 and 7, the order of overall performance is anti-PDL1 x EGFR BsAb1 >= anti-PDL1 x EGFR BsAb2 > other bispecific antibodies.

[0118] Example 8. Pharmacokinetic study of anti-PDL1 x EGFR dual antibodies Five SD rats weighing approximately 200g were taken for each group, and 3mg of the antibody anti-PDL1×EGFR BsAb2 was injected into each rat via the tail vein. The time points for sampling after administration were 0, 3, 24, 48, 96, 168, and 336 hours. Blood was collected from the orbit, allowed to clot naturally, and then centrifuged at 8000rpm / min to obtain serum.

[0119] The serum drug concentration of anti-PDL1×EGFR BsAb2 will be detected by the following method.

[0120] 1) Coat ELISA plates with Protein A to detect antibody Fab segments. Coat with Protein A, the amount of coating is 100ng / well, and coat overnight at 4℃. The next day, wash the plate twice with PBST, then block with PBS+2%BSA at 37℃ for 2 hours. Wash the plate twice with PBST. Anti-PDL1×EGFR BsAb2 standard starts at 1000ng / mL and is serially diluted 2-fold with 12 gradients. Rat serum samples are diluted 1000-4000 times. The above two groups of samples were added to the sealed ELISA plate, incubated for 1 hour, washed twice with PBST, added HRP-labeled mouse anti-human Fab antibody, and left to stand at 37°C for 30 minutes. After washing the plate three times with PBST, pat dry as much of the droplets remaining on the absorbent paper as possible, add 100 μl of TMB to each well, and leave in a dark place at room temperature (20±5°C) for 5 minutes. Add 50 μl of 2MH to each well. 2 SO 4 The substrate reaction is stopped by adding stop solution and the OD value is read at 450 nm in a microplate reader. The calculated half-life is 304 hours.

[0121] 2) EGFR-ECD-hFc coated ELISA plate, 50ng / well. The remaining method is the same as above. The calculated half-life is 275 hours.

[0122] 3) PDL1-ECD-hFc coated ELISA plate, 100 ng / well. The remaining method is the same as above. The calculated half-life is 232 hours.

[0123] As shown in Tables 1 to 3, the calculated half-life results from the ELISA results of the above three groups were similar, indicating that the analytical data were reliable.

[0124] [Table 3]

[0125] [Table 4]

[0126] [Table 5]

[0127] Example 9. Biacore8K measurement of the affinity of anti-PDL1 x EGFR bispecific antibodies to antigens Measure the binding kinetic parameters of anti-PDL1×EGFR bispecific antibody and antigen PDL1-ECD-his using Protein A capture method. Bispecific antibody with a concentration of 2 μg / ml is bound to Protein A chip, and antigen PDL1-ECD-his is serially diluted 2-fold from 50 nM with 1×HBS-EP working solution, and seven concentration gradients are set up to bind to antibody, and dissociate with HBS-EP working solution (as shown in Figure 9).

[0128] Measure the kinetic parameters of binding of anti-PDL1×EGFR bispecific antibody and antigen EGFR-ECD-his using Protein A capture method. The bispecific antibody with a concentration of 2 μg / ml is bound to Protein A chip, and the antigen EGFR-ECD-his is serially diluted 2-fold from 25 nM with 1×HBS-EP working solution, and seven concentration gradients are set up to bind to the antibody, and dissociate with HBS-EP working solution (for example, as shown in Figure 8).

[0129] The kinetic parameters of binding to anti-PDL1xEGFR BsAb1 and control monoclonal antibodies, as well as to EGFR-his, PDL1-ECD-his, are shown in the tables below (Tables 4, 5).

[0130] The data results show that there is no significant difference in the affinity dissociation constants of the anti-PDL1 x EGFR bispecific antibodies compared to the positive control.

[0131] [Table 6]

[0132] [Table 7]

[0133] Example 10. Synergistic effect of anti-PDL1 x EGFR bispecific antibodies at the cellular level Cell plating in 96-well plates: SW48 cells cultured in logarithmic growth phase were digested with peptic sphingosine, and then replaced with RPMI1640 medium supplemented with 5% FBS to adjust the cell density to 100,000 cells / mL. 100 μL / well was used to plate cells in a 96-well plate, i.e., 10,000 cells / well. At 37°C, 5% CO 2 Incubate in the incubator overnight.

