Antigen-binding proteins targeting PD-L1 and CD40 and their production and application

Abstract

The present invention discloses an antigen-binding protein targeting PD-L1 and CD40 and its preparation and application, which blocks the PD-1 / PD-L1 inhibitory signal pathway and acts on the activated CD40 receptor, thereby simultaneously activating antigen-presenting cells and lymphocytes, achieving significant synergistic anti-tumor effects, and the activation of immune cells by the antigen-binding protein targeting PD-L1 and CD40 only occurs at the tumor microenvironment site, significantly eliminating the problems of systemic drug toxicity and side effects, so that the antigen-binding protein targeting PD-L1 and CD40 exerts its excellent anti-tumor effects under very safe preconditions.

Description

[Technical field]

[0001] This application claims priority to Chinese patent application No. 202210351186X, filed on April 2, 2022. This application incorporates all of the above Chinese patent applications by reference.

[0002] The present invention relates to the field of biopharmaceuticals, in particular to a bispecific antigen-binding protein targeting PD-L1 and CD40 and its production and application. [Background technology]

[0003] CD40 is a glycosylated type I transmembrane protein and a member of the tumor necrosis factor receptor superfamily (TNFRSF), also called tumor necrosis factor receptor superfamily member 5 (TNFRSF5). CD40 is expressed on the surface of a series of antigen-presenting cells (APCs), including monocytes, dendritic cells (DCs), B cells, and macrophages. Its ligand, CD40L, is expressed primarily on the surface of lymphocytes, including T cells, B cells, and NK cells, and is usually present in trimeric and multimeric forms. CD40 and CD40L are a pair of costimulatory molecules, and activation of CD40 downstream signaling pathways requires the formation of trimeric and multimeric forms of CD40L crosslinked to CD40. They interact with the cell surface, causing CD40 to redistribute to membrane lipid rafts, generating a conformational change, and CD40 recruits TNFR-associated factors (TRAFs) in the cytoplasm via the intracellular terminal domain, promoting intracellular signal transduction, thereby activating different signal pathways, such as the classical and non-classical nuclear factor κB pathways, p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase (PI3K) and phospholipase Cγ pathways, and further regulating apoptosis, cell cycle progression, cytokine production and cell surface immune regulator expression by genes targeted by these signal pathways. Thus, activating CD40 can increase antigen presentation, promote cytokine secretion, activate lymphocytes, and stimulate and activate the human innate and adaptive immune systems, creating a synergistic effect and combating the development and progression of cancer.

[0004] CD40 is also widely expressed on tumor cells, and it is expressed on almost all B-cell malignancies and a wide range of solid tumors, including melanoma, lung cancer, breast cancer, colon cancer, prostate cancer, pancreatic cancer, renal cancer, ovarian cancer, and head and neck cancer. CD40 expressed on the surface of tumor cells can mediate tumor cell death. In the absence of immune helper cells, CD40 expressed on the surface of various tumor cells mediates direct cytotoxicity through CD40L crosslinking. In vitro, it is demonstrated that CD40 crosslinking by CD40L can induce tumor cell apoptosis and inhibit the proliferation of solid tumor cells and B malignant tumor cells. In vivo, activation of CD40 also mediates tumor inhibitory effects. In immunodeficient mice, evidence shows that interference with CD40 on the surface of tumor cells through CD40L can inhibit the proliferation of breast cancer cell transplants or B lymphocyte transplants, even in the absence of lymphocyte activation.

[0005] Therefore, the mechanism of CD40-mediated tumor cell death may be dual, i.e., the stimulation of tumor cell killing by the immune system and the direct tumor cell cytotoxicity can synergistically play an antitumor effect. Agonistic anti-CD40 antibodies, like CD40L, can crosslink and activate CD40 on the surface of immune cells and tumor cells, and play a significant antitumor effect. Such antitumor effects have been demonstrated in preclinical animal models and clinical trials in tumor patients, and can be combined with both chemotherapeutic agents such as gemcitabine and paclitaxel, and immunomodulators such as PD-1 antibodies and CTLA-4 antibodies to produce synergistic antitumor effects.

[0006] Currently, in terms of antitumor, there are nearly 20 CD40 antibody drugs in clinical trials, but the earliest products are only in phase 2 clinical trials, and there is no product on the market. The most major problems that CD40 antibodies face in clinical practice include low objective remission rates, significant toxic side effects, and low tolerated doses. The possible causes of these results are reduced agonist activity, such as Celldex's product CDX-1140. In its phase 1 clinical trial, 42 patients who received monotherapy did not achieve complete or partial remission, and only one of 20 patients achieved partial remission when combined with rhFLT3L. The product has weak activation against DC cells in vitro and cannot enhance agonist activity when crosslinked. The causes include the fact that CD40 antibodies are not selectively activated, resulting in systemic toxic side effects. For example, in clinical trials, Roche's CD40 antibody Selicrelumab caused toxic side effects such as significant cytokine release syndrome and liver toxicity in some test patients when the dose exceeded 0.2 mg / kg, and Apexigen's CD40 antibody APX005M caused significant neutropenia, and further sepsis and septic shock in some test patients when the dose exceeded 0.3 mg / kg in clinical trials. In summary, currently, clinical CD40 antibodies face problems and challenges in that the therapeutic window between efficacy and safety is too small. Therefore, the development of other CD40 antibodies offers the possibility of treating various tumors and has high scientific and commercial value.

[0007] PD-L1, or programmed death ligand 1, also known as CD274 or B7H1, is a glycosylated type I transmembrane protein and is one of the ligands of PD-1, or programmed death protein 1. Under normal circumstances, PD-L1 is widely expressed on antigen-presenting cells, such as dendritic cells, macrophages, and B cells, and other immune cells. When PD-L1 binds to the PD-1 receptor expressed on the surface of T cells, it inhibits T cell activation and cytokine secretion, and plays an important role in inducing immune contraction in the body and maintaining immune tolerance. On the other hand, PD-L1 is abundantly expressed on the surface of many cancer cells, including renal cell carcinoma, breast cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, papillary thyroid carcinoma, and testicular cancer. Binding of PD-L1 on tumor cells to PD-1 on tumor-infiltrating T cells (TILs) activates Src homology region 2 domain phosphatases (SHPs), leading to inhibition of the T cell receptor (TCR) pathway and inhibition of T cell activity. Furthermore, tumor cells exploit the PD-L1 / PD-1 signaling pathway to disrupt immune monitoring and promote cancer cell survival.

[0008] Therefore, blocking the PD-L1 / PD-1 signaling axis by antibodies can reactivate immune cells such as T cells that are inhibited and exhausted in the tumor microenvironment, thereby eliminating cancer cells and re-establishing immune balance. Based on the above findings, therapeutic PD-L1 antibodies (e.g., atezolizumab, avelumab, and durvalumab) and PD-1 antibodies (e.g., nivolumab, pembrolizumab, and cemiplimab) have already been developed and marketed, and have obtained 10% to 40% clinical responses in clinical trials against various cancers, including melanoma, small cell lung cancer, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, classical Hodgkin lymphoma, and Merkel cell carcinoma.

[0009] However, although anti-PD-L1 / PD-1 therapy has shown impressive effects in tumor therapy, especially in solid tumor therapy, only a small proportion of patients have sustained responses, and some patients who initially responded to the therapy eventually developed acquired resistance. At the same time, most patients still do not respond to anti-PD-L1 / PD-1 therapy, i.e., natural resistance. Therefore, it is urgent to develop an immunotherapy with better therapeutic effect that can produce a synergistic anti-tumor effect with anti-PD-L1 / PD-1 therapy to overcome natural resistance and acquired resistance, and at present, this method has attracted attention in tumor immunotherapy.

[0010] Currently, there is no antigen-binding protein that targets both PD-L1 and CD40 on the market, and there is a great market demand for an antigen-binding protein that targets both PD-L1 and CD40. Summary of the Invention [Problem to be solved by the invention]

[0011] In the prior art, antigen-binding proteins targeting PD-L1 and CD40 have technical shortcomings such as limited efficacy and low safety, and the present invention provides an antigen-binding protein targeting PD-L1 and CD40, and its preparation and application. The antigen-binding protein targeting PD-L1 and CD40 blocks the PD-1 / PD-L1 inhibitory signal pathway and acts on the activated CD40 receptor, thereby simultaneously activating antigen-presenting cells and lymphocytes, achieving a significant synergistic anti-tumor effect, and the activation of immune cells by the antigen-binding protein targeting PD-L1 and CD40 only occurs at the tumor microenvironment site, significantly eliminating the problems of systemic drug toxicity and side effects, so that the antigen-binding protein targeting PD-L1 and CD40 exerts its excellent anti-tumor effect under the precondition of being very safe. [Means for solving the problem]

[0012] In order to solve the above technical problems, in a first aspect of the present invention, there is provided an antigen binding protein targeting PD-L1 and CD40, the antigen binding protein comprising a first protein functional domain and a second protein functional domain, the first protein functional domain comprising an antigen binding protein targeting CD40, and the second protein functional domain comprising an antigen binding protein targeting PD-L1, wherein: The antigen binding protein targeting CD40 comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises LCDR1, LCDR2 and LCDR3, and wherein the VH comprises HCDR1, HCDR2 and HCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:38 or variant 1 having 3, 2 or 1 amino acid mutations in SEQ ID NO:38, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:43 or variant 2 having 3, 2 or 1 amino acid mutations in SEQ ID NO:43, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:48 or variant 3 having 3, 2 or 1 amino acid mutations in SEQ ID NO:48, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:8 or variant 4 having 3, 2 or 1 amino acid mutations in SEQ ID NO:8, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: and said HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18 or variant 5 having 3, 2 or 1 amino acid mutations in SEQ ID NO: 18, and said HCDR4 comprises the amino acid sequence shown in SEQ ID NO: 27 or variant 6 having 3, 2 or 1 amino acid mutations in SEQ ID NO: 27.

[0013] In some technical solutions of the present invention, the antigen-binding protein targeting CD40 comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprises LCDR1, LCDR2 and LCDR3, the VH comprises HCDR1, HCDR2 and HCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 38, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 43, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 48, or a variant of SEQ ID NO: 48 having 3, 2 or 1 amino acid mutations, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0014] In some technical solutions of the present invention, the variant 3 comprises an amino acid sequence having a PTM site mutation in the amino acid sequence shown in SEQ ID NO: 48, preferably an amino acid sequence having an amino acid mutation at position 4 and / or position 5 of the amino acid sequence shown in SEQ ID NO: 48, and the amino acid mutation is preferably an amino acid substitution, more preferably a conservative amino acid substitution. Preferably, the variant 3 is an amino acid sequence having an N4A / F / Y / V / N and / or S5N mutation in the amino acid sequence shown in SEQ ID NO: 48. More preferably, the variant 3 is an amino acid sequence shown in any one of SEQ ID NOs: 49 to 53.

[0015] In some technical solutions of the present invention, in the antigen-binding protein targeting CD40, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 38, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 43, the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 48 to 53, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27, and specific CDR combinations are shown in Table 1-1.

[0016] [Table 1]

[0017] In some technical solutions of the present invention, in the antigen-binding protein targeting CD40, the VL comprises an amino acid sequence shown in SEQ ID NO: 68 or an amino acid sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 68, and the VH comprises an amino acid sequence shown in SEQ ID NO: 60 or an amino acid sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 60. More preferably, the VL comprises an amino acid sequence shown in any one of SEQ ID NOs: 68 to 73, and the VH comprises an amino acid sequence shown in SEQ ID NO: 60, and specific combinations of VL and VH are shown in Table 1-2.

[0018] [Table 2]

[0019] In some technical solutions of the present invention, the antigen-binding protein targeting CD40 is a full-length antibody, the full-length antibody comprises a light chain and a heavy chain, the light chain comprises a light chain constant region (CL), the light chain constant region is preferably a human-derived antibody light chain constant region, and the human-derived antibody light chain constant region is more preferably a κ subtype light chain constant region, the heavy chain comprises a heavy chain constant region (CH), and the heavy chain constant region is preferably a human-derived antibody heavy chain constant region, more preferably a hIgG1, hIgG2, hIgG3 or hIgG4 subtype heavy chain constant region, and even more preferably a hIgG1 subtype heavy chain constant region.

[0020] In some technical solutions of the present invention, the antigen-binding protein targeting CD40 is a full-length antibody, the full-length antibody comprises a light chain and a heavy chain, the light chain comprises an amino acid sequence shown in any one of SEQ ID NOs: 87-92, and the heavy chain comprises an amino acid sequence shown in SEQ ID NO: 78 or 84. Preferably, the light chain comprises an amino acid sequence shown in SEQ ID NO: 87, the heavy chain comprises an amino acid sequence shown in SEQ ID NO: 78, or the light chain comprises an amino acid sequence shown in any one of SEQ ID NOs: 88-92, and the heavy chain comprises an amino acid sequence shown in SEQ ID NO: 84, and specific combinations of light chains and heavy chains are shown in Tables 1-3.

[0021] [Table 3]

[0022] In some technical solutions of the present invention, the antigen binding protein targeting PD-L1 comprises a heavy chain variable region (VH), the VH comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:5 or variant 7 of SEQ ID NO:5 having 3, 2 or 1 amino acid mutations, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:16 or variant 8 of SEQ ID NO:16 having 3, 2 or 1 amino acid mutations, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:25 or variant 9 of SEQ ID NO:25 having 3, 2 or 1 amino acid mutations.

[0023] In some technical solutions of the present invention, the variant 7 is an amino acid sequence of SEQ ID NO: 5 with an amino acid mutation at position 3 and / or 6, the variant 8 is an amino acid sequence of SEQ ID NO: 16 with an amino acid mutation at position 1, 3 and / or 6, and the variant 9 is an amino acid sequence of SEQ ID NO: 25 with an amino acid mutation at position 4, 8 and / or 11, and the amino acid mutation is preferably an amino acid substitution, more preferably a conservative amino acid substitution. More preferably, the variant 7 is an amino acid sequence of SEQ ID NO: 5 with an N3T / D and / or N5S mutation, the variant 8 is an amino acid sequence of SEQ ID NO: 16 with a W1R, D3T and / or K5E mutation, and the variant 9 is an amino acid sequence of SEQ ID NO: 25 with an I4L, V8I and / or A11D mutation. Preferably, said variant 7 has the amino acid sequence shown in SEQ ID NO: 7 or 9, said variant 8 has the amino acid sequence shown in SEQ ID NO: 19, and said variant 9 has the amino acid sequence shown in SEQ ID NO: 28, 29 or 30.

[0024] In some technical solutions of the present invention, in the antigen binding protein targeting PD-L1, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 5, 7 or 9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 16 or 19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 25, 28, 29 or 30.

[0025] In some technical solutions of the present invention, in the antigen binding protein targeting PD-L1, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:5, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:16, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:25; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:7, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:16, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:25; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:7, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:16, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:25; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:7, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:28; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: or said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:19 and said HCDR3 comprises the amino acid sequence shown in SEQ ID NO:29; or said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:19 and said HCDR3 comprises the amino acid sequence shown in SEQ ID NO:30; or said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:19 and said HCDR3 comprises the amino acid sequence shown in SEQ ID NO:28.

[0026] In some technical solutions of the present invention, in the antigen-binding protein targeting PD-L1, the VH comprises the amino acid sequence of SEQ ID NO: 57 or an amino acid sequence having at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 57. More preferably, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 57 to 65.

[0027] In some technical solutions of the present invention, the first protein functional domain is an immunoglobulin comprising an antigen binding protein that targets CD40, and the second protein functional domain comprises the VH of one, two or more antigen binding proteins that target PD-L1.

[0028] In some technical solutions of the present invention, the VH of the PD-L1-targeting antigen-binding protein is directly linked to the immunoglobulin, or the VH is linked to the immunoglobulin via a linker, or if the number of VHs is greater than 1, each VH is linked to the immunoglobulin directly or via a linker, respectively.

[0029] In some technical solutions of the present invention, when the VH is linked to the immunoglobulin via a linker, the linker is selected from GS, GGS, and any one of the amino acid sequences shown in SEQ ID NO: 100 to 106.

[0030] In some technical solutions of the present invention, the second protein functional region comprises the VHs of two antigen-binding proteins targeting PD-L1. Preferably, the two VHs comprised in the second protein functional region are the same and / or the C-terminus of the two VHs is linked to the N-terminus of two heavy chains of the immunoglobulin via a linker as shown in GGS or SEQ ID NO: 5, respectively.

[0031] In some technical solutions of the present invention, the first protein functional region comprises two light chains having the amino acid sequence shown in any one of SEQ ID NOs: 88 to 92 and two heavy chains having the amino acid sequence shown in SEQ ID NO: 84; the second protein functional region comprises two VHs having the amino acid sequence shown in any one of SEQ ID NOs: 79 to 83, and the C-terminus of the two VHs is linked to the N-terminus of the two heavy chains of the first protein functional region via a linker shown in GGS or SEQ ID NO: 5.

[0032] Suitably, the antigen binding protein that targets PD-L1 and CD40 comprises: a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:89, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:93, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:88, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:94, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:94, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:90, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:93, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:92, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:93, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:88, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:95, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:96 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:88, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:97 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:95 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:93 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:96 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:89, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:97 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:90, a first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 97, or a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 88;The present invention relates to a method for producing a polypeptide comprising the steps of: a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:98, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:88; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:99, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:98, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91, or a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:99.

[0033] In order to solve the above technical problems, in a second aspect of the present invention, there is provided an antigen binding protein targeting PD-L1 and CD40, the antigen binding protein comprising a first protein functional domain and a second protein functional domain, the first protein functional domain comprising an antigen binding protein targeting CD40, and the second protein functional domain comprising an antigen binding protein targeting PD-L1, wherein: The antigen binding protein targeting PD-L1 comprises a heavy chain variable region (VH), the VH comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:5 or variant 7 of SEQ ID NO:5 having 3, 2 or 1 amino acid mutations, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:16 or variant 8 of SEQ ID NO:16 having 3, 2 or 1 amino acid mutations, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:25 or variant 9 of SEQ ID NO:25 having 3, 2 or 1 amino acid mutations.

