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 202210351186X, filed on 2022 / 4 / 2. This application references the entirety of the aforementioned Chinese patent application.

[0002] This invention relates to the field of biopharmaceuticals, and more particularly to bispecific antigen-binding proteins targeting PD-L1 and CD40, as well as their production and application. [Background technology]

[0003] CD40 is a glycosylated type I transmembrane protein, a member of the tumor necrosis factor receptor superfamily (TNFRSF), and is also referred to as tumor necrosis factor receptor superfamily member 5 (TNFRSF5). CD40 is expressed on the surface of a range of antigen-presenting cells (APCs), including monocytes, dendritic cells (DCs), B cells, and macrophages. Its ligand, CD40L, is mainly expressed on the surface of lymphocytes including T cells, B cells, and NK cells, and normally exists in the form of trimers and multimers. CD40 and CD40L are a pair of costimulatory molecules, and activation of the downstream signaling pathway of CD40 requires the formation of cross-linking between CD40L in trimeric and multimeric forms and CD40. They interact on the cell surface, inducing the redistribution of CD40 to membrane lipid rafts and triggering conformational changes. CD40 recruits TNFR-associated factors (TRAFs) in the cytoplasm via its intracellular terminal domain and promotes intracellular signal transduction, thereby activating different signaling pathways, such as the canonical and non-canonical nuclear factor κB pathways, p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase (PI3K) and phospholipase Cγ pathways. Genes targeted by these signaling pathways further regulate apoptosis, cell cycle progression, cytokine production, and the expression of cell surface immunoregulatory factors. Thus, activating CD40 can increase antigen presentation, promote cytokine secretion, activate lymphocytes, stimulate and activate the human innate and adaptive immune systems, produce a synergistic effect, and counteract the occurrence and progression of cancer.

[0004] CD40 is widely expressed in tumor cells, including almost all B-cell malignancies and a wide range of solid tumors, such as melanoma, lung cancer, breast cancer, colon cancer, prostate cancer, pancreatic cancer, kidney 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 via CD40L crosslinking mediates direct cytotoxicity. In vitro, CD40 crosslinking by CD40L has been shown to induce apoptosis in tumor cells and inhibit the proliferation of solid tumor cells and B-cell malignancies. In vivo, CD40 activation also mediates tumor inhibitory effects. In immunodeficient mice, evidence shows that interfering with CD40 on the surface of tumor cells via CD40L can inhibit the proliferation of breast cancer cell transplanted tumors or B-lymphocyte transplanted tumors, even without lymphocyte activation.

[0005] Therefore, the mechanism of CD40-mediated tumor cell death may be dual, meaning that the immune system's stimulating action against tumor cells and the direct tumor cell-toxic effect can synergistically produce an antitumor effect. Agonist-type anti-CD40 antibodies, like CD40L, can cross-link and activate CD40 on the surface of immune cells and tumor cells, thereby producing a significant antitumor effect. Such antitumor effects have been demonstrated in preclinical animal models and clinical trials in tumor patients, and can produce a synergistic antitumor effect when used in combination with both chemotherapeutic agents such as gemcitabine and paclitaxel, and immunomodulatory agents such as PD-1 antibodies and CTLA-4 antibodies.

[0006] Currently, in the field of anti-tumor therapy, nearly 20 CD40 antibody drugs are in the clinical trial stage, but the earliest developed product is only in Phase II clinical trial, and no product has been commercialized yet. The main problems encountered by CD40 antibodies in clinical application include low objective response rate, significant toxic side effects, and low tolerable dose. One of the causes that may lead to these results is reduced agonist activity, as seen in Celldex's product CDX-1140. In its Phase I clinical trial, no complete or partial remission occurred in 42 patients treated with the single agent; in the combination therapy with rhFLT3L, only 1 out of 20 patients achieved partial remission. This product has weak activation activity against DC cells in vitro, and cannot enhance agonist activity even when crosslinking is performed. Another cause is that CD40 antibodies cannot be selectively activated, leading to systemic toxic side effects. For example, in clinical trials of Roche's CD40 antibody Selicrelumab, when the administered dose exceeds 0.2 mg / kg, significant toxic side effects such as cytokine release syndrome and liver toxicity occur in some tested patients; in clinical trials of Apexigen's CD40 antibody APX005M, when the administered dose exceeds 0.3 mg / kg, significant neutropenia, and even subsequent sepsis and septic shock are induced in some tested patients. In summary, currently clinically studied CD40 antibodies are facing the problem and challenge of an excessively narrow therapeutic window between efficacy and safety. Therefore, the development of novel CD40 antibodies provides possibilities for the treatment of various tumors, and has high scientific and market value.

[0007] PD-L1, also known as programmed death ligand 1, CD274 or B7H1, is a glycosylated type I transmembrane protein and one of the ligands for 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, as well as 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, inducing immunoconstriction in the body and playing a crucial role in maintaining immune tolerance. On the other hand, PD-L1 is expressed in large quantities 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. The binding of PD-L1 on tumor cells to PD-1 on tumor-infiltrating T cells (TILs) activates phosphatases (SHPs) in the Src homology domain 2, leading to inhibition of the T cell receptor (TCR) pathway and T cell activity. Furthermore, tumor cells utilize 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 with antibodies can reactivate immune cells such as T cells that have been inhibited and depleted in the tumor microenvironment, thereby eliminating cancer cells and restoring the 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 achieved clinical responses of 10% to 40% for 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, while anti-PD-L1 / PD-1 therapy has shown impressive effects in tumor treatment, particularly in solid tumors, sustained responses occurred in only a small number of patients, and some patients who initially responded to treatment eventually developed acquired resistance. Moreover, the majority of patients still do not respond to anti-PD-L1 / PD-1 therapy, i.e., they exhibit spontaneous resistance. Therefore, there is an urgent need to develop more effective immunotherapies that can produce synergistic antitumor effects with anti-PD-L1 / PD-1 therapy to overcome spontaneous and acquired resistance, and this approach is currently attracting attention in tumor immunotherapy.

[0010] Currently, there are no commercially available antigen-binding proteins that target both PD-L1 and CD40, and there is a growing market demand for antigen-binding proteins that target both PD-L1 and CD40. [Overview of the project] [Problems that the invention aims to solve]

[0011] In contrast to the limited effectiveness and low safety of antigen-binding proteins targeting PD-L1 and CD40 in conventional technologies, the present invention provides antigen-binding proteins targeting PD-L1 and CD40, as well as their production and application. The antigen-binding proteins targeting PD-L1 and CD40 block the PD-1 / PD-L1 inhibitory signaling pathway and act on activated CD40 receptors, simultaneously activating antigen-presenting cells and lymphocytes, thereby achieving a remarkable synergistic antitumor effect. Furthermore, the activation of immune cells by the antigen-binding proteins targeting PD-L1 and CD40 occurs only in the tumor microenvironment, significantly eliminating systemic drug toxicity and side effects. As a result, the antigen-binding proteins targeting PD-L1 and CD40 exert their excellent antitumor effect under extremely safe conditions. [Means for solving the problem]

[0012] To solve the above technical problems, a first aspect of the present invention provides an antigen-binding protein targeting PD-L1 and CD40, wherein the antigen-binding protein comprises a first protein functional region and a second protein functional region, the first protein functional region comprising an antigen-binding protein targeting CD40, and the second protein functional region comprising an antigen-binding protein targeting PD-L1, where, The antigen-binding protein targeting CD40 comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprising LCDR1, LCDR2, and LCDR3, the VH comprising HCDR1, HCDR2, and HCDR3, the LCDR1 comprising 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 comprising 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 comprising 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 comprising 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 comprising SEQ ID NO: The HCDR3 comprises a mutant 5 having 3, 2, or 1 amino acid mutations in the amino acid sequence shown in 18 or SEQ ID NO: 18, and the HCDR3 comprises a mutant 6 having 3, 2, or 1 amino acid mutations in the amino acid sequence shown in SEQ ID NO: 27 or SEQ ID NO: 27.

[0013] In some technical proposals of the present invention, 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 LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 38, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 43, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 48, or a mutant 3 having 3, 2, or 1 amino acid mutations in SEQ ID NO: 48, and HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0014] In some technical proposals of the present invention, the variant 3 includes 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 in which an amino acid mutation occurs at the 4th and / or 5th position of the amino acid sequence shown in SEQ ID NO: 48, wherein the amino acid mutation is preferably an amino acid substitution, and more preferably a conserved amino acid substitution. Preferably, the variant 3 is an amino acid sequence in which an N4A / F / Y / V / N and / or S5N mutation occurs 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 NO: 49 to 53.

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

[0016] [Table 1]

[0017] In some technical applications of the present invention, in the antigen-binding protein targeting CD40, the VL includes the 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 includes the 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 includes the amino acid sequence shown in any one of SEQ ID NO: 68 to 73, and the VH includes the amino acid sequence shown in SEQ ID NO: 60, with specific combinations of VL and VH being shown in Table 1-2.

[0018] [Table 2]

[0019] In some technical proposals 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, 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), 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 an hIgG1 subtype heavy chain constant region.

[0020] In some technical applications of the present invention, 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 an amino acid sequence shown in any one of SEQ ID NO: 87 to 92, and the heavy chain comprising 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 and 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 NO: 88 to 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 Table 1-3.

[0021] [Table 3]

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

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

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

[0025] In some technical applications 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: 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: The amino acid sequence is as shown in 25, or the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 29, or the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 30, or the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 28.

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

[0027] In some technical applications 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 applications 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, either directly or via a linker.

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

[0030] In some technical applications of the present invention, the second protein functional region comprises the VHs of two PD-L1-targeting antigen-binding proteins. Preferably, the two VHs included in the second protein functional region are identical and / or the C-terminuses of the two VHs are linked to the N-terminuses of the two heavy chains of the immunoglobulin via linkers shown in GGS or SEQ ID NO: 5, respectively.

[0031] In some technical applications of the present invention, the first protein functional region comprises two light chains containing the amino acid sequence shown in any one of SEQ ID NO: 88 to 92 and two heavy chains containing the amino acid sequence shown in SEQ ID NO: 84, and the second protein functional region comprises two VHs containing the amino acid sequence shown in any one of SEQ ID NO: 79 to 83, and the C-terminuses through which the two VHs have passed are linked to the N-terminuses of the two heavy chains of the first protein functional region, respectively, via a GGS or a linker shown in SEQ ID NO: 5.

[0032] Preferably, the antigen-binding proteins that target PD-L1 and CD40 are: The amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 89, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 93, or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 88, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 94, or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 91, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 94, or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 90, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 93, or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 92, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 93, or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 88, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 95, or the amino acid sequence is shown in SEQ ID NO: The second polypeptide chain shown in 88, the amino acid sequence of the first polypeptide chain shown in SEQ ID NO: 96, or the amino acid sequence of the second polypeptide chain shown in SEQ ID NO: 88, the amino acid sequence of the first polypeptide chain shown in SEQ ID NO: 97, or the amino acid sequence of the second polypeptide chain shown in SEQ ID NO: 91, the amino acid sequence of the first polypeptide chain shown in SEQ ID NO: 95, or the amino acid sequence of the second polypeptide chain shown in SEQ ID NO: 91, the amino acid sequence of the first polypeptide chain shown in SEQ ID NO: 93, or the amino acid sequence of the second polypeptide chain shown in SEQ ID NO: 91, the amino acid sequence of the first polypeptide chain shown in SEQ ID NO: 96, or the amino acid sequence of the second polypeptide chain shown in SEQ ID NO: 89, the amino acid sequence of the first polypeptide chain shown in SEQ ID NO: 97, or the amino acid sequence of the second polypeptide chain shown in SEQ ID NO: 90, the amino acid sequence of SEQ ID NO: The first polypeptide chain shown in 97, or the amino acid sequence is SEQ ID NO: The second polypeptide chain shown in 88, or the amino acid sequence is SEQ ID NO:It contains a first polypeptide chain and a second polypeptide chain, which are either a first polypeptide chain shown in 98, or a second polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 88, a first polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 99, or a second polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 91, or a first polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 98, or a second polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 91, and a first polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 99.