[0134] Administration the next day: the concentration of each antibody was 200 nM, human PBMC was 50,000 cells / well, and the final volume was 200 μL / well. 37° C., 5% CO 2 Continue culturing in the incubator for 7 days.

[0135] Plate reading: After 7 days of treatment, remove 150μL of supernatant with a pipette, then add the remainder to 50μL / well of cell titer Glo, incubate for 10 minutes, read Luminescence, analyze and blot data with GraphPad Prism7.

[0136] The experimental results are shown in Figure 10, which shows that the killing activity of anti-PDL1 x EGFR BsAb1 is significantly greater than that of anti-PD-L1 monoclonal antibody and anti-EGFR monoclonal antibody, indicating that anti-PDL1 x EGFR BsAb1 exerts a synergistic killing effect.

[0137] Example 11. Antitumor effect of anti-PDL1 x EGFR bispecific antibodies in mouse xenograft tumor models 1. Antitumor effect of anti-PDL1 × EGFR bispecific antibody in SW48 xenograft tumor model This experiment is used to evaluate the anti-EGFR activity of anti-PDL1 × EGFR bispecific antibodies in animal models. SW48 colon cancer cells express EGFR on their surface, and anti-EGFR antibodies can effectively inhibit the proliferation of SW48 cells.

[0138] In vitro cultured colon cancer SW48 cells were harvested and cultured in serum-free medium at a cell concentration of 3 × 10 7 Adjust to 100 cells / mL. Under sterile conditions, inoculate 100 µL of the cell suspension subcutaneously into the flank of BALB / C nude mice. Measure the length and width of the xenograft tumor with a caliper, calculate the tumor volume, and determine whether the tumor is 100–200 mm. 3 After reaching maturity, the animals are randomly divided into groups.

[0139] The dose of the test sample anti-PDL1×EGFR BsAb1 was 13.3 mg / kg, and the dose of the positive control anti-EGFR monoclonal antibody was 10 mg / kg. The control group was administered the same amount of PBS. The administration method was intraperitoneal administration, the administration volume was 0.2 mL / mouse (20 g), and the administration was performed twice a week for three consecutive weeks.

[0140] The volume of xenograft tumors was measured twice a week, and the body weight of the mice was measured and recorded. The tumor volume (TV) was calculated as TV=1 / 2×length×width. 2 Based on the measurement results, the relative tumor volume (RTV) was calculated, and the formula was RTV=Vt / V0. Here, V0 is the tumor volume measured at the time of administration (i.e., d0) after group division, and Vt is the tumor volume at each measurement. The evaluation index of antitumor activity is the relative tumor growth rate T / C(%), and the calculation formula is T / C(%)=(TRTV / CRTV)×100 (TRTV: treatment group RTV, CRTV: negative control group RTV), and the tumor inhibition rate=1-T / C(%). Efficacy evaluation standard: T / C(%)>40% is ineffective, and T / C(%)≦40 and p≦0.05 after statistical processing are effective.

[0141] The experimental results are shown in FIG. 11, and there is no significant difference between anti-PDL1×EGFR BsAb1 and the positive control anti-EGFR monoclonal antibody.

[0142] 2. Antitumor effect of anti-PDL1×EGFR bispecific antibody in B-hPD-L1 humanized mouse MC38-hPD-L1 xenograft tumor model This experiment is used to evaluate the anti-PD-L1 activity of anti-PDL1 x EGFR bispecific antibodies in an animal model.

[0143] Mouse colon cancer MC38 cells were incubated at 37℃, 5% CO 2 The cells were cultured in a 1000-mL incubator using Dulbecco's Modified Eagle's (DMEM) medium containing 10% inactivated fetal bovine serum. MC38-hPDL1 cells (genetically modified with Biocytogen) overexpress human PD-L1 and simultaneously knock out mouse PD-L1. MC38-hPDL1 colon cancer cells suspended in PBS were cultured at 5 × 10 5 The cells were subcutaneously inoculated into the right flank of B-hPD-L1 humanized mice (Biocytogen, strain C57BL / 6) at a concentration of 0.1 mL / cell and a volume of 0.1 mL / mouse. The average tumor volume was approximately 138 mm 3 If the tumor volume reaches 100 mg / kg, select 8 mice with appropriate individual tumor volumes for the experiment.