[0034] In some technical solutions of the present invention, the variant 7 is an amino acid sequence of SEQ ID NO: 5 with an amino acid mutation at position 3 and / or 6, the variant 8 is an amino acid sequence of SEQ ID NO: 16 with an amino acid mutation at position 1, 3 and / or 6, and the variant 9 is an amino acid sequence of SEQ ID NO: 25 with an amino acid mutation at position 4, 8 and / or 11, and the amino acid mutation is preferably an amino acid substitution, more preferably a conservative amino acid substitution. More preferably, the variant 7 is an amino acid sequence of SEQ ID NO: 5 with an N3T / D and / or N5S mutation, the variant 8 is an amino acid sequence of SEQ ID NO: 16 with a W1R, D3T and / or K5E mutation, and the variant 9 is an amino acid sequence of SEQ ID NO: 25 with an I4L, V8I and / or A11D mutation. Preferably, said variant 7 has the amino acid sequence shown in SEQ ID NO: 7 or 9, said variant 8 has the amino acid sequence shown in SEQ ID NO: 19, and said variant 9 has the amino acid sequence shown in SEQ ID NO: 28, 29 or 30.

[0035] In some technical solutions of the present invention, in the antigen binding protein targeting PD-L1, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 5, 7 or 9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 16 or 19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 25, 28, 29 or 30.

[0036] In some technical solutions of the present invention, in the antigen binding protein targeting PD-L1, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:5, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:16, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:25; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:7, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:16, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:25; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:7, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:16, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:25; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:7, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:28; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: or said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:19 and said HCDR3 comprises the amino acid sequence shown in SEQ ID NO:29; or said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:19 and said HCDR3 comprises the amino acid sequence shown in SEQ ID NO:30; or said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:19 and said HCDR3 comprises the amino acid sequence shown in SEQ ID NO:28.

[0037] In some technical solutions of the present invention, in the antigen-binding protein targeting PD-L1, the VH comprises the amino acid sequence of SEQ ID NO: 57 or an amino acid sequence having at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 57. More preferably, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 57 to 65.

[0038] In some technical solutions of the present invention, the antigen-binding protein targeting CD40 comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprises LCDR1, LCDR2 and LCDR3, the VH comprises HCDR1, HCDR2 and HCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 38 or variant 1 having 3, 2 or 1 amino acid mutations in SEQ ID NO: 38, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 43 or variant 2 having 3, 2 or 1 amino acid mutations in SEQ ID NO: 43, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 48 or variant 3 having 3, 2 or 1 amino acid mutations in SEQ ID NO: 48, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8 or variant 4 having 3, 2 or 1 amino acid mutations in SEQ ID NO: 8, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: and said HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18 or variant 5 having 3, 2 or 1 amino acid mutations in SEQ ID NO: 18, and said HCDR4 comprises the amino acid sequence shown in SEQ ID NO: 27 or variant 6 having 3, 2 or 1 amino acid mutations in SEQ ID NO: 27.

[0039] In some technical solutions of the present invention, the antigen-binding protein targeting CD40 comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprises LCDR1, LCDR2 and LCDR3, the VH comprises HCDR1, HCDR2 and HCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 38, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 43, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 48, or a variant of SEQ ID NO: 48 having 3, 2 or 1 amino acid mutations, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0040] In some technical solutions of the present invention, the variant 3 comprises an amino acid sequence having a PTM site mutation in the amino acid sequence shown in SEQ ID NO: 48, preferably an amino acid sequence having an amino acid mutation at position 4 and / or position 5 of the amino acid sequence shown in SEQ ID NO: 48, and the amino acid mutation is preferably an amino acid substitution, more preferably a conservative amino acid substitution. Preferably, the variant 3 is an amino acid sequence having an N4A / F / Y / V / N and / or S5N mutation in the amino acid sequence shown in SEQ ID NO: 48. More preferably, the variant 3 is an amino acid sequence shown in any one of SEQ ID NOs: 49 to 53.

[0041] In some technical solutions of the present invention, in the antigen-binding protein targeting CD40, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 38, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 43, the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 48 to 53, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27, and specific CDR combinations are shown in Table 1-1.

[0042] In some technical solutions of the present invention, in the antigen-binding protein targeting CD40, the VL comprises an amino acid sequence shown in SEQ ID NO: 68 or an amino acid sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 68, and the VH comprises an amino acid sequence shown in SEQ ID NO: 60 or an amino acid sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 60. More preferably, the VL comprises an amino acid sequence shown in any one of SEQ ID NOs: 68 to 73, and the VH comprises an amino acid sequence shown in SEQ ID NO: 60, and specific combinations of VL and VH are shown in Table 1-2.

[0043] In some technical solutions of the present invention, the antigen-binding protein targeting CD40 is a full-length antibody, the full-length antibody comprises a light chain and a heavy chain, the light chain comprises a light chain constant region (CL), the light chain constant region is preferably a human-derived antibody light chain constant region, and the human-derived antibody light chain constant region is more preferably a κ subtype light chain constant region, the heavy chain comprises a heavy chain constant region (CH), and the heavy chain constant region is preferably a human-derived antibody heavy chain constant region, more preferably a hIgG1, hIgG2, hIgG3 or hIgG4 subtype heavy chain constant region, and even more preferably a hIgG1 subtype heavy chain constant region.

[0044] In some technical solutions of the present invention, the antigen-binding protein targeting CD40 is a full-length antibody, the full-length antibody comprises a light chain and a heavy chain, the light chain comprises an amino acid sequence shown in any one of SEQ ID NOs: 87-92, and the heavy chain comprises an amino acid sequence shown in SEQ ID NO: 78 or 84. Preferably, the light chain comprises an amino acid sequence shown in SEQ ID NO: 87, the heavy chain comprises an amino acid sequence shown in SEQ ID NO: 78, or the light chain comprises an amino acid sequence shown in any one of SEQ ID NOs: 88-92, and the heavy chain comprises an amino acid sequence shown in SEQ ID NO: 84, and specific combinations of light chains and heavy chains are shown in Tables 1-3.

[0045] In some technical solutions of the present invention, the first protein functional domain is an immunoglobulin comprising an antigen binding protein that targets CD40, and the second protein functional domain comprises the VH of one, two or more antigen binding proteins that target PD-L1.

[0046] In some technical solutions of the present invention, the VH of the PD-L1-targeting antigen-binding protein is directly linked to the immunoglobulin, or the VH is linked to the immunoglobulin via a linker, or if the number of VHs is greater than 1, each VH is linked to the immunoglobulin directly or via a linker, respectively.

[0047] In some technical solutions of the present invention, when the VH is linked to the immunoglobulin via a linker, the linker is selected from GS, GGS, and any one of the amino acid sequences shown in SEQ ID NO: 100 to 106.

[0048] In some technical solutions of the present invention, the second protein functional region comprises the VHs of two antigen-binding proteins targeting PD-L1. Preferably, the two VHs comprised in the second protein functional region are the same and / or the C-terminus of the two VHs is linked to the N-terminus of two heavy chains of the immunoglobulin via a linker as shown in GGS or SEQ ID NO: 5, respectively.

[0049] In some technical solutions of the present invention, the first protein functional region comprises two light chains having the amino acid sequence shown in any one of SEQ ID NOs: 88 to 92 and two heavy chains having the amino acid sequence shown in SEQ ID NO: 84; the second protein functional region comprises two VHs having the amino acid sequence shown in any one of SEQ ID NOs: 79 to 83, and the C-terminus of the two VHs is linked to the N-terminus of the two heavy chains of the first protein functional region via a linker shown in GGS or SEQ ID NO: 5.

[0050] Suitably, the antigen binding protein that targets PD-L1 and CD40 comprises: a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:89, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:93, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:88, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:94, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:94, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:90, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:93, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:92, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:93, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:88, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:95, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:96 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:88, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:97 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:95 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:93 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:96 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:89, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:97 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:90, a first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 97, or a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 88;The present invention relates to a method for producing a polypeptide comprising the steps of: a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:98, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:88; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:99, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91; a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:98, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91, or a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:99.

[0051] In the present invention, the variant 1, variant 2, variant 3, variant 4, variant 5, variant 6, variant 7, variant 8 and variant 9 are merely for distinguishing different variants, the first protein functional region and the second protein functional region are merely for distinguishing different protein functions, the first polypeptide chain and the second polypeptide chain are merely for distinguishing different polypeptide chains, and none of the numbers and numerals have any actual meaning.

[0052] The amino acid sequences of the CDRs in the present invention are determined according to the Chothia definition rules.

[0053] In order to solve the above technical problems, in another aspect of the present invention, a chimeric antigen receptor (CAR) is provided, wherein the chimeric antigen receptor comprises an antigen binding protein that targets PD-L1 and CD40 as described in the present invention.

[0054] In order to solve the above technical problems, in another aspect of the present invention, there is provided an isolated nucleic acid, wherein the isolated nucleic acid encodes an antigen binding protein that targets PD-L1 and CD40 according to the present invention.

[0055] In order to solve the above technical problems, in another aspect of the present invention, there is provided a recombinant expression vector, comprising the isolated nucleic acid according to the present invention. Preferably, the recombinant expression vector comprises a eukaryotic expression vector and / or a prokaryotic expression vector.

[0056] In order to solve the above technical problem, in another aspect of the present invention, there is provided a transformant, the transformant comprising the isolated nucleic acid according to the present invention or the recombinant expression vector according to the present invention. Suitably, the host cell of the transformant is a prokaryotic cell and / or a eukaryotic cell, the prokaryotic cell is preferably an E. coli cell such as TG1, BL21, and the eukaryotic cell is preferably a HEK293 cell or a CHO cell.

[0057] In order to solve the above technical problems, in another aspect of the present invention, there is provided a method for producing an antigen binding protein targeting PD-L1 and CD40 described in the present invention, the method comprising the steps of culturing a transformant described in the present invention, and obtaining the antigen binding protein targeting PD-L1 and CD40 from the culture.

[0058] In order to solve the above technical problems, in another aspect of the present invention, there is provided an antibody-drug conjugate (ADC), which comprises an antigen-binding protein targeting PD-L1 and CD40 according to the present invention, and a cytotoxic agent or tag. Preferably, the cytotoxic agent is MMAF or MMAE, and the tag is a fluorescent agent.

[0059] In order to solve the above technical problem, in another aspect of the present invention, there is provided a genetically modified cell, which expresses a chimeric antigen receptor according to the present invention. Suitably, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell, more preferably an immune cell such as a T cell or a NK cell.

[0060] In order to solve the above technical problems, in another aspect of the present invention, there is provided a pharmaceutical composition, comprising one or more of the antigen binding protein targeting PD-L1 and CD40 described in the present invention, the chimeric antigen receptor described in the present invention, the isolated nucleic acid described in the present invention, the recombinant expression vector described in the present invention, the genetically modified cell described in the present invention, and the antibody drug conjugate described in the present invention, and / or a pharma- ceutical acceptable carrier. Preferably, the pharmaceutical composition further comprises another anti-tumor drug as an active ingredient. The anti-tumor drug includes, but is not limited to, a chemotherapeutic drug, a nucleotide drug, a small molecule targeted drug, a monoclonal antibody drug, a bi / multispecific antibody drug, a recombinant protein drug, an immunomodulatory drug, a cell therapy drug, and a gene therapy drug.

[0061] In order to solve the above technical problems, in another aspect of the present invention, a detection reagent is provided, the detection reagent comprises an antigen binding protein targeting PD-L1 and CD40 as described in the present invention and / or an antibody drug conjugate as described in the present invention.Preferably, the detection reagent is in a liquid dosage form, a gas dosage form, a solid dosage form and a semi-solid dosage form.More preferably, the detection reagent further comprises a secondary antibody, CD40 or a derivative thereof, the secondary antibody, for example, an anti-human IgG antibody, is bound to horseradish peroxidase, and the anti-human IgG antibody is bound to a biotinylated protein.

[0062] In order to solve the above technical problems, in another aspect of the present invention, there is provided a kit-of-parts, comprising Kit A, wherein Kit A contains one or more of the antigen binding protein targeting PD-L1 and CD40 described in the present invention, the chimeric antigen receptor described in the present invention, the pharmaceutical composition described in the present invention, the detection reagent described in the present invention, the genetically modified cell described in the present invention, and the antibody drug conjugate described in the present invention.

[0063] Preferably, the kit of parts further comprises kit B, wherein kit B contains another anti-tumor antibody or a pharmaceutical composition comprising said other anti-tumor antibody, and / or one or more of the group consisting of a hormonal preparation, a targeted small molecule preparation, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic agent, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.

[0064] In order to solve the above technical problems, another aspect of the present invention provides an application of one or more of the antigen binding protein targeting PD-L1 and CD40 described in the present invention, the chimeric antigen receptor described in the present invention, the isolated nucleic acid described in the present invention, the recombinant expression vector described in the present invention, the pharmaceutical composition described in the present invention, the detection reagent described in the present invention, the kit of parts described in the present invention, the genetically modified cell described in the present invention, and the antibody drug conjugate described in the present invention in the manufacture of a medicament for diagnosing, preventing and / or treating a disease associated with PD-L1 and / or CD40. Preferably, the PD-L1 and / or CD40 associated disease is a PD-L1 associated tumor, a CD40 associated tumor, or a PD-L1×CD40 associated tumor. Preferably, the CD40 associated tumor includes a solid tumor and a hematological tumor, and the PD-L1 associated tumor includes a solid tumor and a hematological tumor.More preferably, the CD40-associated tumors include B-lineage NHL, chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), Hodgkin's disease, multiple myeloma, bladder cancer, kidney cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, nasopharyngeal cancer, malignant melanoma, pancreatic cancer, and colon cancer, and the PD-L1-associated tumors include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary longitudinal myeloma, myeloma, pulmonary arterial leukemia, and myeloma. Large B-cell lymphoma, Mantle cell lymphoma (MCL), Small lymphocytic lymphoma (SLL), Large B-cell lymphoma rich in T cells / histiocytes, Multiple myeloma, Myeloid cell leukemia-1 protein (Mcl-1), Myelodysplastic syndrome (MDS), Gastrointestinal (tubal) cancer, Renal cancer, Ovarian cancer, Liver cancer, Lymphoblastic leukemia, Lymphocytic leukemia, Colorectal cancer, Endometrial cancer, Kidney cancer, Prostate cancer, Thyroid cancer, Melanoma, Chondrosarcoma, Neuroblastoma, Pancreatic cancer, Glioblastoma multiforme, Gastric cancer, Bone cancer, Ewing's sarcoma, Cervical cancer, Brain cancer, Gastric cancer, Bladder cancer, Hepatocellular carcinoma, Breast cancer, Colon cancer, Hepatocellular carcinoma (HCC), Clear cell renal cell carcinoma (RCC), Head and neck cancer, Pharyngeal cancer, Hepatobiliary cancer cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light-chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative neoplasm, neuroendocrine tumor, Merkel cell carcinoma, testicular cancer and skin cancer, preferably colon cancer with high PD-L1 expression.

[0065] In order to solve the above technical problems, another aspect of the present invention provides a method for detecting PD-L1 and / or CD40 in a sample, the method comprising contacting the sample with one or more of the antigen binding proteins targeting PD-L1 and CD40 described in the present invention, the detection reagent described in the present invention, and the antibody drug conjugate described in the present invention. The sample includes, for example, a blood sample (e.g., a whole blood sample and a serum sample), and a reagent containing PD-L1 and / or CD40. Suitably, the method is not intended for treatment / diagnosis, for example, in scientific research, to detect the concentration of PD-L1 and / or CD40 standards, whether other reagents are contaminated with PD-L1 and / or CD40, etc.

[0066] In order to solve the above technical problems, in another aspect of the present invention, there is provided a method for diagnosing, treating and / or preventing a disease associated with PD-L1 and / or CD40, the method comprising administering to a patient in need thereof a therapeutically effective amount of one or more of the antigen binding protein targeting PD-L1 and CD40 described in the present invention, the chimeric antigen receptor described in the present invention, the isolated nucleic acid described in the present invention, the recombinant expression vector described in the present invention, the pharmaceutical composition described in the present invention, the detection reagent described in the present invention, the genetically modified cell described in the present invention and the antibody drug conjugate described in the present invention, or diagnosing or treating a patient in need thereof with a kit of parts described in the present invention. Preferably, the PD-L1 and / or CD40 associated disease is a PD-L1 associated tumor, a CD40 associated tumor or a PD-L1 x CD40 associated tumor. Preferably, the CD40 associated tumor includes a solid tumor and a hematological tumor, and the PD-L1 associated tumor includes a solid tumor and a hematological tumor.More preferably, the CD40-associated tumors include B-lineage NHL, chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), Hodgkin's disease, multiple myeloma, bladder cancer, kidney cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, nasopharyngeal cancer, malignant melanoma, pancreatic cancer, and colon cancer, and the PD-L1-associated tumors include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary longitudinal myeloma, myeloma, pulmonary arterial leukemia, and myeloma. Large B-cell lymphoma, Mantle cell lymphoma (MCL), Small lymphocytic lymphoma (SLL), Large B-cell lymphoma rich in T cells / histiocytes, Multiple myeloma, Myeloid cell leukemia-1 protein (Mcl-1), Myelodysplastic syndrome (MDS), Gastrointestinal (tubal) cancer, Renal cancer, Ovarian cancer, Liver cancer, Lymphoblastic leukemia, Lymphocytic leukemia, Colorectal cancer, Endometrial cancer, Kidney cancer, Prostate cancer, Thyroid cancer, Melanoma, Chondrosarcoma, Neuroblastoma, Pancreatic cancer, Glioblastoma multiforme, Gastric cancer, Bone cancer, Ewing's sarcoma, Cervical cancer, Brain cancer, Gastric cancer, Bladder cancer, Hepatocellular carcinoma, Breast cancer, Colon cancer, Hepatocellular carcinoma (HCC), Clear cell renal cell carcinoma (RCC), Head and neck cancer, Pharyngeal cancer, Hepatobiliary cancer cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light-chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative neoplasm, neuroendocrine tumor, Merkel cell carcinoma, testicular cancer and skin cancer, preferably colon cancer with high PD-L1 expression.