[0033] To solve the above technical problems, a second aspect of the present invention provides an antigen-binding protein targeting PD-L1 and CD40, wherein the antigen-binding protein comprises a first protein functional region and a second protein functional region, the first protein functional region comprising an antigen-binding protein targeting CD40, and the second protein functional region comprising an antigen-binding protein targeting PD-L1, where, The PD-L1 targeting antigen-binding protein includes a heavy chain variable region (VH), the VH includes HCDR1, HCDR2, and HCDR3, the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5 or a mutant 7 having 3, 2, or 1 amino acid mutations in SEQ ID NO: 5, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 16 or a mutant 8 having 3, 2, or 1 amino acid mutations in SEQ ID NO: 16, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 25 or a mutant 9 having 3, 2, or 1 amino acid mutations in SEQ ID NO: 25.

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

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

[0036] In some technical applications 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: 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: The amino acid sequence is as shown in 25, or the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 29, or the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 30, or the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 28.

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

[0038] In some technical applications of the present invention, 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 LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 38 or mutant 1 having 3, 2, or 1 amino acid mutations in SEQ ID NO: 38, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 43 or mutant 2 having 3, 2, or 1 amino acid mutations in SEQ ID NO: 43, LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 48 or mutant 3 having 3, 2, or 1 amino acid mutations in SEQ ID NO: 48, and HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8 or mutant 4 having 3, 2, or 1 amino acid mutations in SEQ ID NO: 8, and HCDR2 comprises SEQ ID NO: The HCDR3 comprises a mutant 5 having 3, 2, or 1 amino acid mutations in the amino acid sequence shown in 18 or SEQ ID NO: 18, and the HCDR3 comprises a mutant 6 having 3, 2, or 1 amino acid mutations in the amino acid sequence shown in SEQ ID NO: 27 or SEQ ID NO: 27.

[0039] In some technical proposals of the present invention, 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 LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 38, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 43, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 48, or a mutant 3 having 3, 2, or 1 amino acid mutations in SEQ ID NO: 48, and HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0040] In some technical proposals of the present invention, the variant 3 includes 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 in which an amino acid mutation occurs at the 4th and / or 5th position of the amino acid sequence shown in SEQ ID NO: 48, wherein the amino acid mutation is preferably an amino acid substitution, and more preferably a conserved amino acid substitution. Preferably, the variant 3 is an amino acid sequence in which an N4A / F / Y / V / N and / or S5N mutation occurs 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 NO: 49 to 53.

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

[0042] In some technical applications of the present invention, in the antigen-binding protein targeting CD40, the VL includes the 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 includes the 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 includes the amino acid sequence shown in any one of SEQ ID NO: 68 to 73, and the VH includes the amino acid sequence shown in SEQ ID NO: 60, with specific combinations of VL and VH being shown in Table 1-2.

[0043] In some technical proposals 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, 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), 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 an hIgG1 subtype heavy chain constant region.

[0044] In some technical applications of the present invention, 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 an amino acid sequence shown in any one of SEQ ID NO: 87 to 92, and the heavy chain comprising 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 and 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 NO: 88 to 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 Table 1-3.

[0045] In some technical applications 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 applications 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, either directly or via a linker.

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

[0048] In some technical applications of the present invention, the second protein functional region comprises the VHs of two PD-L1-targeting antigen-binding proteins. Preferably, the two VHs included in the second protein functional region are identical and / or the C-terminuses of the two VHs are linked to the N-terminuses of the two heavy chains of the immunoglobulin via linkers shown in GGS or SEQ ID NO: 5, respectively.

[0049] In some technical applications of the present invention, the first protein functional region comprises two light chains containing the amino acid sequence shown in any one of SEQ ID NO: 88 to 92 and two heavy chains containing the amino acid sequence shown in SEQ ID NO: 84, and the second protein functional region comprises two VHs containing the amino acid sequence shown in any one of SEQ ID NO: 79 to 83, and the C-terminuses through which the two VHs have passed are linked to the N-terminuses of the two heavy chains of the first protein functional region, respectively, via a GGS or a linker shown in SEQ ID NO: 5.

[0050] Preferably, the antigen-binding proteins that target PD-L1 and CD40 are: The amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 89, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 93, or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 88, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 94, or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 91, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 94, or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 90, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 93, or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 92, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 93, or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 88, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 95, or the amino acid sequence is shown in SEQ ID NO: The second polypeptide chain shown in 88, the amino acid sequence of the first polypeptide chain shown in SEQ ID NO: 96, or the amino acid sequence of the second polypeptide chain shown in SEQ ID NO: 88, the amino acid sequence of the first polypeptide chain shown in SEQ ID NO: 97, or the amino acid sequence of the second polypeptide chain shown in SEQ ID NO: 91, the amino acid sequence of the first polypeptide chain shown in SEQ ID NO: 95, or the amino acid sequence of the second polypeptide chain shown in SEQ ID NO: 91, the amino acid sequence of the first polypeptide chain shown in SEQ ID NO: 93, or the amino acid sequence of the second polypeptide chain shown in SEQ ID NO: 91, the amino acid sequence of the first polypeptide chain shown in SEQ ID NO: 96, or the amino acid sequence of the second polypeptide chain shown in SEQ ID NO: 89, the amino acid sequence of the first polypeptide chain shown in SEQ ID NO: 97, or the amino acid sequence of the second polypeptide chain shown in SEQ ID NO: 90, the amino acid sequence of SEQ ID NO: The first polypeptide chain shown in 97, or the amino acid sequence is SEQ ID NO: The second polypeptide chain shown in 88, or the amino acid sequence is SEQ ID NO:It contains a first polypeptide chain and a second polypeptide chain, which are either a first polypeptide chain shown in 98, or a second polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 88, a first polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 99, or a second polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 91, or a first polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 98, or a second polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 91, and a first polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 99.

[0051] In this invention, mutant 1, mutant 2, mutant 3, mutant 4, mutant 5, mutant 6, mutant 7, mutant 8, and mutant 9 are merely for distinguishing different mutants, the first protein functional region and the second protein functional region are merely for distinguishing different protein functions, and the first polypeptide chain and the second polypeptide chain are merely for distinguishing different polypeptide chains; the numbers and designations do not represent any actual meaning.

[0052] The amino acid sequence of the CDR in this invention is determined according to the Chothia definition rules.

[0053] To solve the above technical problems, another aspect of the present invention provides a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an antigen-binding protein that targets PD-L1 and CD40 as described in the present invention.

[0054] To solve the above technical problems, another aspect of the present invention provides isolated nucleic acids that encode antigen-binding proteins targeting PD-L1 and CD40 as described in the present invention.

[0055] To solve the above technical problems, another aspect of the present invention provides a recombinant expression vector comprising an isolated nucleic acid described in the present invention. Preferably, the recombinant expression vector comprises a eukaryotic cell expression vector and / or a prokaryotic cell expression vector.

[0056] To solve the above technical problems, another aspect of the present invention provides a transformant comprising an isolated nucleic acid or a recombinant expression vector as described in the present invention. Preferably, 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] To solve the above technical problems, another aspect of the present invention provides a method for producing antigen-binding proteins targeting PD-L1 and CD40 as described in the present invention, the method comprising the steps of culturing the transformant described in the present invention and obtaining antigen-binding proteins targeting PD-L1 and CD40 from the culture.

[0058] To solve the above technical problems, another aspect of the present invention provides an antibody-drug conjugate (ADC) comprising an antigen-binding protein targeting PD-L1 and CD40 as described in 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] To solve the above technical problems, another aspect of the present invention provides genetically modified cells that express the chimeric antigen receptor described in the present invention. Preferably, the genetically modified cells are eukaryotic cells, preferably isolated human cells, and more preferably immune cells such as T cells or NK cells.

[0060] To solve the above technical problems, another aspect of the present invention provides a pharmaceutical composition comprising one or more of the following: 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 genetically modified cell as described in the present invention, and an antibody-drug conjugate as described in the present invention, and / or a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition further comprises another antitumor drug as an active ingredient. The antitumor drug includes, but is not limited to, chemotherapeutic agents, nucleotide drugs, small molecule targeted drugs, monoclonal antibody drugs, bispecific / multispecific antibody drugs, recombinant protein drugs, immunomodulators, cell therapies, and gene therapies.

[0061] To solve the above technical problems, another aspect of the present invention provides a detection reagent comprising 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 liquid, gaseous, solid, and semi-solid dosage forms. More preferably, the detection reagent further comprises a secondary antibody, CD40, or a derivative thereof, wherein the secondary antibody, for example, an anti-human IgG antibody, binds to horseradish peroxidase, and an anti-human IgG antibody binds to a biotinylated protein.

[0062] To solve the above technical problems, in another aspect of the present invention, a parts kit is provided, the parts kit comprising Kit A, which contains one or more of the following: 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, a pharmaceutical composition as described in the present invention, a detection reagent as described in the present invention, genetically modified cells as described in the present invention, and an antibody-drug conjugate as described in the present invention.

[0063] Preferably, the parts kit further comprises kit B, which contains other antitumor antibodies or pharmaceutical compositions comprising the other antitumor antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, contrast agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0064] To solve the above technical problems, another aspect of the present invention provides applications in the manufacture of drugs for diagnosing, preventing and / or treating diseases related to PD-L1 and / or CD40, one or more of the following: antigen-binding proteins targeting PD-L1 and CD40 as described in the present invention, chimeric antigen receptors as described in the present invention, isolated nucleic acids as described in the present invention, recombinant expression vectors as described in the present invention, pharmaceutical compositions as described in the present invention, detection reagents as described in the present invention, parts kits as described in the present invention, genetically modified cells as described in the present invention, and antibody-drug conjugates as described in the present invention. Preferably, the PD-L1 and / or CD40-related diseases are PD-L1-related tumors, CD40-related tumors, or PD-L1 × CD40-related tumors. Preferably, the CD40-related tumors include solid tumors and hematological tumors, and the PD-L1-related tumors include solid tumors and hematological tumors.More preferably, the CD40-related tumors include B-series 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, while the PD-L1-related 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), and primary longitudinal leukemia. Large cell B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), large B-cell lymphoma rich in T cells / histiocytes, multiple myeloma, myelocellular leukemia-1 protein (MCL-1), myelodysplastic syndrome (MDS), gastrointestinal cancer, kidney 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 tract cancer This includes cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative neoplasm, neuroendocrine neoplasm, Merkel cell carcinoma, testicular cancer, and skin cancer, and preferably PD-L1 high-expression colon cancer.

[0065] 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 the step of contacting the sample with one or more of the antigen-binding proteins targeting PD-L1 and CD40 as described in the present invention, the detection reagents as described in the present invention, and the antibody-drug conjugates as described in the present invention. The sample includes, for example, a blood sample (e.g., whole blood sample and serum sample) and a reagent containing PD-L1 and / or CD40. Preferably, the method is not intended for therapeutic / diagnostic purposes, but 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] To solve the above technical problems, another aspect of the present invention provides a method for diagnosing, treating and / or preventing diseases related to PD-L1 and / or CD40, the method comprising administering to a patient in need one or more of the following to a patient in need: 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 genetically modified cell as described in the present invention, and an antibody-drug conjugate as described in the present invention, or diagnosing or treating a patient in need using a parts kit as described in the present invention. Preferably, the PD-L1 and / or CD40-related disease is a PD-L1-related tumor, a CD40-related tumor, or a PD-L1 × CD40-related tumor. Preferably, the CD40-related tumor includes solid tumors and hematological malignancies, and the PD-L1-related tumor includes solid tumors and hematological malignancies.More preferably, the CD40-related tumors include B-series 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, while the PD-L1-related 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), and primary longitudinal leukemia. Large cell B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), large B-cell lymphoma rich in T cells / histiocytes, multiple myeloma, myelocellular leukemia-1 protein (MCL-1), myelodysplastic syndrome (MDS), gastrointestinal cancer, kidney 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 tract cancer This includes cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative neoplasm, neuroendocrine neoplasm, Merkel cell carcinoma, testicular cancer, and skin cancer, and preferably PD-L1 high-expression colon cancer.