[0144] The dose of anti-PDL1×EGFR BsAb1 was 27mg / kg, and the dose of the positive control anti-EGFR monoclonal antibody was 20mg / kg. The control group was administered with the same amount of PBS. The administration method was intraperitoneal administration, twice a week for three consecutive weeks. The volume of the xenograft tumor was measured twice a week, and the body weight of the mouse was measured and recorded at the same time.

[0145] The experimental results are shown in Figure 12. In the efficacy model of this experiment, compared with the control group (PBS), the test product anti-PDL1 x EGFR BsAb1 had a significant inhibitory effect on the growth of subcutaneous tumors in MC38-hPD-L1 cell-implanted B-hPD-L1 humanized mice at the measured dose, and there was no significant difference compared with the positive control anti-EGFR monoclonal antibody.

[0146] All documents mentioned in the present invention are incorporated by reference in this application as if each document was incorporated by reference individually. Furthermore, after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalents are also included in the scope defined by the appended claims of this application.

Claims

1. 1. An anti-PDL1 x EGFR bispecific antibody, Two polypeptide chains of group (a) or group (b) below and two light chains, (a) the polypeptide chain is, from the N-terminus to the C-terminus (VL-PDL1)-linker1-(VH-PDL1)-linker2-(VH-EGFR)-CH1-CH2-CH3, or (VH-PDL1)-linker1-(VL-PDL1)-linker2-(VH-EGFR)-CH1-CH2-CH3, or (VH-EGFR)-CH1-CH2-CH3-Linker2-(VL-PDL1)-Linker1-(VH-PDL1), or (VH-EGFR)-CH1-CH2-CH3-linker2-(VH-PDL1)-linker1-(VL-PDL1), the light chain comprises from N-terminus to C-terminus (VL-EGFR)-CL; or (b) the polypeptide chain is, from the N-terminus to the C-terminus (VL-EGFR)-linker1-(VH-EGFR)-linker2-(VH-PDL1)-CH1-CH2-CH3, or (VH-EGFR)-linker1-(VL-EGFR)-linker2-(VH-PDL1)-CH1-CH2-CH3, or (VH-PDL1)-CH1-CH2-CH3-Linker2-(VL-EGFR)-Linker1-(VH-EGFR), or (VH-PDL1)-CH1-CH2-CH3-linker2-(VH-EGFR)-linker1-(VL-EGFR), the light chain comprises from N-terminus to C-terminus (VL-PDL1)-CL, wherein the VL-PDL1 is a light chain variable region that binds to PD-L1, VH-PDL1 is a heavy chain variable region that binds to PD-L1; the VL-EGFR is a light chain variable region that binds to EGFR; VH-EGFR is a heavy chain variable region that binds to EGFR; said CH1-CH2-CH3 is a heavy chain constant region and said CL is a light chain constant region; said linker 1 and linker 2 are each independently a flexible peptide linker; the VL-PDL1 and the VH-PDL1 form an antigen-binding site that specifically binds to anti-PD-L1, the VH-EGFR and the VL-EGFR form an antigen-binding site that specifically binds to EGFR; The VL-PDL1 comprises a light chain CDR having an amino acid sequence as shown in SEQ ID NO: 1-3, the VH-PDL1 comprises a heavy chain CDR having an amino acid sequence as shown in SEQ ID NO: 4-6, the VH-EGFR comprises a heavy chain CDR having an amino acid sequence as shown in SEQ ID NO: 7-9, and the VL-EGFR comprises a light chain CDR having an amino acid sequence as shown in SEQ ID NO: 10-12. The PDL1 x EGFR bispecific antibody.

2. (a) two polypeptide chains comprising from N-terminus to C-terminus (VL-PDL1)-linker1-(VH-PDL1)-linker2-(VH-EGFR)-CH1-CH2-CH3; and (b) two light chains comprising from N-terminus to C-terminus (VL-EGFR)-CL. In the two polypeptide chains of (a), VL-PDL1 is a light chain variable region that binds to PD-L1, said linker 1 and linker 2 are each independently a flexible peptide linker; VH-EGFR is a heavy chain variable region that binds to EGFR; said CH1-CH2-CH3 is a heavy chain constant region, and In the two light chains of (b), the VL-EGFR is a light chain variable region that binds to EGFR, and the CL is a light chain constant region; wherein the VL-PDL1 and the VH-PDL1 form an antigen-binding site that specifically binds to PD-L1, and the VH-EGFR and the VL-EGFR form an antigen-binding site that specifically binds to EGFR.