[0067] In order to solve the above technical problems, in another aspect of the present invention, there is provided an antigen binding protein targeting PD-L1 and CD40 as described in the present invention, a chimeric antigen receptor as described in the present invention, an isolated nucleic acid as described in the present invention, a recombinant expression vector as described in the present invention, a pharmaceutical composition as described in the present invention, a detection reagent as described in the present invention, a kit of parts as described in the present invention, a genetically modified cell as described in the present invention and / or an antibody drug conjugate as described in the present invention for diagnosing, preventing and / or treating a disease associated with PD-L1 and / or CD40. Preferably, the PD-L1 and / or CD40 associated disease is a PD-L1 associated tumor, a CD40 associated tumor or a PD-L1×CD40 associated tumor. Preferably, the CD40 associated tumor includes a solid tumor and a hematological tumor, and the PD-L1 associated tumor includes a solid tumor and a hematological tumor.More preferably, the CD40-associated tumors include B-lineage NHL, chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), Hodgkin's disease, multiple myeloma, bladder cancer, kidney cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, nasopharyngeal cancer, malignant melanoma, pancreatic cancer, and colon cancer, and the PD-L1-associated tumors include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary longitudinal myeloma, myeloma, pulmonary arterial leukemia, and myeloma. Large B-cell lymphoma, Mantle cell lymphoma (MCL), Small lymphocytic lymphoma (SLL), Large B-cell lymphoma rich in T cells / histiocytes, Multiple myeloma, Myeloid cell leukemia-1 protein (Mcl-1), Myelodysplastic syndrome (MDS), Gastrointestinal (tubal) cancer, Renal cancer, Ovarian cancer, Liver cancer, Lymphoblastic leukemia, Lymphocytic leukemia, Colorectal cancer, Endometrial cancer, Kidney cancer, Prostate cancer, Thyroid cancer, Melanoma, Chondrosarcoma, Neuroblastoma, Pancreatic cancer, Glioblastoma multiforme, Gastric cancer, Bone cancer, Ewing's sarcoma, Cervical cancer, Brain cancer, Gastric cancer, Bladder cancer, Hepatocellular carcinoma, Breast cancer, Colon cancer, Hepatocellular carcinoma (HCC), Clear cell renal cell carcinoma (RCC), Head and neck cancer, Pharyngeal cancer, Hepatobiliary cancer cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light-chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative neoplasm, neuroendocrine tumor, Merkel cell carcinoma, testicular cancer and skin cancer, preferably colon cancer with high PD-L1 expression.

[0068] In the present invention, "having 3, 2 or 1 amino acid mutations" includes insertion, deletion or substitution of amino acids based on the original amino acid sequence. As an exemplary interpretation, the mutations to the CDRs may include 3, 2 or 1 amino acid mutations, and these CDRs may be optionally mutated with the same or different number of amino acid residues selected, for example, one amino acid mutation in CDR1, and no mutations in CDR2 and CDR3.

[0069] In the present invention, "PTM site mutation" refers to the presence of amino acid mutations at the PTM site in the mutant sequence compared to the original amino acid sequence. The method of designing the mutations varies depending on the antibody sequence and the PTM sequence pattern. One method is to replace the "hot spot" amino acid (e.g., N or S in the NS pattern) with an amino acid with similar physicochemical properties (e.g., mutate N to Q). If the PTM sequence pattern is derived from somatic hypermutation but does not exist in the germline gene sequence, another method is to replace the sequence pattern with the corresponding germline gene sequence.

[0070] In the present invention, the VH, VL or full-length antibody may be mutated based on a limited sequence, where the mutation is a deletion, substitution or addition of one or more amino acid residues in the limited amino acid sequence, and the mutated amino acid sequence has at least 80% sequence identity with the limited amino acid sequence, and maintains or improves the binding activity of the antigen-binding protein of the amino acid sequence containing the mutation, and the at least 80% sequence identity is preferably at least 85% sequence identity, 90% sequence identity, more preferably at least 95% sequence identity, and most preferably at least 99% sequence identity.

[0071] In the present invention, the terms "comprise", "include", and "are" have the same meaning in some specific embodiments.

[0072] Each of the above preferred conditions can be arbitrarily combined in accordance with common knowledge in the art to obtain each of the preferred embodiments of the present invention.

[0073] All of the reagents and materials used in the present invention are commercially available.

[0074] The positive effects of the present invention are as follows: The antigen-binding protein targeting CD40 described in the present invention has the properties of high affinity to CD40 and strong agonism activity on signal pathways, especially the agonism activity is enhanced after crosslinking, so that the antigen-binding protein targeting CD40 described in the present invention has a larger therapeutic window and provides a good basis for significantly improving the response rate of patients in clinical trials under safe and tolerable dose conditions, and the antigen-binding protein targeting PD-L1 described in the present invention has high affinity to PD-L1 and can effectively inhibit the biological activity of PD-L1. Moreover, the antigen-binding protein targeting PD-L1 and CD40 described in the present invention has excellent antitumor effect, good safety and excellent drug formability. The antigen-binding protein targeting PD-L1 and CD40 described in the present invention blocks the PD-1 / PD-L1 inhibitory signal pathway and acts on the activated CD40 receptor, thereby simultaneously activating antigen-presenting cells and lymphocytes, enhancing the presentation of tumor antigens to lymphocytes by antigen-presenting cells, and promoting the response of lymphocytes, thereby achieving a significant synergistic anti-tumor effect. At the same time, the antigen-binding protein targeting PD-L1 and CD40 only activates immune cells at the tumor microenvironment site, significantly eliminating the problems of systemic drug toxicity and side effects. Therefore, the antigen-binding protein can exert its excellent anti-tumor effect under very safe preconditions. At present, there are no new antibody drugs combining these targets on the market at home and abroad. It is expected that this invention will bring new opportunities to the treatment of many tumors. [Brief description of the drawings]

[0075] [Figure 1A] FIG. 1 shows the binding levels of some of the CD40 antibodies of the present invention to Raji cells that highly express human CD40. [Figure 1B] FIG. 1 shows the binding levels of some of the CD40 antibodies of the present invention to Raji cells that highly express human CD40. [Figure 1C] FIG. 1 shows the binding levels of some of the CD40 antibodies of the present invention to Raji cells that highly express human CD40. [Figure 1D]FIG. 1 shows the binding levels of some of the CD40 antibodies of the present invention to Raji cells that highly express human CD40. [Figure 2A] FIG. 2A shows the activating effect of PR003379 on HEK293-hCD40-NFκB fluorescent reporter gene cells in the presence or absence of human CD32B-expressing cell-mediated crosslinking. [Figure 2B] FIG. 2B shows the activating effect of control Selicrelumab on HEK293-hCD40-NFκB fluorescent reporter gene cells in the presence or absence of CD32 B cell-mediated crosslinking. [Figure 2C] FIG. 2C shows the activating effect of other CD40 antibodies on HEK293-hCD40-NFκB fluorescent reporter gene cells in the presence of CD32B cell-mediated cross-linking. [Figure 2D] FIG. 2D shows the activating effect of other CD40 antibodies on HEK293-hCD40-NFκB fluorescent reporter gene cells in the absence of human CD32B-expressing cell-mediated cross-linking. [Figure 3A] Figure 2 shows the binding levels of some PD-L1 antibodies of the invention to CHO-K1 cells, CHO-K1 / hPD-L1, which highly express human PD-L1. [Figure 3B] Figure 2 shows the binding levels of some PD-L1 antibodies of the invention to CHO-K1 cells, CHO-K1 / hPD-L1, which highly express human PD-L1. [Figure 4A] Figure 2 shows the inhibitory effects of some PD-L1 antibodies of the present invention on the PD-1 signal pathway in a reporter gene cell experiment. [Figure 4B] Figure 2 shows the inhibitory effects of some PD-L1 antibodies of the present invention on the PD-1 signal pathway in a reporter gene cell experiment. [Diagram 5] Schematic diagram of the molecular structure of PD-L1×CD40 bispecific binding protein. [Figure 6A] Figure 2 shows the binding levels of some PD-L1 x CD40 bispecific binding proteins of the invention to human PD-L1 cells CHO-K1 / hPD-L1, respectively. [Figure 6B] Figure 2 shows the binding levels of some PD-L1 x CD40 bispecific binding proteins of the invention to human PD-L1 cells CHO-K1 / hPD-L1, respectively. [Figure 7A] Figure 2 shows the binding levels of some PD-L1 x CD40 bispecific binding proteins of the invention to cynomolgus monkey PD-L1 cells CHO-K1 / cyPD-L, respectively. [Figure 7B] Figure 2 shows the binding levels of some PD-L1 x CD40 bispecific binding proteins of the invention to cynomolgus monkey PD-L1 cells CHO-K1 / cyPD-L, respectively. [Figure 8A] Figure 2 shows the binding levels of some PD-L1 x CD40 bispecific binding proteins of the invention to human CD40 cells CHO-K1 / hCD40. [Figure 8B] Figure 2 shows the binding levels of some PD-L1 x CD40 bispecific binding proteins of the invention to human CD40 cells CHO-K1 / hCD40. [Figure 9A] Figure 2 shows the binding levels of some PD-L1 x CD40 bispecific binding proteins of the invention to cynomolgus monkey CD40 cells CHO-K1 / cyCD40. [Figure 9B] Figure 2 shows the binding levels of some PD-L1 x CD40 bispecific binding proteins of the invention to cynomolgus monkey CD40 cells CHO-K1 / cyCD40. [Figure 10A] Figure 2 shows that some PD-L1 x CD40 bispecific binding proteins of the invention do not bind to CHO-K1 cells. [Figure 10B] Figure 2 shows that some PD-L1 x CD40 bispecific binding proteins of the invention do not bind to CHO-K1 cells. [Figure 11A] Figure 2 shows the binding levels of some PD-L1 x CD40 bispecific binding proteins of the invention to NCI-H226 cells with high expression of human PD-L1. [Figure 11B]Figure 2 shows the binding levels of some PD-L1 x CD40 bispecific binding proteins of the invention to NCI-H226 cells with high expression of human PD-L1. [Figure 12A] Figure 2 shows the binding levels of some PD-L1 x CD40 bispecific binding proteins of the invention to human CD40 highly expressing Raji cells. [Figure 12B] Figure 2 shows the binding levels of some PD-L1 x CD40 bispecific binding proteins of the invention to human CD40 highly expressing Raji cells. [Figure 13A] Figure 2 shows the binding levels of some PD-L1 x CD40 bispecific binding proteins of the invention to MDA-MB-231 cells expressing human PD-L1 and human CD40, respectively. [Figure 13B] Figure 2 shows the binding levels of some PD-L1 x CD40 bispecific binding proteins of the invention to MDA-MB-231 cells expressing human PD-L1 and human CD40, respectively. [Figure 14A] Figure 14A and Figure 14B are the first batch of experiments showing the inhibitory effect of PD-L1 x CD40 bispecific binding protein on the PD-1 signal pathway in reporter gene cell experiments. [Figure 14B] Figure 14A and Figure 14B are the first batch of experiments showing the inhibitory effect of PD-L1 x CD40 bispecific binding protein on the PD-1 signal pathway in reporter gene cell experiments. [Figure 14C] Figure 14C and Figure 14D are a second batch of experiments showing the inhibitory effect of PD-L1 x CD40 bispecific binding proteins on the PD-1 signaling pathway in reporter gene cell experiments. [Figure 14D] Figure 14C and Figure 14D are a second batch of experiments showing the inhibitory effect of PD-L1 x CD40 bispecific binding proteins on the PD-1 signaling pathway in reporter gene cell experiments. [Figure 15A]Figure 15A-C show the first batch of experiments showing the activating effect of PD-L1 x CD40 bispecific binding proteins on the CD40 signal pathway in reporter gene cell experiments. [Figure 15B] Figure 15A-C show the first batch of experiments showing the activating effect of PD-L1 x CD40 bispecific binding proteins on the CD40 signal pathway in reporter gene cell experiments. [Figure 15C] Figure 15A-C show the first batch of experiments showing the activating effect of PD-L1 x CD40 bispecific binding proteins on the CD40 signal pathway in reporter gene cell experiments. [Figure 15D] Figure 15D and Figure 15E are the second batch of experiments showing the activating effect of PD-L1 x CD40 bispecific binding proteins on the CD40 signal pathway in reporter gene cell experiments. [Figure 15E] Figure 15D and Figure 15E are the second batch of experiments showing the activating effect of PD-L1 x CD40 bispecific binding proteins on the CD40 signal pathway in reporter gene cell experiments. [Figure 15F] Figure 15F to Figure 15K are the third batch of experiments showing the activating effect of PD-L1 x CD40 bispecific binding proteins on the CD40 signal pathway in reporter gene cell experiments. [Figure 15G] Figure 15F to Figure 15K are the third batch of experiments showing the activating effect of PD-L1 x CD40 bispecific binding proteins on the CD40 signal pathway in reporter gene cell experiments. [Figure 15H] Figure 15F to Figure 15K are the third batch of experiments showing the activating effect of PD-L1 x CD40 bispecific binding proteins on the CD40 signal pathway in reporter gene cell experiments. [Figure 15I] Figure 15F to Figure 15K are the third batch of experiments showing the activating effect of PD-L1 x CD40 bispecific binding proteins on the CD40 signal pathway in reporter gene cell experiments. [Figure 15J]Figure 15F to Figure 15K are the third batch of experiments showing the activating effect of PD-L1 x CD40 bispecific binding proteins on the CD40 signal pathway in reporter gene cell experiments. [Figure 15K] Figure 15F to Figure 15K are the third batch of experiments showing the activating effect of PD-L1 x CD40 bispecific binding proteins on the CD40 signal pathway in reporter gene cell experiments. [Figure 16A] FIG. 14 shows the levels to which some PD-L1×CD40 bispecific binding proteins of the invention activate human DC cells (donor #1) and promote secretion of IL12p40, respectively. [Figure 16B] FIG. 14 shows the levels to which some PD-L1×CD40 bispecific binding proteins of the invention activate human DC cells (donor #1) and promote secretion of IL12p40, respectively. [Figure 17A] FIG. 14 shows the levels to which some PD-L1×CD40 bispecific binding proteins of the invention activate human DC cells (donor #2) and promote secretion of IL12p40, respectively. [Figure 17B] FIG. 14 shows the levels to which some PD-L1×CD40 bispecific binding proteins of the invention activate human DC cells (donor #2) and promote secretion of IL12p40, respectively. [Figure 18A] FIG. 14 shows the levels to which some PD-L1×CD40 bispecific binding proteins of the invention activate human DC cells (donor #3) and promote secretion of IL12p40, respectively. [Figure 18B] FIG. 14 shows the levels to which some PD-L1×CD40 bispecific binding proteins of the invention activate human DC cells (donor #3) and promote secretion of IL12p40, respectively. [Figure 19A] Figures 19A-B show the levels at which PD-L1xCD40 bispecific binding protein activates human DC cells (donor #4) and promotes secretion of IL12p40 by iDC cells. [Figure 19B]Figures 19A-B show the levels at which PD-L1xCD40 bispecific binding protein activates human DC cells (donor #4) and promotes secretion of IL12p40 by iDC cells. [Figure 19C] Figures 19C-D show the levels at which PD-L1xCD40 bispecific binding protein activates human DC cells (donor #4) and promotes secretion of IL12p40 by mDC cells. [Figure 19D] Figures 19C-D show the levels at which PD-L1xCD40 bispecific binding protein activates human DC cells (donor #4) and promotes secretion of IL12p40 by mDC cells. [Figure 20] FIG. 2 shows the level to which PD-L1×CD40 bispecific binding protein stimulates proliferation of human B cells (donor #1). [Figure 21] FIG. 2 shows the level to which PD-L1×CD40 bispecific binding protein promotes proliferation of human B cells (donor #2). [Figure 22A] FIG. 14 shows the level to which some PD-L1×CD40 bispecific binding proteins of the invention promote the proliferation of human B cells (donor #3). [Figure 22B] FIG. 14 shows the level to which some PD-L1×CD40 bispecific binding proteins of the invention promote the proliferation of human B cells (donor #3). [Figure 23A] FIG. 14 shows the level to which some PD-L1×CD40 bispecific binding proteins of the invention promote the proliferation of human B cells (donor #4). [Figure 23B] FIG. 14 shows the level to which some PD-L1×CD40 bispecific binding proteins of the invention promote the proliferation of human B cells (donor #4). [Figure 24A] FIG. 14 shows the level to which some PD-L1×CD40 bispecific binding proteins of the invention promote the proliferation of human B cells (donor #5). [Figure 24B]FIG. 14 shows the level to which some PD-L1×CD40 bispecific binding proteins of the invention promote the proliferation of human B cells (donor #5). [Figure 25A] Figure 25A is the change in tumor volume in mice in vivo anti-tumor effect of PD-L1 x CD40 bispecific binding protein (mouse pharmacodynamic experiment #1). [Diagram 25] Figure 25B is the change in mouse body weight in the mouse in vivo anti-tumor effect of PD-L1 x CD40 bispecific binding protein (mouse pharmacodynamic experiment #1). [Figure 26A] Figure 26A is the change in tumor volume in mice in vivo anti-tumor effect of PD-L1 x CD40 bispecific binding protein (mouse pharmacodynamic experiment #2). [Figure 26B] Figure 26B is the change in mouse body weight in the mouse in vivo anti-tumor effect of PD-L1 x CD40 bispecific binding protein (mouse pharmacodynamic experiment #2). [Figure 27A] Figure 27A is the change in tumor volume in mice in vivo anti-tumor effect of PD-L1 x CD40 bispecific binding protein (mouse pharmacodynamic experiment #3). [Figure 27B] Figure 27B is the change in mouse body weight in the mouse in vivo anti-tumor effect of PD-L1 x CD40 bispecific binding protein (mouse pharmacodynamic experiment #3). [Figure 28] FIG. 1 shows the effects of PD-L1×CD40 bispecific binding protein and selicrelumab on ALT and AST, indicators of mouse liver function, in mouse pharmacodynamic study #3. [Figure 29] FIG. 13 shows the results of staining analysis of mouse liver tissue sections in mouse pharmacodynamic experiment #3. [Figure 30A] Figure 30A is the change in tumor volume in mice in vivo anti-tumor effect of PD-L1 x CD40 bispecific binding protein and Selicrelumab (Mouse Pharmacodynamics Experiment #4). [Figure 30B]Figure 30B shows the change in mouse body weight in the mouse in vivo anti-tumor effect of PD-L1 x CD40 bispecific binding protein and Selicrelumab (Mouse Pharmacodynamics Experiment #4). [Diagram 31] FIG. 1 shows the effects of PD-L1×CD40 bispecific binding protein and selicrelumab on ALT and AST, indicators of mouse liver function, in mouse pharmacodynamic study #4. [Diagram 32] FIG. 1 shows mouse peripheral blood cytokine levels (IL-6, IFN-γ) in mouse pharmacodynamic experiment #4. [Diagram 33] FIG. 1 shows mouse peripheral blood cytokine levels (TNF-α, IL12p40) in mouse pharmacodynamic experiment #4. [Diagram 34] FIG. 13 shows the results of staining analysis of mouse liver tissue sections in mouse pharmacodynamic experiment #4. [Figure 35A] Figure 35A shows the in vivo pharmacokinetics of PR007619PD-L1 x CD40 bispecific binding protein in mice, Figure 35B shows the in vivo pharmacokinetics of PR007556PD-L1 x CD40 bispecific binding protein in mice, Figure 35C shows the in vivo pharmacokinetics of PR007281PD-L1 x CD40 bispecific binding protein in mice, and Figure 35D shows the in vivo pharmacokinetics of PR007276PD-L1 x CD40 bispecific binding protein in mice. In the figures, Total refers to Fc end detection, and Free refers to PD-L1 end detection. [Figure 35B] Figure 35A shows the in vivo pharmacokinetics of PR007619PD-L1 x CD40 bispecific binding protein in mice, Figure 35B shows the in vivo pharmacokinetics of PR007556PD-L1 x CD40 bispecific binding protein in mice, Figure 35C shows the in vivo pharmacokinetics of PR007281PD-L1 x CD40 bispecific binding protein in mice, and Figure 35D shows the in vivo pharmacokinetics of PR007276PD-L1 x CD40 bispecific binding protein in mice. In the figures, Total refers to Fc end detection, and Free refers to PD-L1 end detection. [Figure 35C] Figure 35A shows the in vivo pharmacokinetics of PR007619PD-L1 x CD40 bispecific binding protein in mice, Figure 35B shows the in vivo pharmacokinetics of PR007556PD-L1 x CD40 bispecific binding protein in mice, Figure 35C shows the in vivo pharmacokinetics of PR007281PD-L1 x CD40 bispecific binding protein in mice, and Figure 35D shows the in vivo pharmacokinetics of PR007276PD-L1 x CD40 bispecific binding protein in mice. In the figures, Total refers to Fc end detection, and Free refers to PD-L1 end detection. [Figure 35D] Figure 35A shows the in vivo pharmacokinetics of PR007619PD-L1 x CD40 bispecific binding protein in mice, Figure 35B shows the in vivo pharmacokinetics of PR007556PD-L1 x CD40 bispecific binding protein in mice, Figure 35C shows the in vivo pharmacokinetics of PR007281PD-L1 x CD40 bispecific binding protein in mice, and Figure 35D shows the in vivo pharmacokinetics of PR007276PD-L1 x CD40 bispecific binding protein in mice. In the figures, Total refers to Fc end detection, and Free refers to PD-L1 end detection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] Hereinafter, embodiments of the present invention will be described through specific examples, and a person skilled in the art can easily understand other advantages and effects of the present invention based on the contents disclosed in this specification.