[0067] To solve the above technical problems, another aspect of the present invention provides antigen-binding proteins targeting PD-L1 and CD40 as described in the present invention, chimeric antigen receptors as described in the present invention, isolated nucleic acids as described in the present invention, recombinant expression vectors as described in the present invention, pharmaceutical compositions as described in the present invention, detection reagents as described in the present invention, parts kits as described in the present invention, genetically modified cells as described in the present invention, and / or antibody-drug conjugates as described in the present invention, for diagnosing, preventing and / or treating diseases related to PD-L1 and / or CD40. Preferably, the PD-L1 and / or CD40-related disease is a PD-L1-related tumor, a CD40-related tumor, or a PD-L1 × CD40-related tumor. Preferably, the CD40-related tumor includes solid tumors and hematological malignancies, and the PD-L1-related tumor includes solid tumors and hematological malignancies.More preferably, the CD40-related tumors include B-series 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, while the PD-L1-related 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), and primary longitudinal leukemia. Large cell B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), large B-cell lymphoma rich in T cells / histiocytes, multiple myeloma, myelocellular leukemia-1 protein (MCL-1), myelodysplastic syndrome (MDS), gastrointestinal cancer, kidney 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 tract cancer This includes cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative neoplasm, neuroendocrine neoplasm, Merkel cell carcinoma, testicular cancer, and skin cancer, and preferably PD-L1 high-expression colon cancer.

[0068] In this invention, "having 3, 2, or 1 amino acid mutations" includes the occurrence of amino acid insertions, deletions, or substitutions based on the original amino acid sequence. By exemplary interpretation, mutations in a CDR may include 3, 2, or 1 amino acid mutations, and these CDRs may be selectively mutated with the same or different number of amino acid residues, for example, one amino acid mutation in CDR1, while the amino acids in CDR2 and CDR3 remain unchanged.

[0069] In this invention, a "PTM site mutation" refers to the presence of an amino acid mutation at a PTM site in the mutant sequence compared to the original amino acid sequence. The method for designing the mutation varies depending on the antibody sequence and the PTM sequence pattern. One method involves replacing a "hotspot" amino acid (e.g., N or S in an NS pattern) with an amino acid that has similar physicochemical properties (e.g., mutating N to Q). If the PTM sequence pattern originates from a somatic high-frequency mutation but is not present in a 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 include cases in which mutations are induced based on a limited sequence. The mutation involves the deletion, substitution, or addition of one or more amino acid residues to the limited amino acid sequence, and the mutated amino acid sequence has at least 80% sequence identity with the limited amino acid sequence, maintaining or improving the binding activity of the antigen-binding protein of the amino acid sequence containing the mutation, wherein 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 this invention, "includes," "incorporates," and "is..." have the same meaning in several specific embodiments.

[0072] By arbitrarily combining the above preferred conditions while being consistent with common sense in the art, various preferred examples of the present invention can be obtained.

[0073] All reagents and raw materials used in this invention are commercially available.

[0074] The positive effects of the present invention are as follows: The CD40-targeting antigen-binding proteins described in the present invention have high affinity for CD40 and strong agonistic activity against the signaling pathway, particularly enhanced agonistic activity after crosslinking. As a result, the CD40-targeting antigen-binding proteins described in the present invention have a wider therapeutic range and provide a good basis for significantly improving patient response rates under safe and tolerable dose conditions in clinical trials. The PD-L1-targeting antigen-binding proteins described in the present invention have high affinity for PD-L1 and can effectively inhibit the biological activity of PD-L1. Furthermore, the PD-L1 and CD40-targeting antigen-binding proteins described in the present invention have excellent antitumor effects, good safety, and excellent drug-forming properties. The antigen-binding proteins targeting PD-L1 and CD40 described in this invention block the PD-1 / PD-L1 inhibitory signaling pathway and act on activated CD40 receptors, thereby simultaneously activating antigen-presenting cells and lymphocytes. This enhances the presentation of tumor antigens to lymphocytes by antigen-presenting cells, promotes the lymphocyte response, and yields a remarkable synergistic antitumor effect. Furthermore, the activation of immune cells by these PD-L1 and CD40-targeted antigen-binding proteins occurs only at the tumor microenvironment site, significantly eliminating systemic drug toxicity and side effects. As a result, these antigen-binding proteins exert their excellent antitumor effect under extremely safe conditions. Currently, no new antibody drugs combining these targets are commercially available domestically or internationally, and this invention is expected to bring new opportunities to the treatment of many tumors. [Brief explanation of the drawing]

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

[0076] The embodiments of the present invention will be described below through specific examples, and those skilled in the art will be able to easily understand other advantages and effects of the present invention based on what is disclosed herein.

[0077] In this application, the terms “binding protein” or “antigen-binding protein” typically refer to a protein containing a portion that binds to an antigen, and optionally, a scaffold or skeletal portion that allows the antigen-binding portion to adopt a configuration that facilitates the binding of the antigen-binding protein to the antigen. Typically, this 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 distinguished into hypervariable regions called complementarity-determining regions (CDRs), which are scattered within more conserved regions called framework regions (FRs). Each VH and VL may consist of three CDRs and four FR regions, which may be arranged in the order FR-1, CDR1, FR-2, CDR2, FR-3, CDR3, and FR-4 from the amino group terminus to the carboxyl group terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The three VH CDRs may be represented as HCDR1, HCDR2, and HCDR3, respectively, or as VH CDR1, VH CDR2, and VH CDR3, and the three VL CDRs may be represented as LCDR1, LCDR2, and LCDR3, respectively, or as VL CDR1, VL CDR2, and VL CDR3. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragment, 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, as long as they exhibit the required antigen-binding activity.

[0078] In this application, the amino acid sequences of the CDRs are all shown according to the Chothia definition rules. However, as those skilled in the art will know, in the art, the CDRs of antibodies can be defined in various ways, for example, by the Kabat definition rules based on sequence variability (see Kabat et al., Immunology Protein Sequences, 5th edition, National Institutes of Health, Bethesda, Maryland (1991)) and by the Chothia definition rules based on the position of the structural loop region (see JMol Biol 273:927-48, 1997). In the proposed method of this invention, amino acid residues in the variable domain sequence can also be determined using a Combined definition rule that includes both the Kabat and Chothia definitions. Here, the Combined definition rule combines the ranges of the Kabat and Chothia definitions to obtain a larger range, for details see Table 2 below. As those skilled in the art will understand, unless otherwise specified, the terms “CDR” and “complementarity-determining region” for a given antibody or region thereof (e.g., variable region) should be understood to cover the complementarity-determining region as defined by any one of the known schemes described in the present invention. While the scope claimed in the present invention is a sequence shown according to the Chothia definition rule, corresponding amino acid sequences according to 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 rule) to position bb (Chothia numbering rule) from the N-terminus of the antibody light chain, and Haa-Hbb may refer to the amino acid sequence from position aa (Chothia numbering rule) to position bb (Chothia numbering rule) from the N-terminus of the antibody heavy chain. For example, L24-L34 may refer to the amino acid sequence from position 24-34 of the N-terminus of the antibody light chain according to the Chothia numbering rule, and H26-H32 may refer to the amino acid sequence from position 26-32 of the N-terminus of the antibody heavy chain according to the Chothia numbering rule. As those skilled in the art will know, when encoding a CDR with Chothia, there may be insertion sites at several positions (see http: / / bioinf.org.uk / abs / ).

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

[0081] In this application, the term "fully human-derived antibody" typically refers to an antibody expressed in a genetically engineered antibody gene-deficient animal in which all of the human genes encoding the antibody have been transferred. All parts of the antibody (including the variable and constant regions) are encoded by human-derived genes. Fully human-derived antibodies can significantly reduce the immune side effects of heterologous antibodies against humans. Methods for obtaining fully human-derived antibodies in this field include phage display technology and transgenic mouse technology.

[0082] In this application, the term “specific binding” typically means that an antibody binds to an epitope via its antigen-binding domain, and that such binding requires some degree of 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 it to bind to an epitope via its antigen-binding domain than for it to bind to a random, unrelated epitope. “Epitope” refers to a specific atomic group (e.g., a sugar side chain, a phosphoryl group, a 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" typically refers to the antigen-binding portion in 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. In conventional antibodies, 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 and other constant region domains of the heavy chain to form a heavy chain. In some embodiments, "Fab" also refers to variant structures 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 Fab(cross VH / VL), and in some embodiments, CH1 of Fab is not linked to the hinge region, and instead the C-terminus of CL is linked to the hinge region of the heavy chain, forming the structure Fab(cross Fd / LC).

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

[0085] In this application, the term “antigen-binding fragment” usually refers to any protein functional region that can specifically bind to an antigen, and may be a “Fab,” a “VH,” or other antigen-binding form (e.g., an inducer protein structure such as lipocalins, neuronal adhesion molecules (NCAMs), fibronectin, or ankyrin repeat fragment proteins (DARPins)).

[0086] In this application, the term "tumor antigen" may refer to either 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 but is specific to tumor cells. A tumor-associated antigen is not specific to tumor cells and is 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 removed, and is mainly tumor cells, but may also be immunosuppressive cells, etc.

[0088] In this application, the term "immune effector cells" generally refers to immune cells that are involved in the removal of foreign antigens and the performance of effector functions in the immune response. Examples include plasma cells, cytotoxic T cells, and NK cells.

[0089] In this application, the terms “costimulatory molecule,” “costimulatory molecule antigen,” or “stimulatory antigen” refer to cell surface molecules and their ligands that provide a costimulatory signal for the complete activation of immune cells such as T or B cells, and that play a positive regulatory role in the activation of immune cells. Examples include CD28, 4-1BB, ICOS, OX40, and CD40.

[0090] In this application, the terms "co-inhibitory molecule" or "co-inhibitory molecule antigen" refer to a class of cell surface molecules and their ligands that exert a negative regulatory effect on the function of immune cells. Examples include CTLA-4, PD-L1, and PD-1.

[0091] In this application, the term "PD-L1" typically refers to the programmed death ligand 1 protein, its functional variants, and / or its functional fragments. PD-L1 is also known as differentiation cluster 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, the amino acid sequence of an exemplary full-length human PD-L1 protein can be found under NCBI registry number NP_054862 or UniProt registry number Q9NZQ7, and the sequence of an exemplary full-length cynomolgus monkey PD-L1 protein can be found under NCBI registry number XP_005581836 or UniProt registry number G7PSE7.

[0092] In this application, the term "CD40" typically refers to the tumor necrosis factor receptor superfamily member 5 protein, its functional variants and / or functional fragments, also known as TNFRSF5. CD40 sequences are known in the art. For example, the amino acid sequence of an exemplary human CD40 protein can be found under UniProt registry number P25942, the exemplary cynomolgus monkey CD40 protein sequence can be found under UniProt registry number G7PG38, and the exemplary mouse CD40 protein sequence can be found under UniProt registry number P27512. CD40L is a natural trimer ligand molecule of CD40.

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

[0094] Example 1: Expression purification and physicochemical characterization analysis of recombinant antibodies Example 1.1 Antibody Expression and Purification This example describes a general method for producing antibodies using mammalian host cells (e.g., human embryonic kidney cells HEK293 or Chinese hamster ovary cells CHO and cells derived therefrom), transient transfection expression, and affinity capture isolation. This method is suitable for target antibodies containing an Fc region, which may consist of one or more protein polypeptide chains or be derived from one or more expression plasmids.