2. The anti-PDL1 x EGFR bispecific antibody of claim 1.

3. (a) two polypeptide chains comprising from N-terminus to C-terminus: (VL-PDL1)-Linker1-(VH-PDL1)-Linker2-(VH-EGFR)-CH1-CH2-CH3; and (b) two light chains comprising from N-terminus to C-terminus: (VL-EGFR)-CL; In the two polypeptide chains of (a), the VL-PDL1 is a light chain variable region that binds to PD-L1, the linker 1 is 4 or 5 GGGGS, the VH-PDL1 is a heavy chain variable region that binds to PD-L1, the linker 2 is 3 or 4 GGGGS, the VH-EGFR is a heavy chain variable region that binds to EGFR, and the CH1-CH2-CH3 are heavy chain constant regions; and In the two light chains of (b), the VL-EGFR is a light chain variable region that binds to EGFR, and the CL is a light chain constant region; wherein the VL-PDL1 and the VH-PDL1 form an antigen-binding site that specifically binds to PD-L1, and the VH-EGFR and the VL-EGFR form an antigen-binding site that specifically binds to EGFR. The anti-PDL1 x EGFR bispecific antibody of claim 2.

4. The VL-PDL1 has an amino acid sequence shown in SEQ ID NO: 13, the VH-PDL1 has an amino acid sequence shown in SEQ ID NO: 14, the VH-EGFR has an amino acid sequence shown in SEQ ID NO: 15, and the VL-EGFR has an amino acid sequence shown in SEQ ID NO:

16.

2. The anti-PDL1 x EGFR bispecific antibody of claim 1.

5. the polypeptide chain has the amino acid sequence set forth in SEQ ID NO:17 or SEQ ID NO:18 or SEQ ID NO:23 or SEQ ID NO:24 or SEQ ID NO:25, and the light chain has the amino acid sequence set forth in SEQ ID NO:19; or the polypeptide chain has the amino acid sequence shown in SEQ ID NO: 26 or SEQ ID NO: 27 or SEQ ID NO: 28 or SEQ ID NO: 29, and the light chain has the amino acid sequence shown in SEQ ID NO:

22. The anti-PDL1 x EGFR bispecific antibody according to any one of claims 1 to 4.

6. The heavy chain constant region comprises an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, and the light chain constant region comprises a κ or λ light chain constant region. The anti-PDL1 x EGFR bispecific antibody according to any one of claims 1 to 5.

7. An isolated nucleotide, characterized in that it encodes a bispecific antibody described in any one of claims 1 to 6.

8. An expression vector comprising the nucleotide described in claim 7.

9. A host cell, characterized by containing the expression vector described in claim 8.

10. A method for preparing a bispecific antibody according to any one of claims 1 to 6, comprising the steps of: (a) expressing the bispecific antibody by culturing a host cell according to claim 9 under expression conditions; (b) isolating and purifying the bispecific antibody described in (a).

11. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 6 and a pharma- ceutically acceptable vector.

12. Use of a bispecific antibody according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 11 in the preparation of a medicament for treating cancer.

13. 13. The use according to claim 12, characterized in that the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer, squamous cell carcinoma, head and neck cancer, kidney cancer, bladder cancer, ovarian cancer, breast cancer, melanoma, lung cancer, liver cancer, gastric cancer, lymphoma, leukemia, prostate cancer, bone marrow cancer and other neoplastic malignancies.

14. A bispecific antibody according to any one of claims 1 to 6, or an immunoconjugate thereof, or a pharmaceutical composition according to claim 11, for use in cancer therapy.

15. 15. The bispecific antibody, or immunoconjugate or pharmaceutical composition thereof, according to claim 14, characterized in that the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer, squamous cell carcinoma, head and neck cancer, kidney cancer, bladder cancer, ovarian cancer, breast cancer, melanoma, lung cancer, liver cancer, gastric cancer, lymphoma, leukemia, prostate cancer, bone marrow cancer and other neoplastic malignancies.

16. 1. An immunoconjugate comprising: (a) a bispecific antibody according to any one of claims 1 to 6, and (b) a coupling moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

17. Use of the immunoconjugate described in claim 16, characterized in that it is used for the preparation of a pharmaceutical composition for treating tumors.

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