[0077] In this application, the term "binding protein" or "antigen-binding protein" generally refers to a protein that includes an antigen-binding portion and, optionally, a scaffold or framework portion that allows the antigen-binding portion to assume a conformation that promotes binding of the antigen-binding protein to an antigen. Typically, it may include an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both. The VH and VL regions may be further differentiated into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL may be composed of three CDRs and four FR regions, which may be arranged in the following order from the amino terminus to the carboxy terminus: FR-1, CDR1, FR-2, CDR2, FR-3, CDR3, and FR-4. The variable regions of the heavy and light chains include binding domains that interact with antigens. The three CDRs of VH are represented as HCDR1, HCDR2 and HCDR3, and may be represented as VH CDR1, VH CDR2 and VH CDR3, respectively, and the three CDRs of VL are represented as LCDR1, LCDR2 and LCDR3, and may be represented as VL CDR1, VL CDR2 and VL CDR3, respectively. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv and / or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., so long as they exhibit the required antigen-binding activity.

[0078] In the present application, the amino acid sequences of the CDRs are all shown according to the Chothia definition rules. However, as known to those skilled in the art, the CDRs of antibodies can be defined in various ways in the art, such as the Kabat definition rules based on sequence variability (see Kabat et al., Protein Sequence in Immunology, 5th Edition, National Institutes of Health, Bethesda, MD (1991)) and the Chothia definition rules based on the location of structural loop regions (see JMol Biol 273:927-48, 1997). In the technical proposal of the present invention, the amino acid residues in the variable domain sequence can also be determined using the Combined definition rules, which include the Kabat definition and the Chothia definition. Here, the Combined definition rules combine the scope of the Kabat definition and the Chothia definition to obtain a larger scope based on this, and the details of which are shown in Table 2 below. As will be appreciated by those skilled in the art, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to cover the complementarity determining regions defined by any one of the above known schemes described in the present invention. Although the scope claimed in the present invention is the sequence shown based on the Chothia definition rules, corresponding amino acid sequences based on other CDR definition rules also fall within the scope of protection of the present invention.

[0079] [Table 4] Here, Laa-Lbb may refer to the amino acid sequence from position aa (Chothia numbering rules) to position bb (Chothia numbering rules) from the N-terminus of the antibody light chain, and Haa-Hbb may refer to the amino acid sequence from position aa (Chothia numbering rules) to position bb (Chothia numbering rules) from the N-terminus of the antibody heavy chain. For example, L24-L34 may refer to the amino acid sequence from position 24 to 34 according to the Chothia numbering rules from the N-terminus of the antibody light chain, and H26-H32 may refer to the amino acid sequence from position 26 to 32 according to the Chothia numbering rules from the N-terminus of the antibody heavy chain. As will be appreciated by those skilled in the art, when encoding CDRs with Chothia, there may be insertion sites at several positions (see http: / / bioinf.org.uk / abs / ).

[0080] In this application, the term "monoclonal antibody" refers to an antibody that is usually obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the cluster are the same except for minor natural mutations that may be present. Monoclonal antibodies are usually highly specific to a single antigenic site. And, unlike conventional polyclonal antibody preparations (which usually have different antibodies against different determinants), each monoclonal antibody is against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized in a hybridoma culture and are not contaminated by other immunoglobulins. "Monoclonal" refers to the character of an antibody obtained from a substantially homogeneous antibody population and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in the present invention may be produced in hybridoma cells or by recombinant DNA methods.

[0081] In this application, the term "fully human antibody" generally refers to an antibody expressed in a genetically engineered animal that has all of the human genes encoding the antibody transferred to the animal without the antibody gene. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side reactions of heterologous antibodies against humans. In the art, methods for obtaining fully human antibodies include phage display technology, transgenic mouse technology, etc.

[0082] In this application, the term "specific binding" usually means that an antibody binds to an epitope via its antigen-binding domain, and that the binding requires some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an antigen if it is easier for the antibody to bind to an epitope via its antigen-binding domain than it is to bind to a random, unrelated epitope. An "epitope" refers to a specific atomic group (e.g., sugar side chain, phosphoryl group, sulfonyl group) or amino acid on an antigen that binds to an antigen-binding protein (e.g., an antibody).

[0083] In this application, the term "Fab" generally refers to the antigen-binding portion of a conventional antibody (e.g., IgG), and includes the heavy chain variable region VH, the light chain variable region VL, the heavy chain constant region domain CH1, and the light chain constant region CL of the antibody. In a conventional antibody, the C-terminus of VH is linked to the N-terminus of CH1 to form a heavy chain Fd fragment, the C-terminus of VL is linked to the N-terminus of CL to form a light chain, and the C-terminus of CH1 is linked to the hinge region of the heavy chain and other constant region domains to form a heavy chain. In some embodiments, "Fab" also refers to a variant structure of Fab. For example, in some embodiments, the C-terminus of VH is linked to the N-terminus of CL to form one polypeptide chain, and the C-terminus of VL is linked to the N-terminus of CH1 to form another polypeptide chain, forming the structure of Fab (cross VH / VL), and in some embodiments, the CH1 of Fab is not linked to the hinge region, but instead the C-terminus of CL is linked to the hinge region of the heavy chain to form the structure of Fab (cross Fd / LC).

[0084] In the present application, the term "VH" generally refers to the VH domain of the heavy chain variable region of an antibody, i.e., it may be the heavy chain variable region VH of a conventional antibody (H2L2 structure) of a human or other animal, or it may be the heavy chain variable region VHH of a heavy chain antibody (HCAb structure) of an animal such as a camelid, or it may be the heavy chain variable region VH of a fully human-derived heavy chain antibody (HCAb structure) produced using a Harbour HCAb transgenic mouse.

[0085] In this application, the term "antigen-binding fragment" generally refers to any functional region of a protein capable of specifically binding to an antigen, whether it be "Fab", "VH" or other antigen-binding forms (e.g. derived protein structures such as lipocalins, neural cell adhesion molecule (NCAM), fibronectin, ankyrin repeat fragment proteins (DARPins) etc.).

[0086] In this application, the term "tumor antigen" may be a tumor specific antigen (TSA) or a tumor associated antigen (TAA). A tumor specific antigen refers to an antigen that is not present in normal cells or tissues and is unique to tumor cells. A tumor associated antigen is not unique to tumor cells and is also present in normal cells or tissues, but is highly expressed when tumor cells proliferate.

[0087] In this application, the term "target cells" refers to cells that need to be eliminated, mainly tumor cells, but may also be immunosuppressive cells, etc.

[0088] In this application, the term "immune effector cell" generally refers to immune cells that are involved in the clearance of foreign antigens and performing effector functions in an immune response, such as plasma cells, cytotoxic T cells, NK cells, etc.

[0089] In this application, the term "costimulatory molecule", "costimulatory molecule antigen" or "stimulatory antigen" refers to cell surface molecules and their ligands that provide costimulatory signals for the full activation of immune cells, such as T or B cells, and play a positive regulatory role in immune cell activation, such as CD28, 4-1BB, ICOS, OX40, CD40, etc.

[0090] As used herein, the term "co-inhibitory molecules" or "co-inhibitory molecule antigens" refers to a class of cell surface molecules and their ligands that exert a negative regulatory effect on immune cell function, such as CTLA-4, PD-L1, and PD-1.

[0091] In this application, the term "PD-L1" generally refers to the programmed death-ligand 1 protein, its functional variants and / or its functional fragments. PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), and is a protein encoded by the CD274 gene (in humans). PD-L1 sequences are known in the art. For example, an exemplary full-length human PD-L1 protein amino acid sequence can be found at NCBI Accession No. NP_054862 or UniProt Accession No. Q9NZQ7, and an exemplary full-length cynomolgus PD-L1 protein sequence can be found at NCBI Accession No. XP_005581836 or Uniprot Accession No. G7PSE7.

[0092] In this application, the term "CD40" generally refers to tumor necrosis factor receptor superfamily member 5 protein, its functional variants and / or its functional fragments, also referred to as TNFRSF5. CD40 sequences are known in the art. For example, an exemplary human CD40 protein amino acid sequence can be found at Uniprot Accession No. P25942, an exemplary cynomolgus monkey CD40 protein sequence can be found at Uniprot Accession No. G7PG38, and an exemplary mouse CD40 protein sequence can be found at Uniprot Accession No. P27512. CD40L is the natural trimeric ligand molecule of CD40.

[0093] Working Example The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the examples. The examples do not include detailed descriptions of conventional methods such as methods for constructing vectors and plasmids, inserting protein-encoding genes into such vectors and plasmids, or introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in many publications. The following examples do not specify specific conditions for the experimental methods, but are selected according to conventional methods and conditions or according to product instructions.

[0094] Example 1 Expression, purification and characterization of physicochemical properties of recombinant antibodies Example 1.1 Antibody Expression and Purification This example introduces a general method for producing antibodies by mammalian host cells (e.g., human embryonic kidney cells HEK293 or Chinese hamster ovary cells CHO and cells derived therefrom), techniques such as transient transfection expression and affinity capture separation. This method is suitable for target antibodies that contain an Fc region, and the target antibodies may consist of one or more protein polypeptide chains or may be derived from one or more expression plasmids.

[0095] The amino acid sequence of the antibody polypeptide chain is converted into a nucleotide sequence by codon optimization, the encoded nucleotide sequence is synthesized, and cloned into an expression vector compatible with the host cell. The plasmids encoding the antibody polypeptide chain are co-transfected into a mammalian host cell in a specific ratio, and recombinant antibodies with correct folding and polypeptide chain assembly can be obtained using conventional recombinant protein expression and purification techniques. TM 293-F cells (Thermo, #R79007) were cultured in a FreeStyle TM The cells were cultured in F17 Expression Medium (Thermo, #A1383504). Before the start of transient transfection, the cell concentration was adjusted to 6–8 × 10 5 The cells were then adjusted to 1.2 × 10 cells / mL and cultured in a shaker at 37 °C with 8% CO2 for 24 hours until the cell concentration reached 1.2 × 10 6The cell concentration is 100 μg / mL. 30 mL of cultured cells were prepared. Plasmids (pTT5, NRC) encoding antibody polypeptide chains were mixed at a certain ratio, and a total of 30 μg of plasmid (1 μg:1 mL ratio of plasmid to cells) was dissolved in 1.5 mL Opti-MEM serum-reduced medium (Thermo, #31985088) and sterilized by filtering through a 0.22 μm filter membrane. Next, 1.5 mL of Opti-MEM was taken and dissolved in 120 μL of 1 mg / mL PEI (Polysciences, #23966-2) and left to stand for 5 minutes. PEI was slowly added to the plasmid and incubated at room temperature for 10 minutes, and the plasmid-PEI mixed solution was slowly added dropwise while shaking the culture flask slowly, and the cells were cultured for 5 days in a shaker at 37 °C with 8% CO2. After 5 days, the cell viability was observed. The culture was harvested and centrifuged at 3300g for 10 minutes, after which the supernatant was taken and centrifuged at high speed to remove impurities. TM A gravity column (Bio-Rad, #7311550) containing 100% glycine (GE Healthcare, #71-5020-91) was equilibrated and washed with 2-5 column volumes. The supernatant sample was passed through the column, and the column was washed with 5-10 column volumes of PBS buffer. The target protein was then eluted with 0.1 M glycine at pH 3.5, then neutralized with Tris-HCl at pH 8.0, and finally concentrated with an ultrafiltration tube (Millipore, #UFC901024) and exchanged into PBS buffer or a buffer containing other components to obtain a purified recombinant antibody solution. Finally, a NanoDrop (Thermo, NanoDrop TM The concentration was measured using a 300 mL aliquot dispenser, which was then stored and put on hold.

[0096] Example 1.2 Protein Purity and Aggregation Analysis by SEC-HPLC In this example, analytical size exclusion chromatography (SEC) was used to analyze the purity and aggregate form of protein samples. An analytical chromatography column TSKgel G3000SWxl (Tosoh Bioscience, #08541, 5 μm, 7.8 mm × 30 cm) was connected to a high performance liquid chromatography HPLC (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer (pH 7.4) at room temperature for at least 1 hour. An appropriate amount of protein sample (at least 10 μg) was injected into the system after filtering through a 0.22 μm filter membrane, and the HPLC procedure was set to pass the sample through the chromatography column at a flow rate of 1.0 mL / min with PBS buffer (pH 7.4) for a maximum time of 25 minutes. The HPLC generated an analysis report and reported the retention times of components of different molecular sizes in the sample.

[0097] Example 1.3 Measurement of thermal stability of protein molecules by DSF Differential Scanning Fluorimetry (DSF) is a commonly used high-throughput method to measure the thermal stability of proteins. It reflects the process of protein denaturation by monitoring the change in the fluorescence intensity of the dye bound to the unfolded protein molecules using a real-time fluorescent quantitative PCR instrument, thereby reflecting the thermal stability of the protein molecules. This example uses the DSF method to measure the thermal denaturation temperature (Tm) of protein molecules. 10 μg of protein was added to a 96-well PCR plate (Thermo, #AB-0700 / W), then 2 μL 100× diluted dye SYPROTM (Invitrogen, #2008138) was added, and buffer was added to make the final volume 40 μL / well. The PCR plate was sealed and placed in a real-time fluorescent quantitative PCR instrument (Bio-Rad CFX96 PCR System), first incubated at 25°C for 5 min, then gradually increased in temperature from 25°C to 95°C with a gradient of 0.2°C / 0.2 min, and at the end of the experiment the temperature was reduced to 25°C. The data was analyzed and the Tm of the samples was calculated with Bio-Rad CFX Maestro software using the FRET scanning mode.

[0098] Example 2: Production of CD40 antibody The CD40 antigen is used to immunize laboratory animals to obtain antibody molecules that specifically bind to CD40, and the laboratory animals may be mice, rats, rabbits, sheep, camels, etc. Usually, the obtained antibody molecules are of non-human origin. After obtaining antibodies of non-human origin, it is necessary to carry out humanization operations on these molecules using antibody engineering techniques to reduce immunogenicity and increase drug discovery potential. However, the humanization process of antibodies is technically complex, and humanized molecules often have reduced affinity for antigens. Meanwhile, advances in transgenic technology have made it possible to create genetically engineered mice that have a human immunoglobulin immune repertoire and delete their endogenous mouse immune repertoire. The antibodies produced from such transgenic mice have a fully human sequence, so no further humanization operations are required, greatly improving the efficiency of developing therapeutic antibodies. The Harbour H2L2 mouse (Harbour Antibodies BV) is a transgenic mouse carrying an immune repertoire of human immunoglobulins and the antibodies produced therefrom have fully human antibody variable domains and rat constant domains.

[0099] Immunized mice Harbour H2L2 mice were immunized multiple times with soluble recombinant human CD40 extracellular segment fusion protein (Acrobiosystems, #CD0-H5253). The antigen protein and the immune adjuvant were mixed to form an immunogenic reagent, which was then injected subcutaneously into the groin or intraperitoneally. In each round of immunization, the total injection volume received by each mouse was 100 μL. In the first immunization, each mouse was immunized with the immunogenic reagent prepared by mixing 50 μg of the antigen protein with complete Freund's adjuvant (Sigma, #F5881) in a 1:1 volume ratio. In each subsequent round of boosting, each mouse was immunized with the immunogenic reagent prepared by mixing 25 μg of the antigen protein with Sigma Adjuvant System adjuvant (Sigma, #S6322). The interval between each round of boosting was at least 2 weeks, and there were usually no more than 5 rounds of boosting. The immunization times were days 0, 14, 28, 42, 56, and 70, and antibody titers in mouse serum were detected on days 49 and 77. The final booster was administered at a dose of 25 μg of antigen protein per mouse 3 days before cell fusion.