[0095] The amino acid sequence of the antibody polypeptide chain was converted to a nucleotide sequence using a codon optimization method, the encoded nucleotide sequence was synthesized, and it was cloned into an expression vector compatible with host cells. By simultaneously transfecting mammalian host cells with plasmids encoding the antibody polypeptide chain in a specific proportion, recombinant antibodies with accurate folding and polypeptide chain assembly can be obtained using conventional recombinant protein expression and purification techniques. Specifically, FreeStyle TM 293-F cells (Thermo, #R79007) FreeStyle TM Cells were cultured in F17 Expression Medium (Thermo, #A1383504). Before transient transfection was initiated, the cell concentration was increased to 6–8 × 10⁶. 5 Adjust the concentration to cells / mL and culture in a shaker at 37°C and 8% CO2 for 24 hours until the cell concentration reaches 1.2 × 10⁶. 6The concentration is cells / mL. 30 mL of cultured cells were prepared. A plasmid encoding an antibody polypeptide chain (pTT5, NRC) was mixed in a fixed ratio, and a total of 30 μg of plasmid (plasmid-to-cell ratio of 1 μg:1 mL) was dissolved in 1.5 mL of Opti-MEM serum-reduced medium (Thermo, #31985088), filtered through a 0.22 μm filter membrane, and sterilized. Next, 1.5 mL of Opti-MEM was taken and dissolved in 120 μL of 1 mg / mL PEI (Polysciences, #23966-2), and allowed to stand for 5 minutes. PEI was slowly added to the plasmid, incubated at room temperature for 10 minutes, and the plasmid-PEI mixture was slowly added dropwise while gently shaking the culture flask. The cells were cultured for 5 days in a shaker at 37°C and 8% CO2. Cell viability was observed after 5 days. The culture was collected, centrifuged at 3300 g for 10 minutes, and the supernatant was taken. The supernatant was then centrifuged at high speed to remove impurities. MabSelect was used in PBS pH 7.4 buffer. TM A gravity column (Bio-Rad, #7311550) containing (GE Healthcare, #71-5020-91) was equilibrated and washed with 2-5 times the column volume. The supernatant sample was passed through the column, and the column was washed with 5-10 times the column volume of PBS buffer. Next, the target protein was eluted with 0.1 M glycine at pH 3.5, then the pH was adjusted to neutral with Tris-HCl at pH 8.0, and finally concentrated in an ultrafiltration tube (Millipore, #UFC901024). The solution was then replaced with PBS buffer or a buffer containing other components to obtain a purified recombinant antibody solution. Finally, NanoDrop (Thermo, NanoDrop) TM The concentration was measured using a meter (One), dispensed, stored, and kept awaiting.

[0096] Example 1.2 Analysis of protein purity and aggregates by SEC-HPLC In this example, the purity and aggregate form of protein samples were analyzed using analytical size exclusion chromatography (SEC). 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) system (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 filtered through a 0.22 μm filtration membrane and injected into the system. The HPLC procedure was then set up, and the sample was passed through the chromatography column at a flow rate of 1.0 mL / min using PBS buffer (pH 7.4) for a maximum time of 25 minutes. The HPLC generated an analytical report, which reported the retention times of components of different molecular sizes in the sample.

[0097] Example 1.3 Measurement of the thermal stability of protein molecules using DSF Differential scanning fluorescence (DSF) is a high-throughput method commonly used to measure the thermal stability of proteins. It reflects the process of protein denaturation and thus the thermal stability of protein molecules by monitoring the change in fluorescence intensity of a dye bound to an unfolded protein molecule using a real-time fluorescence quantitative PCR instrument. In this example, the thermal denaturation temperature (Tm) of a protein molecule was measured using the DSF method. 10 μg of protein was added to a 96-well PCR plate (Thermo, #AB-0700 / W), then 2 μL of 100× diluted dye SYPRO™ (Invitrogen, #2008138) was added, and buffer was added to bring the final volume to 40 μL / well. The PCR plate was sealed and placed in a real-time fluorescence quantitative PCR instrument (Bio-Rad CFX96 PCR System). It was first incubated at 25°C for 5 minutes, then gradually heated from 25°C to 95°C with a gradient of 0.2°C / 0.2 minutes, and finally cooled back down to 25°C at the end of the test. Using FRET scanning mode, the data was analyzed with Bio-Rad CFX Maestro software, and the Tm of the sample was calculated.

[0098] Example 2: Production of CD40 antibody Experimental animals are immunized with the CD40 antigen to obtain antibody molecules that specifically bind to CD40. These experimental animals may be mice, rats, rabbits, sheep, camels, etc. Typically, the obtained antibody molecules are of non-human origin. After obtaining non-human antibodies, it is necessary to perform 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. On the other hand, advances in transgenic technology have made it possible to create genetically modified mice that possess an immune repertoire of human immunoglobulins and have a deletion of their endogenous mouse immune repertoire. Antibodies produced from such transgenic mice have a completely human sequence, eliminating the need for further humanization operations and significantly improving the efficiency of therapeutic antibody development. Harbour H2L2 mice (Harbour Antibodies BV) are transgenic mice possessing an immune repertoire of human immunoglobulins, and the antibodies produced from them have a complete human antibody variable domain and a rat constant domain.

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

[0100] Hybrid Mama Screening When a certain level of CD40-specific antibody titer was detected in mouse serum, mouse spleen cells were isolated and fused with myeloma cell lines to obtain hybridoma cells. After multiple rounds of mass screening and cloning of the hybridoma cells, hybridomas expressing the CD40 monoclonal antibody molecule were isolated. The isolated hybridomas expressed antibody molecules having a complete human variable domain and heavy and light chains of the rat constant domain. The above monoclonal antibody was further identified, and several hybridoma clones were selected and sequenced based on parameters such as binding ability to human CD40, binding ability to cynomolgus monkey CD40, and ability to activate the CD40 downstream signaling pathway. During mass screening, selicrelumab (also numbered PR001028 in this invention 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 using conventional hybridoma sequencing methods. In this example, the sequence of the variable domain of the CD40 monoclonal antibody molecule obtained from immunized Harbour H2L2 mice is a human-derived antibody sequence. The CDR sequence of the antibody variable domain can be analyzed by Kabat, Chothia, or other CDR definition rules (Table I).

[0101] Recombinant IgG antibodies and sequence analysis and optimization After obtaining the sequences of the light and heavy chain variable domains encoding the antibody molecule, the sequences of the light and heavy chain variable domains and the corresponding light and heavy chain constant domain sequences of the human antibody are fused and expressed using conventional recombinant DNA technology to obtain the recombinant antibody molecule by the method described above. In this example, the antibody heavy chain variable domain sequence (VH) was gene-synthesized and cloned into a mammalian cell expression plasmid vector encoding the human IgG1 or IgG2 antibody heavy chain constant domain sequence to encode the full-length heavy chain that produces the IgG antibody. The antibody light chain variable domain sequence (VL) was gene-synthesized and cloned into a mammalian cell expression plasmid vector encoding the human antibody Igκ light chain constant domain sequence to encode the full-length light chain that produces the antibody. In this example, a fully human-derived anti-CD40 recombinant IgG antibody was obtained.

[0102] The heavy chain variable domain sequences of antibodies originate from events such as gene rearrangements of germline gene V, D, and J fragments in the heavy chain gene group on chromosomes, and somatic high-frequency mutations. The light chain variable domain sequences originate from events such as gene rearrangements of germline gene V and J fragments in the light chain gene group, and somatic high-frequency mutations. Gene rearrangements and somatic high-frequency mutations are major factors that increase antibody diversity. Antibodies from the same germline V gene fragment can produce different sequences, but overall they exhibit high similarity. Several algorithms, such as IMGT / DomainGapAlign (http: / / imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi) or NCBI / IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ), can be used to estimate the possible germline gene fragments from which gene rearrangements occur, based on the antibody's variable domain sequences.

[0103] The amino acid chains of proteins or polypeptides may undergo chemical modifications called post-translational modifications (PTMs) after translation and synthesis within cells. In the case of antibodies, some PTM sites are highly conserved; for example, the conserved amino acid asparagine (ASN) at position 297 (EU number) of the constant domain of human IgG1 antibodies usually undergoes glycosylation modification to form a sugar chain, and this sugar chain structure is quite important for the antibody structure and associated effector function. However, if PTMs are present in the variable domain of the antibody, particularly in the antigen-binding region (e.g., CDR), the presence of these PTMs can significantly affect antigen binding and alter the physicochemical properties of the antibody. For example, glycosylation, deamidation, isomerization, and oxidation can all increase the instability or heterogeneity of the antibody molecule, potentially increasing the difficulty and risk of antibody development. Therefore, avoiding some potential PTMs is crucial for the development of therapeutic antibodies. Through accumulated experience, it has been discovered that some PTMs are highly correlated with the composition of amino acid sequences, particularly the "patterns" of adjacent amino acid compositions, allowing for the prediction of potential PTMs from the primary amino acid sequence of a protein. For example, the N-linked glycosylation site can be predicted from the sequence pattern NxS / T (position 1 is asparagine, position 2 is any amino acid other than non-proline, and position 3 is serine or threonine). The amino acid sequence patterns that cause PTMs may originate from germline gene sequences, such as the human germline gene fragment IGHV3-33, which naturally contains the glycosylation pattern NST in the FR3 region, or from somatic high-frequency mutations. Specifically, NGS or NLT may be glycosylation sites, NS may be deamide sites, and DG may cause aspartic acid isomerization. By disrupting the amino acid sequence pattern of a PTM through amino acid mutations, the formation of a specific PTM can be reduced or eliminated. The method of designing mutations differs depending on the antibody sequence and the PTM sequence pattern. One approach is to replace the "hotspot" amino acid (for example, N or S in an NS pattern) with an amino acid that has similar physicochemical properties (for example, mutating N to Q).Alternatively, random mutations were performed on the PTM sequence pattern using a mutation library method. In actual operation, various mutation design methods can be used for the same PTM sequence pattern.

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

[0105] Table 4 lists novel antibody molecules obtained by amino acid mutations in the CD40 antibody PR003379. All designed mutant molecules were further validated in subsequent functional experiments after obtaining purified recombinant antibodies according to the method described in 0. The methods for obtaining the antibodies in Table 5 are as follows: The heavy chain variable region (VH) of PR003379 was ligated to an IgG1 subtype antibody heavy chain constant domain sequence containing L234A, L235A, G237A mutations, and cloned into a mammalian cell expression plasmid to encode a full-length heavy chain producing IgG1 (L234A, L235A, G237A). The antibody light chain variable domain sequence (VL) containing a PTM mutation site was gene-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 producing the antibody. Two plasmids were simultaneously transfected into mammalian cells, and after expression, production, and purification, fully human-derived anti-CD40 recombinant IgG1 (L234A, L235A, G237A) antibodies were obtained.

[0106] Table 5 lists the sequences of the CD40 antibodies in the present invention application and the amino acid sequences of CDRs as defined according to the Chothia definition rules. These CD40 antibodies include a 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 protein to CD40-expressing cells In this example, flow cytometry FACS was used to investigate the binding activity of CD40-targeting antibodies or antigen-binding proteins to CD40-expressing cells. Examples include the human CD40-highly expressing CHO-K1 cell line CHO-K1 / hCD40 (Shanghai Ruizhi Chemical), the cynomolgus monkey CD40-highly expressing CHO-K1 cell line CHO-K1 / cyCD40 (Shanghai Ruizhi Chemical), and the human CD40-highly expressing lymphoma cell Raji (ATCC,#CCL-86).

[0111] Specifically, cells expressing CD40 are digested, resuspended in complete medium (F-12K for CHO-K1 cells, RPMI-1640 for Raji cells), and the cell density is increased to 1 × 10⁶. 6The solution was adjusted to cells / mL. 100 μL of cells / well was inoculated into a 96-well V-bottom plate (Corning, #3894), and then 100 μL / well of the target antibody at twice the final concentration was added and mixed uniformly. The maximum final concentration of the antibody was 300 nM, resulting in a total of eight concentrations, which were gradient diluted fivefold. hIgG (Crownbio, #C0002) was used as the negative control, and selicrelumab was the positive control molecule. The concentration settings were the same as those for the subsequent test duo antibodies. The cells were incubated at 4°C in the dark for 1 hour. Then, 100 μL / well of pre-cooled PBS was added, the cells were rinsed twice, and the cells were centrifuged at 500 g at 4°C for 5 minutes, and the supernatant was discarded. A 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 in the dark for 30 minutes. Cells were washed twice with 200 μL / well of pre-cooled PBS, centrifuged at 500 g at 4°C for 5 minutes, and the supernatant was discarded. Finally, cells were resuspended in 200 μL / well of pre-cooled PBS. Fluorescence emission signal values ​​were read using a BD FACS CANTO II flow cytometer or ACEA NovoCyte flow cytometer, and the data were processed and analyzed using FlowJo v10 software (FlowJo, LLC).

[0112] Data processing and graph analysis were performed using the GraphPad Prism 8 software, and the binding curves and EC of antibodies to target cells were determined by 4-parameter nonlinear fitting. 50 We obtained parameters such as values.

[0113] Figures 1A to 1D and Table 6 show the binding activity of the CD40 antibody PR003379 and its variant molecules to Raji cells. The results showed that PR003379 and its variant molecules could strongly bind to Raji cells, and the binding activity of PR003379 was stronger than that of selicrelumab.

[0114] [Table 8]

[0115] Example 4 Activity of CD40 antigen-binding protein in fluorescent reporter gene assay In this example, the activation effect of CD40-targeted antibodies or antigen-binding proteins on CD40 with and without crosslinking was investigated via activation assay using HEK293-hCD40-NFκB fluorescent reporter gene cells (BPS Bioscience, #60626). Cells with high expression of human CD32B were used to perform Fc-mediated crosslinking on the molecules to be detected, and whether crosslinking can further improve the activation ability against CD40 was investigated.

[0116] CHO-K1 cells expressing human CD32B, CHO-K1 / hCD32B (GenScript, #M00600), and CHO-K1 cells were plated at 1×10 4 cells / well, 100 μL / well in a 96-well plate (PerkinElmer, #6005181), and incubated overnight at 37°C in an environment of 5% CO2. The supernatant was removed, and HEK293-hCD40-NFκB reporter gene cells (BPS bioscience, #60626) expressing human CD40 and the NFκB-luciferase reporter gene were collected, added to a 96-well plate at 5×10 4 cells / well, 50 μL / well. The diluted antibody solution to be detected was added at 50 μL / well, with a starting concentration of 100 nM, diluted 5-fold serially to a total of 8 concentrations. The plate was incubated for 6 hours at 37°C in an environment of 5% CO2. ONE-Glo TM luciferase reagent (Promega, #E6110) was added, the plate was incubated at room temperature for 5 minutes, and the luminescence value was detected with a microplate reader. Data processing and graph analysis were performed using GraphPad Prism 8 software, and through four-parameter non-linear fitting, the curve of antibody concentration-dependent relative luminescence signal unit (RLU) and EC 50Parameters such as values ​​were obtained. Samples incubated with CD40 antibody and CHO-K1 / hCD32B cells were called "crosslinked," while samples incubated with CD40 antibody and CHO-K1 cells were not crosslinked.

[0117] As shown in Figures 2A to 2B and Table 7, PR003379 exhibited a clearer "crosslinking enhancement" effect than the control antibody Selicrelumab. Specifically, under CD32B-mediated antibody crosslinking conditions, PR003379 significantly amplified the signal of the fluorescent reporter gene and significantly enhanced the activating effect of the CD40 molecule on downstream signaling molecules.

[0118] Figures 2C to 2D and Table 8 show the enhancement factor of fluorescence intensity of activation of reporter gene cells by the PR003379 mutant molecule, under conditions with and without cell-mediated crosslinking expressing human CD32B. The results show that the PR003379 mutant molecule significantly enhanced the activating effect on the CD40 molecule in the case of crosslinking, and its EC 50 The value is significantly lower in the case of crosslinking than in the case of non-crosslinking (multiple (non-crosslinked EC) 50 / Crosslinked EC 50 It was shown that )」> 1).

[0119] [Table 9]

[0120] [Table 10]

[0121] Example 5: Production of PD-L1 heavy chain antibody The Harbour HCAb mouse (Harbour Antibodies BV, WO2010 / 109165A2) is a transgenic mouse with a human immunoglobulin immune repertoire that can produce antibodies (HCAb) containing only the heavy chain, and these antibodies are only half the size of conventional IgG antibodies. The antibodies produced from it contain only the human antibody heavy chain variable domain and the mouse Fc constant domain. Due to the absence of a light chain, these antibodies largely solve the problems of light chain mismatch and heterodimerization, enabling this technology platform to develop products that are difficult to achieve with conventional antibody platforms.

[0122] Immunized mice Harbour HCAb mice aged 6-8 weeks were immunized multiple times with soluble recombinant human PD-L1-mFc fusion protein (Novo Protein Inc., #CM06). Each mouse received a total of 100 μL of immunization via subcutaneous or intraperitoneal injection in the inguinal region. In the initial immunization, each mouse was immunized with an immunogenic reagent prepared by mixing 50 μg of antigen protein with complete Freund's adjuvant (Sigma, #F5881) in a 1:1 volume ratio. In subsequent rounds of immunization, each mouse was immunized with an immunogenic reagent prepared by mixing 25 μg of antigen protein with Ribi adjuvant (Sigma Adjuvant System, #S6322). The interval between each round of immunization was at least two weeks, and typically there were no more than five rounds of immunization. Immunization times were on days 0, 14, 28, 42, 56, and 70, and antibody titers in mouse serum were detected on days 49 and 77. A final booster immunization was performed with a dose of 25 μg of antigen protein per mouse five days before isolation of HCAb mouse spleen B cells.

[0123] Construction of an HCAb library and high-throughput transient expression When the titer of PD-L1-specific antibodies in mouse serum was detected to have reached a certain level, mouse spleen cells were isolated, 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. The human VH gene was expanded from the plasma cells and B cell populations using conventional molecular biological methods, and the expanded human VH gene fragment was 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 Screening Using the Mirrorball high-throughput no-wash antibody mass screening platform (SPT Labtech), supernatant expressing HCAb was mass screened, and the binding of this supernatant to the CHO-K1 / hPD-L1 (GenScript, #M00543) stable cell line overexpressing human PD-L1 was detected. A positive antibody PR000151 (Atezolizumab analog) was used as a positive control.

[0125] Specifically, CHO-K1 / hPD-L1 cells were washed with serum-free F12K medium (Thermofisher, #21127022), and 1 × 10⁶ cells were used. 6 The cells were resuspended to a concentration of 1.0 × 10⁶ / mL. Draq5 fluorescent probe (CTS, #4048L) (1 μL Draq5 ~ 1 mL CHO-K1 / hPD-L1 cells, 1:1000 dilution) was added, and the cells were incubated in the dark for 30 minutes. After centrifuging the cells, they were washed with culture medium, and the cell density was reduced to 1.0 × 10⁶. 5The solution was adjusted to cells / mL. Next, a secondary antibody (Alexa Fluor® 488 AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson ImmunoResearch, #109-545-098) diluted 1:1000 was added. 30 μL of this mixture per well was added to a 384-well plate (Greiner, #781091). 10 μL of positive control or HCAb expression supernatant was then added to the 384-well plate and incubated for 2 hours. Fluorescence values ​​were read on a Mirrorball.

[0126] The positive antibody supernatant was further identified, and binding to CHO-K1 / hPD-L1 cells was detected by FACS. Cross-binding activity with cynomolgus monkey PD-L1 protein (Acro biosystems, #PD1-C52H4) was detected by ELISA. HCAb was obtained by mass screening using conventional sequencing methods and then sequenced 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 fusion-expressed to obtain a fully human recombinant HCAb antibody molecule.

[0127] Sequence analysis and optimization Table 9 lists the preferred recombinant heavy chain antibody molecule PR000960 for anti-PD-L1 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 drug formation in later stages of development. To remove this N-glycosylation modification site, amino acid mutations were performed on PR000960 to obtain a new antibody molecule PR002082 (Table 10).

[0128] Improvement of PD-L1 binding affinity through affinity maturation technology To further improve the binding affinity of PR002082 to PD-L1, in vitro affinity maturation was performed on the PR002082 molecule. A yeast display antibody mutant library was established by randomly inducing mutations in the CDR region, and the PR002082 molecular mutant library was mass-screened using the BD FACS AriaIII sorting platform.

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

[0130] Next, the molecules sorted in the previous three rounds were sequenced, mutation sites were identified and randomly combined to establish a combined mutation library. Then, using FACS, we continued sorting for mutant molecules with higher affinity at lower antigen concentrations.

[0131] Recombinant HCAb antibody molecules were produced by synthesizing the VH mutant molecule obtained through affinity maturation and cloning it into a mammalian cell expression plasmid vector encoding the human IgG1 heavy chain Fc sequence. 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 after obtaining purified recombinant antibodies according to the method described in 0.

[0133] Table 12 lists the sequences of the PD-L1 antibodies in the present invention application and the amino acid sequences of the CDRs defined according to the Chothia definition rules. These PD-L1 antibodies include a 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 protein to PD-L1-expressing cells In this example, flow cytometry FACS was used to investigate the binding activity of antibodies or antigen-binding proteins targeting PD-L1 to cells expressing PD-L1. Examples include the CHO-K1 / hPD-L1 cell line (Shanghai Ruizhi Chemical) which highly expresses human PD-L1, the CHO-K1 / cyPD-L1 cell line (Shanghai Ruizhi Chemical) which highly expresses cynomolgus monkey PD-L1, the NCI-H226 lung cancer cell line (ATCC,#CRL-5826) which highly expresses human PD-L1, and the MDA-MB-231 breast cancer cell line (ATCC,#CRM-HTB-26) which highly expresses human PD-L1.

[0139] Specifically, cells expressing PD-L1 were digested and resuspended in PBS buffer containing 2% BSA. The cell density was set to 1 × 10⁶. 6 The cells were adjusted to the required concentration per mL. 100 μL of cells / well was inoculated into a 96-well V-bottom plate (Corning, #3894), and then 100 μL / well of the target antibody, which had been gradient-diluted to a 4-fold concentration (twice the final concentration), was added. The cells were incubated at 4°C for 2 hours in the dark. Then, 100 μL / well of pre-cooled 2% BSA-containing PBS was added, the cells were rinsed twice, and the mixture was centrifuged at 500 g at 4°C for 5 minutes, discarding the supernatant. Next, 100 μL / well of the fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-098, 1:500 dilution) was added, and the mixture was incubated at 4°C for 1 hour in the dark. Cells were washed twice with 100 μL / well of pre-cooled 2% BSA-containing PBS, centrifuged at 500 g at 4°C for 5 minutes, and the supernatant was discarded. Finally, cells were resuspended in 200 μL / well of pre-cooled 2% BSA-containing PBS buffer. Fluorescence emission signal values ​​were read using a BD FACS CANTO II flow cytometer or ACEA NovoCyte flow cytometer, and the data were processed and analyzed using FlowJo v10 software (FlowJo, LLC).

[0140] Data processing and graph analysis were performed using the GraphPad Prism 8 software, and the binding curves and EC of antibodies to target cells were determined by 4-parameter nonlinear fitting. 50 We obtained parameters such as values.