[0100] Hybridoma Screening When the CD40-specific antibody titer in the mouse serum was detected to reach a certain level, the spleen cells of the mouse were taken out and fused with a myeloma cell line to obtain hybridoma cells, and the hybridoma cells were subjected to multiple rounds of mass screening and cloning, after which hybridomas expressing CD40 monoclonal antibody molecules were isolated. The isolated hybridomas expressed antibody molecules with complete human variable domains and heavy and light chains of rat constant domains. The monoclonal antibodies were further identified, and several hybridoma clones were selected and sequenced based on parameters such as the ability to bind to human CD40, the ability to bind to cynomolgus monkey CD40, and the ability to activate the CD40 downstream signal pathway. During mass screening, Selicrelumab (also numbered PR001028 in the present application) was used as a positive control antibody. The nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence were obtained by conventional hybridoma sequencing means. In this example, the sequences of the variable domains of the CD40 monoclonal antibody molecules obtained from immunized Harbour H2L2 mice are human-derived antibody sequences. The CDR sequences of the antibody variable domains can be analyzed according to Kabat or Chothia or other CDR definition rules (Table I).

[0101] Recombinant IgG antibody and sequence analysis and optimization After obtaining the sequences of light and heavy chain variable domains encoding an antibody molecule, conventional recombinant DNA technology is used to fuse and express the sequences of light and heavy chain variable domains and the sequences of light and heavy chain constant domains of the corresponding human antibody, and a recombinant antibody molecule can be obtained by the above method. In this example, an antibody heavy chain variable domain sequence (VH) was genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding a human IgG1 or IgG2 antibody heavy chain constant domain sequence to encode a full-length heavy chain that produces an IgG antibody. An antibody light chain variable domain sequence (VL) was genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding a human antibody Igκ light chain constant domain sequence to encode a full-length light chain that produces an antibody. In this example, a fully human-derived anti-CD40 recombinant IgG antibody was obtained.

[0102] The heavy chain variable domain sequence of an antibody is derived from events such as gene rearrangement and somatic hypermutation of germline gene V, D, and J gene segments of a heavy chain gene group in a chromosome, and the light chain variable domain sequence is derived from events such as gene rearrangement and somatic hypermutation of germline gene V and J gene segments of a light chain gene group. Gene rearrangement and somatic hypermutation are the main factors that increase the diversity of antibodies. Although antibodies from the same germline V gene segment can produce different sequences, they are generally highly similar. Using several algorithms, such as IMGT / DomainGapAlign (http: / / imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi) or NCBI / IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ), the possible germline gene segments when gene rearrangement occurs can be predicted from the variable domain sequence of an antibody.

[0103] After translational synthesis in cells, the amino acid chain of a protein or polypeptide may introduce chemical modifications called post-translational modifications (PTMs). In the case of antibodies, some PTM sites are very conserved; for example, the conserved amino acid asparagine Asn at position 297 (EU numbering) of the constant domain of human IgG1 antibodies is usually glycosylated to form a glycan, and the glycan structure is quite important for antibody structure and related effector functions. However, if PTMs are present in the variable domain of an antibody, particularly in the antigen-binding region (e.g., CDR), the presence of these PTMs may have a significant effect on antigen binding and may also change the physicochemical properties of the antibody. For example, glycosylation, deamidation, isomerization, oxidation, etc., may all increase the instability or heterogeneity of the antibody molecule, thereby increasing the difficulty and risk of antibody development. Therefore, avoiding some potential PTMs is very important for the development of therapeutic antibodies. With the accumulation of experience, it has been discovered that some PTMs are highly correlated with the composition of amino acid sequences, especially the "pattern" of the composition of adjacent amino acids, which allows the prediction of potential PTMs from the primary amino acid sequence of a protein. For example, the sequence pattern of NxS / T (position 1 is asparagine, position 2 is any amino acid other than non-proline, and position 3 is serine or threonine) predicts N-linked glycosylation sites. The amino acid sequence pattern that causes PTMs may be derived from germline gene sequences, for example, human germline gene fragment IGHV3-33, which naturally has a glycosylation pattern NST in the FR3 region, or may be derived from somatic hypermutation. Specifically, NGS or NLT may be glycosylation sites, NS may be deamidation sites, and DG may cause isomerization of aspartic acid. The formation of certain PTMs can be reduced or eliminated by disrupting the amino acid sequence pattern of the PTM by amino acid mutation. The method of designing the mutations varies depending on the antibody sequence and the PTM sequence pattern. One approach is to replace "hot spot" amino acids (eg, N or S in an NS pattern) with amino acids that have similar physicochemical properties (eg, mutating N to Q).Alternatively, the PTM sequence pattern was randomly mutated using a mutation library method. In practical operation, various mutation design methods can be used for the same PTM sequence pattern.

[0104] Table 3 lists the anti-CD40 hybridoma cloning and recombinant antibody molecule (PR003379) obtained in this example, as well as the analyzed VH and VL germline gene V gene fragments and potential PTM sites.

[0105] Table 4 lists the new antibody molecules obtained by performing amino acid mutations on the CD40 antibody PR003379. All designed mutant molecules were further verified in subsequent functional experiments by obtaining purified recombinant antibodies according to the method described in 0. The antibodies in Table 5 were obtained as follows: the heavy chain variable region (VH) of PR003379 was linked to an antibody heavy chain constant domain sequence of IgG1 subtype containing L234A, L235A, G237A mutations and cloned into a mammalian cell expression plasmid to encode a full-length heavy chain that produces IgG1 (L234A, L235A, G237A). The antibody light chain variable domain sequence (VL) with PTM mutation sites was genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding a human antibody Igκ light chain constant domain sequence to encode a full-length light chain that produces an antibody. The two plasmids were co-transfected into mammalian cells, and a fully human anti-CD40 recombinant IgG1 (L234A, L235A, G237A) antibody was obtained through expression, production and purification.

[0106] Table 5 lists the sequences of the CD40 antibodies in the present application and the amino acid sequences of the CDRs defined according to the Chothia definition rules. These CD40 antibodies include the positive control antibody, the CD40 antibody PR003379 of the present invention and its mutant molecules.

[0107] [Table 5]

[0108] [Table 6]

[0109] [Table 7]

[0110] Example 3 Binding of CD40 antigen-binding proteins to CD40-expressing cells In this example, flow cytometry (FACS) was used to examine the binding activity of antibodies or antigen-binding proteins targeting CD40 to cells expressing CD40, such as the CHO-K1 cell line CHO-K1 / hCD40 (Shanghai Ruizhi Chemical) that highly expresses human CD40, the CHO-K1 cell line CHO-K1 / cyCD40 (Shanghai Ruizhi Chemical) that highly expresses cynomolgus monkey CD40, and the lymphoma cell line Raji (ATCC, #CCL-86) that highly expresses human CD40.

[0111] Specifically, CD40-expressing cells were digested and resuspended in complete medium (F-12K for CHO-K1 cells, RPMI-1640 for Raji cells) to a cell density of 1 × 10 6The cells were adjusted to 100 μL / mL. 100 μL cells / well were seeded into a 96-well V-bottom plate (Corning, #3894), and then 100 μL / well of the detection antibody, which was twice the final concentration, was added and mixed evenly. Here, the highest final concentration of the antibody was 300 nM, and a total of 8 concentrations were gradient diluted with 5-fold concentration, hIgG (Crownbio, #C0002) was used as a negative control, and the positive control molecule was Selicrelumab, and the concentration settings were all the same as the subsequent test double antibodies. The cells were incubated at 4 °C for 1 hour in the dark. Then, 100 μL / well of pre-cooled PBS was added, the cells were rinsed twice, and centrifuged at 500 g at 4 °C for 5 minutes, and the supernatant was discarded. Fluorescent secondary antibody (Goat anti-human IgG (H+L) secondary antibody, Alexa Fluor® 488 conjugate, Invitrogen, #A11013, 1:1000 dilution) was added at 100 μL / well and incubated at 4° C., protected from light, for 30 min. Cells were washed twice with 200 μL / well pre-chilled PBS, centrifuged at 500 g for 5 min at 4° C., and the supernatant was discarded. Finally, cells were resuspended in 200 μL / well pre-chilled PBS. Fluorescence signal values ​​were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed with the software FlowJo v10 (FlowJo, LLC).

[0112] Data processing and graph analysis were performed using the software GraphPad Prism 8, and the binding curves and EC values ​​of the antibodies to the target cells were calculated by four-parameter nonlinear fitting. 50 The parameters such as values ​​were obtained.

[0113] Figure 1A-Figure 1D and Table 6 show the binding activity of CD40 antibody PR003379 and its mutant molecules to Raji cells. The results show that PR003379 and its mutant molecules can strongly bind to Raji cells, and the binding activity of PR003379 is stronger than that of Selicrelumab.

[0114] [Table 8]

[0115] Example 4 Activity of CD40 antigen-binding protein in fluorescent reporter gene experiments In this example, the activation effect of CD40-targeting antibodies or antigen-binding proteins on CD40 with and without crosslinking was examined by activation experiments using HEK293-hCD40-NFκB fluorescent reporter gene cells (BPS Bioscience, #60626). Using cells highly expressing human CD32B, Fc-mediated crosslinking was performed on the target molecule to examine whether crosslinking could further enhance the activation ability of CD40.

[0116] CHO-K1 cells expressing human CD32B, CHO-K1 / hCD32B (GenScript, #M00600), and CHO-K1 cells were cultured at 1 × 10 4 The cells were seeded in a 96-well plate (PerkinElmer, #6005181) at 100 μL / well and incubated overnight at 37°C in a 5% CO environment. The supernatant was removed, and HEK293-hCD40-NFκB reporter gene cells (BPS bioscience, #60626) expressing human CD40 and NFκB-luciferase reporter gene were harvested and 5 × 10 4 50 μL / well of the detection target antibody dilution solution was added to a 96-well plate at 50 μL / well, starting at 100 nM, and diluted 5-fold for a total of 8 concentrations. The plate was incubated at 37°C in 5% CO2 for 6 hours. TM Luciferase reagent (Promega, #E6110) was added, incubated at room temperature for 5 minutes, and luminescence was detected using a microplate reader. Data processing and graph analysis were performed using the software GraphPad Prism 8, and the curves of antibody concentration-dependent relative fluorescence signal units (RLU) and EC 50Parameters such as CD40 antibody titers and CHO-K1 / hCD32B cell incubation were obtained. Samples of CD40 antibody incubation with CHO-K1 / hCD32B cells were called "crosslinked," whereas samples of CD40 antibody incubation with CHO-K1 cells were not crosslinked.

[0117] As shown in Figures 2A to 2B and Table 7, PR003379 had a more obvious "cross-linking enhancement" effect than the control antibody Selicrelumab, i.e., under the conditions of CD32B-mediated antibody cross-linking, PR003379 could significantly amplify the signal of the fluorescent reporter gene, indicating that it significantly enhanced the activation effect of CD40 molecules on downstream signal molecules.

[0118] Figures 2C-D and Table 8 show the fold enhancement of fluorescence intensity of activation by PR003379 mutant molecules on reporter gene cells with or without cell-mediated crosslinking of human CD32B expressing cells. The results show that PR003379 mutant molecules can significantly enhance the activation effect on CD40 molecules in the case of crosslinking, and their EC 50 The value is significantly lower in the cross-linked case than in the non-cross-linked case ("Fold (Non-cross-linked EC 50 / Crosslinked EC 50 )" > 1).

[0119] [Table 9]

[0120] [Table 10]

[0121] Example 5 Preparation of PD-L1 heavy chain antibody Harbour HCAb mouse (Harbour Antibodies BV, WO2010 / 109165A2) is a transgenic mouse with human immunoglobulin immune repertoire, capable of producing antibodies with only heavy chains (HCAb), the size of which is only half that of conventional IgG antibodies. The antibodies produced from it have only human antibody heavy chain variable domains and mouse Fc constant domains. Due to the feature of not containing light chains, the antibodies almost solve the problems of light chain mismatch and heterodimerization, and this technology platform allows the development of products that are difficult to achieve with conventional antibody platforms.

[0122] Immunized mice Soluble recombinant human PD-L1-mFc fusion protein (Novo Protein Inc., #CM06) was used to immunize 6-8 week-old Harbour HCAb mice for multiple rounds. The total injection volume each mouse received per immunization was 100μL by subcutaneous injection in the groin or intraperitoneal injection. In the first immunization, each mouse was immunized with an immunogenic reagent prepared by mixing 50μg of antigen protein with complete Freund's adjuvant (Sigma, #F5881) at a 1:1 volume ratio. In each subsequent round of boosting, each mouse was immunized with an immunogenic reagent prepared by mixing 25μg of antigen protein with Ribi adjuvant (Sigma Adjuvant System, #S6322). The interval between each round of boosting was at least 2 weeks, and there were usually no more than 5 rounds of boosting. The immunization times were days 0, 14, 28, 42, 56, and 70, and the antibody titers in the mouse serum were detected on days 49 and 77. The final booster immunization was performed at a dose of 25 μg of antigen protein per mouse 5 days before HCAb mouse splenic B cell isolation.

[0123] Construction of HCAb library and high-throughput transient expression When the titer of PD-L1-specific antibodies in mouse serum was detected to reach a certain level, the mouse spleen cells were removed, B cells were isolated, and CD138-positive plasma cells and human PD-1 antigen-positive B cell populations were sorted using a BD FACS AriaII Cell Sorter. Human VH genes were expanded from plasma cells and B cell populations using conventional molecular biology methods, and the expanded human VH gene fragments were constructed into a mammalian cell expression plasmid pCAG vector encoding the human IgG1 antibody heavy chain Fc region sequence. The plasmid was transfected into mammalian host cells (e.g., human embryonic kidney cells HEK293) and expressed to obtain a fully human-derived HCAb antibody supernatant.

[0124] Mirrorball High Throughput Mass Cleaning The Mirrorball high-throughput no-wash antibody mass screening platform (SPT Labtech) was used to mass screen the supernatants expressing HCAbs to detect their binding to CHO-K1 / hPD-L1 (GenScript, #M00543) stable cell line overexpressing human PD-L1, and the positive antibody PR000151 (Atezolizumab analogue) was used as a positive control.

[0125] Specifically, CHO-K1 / hPD-L1 cells were washed with serum-free F12K medium (Thermofisher, #21127022) and incubated at 1 × 10 6 The cells were resuspended at 1.0 × 10 / mL. Draq5 fluorescent probe (CTS, #4048L) (1:1000 dilution of 1 μL Draq5 in 1 mL CHO-K1 / hPD-L1 cells) was added and incubated for 30 min in the dark. After centrifugation, the cells were washed with medium and the cell density was adjusted to 1.0 × 10 5The mixture was adjusted to cells / mL. Secondary antibody (Alexa Fluor® 488 AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson ImmunoResearch, #109-545-098) diluted 1:1000 was then added. 30 μL of the mixture was added per well to a 384-well plate (Greiner, #781091). 10 μL of positive control or HCAb-expressing supernatant was added to the 384-well plate and incubated for 2 hours. Fluorescence was read on a Mirrorball.

[0126] The positive antibody supernatants were further identified and detected for binding to CHO-K1 / hPD-L1 cells by FACS, and cross-binding activity with cynomolgus PD-L1 protein (Acro biosystems, #PD1-C52H4) by ELISA. HCAbs were obtained by mass screening and sequencing using conventional sequencing methods to obtain the nucleotide sequence encoding the corresponding VH and the corresponding amino acid sequence. The VH sequence of the HCAb antibody and the human IgG1 heavy chain Fc sequence were fused and expressed to obtain a fully human recombinant HCAb antibody molecule.

[0127] Sequence analysis and optimization Table 9 lists the preferred anti-PD-L1 recombinant heavy chain antibody molecule PR000960 obtained in this example, as well as the VH germline gene V gene fragments and potential post-translational modification PTM sites analyzed. PR000960 has an N-glycosylation modification site in HCDR1, which may affect druggability in late-stage development. To remove the N-glycosylation modification site, amino acid mutations were made to PR000960 to obtain a new antibody molecule PR002082 (Table 10).

[0128] Improving PD-L1 binding affinity through affinity maturation technology To further improve the binding affinity of PR002082 to PD-L1, the PR002082 molecule was subjected to in vitro affinity maturation. A yeast display antibody mutation library was established by random mutation of the CDR regions, and the PR002082 molecule mutation library was mass screened using the BD FACS AriaIII sorting platform.

[0129] First, mutations were randomly introduced into the three CDRs of PR002082 to establish three yeast display mutant libraries (CDR1, CDR2, CDR3), which were then enriched by MACS sorting, and then multiple rounds of sorting were performed using flow FACS to enrich for mutant molecules with high affinity. In the first round, yeast cells with higher binding affinity were sorted using 0.1 nM biotinylated PD-L1-his protein, and then cultured and induced. In the second round, yeast cells with higher binding affinity were further sorted using 0.01 nM biotinylated PD-L1-his protein, and then cultured and induced. In the third round, the concentration of antigen during sorting was continuously decreased, and the final round of sorting was performed with 0.003 nM biotinylated PD-L1-his protein.

[0130] We then sequenced the molecules sorted in the previous three rounds, found mutation sites, and randomly combined them to establish a combinatorial mutation library, and continued to sort mutant molecules with higher affinity at lower antigen concentrations using FACS.

[0131] The VH of the mutant molecules obtained by affinity maturation were gene synthesized and cloned into a mammalian cell expression plasmid vector encoding a human IgG1 heavy chain Fc sequence to produce recombinant HCAb antibody molecules. Table 11 lists the mutant molecules obtained from PR002082 through the affinity maturation process.

[0132] All mutant molecules (Tables 10 and 11) were further validated in subsequent functional experiments to obtain purified recombinant antibodies according to the methods described in 0.

[0133] Table 12 lists the sequences of the PD-L1 antibodies in the present application and the amino acid sequences of the CDRs defined according to the Chothia definition rules. These PD-L1 antibodies include the positive control antibody, the PD-L1 antibody PR000960 of the present invention and mutant molecules derived therefrom.