[0141] Figures 3A and 3B show the binding curves of the PD-L1 heavy chain antibodies PR000960, PR002082, and their variant molecules to CHO-K1 / hPD-L1 cells, and Table 13 shows the EC of the corresponding curves. 50The values ​​and maximum MFI signal values ​​are listed. According to the results, PR000960 and PR002082 have binding ability to human PD-L1 equivalent to the control antibody Atezolizumab. PR000960 Its ability to bind affinity-mature mutant molecules is slightly superior to that of PR002082.

[0142] [Table 15]

[0143] Example 7 Activity of PD-L1 antigen-binding protein in a fluorescent reporter gene experiment This example investigated the inhibitory effect of antibodies or antigen-binding proteins targeting PD-L1 on the PD-1 signaling pathway using Jurkat-NFAT-hPD1 fluorescent reporter gene cell activation experiments (Waha Pharmaceutical). The PD-L1 antibody or antigen-binding protein blocked the interaction between PD-L1 and PD-1, thereby reversing the inhibitory effect of PD-1 on T cells.

[0144] 293T cells 293T-OS8-hPDL1 (Beijing Kangyuan Bochuang, #KC-1148) overexpressing PD-L1 and OS8 (CD3 single-chain antibody transmembrane protein) or Jurkat reporter cells Jurkat-NFAT-hPD1 (Wa Haku Pharmaceutical) co-expressing PD-1 and NFAT-luciferase reporter genes were cultured and collected, and the density of Jurkat-NFAT-hPD1 was set to 1 × 10⁻⁶. 6 Cells / mL, density of 293T-OS8-hPDL1 at 5 × 10 5 The cells were adjusted to the specified cell / mL. Equal volumes of cell suspension were mixed and added to a 96-well U-bottom plate at 100 μL / well, where the ratio of effector cells Jurkat-NFAT-hPD1 to target cells 293T-OS8-hPDL1 was 2:1. 50 μL / well of pre-gradient diluted target antigen-binding protein was added. The 96-well plate was placed in a cell incubator and incubated at 37°C and 5% CO2 for 6 hours. ONE-Glo TMAdd 50 μL / well of luciferase solution (Promega, #E6120), gently tap the plate to mix uniformly, allow to react at room temperature in the dark for 10 minutes, then transfer 100 μL of the liquid to a 96-well flat-bottom plate (PerkinElmer, #6005225), and finally to Envision TM The fluorescence values ​​were read using a plate reader (PerkinElmer).

[0145] Data processing and graph analysis were performed using the GraphPad Prism 8 software, and the binding curves and EC of antibodies to target cells were determined by 4-parameter nonlinear fitting. 50 We obtained parameters such as values.

[0146] Figures 4A and 4B and Table 14 show that the PD-L1 heavy chain antibodies PR000960, PR002082, and their mutant molecules blocked the PD-1 signaling pathway in fluorescence reporter gene experiments and further restored the NFAT signal of Jurkat. The results show that PR000960 has comparable inhibitory activity to the control antibody Atezolizumab, while PR002082 exhibits slightly superior inhibitory activity against affinity-mature mutant molecules.

[0147] [Table 16]

[0148] Example 8: Construction of a PD-L1 × 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 Figure 5, the Fab terminus originates from conventional antibody A, and VH_A and VL_A are the heavy chain variable region and light chain variable region of antibody A, respectively. The VH terminus originates 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 or origin sequence of the IgG antibody, and L is the linking peptide. The binding protein in the shown structure contains two different polypeptide chains: a light chain containing VL_A-CL from the amino group terminus to the carboxyl group terminus, and a heavy chain containing VH_B-L-VH_A-CH1-h-CH2-CH3 from the amino group terminus to the carboxyl group terminus. In some embodiments, the heavy chain VH_B and VH_A are directly fused and linked, i.e., the length of L is 0; in some other embodiments, the heavy chain VH_B is linked to 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 CD40-binding H2L2(IgG) antibodies PR006495, PR006496, PR006497, PR006504, and PR006511, while VH_B is bound to antigen-binding fragment VH of PD-L1-binding heavy chain antibodies PR005878, PR006245, PR006246, PR006247, and PR006248. Based on these PD-L1 and / or CD40 antigen-binding fragments, several PD-L1×CD40 bispecific antigen-binding proteins with the structures shown in Figure 5 were constructed (listed in Table 16). These molecules have similar structures and use the human IgG1 constant region sequence. To eliminate Fc-mediated effector function like ADCC, three amino acid mutations L234A / L235A / G237A (Eu code) were introduced into the Fc region. Table 18 lists the sequence numbers of the polypeptide chain amino acid sequences of PD-L1×CD40. For these PD-L1×CD40 molecules, corresponding protein samples were prepared and analyzed according to the method described in 0, and the results are summarized in Table 17. The PD-L1×CD40 molecules constructed in this example all exhibited high yield and purity (see Example 1 for methods of expression and purity detection of PD-L1×CD40 molecules), with most protein samples exceeding 95% purity.

[0152] [Table 18]

[0153] [Table 19]

[0154] [Table 20]

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

[0156] Table 19 summarizes the corresponding results of binding of PD-L1×CD40 bispecific binding protein and control molecules to various cells. The corresponding figure shows the binding curve of the antigen-binding protein to the target cell, and the corresponding table shows the binding strength parameter (EC) fitted to the corresponding curve. 50The values ​​(and maximum MFI signal values) are listed. Figures 10A and 10B show that PD-L1×CD40 does not bind to CHO-K1 cells. Figures 6A and 6B and Table 20 show the binding of PD-L1×CD40 to CHO-K1 / hPD-L1 cells. Figures 7A and 7B and Table 21 show the binding of PD-L1×CD40 to cynomolgus monkey PD-L1 cells. Figures 8A and 8B and Table 22 show the binding of PD-L1×CD40 to CHO-K1 / hCD40 cells. Figures 9A and 9B and Table 23 show the binding of PD-L1×CD40 to cynomolgus monkey CD40 cells. Figures 11A and 11B and Table 24 show the binding of PD-L1×CD40 to NCI-H226 cells. Figures 12A and 12B and Table 25 show the binding of PD-L1×CD40 to Raji cells. Figures 13A and 13B and Table 26 show the binding of PD-L1×CD40 to MDA-MB-231 cells.

[0157] The results showed that all PD-L1 × CD40 biantibodies were effective against PD-L1-expressing cell lines, such as CHO-K1 / hPD-L1 (EC). 50 (It is approximately 1 nM), CHO-K1 / cy PD-L1 (EC 50 (where the mass is less than 1 nM) and NCI-226 (EC 50 It can efficiently bind to cells (where the target is less than 1 nM), and its binding activity is equivalent to that of the positive reference atezolizumab. The PD-L1×CD40 biantibody does not bind to CHO-K1 cells that do not express the target, and the PD-L1×CD40 biantibody also binds to MDA-MB-231 cells (EC). 50 It efficiently binds to (a molecule with a molecular weight less than 1 nM), and its binding action is significantly stronger than that of the positive reference atezolizumab (higher MFI signal).

[0158] Furthermore, all PD-L1×CD40 biantibodies are CD40-expressing cell lines, such as CHO-K1 / hCD40(EC). 50 (It is approximately 1-2 nM), CHO-K1 / cyCD40 (EC 50 (The magnitude is approximately 1-4 nM) and Raji (EC 50It can efficiently bind to cells with a molecular weight of approximately 1-2 nM, and its binding activity is stronger than or equivalent to that of the positive reference selicrelumab. The PD-L1 × CD40 biantibody also efficiently bound to MDA-MB-231 cells, and its binding activity 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 the PD-1 signaling pathway by PD-L1×CD40 in a fluorescent reporter gene experiment. In this example, the method described in 0 was used to test the inhibitory effect of the PD-L1×CD40 bispecific binding protein on the PD-1 signaling pathway. The results are shown in Figure 14A to Figure 14D and Table 27. All PD-L1×CD40 bispecific antibodies can effectively block the PD-1 / PD-L1 interaction and activate TCR and NFAT signals, with an activity comparable to that of the positive reference antibody Atezolizumab (EC 50 is about 0.1 to 0.3 nM).

[0168]

Table 29

[0169] Example 11 Activation of CD40 Signaling Pathway by PD-L1×CD40 in Fluorescent Reporter Gene Assay In this example, the method described in 0 was used to test the activation effect of PD-L1×CD40 bispecific binding protein on CD40 under PD-L1-mediated cross-linking and non-cross-linking conditions. Cells CHO-K1 / hPD-L1 with high PD-L1 expression were used to cross-link the molecules to be detected, and it was investigated whether cross-linking can further enhance the activation ability against CD40. In this experiment, CHO-K1 / hPD-L1 cells with high PD-L1 expression were able to cross-link with PD-L1×CD40 bispecific antibody molecules via PD-L1, while CHO-K1 cells that do not express PD-L1 or CD40 could not bind to the molecules to be detected. The activation of the HEK293-hCD40-NFκB reporter gene system was tested respectively after the molecules to be detected were incubated with CHO-K1 / hPD-L1 cells or incubated with CHO-K1 cells.

[0170] The results are shown in Figure 15A to Figure 15K and Table 28. The CD40 antibody Selicrelumab can activate the downstream signaling pathway of CD40, and such activation does not depend on the presence of exogenous cross-linking, with EC 50is about 0.2 nM. On the other hand, PD-L1×CD40 bispecific antibodies such as PR007151 and PR007619, under the condition where PD-L1 cross-linking exists (EC 50 is about 0.03 nM), the activation effect on the downstream signaling pathway of CD40 is significantly stronger than that under the condition without PD-L1 cross-linking (EC 50 is about 0.3 to 0.4 nM). Other experiments, such as the test for PR006919 and PR007070, and the tests for PR007268, PR007269, PR007273, PR007275 and PR007276, all have similar results, that is, the PD-L1×CD40 bispecific antibody can better activate the CD40 downstream signaling pathway through the cross-linking of CD40 by 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 protein on dendritic cells (DC) in the case of PD-L1-mediated cross-linking and non-cross-linking was investigated.

[0173] In this example, dendritic cells (DCs) were induced using PBMC-derived monocytes, followed by activation experiments. 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 of IL-4 (R&D Systems, #204-IL) and 100 ng / mL of GM-CSF (R&D Systems, #215-GM) cytokines were added. After induction for 6 days, immature dendritic cells (iDCs) were formed. Half of these iDCs were then treated with 1 μg / mL of LPS (Sigma, #L2880-10MG) and induced for 24 hours to form mature dendritic cells (mDCs). mDCs from donors #1 and #2 were used directly 24 hours after LPS induction, while mDCs from donor #4 could be used after washing off the LPS, re-adding RPMI1640 complete medium with 50 ng / mL of IL-4 and 100 ng / mL of GM-CSF cytokines, and allowing to stand for 24 hours.

[0174] The day before mDC induction was complete, CHO-K1 and CHO-K1 / hPD-L1 cells were seeded into 96-well flat-bottom plates (Corning, #3599). First, CHO-K1 and CHO-K1 / hPD-L1 cells were collected, and 50 μg / mL of mitomycin C (Selleckchem, #S8146) was added, followed by incubation at 37°C for 1 hour to inhibit cell proliferation. After washing, the cells were cultured in F-12K complete medium to a cell density of 1 × 10⁶. 5 The antibody was adjusted to cells / mL and added to a 96-well flat-bottom plate at 100 μL / well, and finally incubated overnight in a cell incubator. The following day, the target antibody was diluted to 2-fold the final concentration in RPMI1640 complete medium, followed by 10-fold or 3-fold gradient dilution. Selicrelumab and hIgG1 were used as positive and negative controls, respectively. After discarding the medium for CHO-K1 and CHO-K1 / hPD-L1 cells, 100 μL / well of antibody was added. Subsequently, iDCs and mDCs were collected and the cell density was set to 5 × 10⁶. 5The solution was adjusted to cells / mL, added to a 96-well plate at 100 μL / well, mixed with the aforementioned target antibody, and finally cultured in a cell incubator. After 3 days, the cell supernatant was collected, and the secretion level of IL-12p40 was detected using a human IL-12 / -23(p40) ELISA kit (MABTECH, #3450-1H-20). Data processing and graph analysis were performed using GraphPad Prism 8 software to show the relationship between IL-12p40 secretion and antibody concentration.