[0134] [Table 11]

[0135] [Table 12]

[0136] [Table 13]

[0137] [Table 14]

[0138] Example 6 Binding of PD-L1 antigen-binding proteins to PD-L1-expressing cells In this example, flow cytometry (FACS) was used to examine the binding activity of antibodies or antigen-binding proteins targeting PD-L1 to cells expressing PD-L1, such as CHO-K1 cell line CHO-K1 / hPD-L1 (Shanghai Ruizhi Chemical) that highly expresses human PD-L1, CHO-K1 cell line CHO-K1 / cyPD-L1 (Shanghai Ruizhi Chemical) that highly expresses cynomolgus monkey PD-L1, lung cancer cell NCI-H226 (ATCC, #CRL-5826) that highly expresses human PD-L1, and breast cancer cell MDA-MB-231 (ATCC, #CRM-HTB-26) that highly expresses human PD-L1.

[0139] Specifically, PD-L1-expressing cells were digested and resuspended in PBS buffer containing 2% BSA. The cell density was 1 × 10 6 The cells were adjusted to 1:500 cells / mL. 100 μL cells / well were seeded into a 96-well V-bottom plate (Corning, #3894), and then 100 μL / well of the detection antibody gradient diluted at 4-fold concentration, which is twice the final concentration, was added. The cells were incubated at 4°C for 2 hours, protected from light. Then, 100 μL / well of pre-cooled 2% BSA in PBS was added, the cells were rinsed twice, centrifuged at 500g for 5 minutes at 4°C, and the supernatant was discarded. Next, fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-098, diluted 1:500) was added at 100 μL / well, and incubated at 4°C for 1 hour, protected from light. The cells were washed twice with 100 μL / well of pre-chilled 2% BSA in PBS, centrifuged at 500 g for 5 min at 4° C., and the supernatant was discarded. Finally, the cells were resuspended with 200 μL / well of pre-chilled 2% BSA in PBS buffer. Fluorescence signal values ​​were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and data were processed and analyzed using the software FlowJo v10 (FlowJo, LLC).

[0140] Data processing and graph analysis were performed using the software GraphPad Prism 8, and the binding curves and EC values ​​of the antibodies to the target cells were calculated by four-parameter nonlinear fitting. 50 The parameters such as values ​​were obtained.

[0141] Figure 3A and Figure 3B show the binding curves of PD-L1 heavy chain antibodies PR000960, PR002082 and their variant molecules to CHO-K1 / hPD-L1 cells, and Table 13 shows the EC 50The values ​​and maximum MFI signals are listed. The results show that PR000960 and PR002082 have similar binding ability to human PD-L1 as the control antibody Atezolizumab, and PR002082 has slightly better binding ability than the affinity matured mutant molecule PR002082.

[0142] [Table 15]

[0143] Example 7 Activity of PD-L1 antigen-binding protein in fluorescent reporter gene experiments In this example, the inhibitory effect of antibodies or antigen-binding proteins targeting PD-L1 on the PD-1 signal pathway was investigated using Jurkat-NFAT-hPD1 fluorescent reporter gene cell (Wahaku Pharmaceuticals) activation experiments. PD-L1 antibodies or antigen-binding proteins blocked the interaction between PD-L1 and PD-1, thereby relieving the inhibitory effect of PD-1 on T cells.

[0144] 293T cells overexpressing PD-L1 and OS8 (CD3 single chain antibody transmembrane protein) 293T-OS8-hPDL1 (Beijing Kangyuan Boxing, #KC-1148) or Jurkat reporter cells coexpressing PD-1 and NFAT-luciferase reporter gene Jurkat-NFAT-hPD1 (Haku Pharmaceuticals) were cultured and harvested at a density of 1 × 10 6 The density of 293T-OS8-hPDL1 cells / mL was 5 × 10 5 The same volume of cell suspension was mixed and added to a 96-well U-bottom plate at 100μL / well, where the ratio of effector cells Jurkat-NFAT-hPD1 and target cells 293T-OS8-hPDL1 was 2:1. 50μL / well of pre-gradient diluted antigen-binding protein to be detected was added. The 96-well plate was placed in a cell incubator and incubated at 37℃ and 5% CO2 for 6 hours. ONE-Glo TMLuciferase solution (Promega, #E6120) was added at 50 μL / well, the plate was gently tapped to mix evenly, and the reaction was allowed to proceed at room temperature for 10 minutes in the dark. Then, 100 μL of the liquid was transferred to a 96-well flat-bottom plate (PerkinElmer, #6005225), and finally, Envision TM Fluorescence values ​​were read on a plate reader (PerkinElmer).

[0145] Data processing and graph analysis were performed using the software GraphPad Prism 8, and the binding curves and EC values ​​of the antibodies to the target cells were calculated by four-parameter nonlinear fitting. 50 The parameters such as values ​​were obtained.

[0146] Figure 4A and Figure 4B and Table 14 show that the PD-L1 heavy chain antibodies PR000960, PR002082 and their mutant molecules could block the PD-1 signal pathway in the fluorescent reporter gene experiment and restore NFAT signaling in Jurkat. The results showed that PR000960 had the same inhibitory activity as the control antibody Atezolizumab, and the inhibitory effect of the affinity matured mutant molecule PR002082 was slightly better than that of PR002082.

[0147] [Table 16]

[0148] Example 8 Construction of PD-L1 x CD40 bispecific antigen-binding protein In this example, a PD-L1×CD40 bispecific antigen-binding protein with the structure shown in Figure 5 was constructed using a CD40 antibody from 0 and a PD-L1 heavy chain antibody from 0.

[0149] As shown in FIG. 5, the Fab end is derived from conventional antibody A, and VH_A and VL_A are the heavy and light chain variable regions of antibody A, respectively. The VH end is derived from heavy chain antibody B, and VH_B is the heavy chain variable region of heavy chain antibody B. CL is the light chain constant region domain. CH1, CH2, and CH3 are the first, second, and third domains of the heavy chain constant region, respectively. h is the hinge region of an IgG antibody or a derived sequence, and L is a connecting peptide. The binding protein of the shown structure comprises two distinct polypeptide chains: a light chain comprising, from the amino terminus to the carboxy terminus, VL_A-CL, and a heavy chain comprising, from the amino terminus to the carboxy terminus, VH_B-L-VH_A-CH1-h-CH2-CH3. In some embodiments, the VH_B and VH_A of the heavy chain are linked by a direct fusion, i.e., the length of L is 0, and in some other embodiments, the VH_B of the heavy chain is linked to the VH_A via a linking peptide L, where L may be a sequence in Table 15.

[0150] [Table 17]

[0151] In this example, VH_A and VL_A in Figure 5 are derived from antigen-binding fragments Fab of H2L2 (IgG) antibodies PR006495, PR006496, PR006497, PR006504, PR006511 that bind to CD40, and VH_B binds to antigen-binding fragments VH of heavy chain antibodies PR005878, PR006245, PR006246, PR006247, PR006248 that bind to PD-L1. Based on these antigen-binding fragments of PD-L1 and / or CD40, several PD-L1 x CD40 bispecific antigen-binding proteins with the structures shown in Figure 5 were constructed (listed in Table 16). These molecules have similar structures, use human IgG1 constant region sequences, and introduce three amino acid mutations L234A / L235A / G237A (Eu code) in the Fc region to eliminate Fc-mediated effector functions such as ADCC. Table 18 lists the SEQ ID NOs for the polypeptide chain amino acid sequences of PD-L1×CD40. For these PD-L1×CD40 molecules, the corresponding protein samples were prepared and analyzed according to the methods described in 0 and are summarized in Table 17. All of the PD-L1×CD40 molecules constructed in this example had high yield and purity (see Example 1 for the expression and purity detection method of PD-L1×CD40 molecule), with the purity of most of the protein samples exceeding 95%.

[0152] [Table 18]

[0153] [Table 19]

[0154] [Table 20]

[0155] Example 9 Binding of PD-L1×CD40 to cells expressing PD-L1 or CD40 In this example, the binding ability of the PD-L1×CD40 bispecific binding protein to several cells expressing PD-L1 or CD40 was tested using the methods described in 0 and 0. These cells were CHO-K1 cells that do not express PD-L1 or CD40, a CHO-K1 cell line CHO-K1 / hPD-L1 (Shanghai Ruizhi Chemical) that highly expresses human PD-L1, a CHO-K1 cell line CHO-K1 / cyPD-L1 (Shanghai Ruizhi Chemical) that highly expresses cynomolgus monkey PD-L1, a CHO-K1 cell line CHO-K1 / hCD40 (Shanghai Ruizhi Chemical) that highly expresses human CD40, and a cynomolgus monkey These include the CHO-K1 cell line CHO-K1 / cyCD40 (Shanghai Ruizhi Chemical), which highly expresses CD40, the lymphoma cell line Raji (ATCC, #CCL-86), which highly expresses human CD40, the lung cancer cell line NCI-H226 (ATCC, #CRL-5826), which highly expresses human PD-L1, and the breast cancer cell line MDA-MB-231 (ATCC, #CRM-HTB-26), which highly expresses both human PD-L1 and CD40.

[0156] Table 19 summarizes the corresponding results of the binding of PD-L1×CD40 bispecific binding proteins and control molecules to various cells, with the corresponding example figures showing the binding curves of the antigen binding proteins binding to the target cells and the corresponding tables showing the avidity parameters (EC 50The MFI signal (maximum and MFI signal value) is listed in Table 1. Figure 10A and Figure 10B show that PD-L1xCD40 does not bind to CHO-K1 cells. Figure 6A and Figure 6B and Table 20 show the binding of PD-L1xCD40 to CHO-K1 / hPD-L1 cells. Figure 7A and Figure 7B and Table 21 show the binding of PD-L1xCD40 to cynomolgus PD-L1 cells. Figure 8A and Figure 8B and Table 22 show the binding of PD-L1xCD40 to CHO-K1 / hCD40 cells. Figure 9A and Figure 9B and Table 23 show the binding of PD-L1xCD40 to cynomolgus CD40 cells. Figure 11A and Figure 11B and Table 24 show the binding of PD-L1xCD40 to NCI-H226 cells. Figures 12A and 12B and Table 25 show the binding of PD-L1xCD40 to Raji cells. Figures 13A and 13B and Table 26 show the binding of PD-L1xCD40 to MDA-MB-231 cells.

[0157] The results showed that all PD-L1×CD40 dual antibodies were able to inhibit PD-L1-expressing cell lines, e.g., CHO-K1 / hPD-L1 (EC 50 is approximately 1 nM), CHO-K1 / cy PD-L1 (EC 50 <1 nM) and NCI-226 (EC 50 The PD-L1×CD40 bibody can efficiently bind to the target-expressing CHO-K1 cells (<1 nM) with a binding activity equivalent to that of the positive reference Atezolizumab. The PD-L1×CD40 bibody does not bind to CHO-K1 cells, which do not express the target, and the PD-L1×CD40 bibody also binds to MDA-MB-231 cells (EC 50 The binding activity of the antibody was significantly stronger (higher MFI signal) than that of the positive reference Atezolizumab.

[0158] Furthermore, all PD-L1×CD40 dual antibodies were specifically targeted to CD40-expressing cell lines, e.g., CHO-K1 / hCD40(EC 50 is approximately 1-2 nM), CHO-K1 / cyCD40 (EC 50 is approximately 1-4 nM) and Raji (EC 50The PD-L1×CD40 bibody also efficiently bound to MDA-MB-231 cells, with a binding activity that was significantly stronger than that of the positive reference, Selicrelumab.

[0159] [Table 21]

[0160] [Table 22]

[0161] [Table 23]

[0162] [Table 24]

[0163] [Table 25]

[0164] [Table 26]

[0165] [Table 27]

[0166] [Table 28]

[0167] Example 10. Inhibition of PD-1 signaling pathway by PD-L1×CD40 in fluorescent reporter gene experiments In this example, the method described in 0 was used to test the inhibitory effect of PD-L1×CD40 bispecific binding proteins on the PD-1 signal pathway. The results are shown in Figures 14A-14D and Table 27. All PD-L1×CD40 bispecific antibodies could effectively block PD-1 / PD-L1 interaction and activate TCR and NFAT signaling, and their activity was higher than that of the positive reference antibody Atezolizumab (EC 50 is approximately 0.1 to 0.3 nM).

[0168] [Table 29]

[0169] Example 11 Activation of the CD40 signaling pathway by PD-L1×CD40 in fluorescent reporter gene experiments In this example, the method described in 0 was used to test the activation effect of PD-L1×CD40 bispecific binding protein on CD40 with and without PD-L1-mediated crosslinking. The PD-L1 highly expressing cells CHO-K1 / hPD-L1 were used to crosslink the target molecule to determine whether crosslinking could further enhance the activation ability of CD40. In this experiment, CHO-K1 / hPD-L1 cells with high PD-L1 expression were able to crosslink the PD-L1×CD40 bibody molecule by PD-L1, but CHO-K1 cells that do not express PD-L1 or CD40 were unable to bind to the target molecule. The activation of the target molecule on the HEK293-hCD40-NFκB reporter gene system was tested after incubation with CHO-K1 / hPD-L1 cells or CHO-K1 cells, respectively.

[0170] The results are shown in Figures 15A to 15K and Table 28. The CD40 antibody Selicrelumab can activate the downstream signaling pathway of CD40, and such activation is independent of the presence of exogenous crosslinking, and inhibits EC 50On the other hand, PD-L1 × CD40 dual antibodies such as PR007151 and PR007619 showed no significant increase in EC 50 The activation effect on the downstream signaling pathway of CD40 was observed in the absence of PD-L1 cross-linking (EC 50 The activation effect was significantly stronger than that at a concentration of about 0.3-0.4 nM. Other experiments, such as the tests on PR006919 and PR007070, and the tests on PR007268, PR007269, PR007273, PR007275 and PR007276, all showed similar results, that is, the PD-L1 × CD40 bibody could better activate the downstream signaling pathway of CD40 by cross-linking CD40 with PD-L1.

[0171] [Table 30]

[0172] Example 12 Activation effect of PD-L1×CD40 on DC cells In this example, the activation effect of PD-L1×CD40 bispecific binding proteins on dendritic cells (DCs) with and without PD-L1-mediated crosslinking was investigated.

[0173] In this example, DCs were induced using monocytes derived from PBMCs, and then activation experiments were performed. First, monocytes were sorted from PBMCs using a human CD14 sorting kit (Miltenyi, #130-050-201), cultured in RPMI1640 complete medium, and 50 ng / mL IL-4 (R&D Systems, #204-IL) and 100 ng / mL GM-CSF (R&D Systems, #215-GM) cytokines were added. After induction for 6 days, immature dendritic cells (iDCs) were produced. Half of the iDCs were added with 1 μg / mL LPS (Sigma, #L2880-10MG), and after induction for 24 hours, they became mature dendritic cells (mDCs). mDCs from donors #1 and #2 were used directly 24 hours after LPS induction, whereas mDCs from donor #4 were used after washing off LPS and re-supplementing with RPMI1640 complete medium and 50 ng / mL IL-4 and 100 ng / mL GM-CSF cytokines for 24 hours.

[0174] The day before completion of mDC induction, CHO-K1 and CHO-K1 / hPD-L1 cells were seeded in 96-well flat-bottom plates (Corning, #3599). First, CHO-K1 and CHO-K1 / hPD-L1 cells were harvested, and then 50 μg / mL mitomycin C (Selleckchem, #S8146) was added and incubated at 37°C for 1 hour to inhibit cell proliferation. After washing, the cells were incubated at a cell density of 1 × 10 in complete F-12K medium. 5 The cells were adjusted to 100μL / well and added to a 96-well flat-bottom plate at 100μL / well, and finally placed in a cell incubator overnight. The next day, the detection antibodies were diluted to 2x the final concentration in RPMI1640 complete medium, followed by 10x or 3x gradient dilutions. Selicrelumab and hIgG1 were used as positive and negative controls, respectively. After discarding the medium of CHO-K1 and CHO-K1 / hPD-L1 cells, 100μL / well of antibodies were added. Then, iDCs and mDCs were harvested, and the cell density was adjusted to 5×10 5The cells were adjusted to 100 cells / mL, added to a 96-well plate at 100 μL / well, mixed with the above-mentioned detection antibody, and finally cultured in a cell incubator. After 3 days, the cell supernatant was collected and the secretion level of IL12p40 was detected using a human IL-12 / -23(p40) ELISA kit (MABTECH, #3450-1H-20). Data processing and graph analysis were performed using the software GraphPad Prism 8 to show the amount of IL12p40 secretion related to the antibody concentration.

[0175] In this example, the PD-L1 x CD40 bibody was tested multiple times using different types of DC cells derived from multiple donors, and the results are shown in Figure 16A and 16B, Figure 17A and 17B, Figure 18A and 18B, and Figure 19A-D. Table 29 summarizes the key experimental parameters for each figure, including DC origin, target cells for crosslinking, and molecules tested.

[0176] The results showed that the positive reference antibody Selicrelumab could effectively activate both iDC and mDC cells, and this activation effect was not affected by the tumor antigen PD-L1. Meanwhile, PD-L1×CD40 bi-antibodies, such as PR006919, PR007070 and PR007151, all conditionally activate iDC and mDC cells, i.e., in the presence of PD-L1 cross-linking, they can efficiently activate DCs, while in the absence of PD-L1 cross-linking, they do not activate DCs or activate them less. Such selective activation properties of PD-L1×CD40 bi-antibodies are not found in Selicrelumab, providing a basis for the bi-antibody molecule to exert its efficacy effectively and safely. In addition, in experiments with several donor DCs, the activation effect of PD-L1×CD40 bi-antibody was significantly stronger than that of Selicrelumab, as shown in Figures 17A to 17B.

[0177] [Table 31]

[0178] Example 13 Activation effect of PD-L1×CD40 on B cells In this example, the effect of PD-L1×CD40 bispecific binding proteins on B cell proliferation with and without PD-L1-mediated crosslinking was investigated.