[0175] In this example, PD-L1×CD40 biantibody was tested multiple times using different species of DC cells derived from multiple donors. The results are shown in Figures 16A and 16B, 17A and 17B, 18A and 18B, and 19A to 19D. Table 29 summarizes the important experimental parameters corresponding to each figure, such as DC origin, cross-linking target cells, and test molecules.

[0176] The results showed that the positive reference antibody Selicrelumab could effectively activate both iDC and mDC cells, and this activation was not affected by the tumor antigen PD-L1. On the other hand, PD-L1×CD40 biantibodies, such as PR006919, PR007070, and PR007151, all conditionally activated iDC and mDC cells; that is, they could efficiently activate DCs when PD-L1 crosslinks were present, but did not activate DCs or activated them to a lesser degree when PD-L1 crosslinks were absent. This selective activating property of PD-L1×CD40 biantibodies is not present in Selicrelumab and provides a basis for the biantibody molecule to exert its efficacy effectively and safely. Furthermore, in experiments with several donor DCs, the activating effect of PD-L1×CD40 biantibodies 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 effects of PD-L1 × CD40 bispecific binding protein on B cell proliferation were investigated in both PD-L1-mediated crosslinking and non-crosslinking cases.

[0179] One day prior, CHO-K1 and CHO-K1 / hPD-L1 cells were inoculated into 96-well flat-bottom plates (Corning, #3599). First, CHO-K1 and CHO-K1 / hPD-L1 cells were collected, and 50 μg / mL of mitomycin C (Selleckchem, #S8146) was added, followed by incubation at 37°C for 1 hour to inhibit cell proliferation. After washing, the cells were incubated in F-12K complete medium to a cell density of 1 × 10⁶. 5 The solution was adjusted to cells / mL and added to a 96-well flat-bottom plate at 100 μL / well, then incubated overnight in a cell incubator. The following day, the target antibody was diluted to 2-fold in RPMI1640 complete medium, then subjected to a 10-fold gradient dilution. Selicrelumab and hIgG1 were used as positive and negative controls, and the working concentrations of the target antibody were 100 nM, 10 nM, and 1 nM. After discarding the medium for CHO-K1 and CHO-K1 / hPD-L1 cells, the antibody was added at 100 μL / well. Subsequently, B cells were sorted from donor PBMCs using a human CD19 sorting kit (Miltenyi, #130-050-301), labeled with 1 μM CFSE (Invitrogen, #34554) at 37°C for 20 minutes, washed twice, and the cell density was reduced to 1 × 10⁶. 6The solution was adjusted to cells / mL, added to a 96-well plate at 100 μL / well, mixed with the aforementioned 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 500 g at 4°C for 4 minutes, discarded the supernatant, added 100 μL / well of dead cell dye (Invitrogen, #L34976A, 1:1000 dilution), and incubated at 4°C in the dark for 15 minutes. The cells were washed twice with 200 μL / well of PBS, resuspended with 100 μL of Fc Block antibody (BD, #564220, 1:50 dilution), and incubated at 4°C in the dark for 15 minutes. Subsequently, 2 μL of anti-human CD19 antibody (Biolegend, #302234) was added per well, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were then washed twice with 200 μL / well of 2% FBS PBS, and finally resuspended with 100 μL of 2% FBS PBS. Fluorescence emission signal values ​​were read using a BD FACS CANTO II flow cytometer. Data processing and graph analysis were performed using GraphPad Prism 8 software.

[0180] In this example, PD-L1×CD40 biantibody was tested multiple times using B cells derived from multiple donors, and the results are shown in Figures 20A and 20B, 21A and 21B, 22A and 22B, 23A and 23B, and 24A and 24B. Table 30 summarizes the important experimental parameters corresponding to each figure, such as B cell origin, cross-linking target cells, and test molecules.

[0181] The results showed that the positive reference antibody Selicrelumab was able to effectively promote B cell proliferation, but this promoting effect was not affected by the tumor antigen PD-L1. On the other hand, PD-L1×CD40 biantibodies, such as PR006919, PR007070, and PR007151, all conditionally activated B cell proliferation; that is, they efficiently promoted B cell proliferation when PD-L1 crosslinking was present, but had little to no effect when PD-L1 crosslinking was absent. This selective activating characteristic of PD-L1×CD40 biantibodies is not present in Selicrelumab and provides a basis for the effective and safe exertion of the biantibody molecule. Furthermore, in several donor B cell proliferation experiments, the proliferation-promoting effect of PD-L1×CD40 biantibodies was significantly stronger than that of Selicrelumab, as shown in parts of Figures 24A and 24B.

[0182] [Table 32]

[0183] Example 14: Study on the antitumor activity and safety of PD-L1×CD40 In this example, the antitumor effect of the 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. The hPD1×hCD40-C57BL / 6J mouse is a PD1 / CD40 humanized transgenic mouse obtained by knocking in human PD-1 and human CD40 genes into C57BL / 6J mice (provided by Shanghai Southern Model Biotechnology Co., Ltd.).

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

[0185] Example 14.1 Mouse Pharmacodynamic Experiment #1 In the first experiment, when the average tumor volume reached 110 mm 3 , individual mice with moderate tumor volume were selected, and grouping and administration were carried out according to the protocol in Table 31.

[0186]

Table 33

[0187] The results are as shown in Figure 25A and Figure 25B. On day 21 after the start of administration, the average tumor volume of the control group Group 1 (Isotype) was 2210.35±487.58 mm 3 . The tumor volumes of administration groups Group 2 (Selicrelumab), Group 4 (Selicrelumab+Atezolizumab), Group 5 (PR007070) and Group 6 (PR007151) were 239.07±73.25 mm 3 , 52.33±18.99 mm 3 , 647.92±176.36 mm3 and 507.58±116.91mm 3 The corresponding tumor inhibition rates were 93.89%, 102.78%, 74.41%, and 81.10%, respectively, showing a statistically significant difference compared to Group 1 (Isotype) (P<0.01). The tumor volume for Group 3 (Atezolizumab) was 1563.33±325.77 mm². 3 The tumor inhibition rate was 30.81%.

[0188] On day 3 after the start of administration, the body weight of mice in Group 2 (Selicrelumab) and Group 4 (Selicrelumab + Atezolizumab) decreased significantly (by nearly 10%), while the body weight of mice in Group 3 (Atezolizumab) remained normal. This indicates that selicrelumab has a significant side effect and toxicity that causes significant body weight loss in mice. On the other hand, the body weight of mice in the PD-L1 x CD40 dual antibody groups, Group 5 (PR007070) and Group 6 (PR007151), remained normal, demonstrating good safety.

[0189] The results above demonstrate that the PD-L1×CD40 biantibodies PR007070 and PR007151 exhibit significant antitumor activity and are considerably superior to atezolizumab. On the other hand, in terms of changes in mouse body weight, the safety of the PD-L1×CD40 biantibodies PR007070 and PR007151 was significantly better than that of selicrelumab or the combination of selicrelumab and atezolizumab.

[0190] Example 14.2 Mouse Pharmacodynamic Experiment #2 In the second experiment, the average tumor volume was 89 mm². 3 When this condition was reached, mice with a moderate tumor volume were selected and divided into groups and administered according to the protocol in Table 32.

[0191] [Table 34]

[0192] The results are shown in Figures 26A and 26B. On day 18 after the start of administration, the mean tumor volume of the control group Group 1 (hIgG 1, 10 mg / kg) was 1927.77 ± 200.44 mm². 3 The tumor volumes for Group 2 (PR007619, 12 mg / kg) and Group 3 (PR007619, 6 mg / kg) were 333.00 ± 103.06 mm², respectively. 3 538.85±224.35mm 3 The corresponding tumor inhibition rates were 82.73% and 72.05%, respectively. The tumor volumes for Group 4 (PR007281, 12 mg / kg) and Group 5 (PR007281, 6 mg / kg) were 602.40 ± 278.52 mm², respectively. 3 , 68.66±11.68mm 3 The corresponding tumor inhibition rates were 68.75% and 96.44%, respectively. The tumor volumes for Group 6 (PR007556, 12 mg / kg) and Group 7 (PR007556, 6 mg / kg) were 347.56 ± 167.80 mm², respectively. 3 , 258.51±102.12mm 3 The corresponding tumor inhibition rates were 81.97% and 86.59%, respectively. The tumor volumes for Group 8 (PR007276, 12 mg / kg) and Group 9 (PR007276, 6 mg / kg) were 200.34 ± 59.12 mm², 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 treated mice remained normal, and the drug did not have any apparent effect on the mice's condition.

[0193] Based on these results, the PD-L1 x CD40 biantibodies PR007619, PR007281, PR007556, and PR007276 all demonstrated excellent antitumor activity and safety.

[0194] Example 14.3 Mouse pharmacodynamic experiment #3 and mouse hepatotoxicity study In the third experiment, the average tumor volume was 85 mm². 3 When this condition was reached, mice with a moderate tumor volume were selected and divided into groups and administered according to the protocol in Table 33.

[0195] 24 hours after the last dose, mouse serum (36 samples in total) was collected from each group and used for blood biochemistry index tests to detect the content of ALT and AST (alanine aminotransferase and aspartate aminotransferase) in the mouse serum. Liver tissue from 36 mice was also collected and embedded in paraffin. Six consecutive sections were taken from each liver tissue, and then H&E staining was performed. Finally, three stained sections were selected and scanned and microscopically analyzed.

[0196] [Table 35]

[0197] The results of the antitumor effect are shown in Figures 27A and 27B. On day 22 after the start of administration, the tumor volume of the control group 1 (hIgG1, 3.6 mg / kg) was 1865.24 ± 125.47 mm². 3 The tumor volumes for 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 mm², 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 for Group 6 (Selicrelumab, 5 mg / kg) was 339.68 ± 72.9 mm². 3The corresponding tumor inhibition rate was 81.79%. Throughout the experiment, the body weight status of mice in the hIgG1 control group and the PR007619 administered group was normal, indicating that the drug did not have a significant effect on the mouse condition. However, some degree of weight loss was observed in the selicrelumab administered group of mice, indicating that selicrelumab has certain side effects and toxicity.

[0198] These results demonstrate that the PD-L1 x CD40 biantibody PR007619 possesses dose-dependent tumor inhibitory activity and exhibits significant inhibitory effects. Furthermore, PR007619 has a better safety profile than the control molecule, selicrelumab.

[0199] The results of the blood biochemistry index tests are shown in Figure 28. The corresponding mean ALT levels for Groups 1 to 6 were 34±8.83 U / L, 46±24.64 U / L, 34.5±7.04 U / L, 32.5±9.38 U / L, 31.5±1.64 U / L, and 128.7±76.2 U / L, respectively. In Group 6 (Selicrelumab, 5 mg / kg), the levels were significantly elevated compared to Group 1 (control group) (P=0.0128), while in Groups 2 to 5 (groups administered different doses of PR007619), there were no significant differences compared to Group 1 (control group). Similarly, the corresponding mean AST levels for each group from Group 1 to Group 6 were 180±40.3 U / L, 197.5±95.9 U / L, 120.5±41.2 U / L, 120±61.3 U / L, 123.5±16.69 U / L, and 326.17±153.6 U / L, respectively. Here, Group 6 (Selicrelumab, 5 mg / kg) showed a significant increase compared to Group 1 (control group) (P=0.0478), while Groups 2 to 5 (groups receiving different doses of PR007619) did not show any significant difference compared to Group 1 (control group).