[0179] One day before, CHO-K1 and CHO-K1 / hPD-L1 cells were seeded in 96-well flat-bottom plates (Corning, #3599). First, CHO-K1 and CHO-K1 / hPD-L1 cells were harvested and incubated at 37°C for 1 hour with 50 μg / mL mitomycin C (Selleckchem, #S8146) to inhibit cell proliferation. After washing, the cells were incubated in F-12K complete medium at a cell density of 1 × 10 5 The cells were adjusted to 100 cells / mL and added to a 96-well flat-bottom plate at 100 μL / well and placed in a cell incubator overnight. The next day, the detection antibodies were diluted to 2x the final concentration in RPMI1640 complete medium, followed by 10-fold gradient dilution, with Selicrelumab and hIgG1 used as positive and negative controls, with the detection antibody working concentrations of 100 nM, 10 nM, and 1 nM. After discarding the medium of CHO-K1 and CHO-K1 / hPD-L1 cells, the antibodies were added at 100 μL / well. Then, B cells were sorted from donor PBMCs using a human CD19 sorting kit (Miltenyi, #130-050-301), and 1 μM CFSE (Invitrogen, #34554) was added and labeled at 37 °C for 20 min, and the cell density was adjusted to 1 × 10 6The cells were adjusted to cells / mL, added to a 96-well plate at 100 μL / well, mixed with the aforementioned detection target antibody, and finally cultured in a cell incubator. After 6 days, the cells were transferred to a 96-well U-bottom plate (Corning, #3799), centrifuged at 500g for 4 minutes at 4°C, the supernatant was discarded, and 100 μL / well of dead cell dye (Invitrogen, #L34976A, 1:1000 dilution) was added and incubated at 4°C for 15 minutes in the dark. The cells were washed twice with 200 μL / well of PBS, and 100 μL of Fc Block antibody (BD, #564220, 1:50 dilution) was added to resuspend the cells, and incubated at 4°C for 15 minutes in the dark. Then, 2 μL of anti-human CD19 antibody (Biolegend, #302234) was added per well, incubated at 4°C for 30 minutes in the dark, washed twice with 200 μL / well of 2% FBS PBS, and finally resuspended in 100 μL of 2% FBS PBS. Fluorescence signal values ​​were read using a BD FACS CANTOII flow cytometer. Data processing and graph analysis were performed using the software GraphPad Prism 8.

[0180] In this example, the PD-L1 x CD40 biantibody was tested multiple times using B cells from multiple donors, and the results are shown in Figure 20A and Figure 20B, Figure 21A and Figure 21B, Figure 22A and Figure 22B, Figure 23A and Figure 23B, and Figure 24A and Figure 24B. Table 30 summarizes the key experimental parameters for each figure, including B cell source, target cells for crosslinking, and molecules tested.

[0181] The results showed that the positive reference antibody Selicrelumab could effectively promote B cell proliferation, but this promoting effect was not affected by the tumor antigen PD-L1. Meanwhile, PD-L1 × CD40 bi-antibodies, such as PR006919, PR007070 and PR007151, all conditionally activated B cell proliferation, i.e., in the presence of PD-L1 cross-linking, they could effectively promote B cell proliferation, but in the absence of PD-L1 cross-linking, the effect was little or very weak. Such selective activating properties of PD-L1 × CD40 bi-antibodies are not found in Selicrelumab, providing a basis for the bi-antibody molecule to exert its efficacy effectively and safely. In addition, in some donor B cell proliferation experiments, the proliferation promoting effect of PD-L1 × CD40 bi-antibody was significantly stronger than that of Selicrelumab, as shown in part of Figure 24A and Figure 24B.

[0182] [Table 32]

[0183] Example 14 Antitumor activity and safety study of PD-L1×CD40 In this example, the antitumor effect of PD-L1×CD40 bispecific binding protein was investigated in the hPD1×hCD40-C57BL / 6J mouse MC38-hPDL1 tumor model. MC38-hPDL1 cells are MC38 mouse colon cancer cells that overexpress human PD-L1. hPD1×hCD40-C57BL / 6J mice are PD1 / CD40 humanized transgenic mice obtained by knocking in human PD-1 and human CD40 genes into C57BL / 6J mice (provided by Shanghai Nanfang Model Biotechnology Company).

[0184] MC38-hPDL1 cells were cultured in a 37°C, 5% CO2 incubator in DMEM medium containing 10% inactivated fetal bovine serum. They were split and passaged every 3-4 days when the petri dish was filled with cell growth. MC38-hPDL1 cells in the logarithmic growth phase were taken and resuspended in PBS, and a cell count was performed to determine the cell concentration at 1.0 × 10 7 The cell suspension was adjusted to 1.0 × 10 cells / mL and inoculated subcutaneously into the right armpit of hPD1 × hCD40-C57BL / 6J mice at 100 μL per mouse using a 1 mL syringe. 6 Tumor cells were inoculated. When the average tumor volume reached a certain volume, mice with medium tumor volume were selected and randomly divided into each experimental group with N mice per group according to the tumor volume. The administration started on the day of grouping, and was administered twice a week (BIW) for a total of 7 times, and the administration route was intraperitoneal injection. The tumor volume was measured on the day of administration, and the weight of the mice was recorded.

[0185] Example 14.1 Mouse Pharmacodynamic Study #1 In the first experiment, the average tumor volume was 110 mm 3 When the tumor volume reached 100%, mice with medium tumor volume were selected and divided into groups and administered according to the protocol in Table 31.

[0186] [Table 33]

[0187] The results are shown in Figures 25A and 25B. On the 21st day after the start of administration, the average tumor volume in the control group, Group 1 (Isotype), was 2210.35 ± 487.58 mm. 3 The tumor volumes in the treatment groups Group 2 (Selicrelumab), Group 4 (Selicrelumab + Atezolizumab), Group 5 (PR007070) and Group 6 (PR007151) were 239.07 ± 73.25 mm, respectively. 3 , 52.33±18.99mm 3 , 647.92±176.36mm3 and 507.58±116.91mm 3 The corresponding tumor inhibition rates were 93.89%, 102.78%, 74.41% and 81.10%, respectively, which were statistically significant compared with Group 1 (Isotype) (P<0.01). The tumor volumes of Group 3 (Atezolizumab) were 1563.33±325.77mm, respectively. 3 and the tumor inhibition rate is 30.81%.

[0188] On the third day after the start of administration, the body weight of mice in Group 2 (Selicrelumab) and Group 4 (Selicrelumab + Atezolizumab) was significantly reduced (by nearly 10%), while the body weight of mice in Group 3 (Atezolizumab) was normal. This indicates that Selicrelumab has side effects and toxicity that significantly cause weight loss in mice. On the other hand, the body weight of mice in the PD-L1 × CD40 double antibody groups Group 5 (PR007070) and Group 6 (PR007151) were both normal, indicating good safety.

[0189] These results indicate that the PD-L1×CD40 dual antibodies PR007070 and PR007151 have significant antitumor activity and are significantly superior to Atezolizumab. On the other hand, the safety profile of the PD-L1×CD40 dual antibodies PR007070 and PR007151 in terms of changes in mouse body weight was significantly superior to that of Selicrelumab and the combination of Selicrelumab and Atezolizumab.

[0190] Example 14.2 Mouse Pharmacodynamic Study #2 In the second experiment, the mean tumor volume was 89 mm 3 When the tumor volume reached 100%, mice with intermediate tumor volume were selected and grouped and administered according to the protocol in Table 32.

[0191] [Table 34]

[0192] The results are shown in Figures 26A and 26B. On the 18th day after the start of administration, the average tumor volume in the control group, Group 1 (hIgG1, 10 mg / kg), was 1927.77 ± 200.44 mm. 3 The tumor volumes in Group 2 (PR007619, 12 mg / kg) and Group 3 (PR007619, 6 mg / kg) were 333.00 ± 103.06 mm, respectively. 3 , 538.85±224.35mm 3 The corresponding tumor inhibition rates were 82.73% and 72.05%, respectively. The tumor volumes of Group 4 (PR007281, 12 mg / kg) and Group 5 (PR007281, 6 mg / kg) were 602.40 ± 278.52 mm, respectively. 3 , 68.66±11.68mm 3 The corresponding tumor inhibition rates were 68.75% and 96.44%, respectively. The tumor volumes in Group 6 (PR007556, 12 mg / kg) and Group 7 (PR007556, 6 mg / kg) were 347.56 ± 167.80 mm, respectively. 3 , 258.51±102.12mm 3 The corresponding tumor inhibition rates were 81.97% and 86.59%, respectively. The tumor volumes in Group 8 (PR007276, 12 mg / kg) and Group 9 (PR007276, 6 mg / kg) were 200.34 ± 59.12 mm, respectively. 3 , 58.33±42.67mm 3 The corresponding tumor inhibition rates were 88.57% and 96.97%, respectively. Throughout the experiment, the body weight status of the mice in the treatment group was normal, and the drug had no obvious effect on the mouse status.

[0193] These results demonstrated that the PD-L1 x CD40 dual antibodies PR007619, PR007281, PR007556, and PR007276 all showed excellent antitumor activity and safety.

[0194] Example 14.3 Mouse Pharmacodynamic Experiment #3 and Mouse Hepatotoxicity Study In the third experiment, the mean tumor volume was 85 mm 3 When the tumor volume reached 100%, mice with intermediate tumor volume were selected and grouped and administered according to the protocol in Table 33.

[0195] 24 hours after the last administration, mouse serum from each group (36 samples in total) was collected and used for blood biochemistry index test to detect the contents of ALT and AST (alanine aminotransferase and aspartate aminotransferase) in mouse serum. Liver tissues from 36 mice were collected and embedded in paraffin. Six consecutive sections were taken from each liver tissue, and then H&E stained. Finally, three stained sections were selected for scanning and microscopic analysis.

[0196] [Table 35]

[0197] The results of the antitumor effect are shown in Figures 27A and 27B. On the 22nd day after the start of administration, the tumor volume in the control group Group 1 (hIgG1, 3.6 mg / kg) was 1865.24 ± 125.47 mm. 3 The tumor volumes in Group 2 (PR007619, 1.8 mg / kg), Group 3 (PR007619, 3.6 mg / kg), Group 4 (PR007619, 6 mg / kg), and Group 5 (PR007619, 10.8 mg / kg) were 1177.74 ± 198.47 mm, respectively. 3 , 829.01±164.87mm 3 , 708.84±208.76mm 3 , 541.99±41.2mm 3 The corresponding tumor inhibition rates were 36.86%, 55.55%, 62%, and 70.94%, respectively. The tumor volume in Group 6 (Selicrelumab, 5 mg / kg) was 339.68 ± 72.9 mm 3The corresponding tumor inhibition rate was 81.79%. Throughout the experiment, the mouse weight status of the hIgG1 control group and the PR007619 treatment group was normal, and the drug had no obvious effect on the mouse status. However, the mice in the Selicrelumab treatment group showed a certain degree of mouse weight loss, indicating that Selicrelumab has certain side effects and toxicity.

[0198] These results demonstrate that the PD-L1 x CD40 dual antibody PR007619 has dose-dependent tumor inhibitory activity and a significant inhibitory effect, and has a better safety profile than the control molecule Selicrelumab.

[0199] The results of the blood biochemistry index test are shown in Figure 28. The corresponding average ALT values ​​of Group 1 to Group 6 were 34±8.83U / L, 46±24.64U / L, 34.5±7.04U / L, 32.5±9.38U / L, 31.5±1.64U / L, and 128.7±76.2U / L, respectively. Here, Group 6 (Selicrelumab, 5mg / kg) was significantly elevated compared with Group 1 (control group) (P=0.0128), while Group 2 to Group 5 (different doses of PR007619 administered groups) had no significant difference compared with Group 1 (control group). Similarly, the corresponding mean AST values ​​in Group 1 to Group 6 were 180±40.3U / L, 197.5±95.9U / L, 120.5±41.2U / L, 120±61.3U / L, 123.5±16.69U / L, and 326.17±153.6U / L, respectively. Here, Group 6 (Selicrelumab, 5mg / kg) was significantly elevated compared with Group 1 (control group) (P=0.0478), while Groups 2 to 5 (different doses of PR007619 administered groups) had no significant difference compared with Group 1 (control group).

[0200] The results of tissue sections are shown in Figure 29. The mice in Group 6 (Selicrelumab, 5 mg / kg) showed obvious damage in the liver tissue, specifically, moderate portal vein multifocal monocytic and lymphocytic infiltration, moderate sinusoidal multifocal Kupffer cell proliferation, significant sinusoidal monocytosis and proliferation, and significant multifocal hepatocyte degeneration and necrosis. On the other hand, the mice in Group 1 (control group) and Groups 2 - 5 (groups administered different doses of PR007619) showed no obvious pathological changes in the liver tissue.

[0201] According to the above blood biochemical indexes and tissue section results, the CD40 monoclonal antibody selicrelumab induced significant acute hepatotoxicity in hPD1×hCD40-C57BL / 6J transgenic mice, while the PD-L1×CD40 dual antibody PR007619 showed excellent safety profile.

[0202] Example 14.4 Mouse Pharmacodynamic Experiment #4 and Mouse Hepatotoxicity Study The fourth experiment aims to investigate the effects of PD-L1 × CD40 dual antibodies on mice, including inhibition of tumor growth, changes in mouse body weight, changes in serum cytokine levels and changes in hepatotoxicity blood biochemical indicators.

[0203] In the fourth experiment, the mean tumor volume was 82 mm 3When the tumor volume reached 100 mg / kg, mice with moderate tumor volume were selected and grouped and administered according to the protocol in Table 34. Each group consisted of 10 mice (5 female mice and 5 male mice). Administration began on the day of grouping, and was administered twice a week (BIW) for a total of four times (days 0, 3, 7, and 10), and the administration route was intraperitoneal injection. Tumor volume was measured during the period (days 0, 2, 5, 9, and 11). 24 hours after the first administration, serum was collected and used to test cytokine levels (IL-6, TNF-α, IFN-γ, and IL12p40). 24 hours after the last administration, serum was collected and used for blood biochemical detection of hepatotoxicity indicators (ALT and AST). Finally, mouse liver tissues (total of 50) were collected and embedded in paraffin, and six consecutive sections were taken from each liver tissue, followed by H&E staining, and finally three stained sections were selected for scanning and microscopic analysis. In addition to the liver, the mouse heart, spleen, lungs, and kidneys were also collected and subjected to paraffin embedding and section staining analysis.

[0204] [Table 36]

[0205] The results of the antitumor effect are shown in Figures 30A and 30B. On the 11th day after the start of administration, the average tumor volume in the control group Group 1 (hIgG1, 10 mg / kg) was 1653.68 ± 153.10 mm 3 The tumor volumes in Group 2 (PR007619, 12 mg / kg), Group 3 (Selicrelumab, 1 mg / kg), Group 4 (Selicrelumab, 2.5 mg / kg) and Group 5 (Selicrelumab, 5 mg / kg) were 552.96 ± 123.59 mm, respectively. 3 , 542.23±79.83mm 3 , 350.60±61.35mm 3 and 272.65±45.48mm 3The corresponding tumor inhibition rates were 66.56%, 67.21%, 78.80% and 83.51%, respectively. Each administration group had a statistical difference compared with Group 1 (P>0.05). On the other hand, according to the weight results, Group 2 (PR007619, 12mg / kg) had no obvious effect on mouse weight, and each dose group of Selicrelumab (Groups 3-5) caused a decrease in mouse weight after administration, the degree of decrease was close to or exceeded 10%, and the higher the dose, the harder it was to recover weight. In particular, the middle and high dose groups of Selicrelumab did not recover weight throughout the administration.

[0206] These results suggest that the PD-L1 × CD40 dual antibody has significant antitumor effects and is safe, but selicrelumab caused weight loss in mice and significant toxic side effects.

[0207] [Table 37]

[0208] The results of the blood biochemistry index test are shown in Figure 31 and Table 35. The corresponding ALT values ​​of Group 1 to Group 5 were 168.3±79.8U / L, 107.6±54.2U / L, 105.1±44.8U / L, 310±189.3U / L and 507.6±150.0U / L, respectively, and the corresponding AST values ​​of Group 1 - Group 5 were 51.6±69.1U / L, 19.7±7.26U / L, 31±19.1 / L, 96.5±86.1U / L and 147.7±73.1U / L, respectively. Here, Group 5 (Selicrelumab, 5 mg / kg) showed a significant improvement in ALT and AST compared with Group 1 control group (P<0.001), and Group 4 (Selicrelumab, 2.5 mg / kg) showed a significant difference in ALT compared with Group 1 control group (P=0.037). However, Group 2 (PR007619, 12 mg / kg) showed no significant difference in both ALT and AST compared with Group 1 control group. This indicates that Selicrelumab caused a significant increase in the hepatotoxic blood biochemistry indexes ALT and AST, but PD-L1 × CD40 dual antibody did not cause an increase in ALT and AST levels in mouse serum.

[0209] [Table 38]

[0210] The results of the peripheral blood cytokine test are shown in Figure 32, Figure 33 and Table 36. The corresponding cytokine IL-6 values ​​in Group 1 to Group 5 were 16.3±8.74pg / mL, 24.8±11.7pg / mL, 1343.8±529.5pg / mL, 1428.2±569.5pg / mL and 2038.0±747.2pg / mL, respectively, and the corresponding cytokine IFN-γ values ​​in Group 1 - Group 5 were 0.54±0.19pg / mL, 1.48±0.98pg / mL, 159.0±90.9pg / mL, 138.9±91.7pg / mL and 147.4±79.44pg / mL, respectively. The corresponding cytokine TNF-α values ​​in Group 1 - Group 5 were 14.1±3.79pg / mL, 17.3±8.08pg / mL, 1186.1±255.7pg / mL, 1274.7±392.3pg / mL and 1311.5±179.9pg / mL, respectively, and the corresponding cytokine IL12p40 values ​​in Group 1 - Group 5 were 1157.1±125.8pg / mL, 1786.8±369.5pg / mL, 92119.7±30959.2pg / mL, 99263.0±24254.9pg / mL and 110786.1±21607.5pg / mL, respectively.

[0211] As can be seen, in the levels of cytokines IL-6, TNF-α and IFN-γ, all of the Selicrelumab dose groups (Groups 3-5) were able to induce a significant increase in the cytokine levels, showing a significant difference compared to the control group of Group 1 (P<0.0001), while the PD-L1×CD40 double antibody (Group 2) did not induce an increase in the cytokine levels, showing no significant difference compared to the control group of Group 1 (P>0.05). In addition, in the levels of cytokines IL12p40, all of the Selicrelumab dose groups (Groups 3-5) were able to induce a significant increase in the IL12p40 levels, showing a significant difference compared to the control group of Group 1 (P<0.0001), while the PD-L1×CD40 double antibody (Group 2) induced a slight increase in IL12p40 (P=0.0004). The combined results showed that selicrelumab, but not the PD-L1 × CD40 dual antibody, induced significant cytokine release syndrome in mouse peripheral blood.

[0212] The results of liver tissue sections are shown in FIG. 34. In Group 1, all 10 animals had livers within normal range. In Group 2, only one animal, B5784, had a small amount of multifocal perivascular lymphocytic infiltration, while the other 9 animals had livers within normal range. In Group 3, all 10 animals had minimal, mild or very mild multifocal perivascular lymphocytic infiltration. In Group 4, the liver of animal A5757 had moderate multifocal perivascular lymphocytic infiltration, while the other animals had moderate multifocal granulomatous inflammation (monocyte infiltration with fibrosis, mostly lymphocytes and macrophages) in the perivascular or venous sinus. In Group 5, all animals had moderate and marked multifocal granulomatous inflammation (monocyte infiltration fibrosis, mostly lymphocytes and macrophages) in the perivascular or venous sinus.

[0213] In this study, compared with the control group (Group 1), the PD-L1 × CD40 bibody (Group 2) had minimal or mild changes in the liver, kidney, lung and spleen, with minimal lymphocytic infiltration, which is a predictable pharmacodynamic response. Compared with the control group (Group 1), each Selicrelumab dose group (Group 3, Group 4 and Group 5) had more adverse changes in these tissues. The renal tubules in these tissues were degenerated and there was more lymphocytic infiltration and / or inflammation, which could expand the predictable pharmacodynamic response and be harmful to the animals. Therefore, the extent and incidence of lesions with the PD-L1 × CD40 bibody was less than with Selicrelumab.

[0214] In conclusion, selicrelumab caused severe cytokine release syndrome, elevated serum indicators of hepatotoxicity, and damage to the liver and other organ tissues in mice, whereas the PD-L1×CD40 biantibody had a strong antitumor effect and an excellent safety profile, and did not cause cytokine release syndrome, elevated serum indicators of hepatotoxicity, or damage to the liver and other organ tissues.

[0215] Example 15 Pharmacokinetic evaluation of PD-L1×CD40 In this example, the pharmacokinetic performance in mice of four bispecific binding protein molecules with similar sequences and identical structures was examined.

[0216] Dosing and blood collection: For each test binding protein molecule, six female C57BL / 6J mice weighing 18-22 grams were selected and administered the test binding protein molecule at a dose of 6 mg / kg via intravenous injection. Whole blood was collected from three mice in one group at 5 minutes, 24 hours (day 1), day 4, and day 10 after dosing, and from three mice in another group at pre-dose, 5 hours after dosing, day 2, day 7, and day 14. Whole blood was allowed to settle until it clotted, then centrifuged, and the separated serum samples were frozen and stored at -80°C until analysis.

[0217] Analytical methods: Two ELISA methods were used to quantitatively measure the drug concentration in mouse serum. ELISA method 1 was the Fc-terminal detection method, in which goat anti-human Fc polyclonal antibody (ROCKLAND, #609-101-017) coated on a 96-well plate was used to capture human Fc-containing antibodies in mouse serum, and HRP-labeled goat anti-human Fc secondary antibody (Jackson, #109-035-098) was added for detection. ELISA method 2 was the functional domain detection method, in which PD-L1 antigen protein (Novoprotein, #C315) coated on a 96-well plate was used to capture antibodies that specifically recognize the antigen in mouse serum, and HRP-labeled goat anti-human Fc secondary antibody (Jackson, #109-035-098) was added for detection. The pharmacokinetic parameters were analyzed using Phoenix WinNonlin software version 8.2, choosing the non-compartment model (NCA).

[0218] As shown in Table 37, the dual antibody molecule PR007556 has a serum half-life t1 / 2 value similar to that of conventional IgG antibodies, and the PD-L1 end detection method showed that its t1 / 2 value was 10.3 days. The dual antibody molecules PR007619, PR007281 and PR007276 have pharmacokinetics similar to that of conventional IgG antibodies, and in the Fc end detection method, PR007619, PR007281 and PR007276 had serum half-life t1 / 2 values ​​of 10.3 days in mice. 1 / 2 In the PD-L1 terminal detection method, PR007619, PR007281 and PR007276 have in vivo serum half-life t1 / 2 values ​​in mice of approximately 13.6 days, 15.1 days and 15.7 days, respectively.

[0219] [Table 39]

[0220] The results are shown in Table 37 and Figures 35A to 35D. These PD-L1×CD40 bibody molecules have serum half-life t values ​​similar to those of conventional IgG antibodies, and their T 1 / 2 The value is 10 to 20 days.

[0221] Although the specific embodiments of the present invention have been described above, it should be understood by those skilled in the art that these are merely examples, and multiple changes or modifications can be made to these embodiments without departing from the principle and essence of the present invention. Therefore, the protection scope of the present invention is limited by the appended claims.

Claims

1. 1. An antigen binding protein that targets PD-L1 and CD40 comprising a first protein functional region and a second protein functional region, said first protein functional region comprising an antigen binding protein that targets CD40 and said second protein functional region comprising an antigen binding protein that targets PD-L1, wherein: The antigen binding protein targeting CD40 comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises LCDR1, LCDR2 and LCDR3, and the VH comprises HCDR1, HCDR2 and HCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:38, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:43 and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:48, or a variant 3 having 3, 2 or 1 amino acid mutations in SEQ ID NO:48, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:8, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:18 and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

27. An antigen-binding protein targeting PD-L1 and CD40, comprising the amino acid sequence shown in FIG.

2. The variant 3 comprises an amino acid sequence having a PTM site mutation in the amino acid sequence shown in SEQ ID NO: 48, preferably an amino acid sequence having an amino acid mutation at position 4 and / or position 5 of the amino acid sequence shown in SEQ ID NO: 48, and the amino acid mutation is preferably an amino acid substitution, more preferably a conservative amino acid substitution. Suitably, the variant 3 is an amino acid sequence having an N4A / F / Y / V / N and / or S5N mutation in the amino acid sequence shown in SEQ ID NO: 48, more suitably, the variant 3 is an amino acid sequence shown in any one of SEQ ID NOs: 49 to 53, and even more suitably, in the antigen-binding protein targeting CD40, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 38, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 43, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

2. The antigen-binding protein targeting PD-L1 and CD40 of claim 1, wherein the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 48 to 53, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

27.

3. 3. The antigen binding protein targeting PD-L1 and CD40 of claim 1 or 2, wherein the VL comprises an amino acid sequence as set forth in SEQ ID NO:68 or an amino acid sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:68, and the VH comprises an amino acid sequence as set forth in SEQ ID NO:60 or an amino acid sequence having at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:60, preferably the VL comprises an amino acid sequence as set forth in any one of SEQ ID NOs:68-73, and the VH comprises an amino acid sequence as set forth in SEQ ID NO:

60.

4. the antigen-binding protein targeting CD40 is a full-length antibody, the full-length antibody comprising a light chain and a heavy chain, the light chain comprising a light chain constant region (CL), the light chain constant region being preferably a human-derived antibody light chain constant region, the human-derived antibody light chain constant region being more preferably a κ subtype light chain constant region, the heavy chain comprising a heavy chain constant region (CH), the heavy chain constant region being preferably a human-derived antibody heavy chain constant region, more preferably a hIgG1, hIgG2, hIgG3 or hIgG4 subtype heavy chain constant region, and even more preferably a hIgG1 subtype heavy chain constant region; 4. The antigen-binding protein targeting PD-L1 and CD40 of any one of claims 1 to 3, wherein the antigen-binding protein targeting CD40 is a full-length antibody, the full-length antibody comprising a light chain and a heavy chain, the light chain comprising the amino acid sequence shown in any one of SEQ ID NOs: 87 to 92, and the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 78 or 84, more preferably the light chain comprising the amino acid sequence shown in SEQ ID NO: 87 and the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 78, or the light chain comprising the amino acid sequence shown in any one of SEQ ID NOs: 88 to 92, and the heavy chain comprising the amino acid sequence shown in SEQ ID NO:

84.

5. The antigen binding protein targeting PD-L1 comprises a heavy chain variable region (VH), the VH comprising HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence shown in SEQ ID NO:5 or variant 7 having 3, 2 or 1 amino acid mutations in SEQ ID NO:5, the HCDR2 comprising the amino acid sequence shown in SEQ ID NO:16 or variant 8 having 3, 2 or 1 amino acid mutations in SEQ ID NO:16, the HCDR3 comprising the amino acid sequence shown in SEQ ID NO:25 or variant 9 having 3, 2 or 1 amino acid mutations in SEQ ID NO:25, preferably variant 7 having an amino acid mutation at position 3 and / or 6 of SEQ ID NO:5, and variant 8 having an amino acid mutation at position 3 of SEQ ID NO:

5. and variant 9 is an amino acid sequence of SEQ ID NO: 25 having an amino acid mutation at position 4, 8 and / or 11, the amino acid mutation being preferably an amino acid substitution, more preferably a conservative amino acid substitution. More preferably, variant 7 is an amino acid sequence of SEQ ID NO: 5 having an N3T / D and / or N5S mutation, variant 8 is an amino acid sequence of SEQ ID NO: 16 having a W1R, D3T and / or K5E mutation, variant 9 is an amino acid sequence of SEQ ID NO: 25 having an I4L, V8I and / or A11D mutation, and even more preferably, variant 7 is the amino acid sequence of SEQ ID NO: 7 or 9, variant 8 is the amino acid sequence of SEQ ID NO: 19, and variant 9 is an amino acid sequence of SEQ ID NO: 25 having an I4L, V8I and / or A11D mutation. The antigen-binding protein targeting PD-L1 and CD40 according to any one of claims 1 to 4, characterized in that it has the amino acid sequence shown in NO: 28, 29 or 30.

6. In the antigen-binding protein targeting PD-L1, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 5, 7 or 9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 16 or 19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 25, 28, 29 or 30; preferably, in the antigen-binding protein targeting PD-L1, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 5, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 16, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 25, or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 16, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 28; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 25; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 29; or the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:

6. The antigen binding protein targeting PD-L1 and CD40 of claim 5, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30; or the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

28.

7. 7. The antigen-binding protein targeting PD-L1 and CD40 of claim 5 or 6, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 57 or an amino acid sequence having at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 57, and preferably the VH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 57-65.

8. the first protein functional region is an immunoglobulin comprising an antigen binding protein that targets CD40, and the second protein functional region comprises the VH of one, two or more antigen binding proteins that target PD-L1; Preferably, the VH of the PD-L1 targeting antigen binding protein is linked directly to the immunoglobulin, or the VH is linked to the immunoglobulin via a linker, or if the number of VHs is greater than one, each VH is linked to the immunoglobulin, respectively, directly or via a linker; More preferably, when the VH is linked to the immunoglobulin via a linker, the linker is selected from the group consisting of GS, GGS, and any one of the amino acid sequences set forth in SEQ ID NOs: 100 to 106. The antigen-binding protein targeting PD-L1 and CD40 of any one of claims 1 to 7.

9. The antigen binding protein targeting PD-L1 and CD40 according to claim 8, wherein the second protein functional region comprises the VHs of the two antigen binding proteins targeting PD-L1, and preferably the two VHs comprised in the second protein functional region are the same and / or the C-terminus of the two VHs is linked to the N-terminus of the two heavy chains of the immunoglobulin via a linker shown in GGS or SEQ ID NO: 5, respectively.

10. The first protein functional region comprises two light chains comprising the amino acid sequence shown in any one of SEQ ID NOs: 88 to 92 and two heavy chains comprising the amino acid sequence shown in SEQ ID NO: 84, the second protein functional region comprises two VHs comprising the amino acid sequence shown in any one of SEQ ID NOs: 79 to 83, and the C-terminus of the two VHs is linked to the N-terminus of the two heavy chains of the first protein functional region via a linker shown in GGS or SEQ ID NO: 5, Suitably, the antigen binding protein that targets PD-L1 and CD40 comprises: a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:89, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:93, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:88, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:94, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:94, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:90, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:93, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:92, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:93, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:95 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:88, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:96 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:88, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:97 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:95 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:93 or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91 a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:96, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:89, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:97, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:90, a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:97, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:88, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:

10. The antigen binding protein targeting PD-L1 and CD40 of any one of claims 1 to 9, comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:88, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:99, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91, the first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:98, or a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO:91, or a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:

99.

11. A chimeric antigen receptor comprising an antigen-binding protein that targets PD-L1 and CD40 according to any one of claims 1 to 10.

12. 11. An isolated nucleic acid, characterized in that said isolated nucleic acid encodes an antigen-binding protein that targets PD-L1 and CD40 according to any one of claims 1 to 10.

13. 13. A recombinant expression vector, comprising the isolated nucleic acid of claim 12, preferably comprising a eukaryotic expression vector and / or a prokaryotic expression vector.

14. A transformant, the transformant comprising the isolated nucleic acid according to claim 12 or the recombinant expression vector according to claim 13, preferably a host cell of the transformant is a prokaryotic cell and / or a eukaryotic cell, the prokaryotic cell is preferably an E. coli cell such as TG1, BL21, and the eukaryotic cell is preferably a HEK293 cell or a CHO cell.

15. A method for producing an antigen-binding protein targeting PD-L1 and CD40 according to any one of claims 1 to 10, comprising the steps of culturing the transformant according to claim 14 and obtaining the antigen-binding protein targeting PD-L1 and CD40 from the culture.

16. 11. An antibody drug conjugate comprising an antigen binding protein targeting PD-L1 and CD40 according to any one of claims 1 to 10, and a cytotoxic agent or tag, preferably wherein the cytotoxic agent is MMAF or MMAE and the tag is a fluorescent agent.

17. A genetically modified cell, characterized in that said genetically modified cell expresses a chimeric antigen receptor according to claim 11, advantageously said genetically modified cell being a eukaryotic cell, preferably an isolated human cell, more preferably an immune cell such as a T cell or a NK cell.

18. 17. A pharmaceutical composition comprising one or more of the antigen binding protein targeting PD-L1 and CD40 according to any one of claims 1 to 10, the chimeric antigen receptor according to claim 11, the isolated nucleic acid according to claim 12, the recombinant expression vector according to claim 13, the genetically modified cell according to claim 17 and the antibody drug conjugate according to claim 16, and / or a pharma- ceutical acceptable carrier, preferably further comprising other anti-tumor drugs as active ingredients, the anti-tumor drugs including, but not limited to, chemotherapeutic drugs, nucleotide drugs, small molecule targeted drugs, monoclonal antibody drugs, bi / multispecific antibody drugs, recombinant protein drugs, immunomodulatory drugs, cell therapy drugs and gene therapy drugs.

19. A detection reagent, comprising an antigen-binding protein targeting PD-L1 and CD40 according to any one of claims 1 to 10 and / or an antibody-drug conjugate according to claim 16, preferably in a liquid dosage form, a gaseous dosage form, a solid dosage form or a semi-solid dosage form, more preferably further comprising a secondary antibody, CD40 or a derivative thereof, wherein the secondary antibody, for example an anti-human IgG antibody, binds to horseradish peroxidase and the anti-human IgG antibody binds to a biotinylated protein.

20. A kit of parts, said kit of parts comprising kit A, said kit A containing one or more of the following: an antigen binding protein targeting PD-L1 and CD40 according to any one of claims 1 to 10, a chimeric antigen receptor according to claim 11, a pharmaceutical composition according to claim 18, a detection reagent according to claim 19, a genetically modified cell according to claim 17 and an antibody drug conjugate according to claim 16; Preferably, the kit of parts further comprises kit B, wherein kit B contains another anti-tumor antibody or a pharmaceutical composition comprising said other anti-tumor antibody, and / or one or more of the group consisting of a hormonal preparation, a targeted small molecule preparation, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic agent, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.

21. The present invention relates to a method for the manufacture of a medicament for diagnosing, preventing and / or treating a disease associated with PD-L1 and / or CD40, comprising administering to said subject one or more of the antigen binding protein targeting PD-L1 and CD40 according to any one of claims 1 to 10, the chimeric antigen receptor according to claim 11, the isolated nucleic acid according to claim 12, the recombinant expression vector according to claim 13, the pharmaceutical composition according to claim 18, the detection reagent according to claim 19, the kit of parts according to claim 20, the genetically modified cell according to claim 17 and the antibody drug conjugate according to claim 16. In the present invention, the PD-L1 and / or CD40-associated disease is preferably a PD-L1-associated tumor, a CD40-associated tumor, or a PD-L1×CD40-associated tumor, and the CD40-associated tumor includes a solid tumor and a blood tumor, and the PD-L1-associated tumor includes a solid tumor and a blood tumor, and more preferably, the CD40-associated tumor is a B-lineage NHL, chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), Hodgkin's disease, multiple myeloma, bladder cancer, kidney cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, nasopharyngeal cancer, malignant melanoma, pancreatic cancer, and colon cancer. The PD-L1 associated tumors include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), primary mediastinal large B cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), T cell / histiocyte rich tumors, and gliomas. large B-cell lymphoma, multiple myeloma, myeloid cell leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), gastrointestinal (tubal) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric cancer, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, pharyngeal cancer, hepatobiliary cancercancer), central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light-chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative neoplasms, neuroendocrine tumors, Merkel cell carcinoma, testicular cancer and skin cancer, preferably PD-L1 highly expressing colon cancer.

22. 13. A method for detecting PD-L1 and / or CD40 in a sample, the method comprising the step of contacting the sample with one or more of an antigen binding protein targeting PD-L1 and CD40 according to any one of claims 1 to 10, a detection reagent according to claim 19 and an antibody drug conjugate according to claim 16, the sample being, for example, a blood sample (e.g. a whole blood sample and a serum sample) and a reagent comprising PD-L1 and / or CD40, preferably the method being not for therapeutic / diagnostic purposes.

23. 20. A method for diagnosing, treating and / or preventing a PD-L1 and / or CD40 associated disease, said method comprising administering to a patient in need thereof a therapeutically effective amount of one or more of the following: an antigen binding protein targeting PD-L1 and CD40 according to any one of claims 1 to 10, a chimeric antigen receptor according to claim 11, an isolated nucleic acid according to claim 12, a recombinant expression vector according to claim 13, a pharmaceutical composition according to claim 18, a detection reagent according to claim 19, a kit of parts according to claim 20, a genetically modified cell according to claim 17, and an antibody drug conjugate according to claim 16, preferably wherein the PD-L1 and / or CD40 associated disease is a PD-L1 associated tumor, a CD40 associated tumor or a PD-L1xCD40 associated tumor.

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