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

[0201] Based on the blood biochemical indicators and tissue section results described above, the CD40 monoclonal antibody Selicrelumab caused a significant acute hepatotoxic reaction in hPD1×hCD40-C57BL / 6J transgenic mice, while the PD-L1×CD40 biantibody PR007619 demonstrated superior safety.

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

[0203] In the fourth experiment, the average tumor volume was 82 mm². 3When the target was reached, mice with moderate tumor volume were selected and divided into groups and administered according to the protocol in Table 34. Each group consisted of 10 mice (5 females and 5 males). Administration began on the day of group division and was administered twice a week (BIW) for a total of 4 doses (days 0, 3, 7, and 10) via intraperitoneal injection. Tumor volume was measured during the period (days 0, 2, 5, 9, and 11). Serum was collected 24 hours after the first dose and used to test cytokine levels (IL-6, TNF-α, IFN-γ, and IL12p40). Serum was collected 24 hours after the last dose and used for blood biochemical detection of hepatotoxicity indicators (ALT and AST). Finally, mouse liver tissue (50 in total) was collected, embedded in paraffin, and 6 consecutive sections were taken from each liver tissue. H&E staining was performed, and finally, 3 stained sections were selected for scanning and microscopic analysis. In addition to the liver, the hearts, spleens, lungs, and kidneys of mice 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 day 11 after the start of administration, the mean tumor volume of the control group Group 1 (hIgG 1, 10 mg / kg) was 1653.68 ± 153.10 mm². 3 The tumor volumes for 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 mm², 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 dose group showed a statistically significant difference compared to Group 1 (P>0.05). On the other hand, regarding body weight results, Group 2 (PR007619, 12 mg / kg) had no significant effect on mouse body weight, while each dose group of selicrelumab (Groups 3-5) caused a decrease in mouse body weight after administration, with the decrease being close to or exceeding 10%, and the higher the dose, the more difficult it was for body weight to recover. In particular, in the medium- and high-dose groups of selicrelumab, body weight never recovered after administration.

[0206] Based on these results, the PD-L1 x CD40 biantibody exhibited significant antitumor efficacy and safety, while selicrelumab caused weight loss in mice and resulted in significant toxic side effects.

[0207] [Table 37]

[0208] The results of the blood biochemistry index tests are shown in Figure 31 and Table 35. The corresponding ALT values ​​for Groups 1 to 5 were 168.3±79.8 U / L, 107.6±54.2 U / L, 105.1±44.8 U / L, 310±189.3 U / L, and 507.6±150.0 U / L, respectively. The corresponding AST values ​​for Groups 1 to 5 were 51.6±69.1 U / L, 19.7±7.26 U / L, 31±19.1 U / L, 96.5±86.1 U / L, and 147.7±73.1 U / L, respectively. In this study, Group 5 (Selicrelumab, 5 mg / kg) showed a significantly greater increase in ALT and AST compared to the Group 1 control group (P<0.001), and Group 4 (Selicrelumab, 2.5 mg / kg) showed a significant difference in ALT compared to the Group 1 control group (P=0.037). However, Group 2 (PR007619, 12 mg / kg) showed no significant difference in either ALT or AST compared to the Group 1 control group. This indicates that selicrelumab caused a significant increase in the hepatotoxic blood biochemical indicators ALT and AST, while the PD-L1 × CD40 biantibody 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 Figures 32 and 33 and Table 36. The corresponding cytokine IL-6 values ​​for each group from Group 1 to Group 5 were 16.3±8.74 pg / mL, 24.8±11.7 pg / mL, 1343.8±529.5 pg / mL, 1428.2±569.5 pg / mL, and 2038.0±747.2 pg / mL, respectively. The corresponding cytokine IFN-γ values ​​for each group from Group 1 to Group 5 were 0.54±0.19 pg / mL, 1.48±0.98 pg / mL, 159.0±90.9 pg / mL, 138.9±91.7 pg / mL, and 147.4±79.44 pg / mL, respectively. The corresponding cytokine TNF-α values ​​for each of the five groups were 14.1±3.79 pg / mL, 17.3±8.08 pg / mL, 1186.1±255.7 pg / mL, 1274.7±392.3 pg / mL, and 1311.5±179.9 pg / mL, respectively. The corresponding cytokine IL12p40 values ​​for each of the five groups from Group 1 to Group 5 were 1157.1±125.8 pg / mL, 1786.8±369.5 pg / mL, 92119.7±30959.2 pg / mL, 99263.0±24254.9 pg / mL, and 110786.1±21607.5 pg / mL, respectively.

[0211] As observed above, in terms of cytokine levels of IL-6, TNF-α, and IFN-γ, each dose group of Selicrelumab (Groups 3-5) was able to induce a significant increase in cytokine levels, showing a statistically significant difference compared to the Group 1 control group (P<0.0001). On the other hand, the PD-L1×CD40 biantibody (Group 2) did not induce an increase in cytokine levels, and there was no statistically significant difference compared to the Group 1 control group (P>0.05). Furthermore, in terms of cytokine levels of IL12p40, each dose group of Selicrelumab (Groups 3-5) was able to induce a significant increase in IL12p40 levels, showing a considerable statistically significant difference compared to the Group 1 control group (P<0.0001). On the other hand, the PD-L1×CD40 biantibody (Group 2) caused only a slight increase in IL12p40 (P=0.0004). The overall results showed that selicrelumab induced a pronounced mouse peripheral blood cytokine release syndrome, while the PD-L1 x CD40 biantibody did not.

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

[0213] In this study, compared to the control group (Group 1), the liver, kidney, lung, and spleen changes in the PD-L1×CD40 biantibody (Group 2) were minimal or mild, and minimal lymphocyte infiltration was a predictable pharmacodynamic response. Compared to the control group (Group 1), each dose group of selicrelumab (Groups 3, 4, and 5) showed more adverse changes in these tissues. The renal tubules in these tissues were degenerated, and there was more lymphocyte infiltration and / or inflammation. This can amplify the predictable pharmacodynamic response and be harmful to animals. Therefore, the degree and incidence of lesions with PD-L1×CD40 biantibody were smaller compared to selicrelumab.

[0214] As described above, selicrelumab caused severe cytokine release syndrome, elevated hepatotoxicity serum indicators, and liver and other organ tissue damage in mice, while the PD-L1×CD40 biantibody exhibited potent antitumor effects and excellent safety, and did not cause cytokine release syndrome, elevated hepatotoxicity serum indicators, or liver and other organ tissue damage.

[0215] Example 15: Pharmacokinetic evaluation of PD-L1 × CD40 In this example, we investigated the pharmacokinetic performance of four bispecific binding protein molecules that have similar sequences and identical structures in mice in vivo.

[0216] Administration and Blood Collection: For each target binding protein molecule, six female C57BL / 6J mice weighing 18-22 grams were selected and administered intravenously to the target binding protein molecule at a dose of 6 mg / kg. Whole blood was collected from three mice in one group 5 minutes, 24 hours (1 day), 4 days, and 10 days after administration, and from three mice in another group before administration and 5 hours, 2 days, 7 days, and 14 days after administration. The whole blood was allowed to coagulate, then centrifuged, and the separated serum samples were frozen and stored at -80°C until analysis.

[0217] Analytical Methods: Drug concentrations in mouse serum were quantitatively measured using two ELISA methods. ELISA Method 1, an Fc-terminal detection method, involved capturing human Fc-containing antibodies in mouse serum using a goat anti-human Fc polyclonal antibody (ROCKLAND, #609-101-017) coated on a 96-well plate, followed by detection using an HRP-labeled goat anti-human Fc secondary antibody (Jackson, #109-035-098). ELISA Method 2, a functional domain detection method, involved capturing antibodies that specifically recognize the PD-L1 antigen protein (Novoprotein, #C315) coated on a 96-well plate, followed by detection using an HRP-labeled goat anti-human Fc secondary antibody (Jackson, #109-035-098). Pharmacokinetic parameters were analyzed using a non-compartmental model (NCA) with Phoenix WinNonlin software version 8.2.

[0218] As shown in Table 37, the biantibody molecule PR007556 has a serum half-life t1 / 2 value similar to that of conventional IgG antibodies, and its t1 / 2 value was shown to be 10.3 days by the PD-L1 terminal detection method. The biantibody molecules PR007619, PR007281, and PR007276 have pharmacokinetics similar to those of conventional IgG antibodies, and by the Fc terminal detection method, PR007619, PR007281, and PR007276 had a serum half-life t1 / 2 of 10.3 days in mouse in vivo. 1 / 2 The values ​​were approximately 13.6 days, 15.1 days, and 15.7 days, respectively. In the PD-L1 terminal detection method, PR007619, PR007281, and PR007276 had serum half-lives (t1 / 2) of approximately 13.5 days, 18.1 days, and 13.7 days in vivo in mice.

[0219] [Table 39]

[0220] The results are shown in Table 37 and Figures 35A to 35D. These PD-L1×CD40 biantibody molecules have serum half-lives (t1 / 2) similar to conventional IgG antibodies, and their T values ​​vary depending on the detection method. 1 / 2 The value is between 10 and 20 days.

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

Claims

1. An antigen-binding protein targeting PD-L1 and CD40, comprising a first protein functional region and a second protein functional region, wherein the first protein functional region comprises an antigen-binding protein targeting CD40, and the second protein functional region comprises an antigen-binding protein targeting PD-L1, and here, 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 LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 38, and LCDR2 comprises the amino acid sequence shown in SEQ ID NO: An antigen-binding protein targeting PD-L1 and CD40, characterized in that it contains the amino acid sequence shown in 43, wherein the LCDR3 contains the amino acid sequence shown in any one of SEQ ID NO: 48 to 53, the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 18, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:

27.

2. The antigen-binding protein that targets CD40, wherein VL comprises an amino acid sequence shown in any one of SEQ ID NO: 68 to 73, and VH comprises an amino acid sequence shown in SEQ ID NO: 60, as described in Claim 1.

3. The antigen-binding protein targeting CD40 is a full-length antibody, and the full-length antibody comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence shown in SEQ ID NO: 87, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 78, or the light chain comprises the amino acid sequence shown in any one of SEQ ID NO: 88 to 92, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 84, as described in Claim 1.

4. In the antigen-binding protein that targets 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: 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: HCDR1 includes the amino acid sequence shown in 9, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 19, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 25, or HCDR1 includes the amino acid sequence shown in SEQ ID NO: 9, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 19, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 29, or HCDR1 includes the amino acid sequence shown in SEQ ID NO: 9, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 19, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 30, or HCDR1 includes the amino acid sequence shown in SEQ ID NO: 9, HCDR2 includes the amino acid sequence shown in SEQ ID NO: The antigen-binding protein targeting PD-L1 and CD40 according to claim 3, characterized in that it contains the amino acid sequence shown in 19, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:

28.

5. The antigen-binding protein targeting PD-L1 and CD40 according to claim 4, characterized in that the VH in the antigen-binding protein targeting PD-L1 includes an amino acid sequence shown in any one of SEQ ID NO: 57 to 65.

6. The first protein functional region is an immunoglobulin containing an antigen-binding protein that targets CD40, and the second protein functional region contains the VH of one, two, or more antigen-binding proteins that target PD-L1. 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, either directly or via a linker. Herein, the linker is selected from any one amino acid sequence shown in GS, GGS, and SEQ ID NO: 100 to 106, characterized in that the antigen-binding protein targeting PD-L1 and CD40 is as described in claim 1.

7. The PD-L1 and CD40 targeting antigen-binding protein according to claim 6, wherein the second protein functional region comprises the VH of two PD-L1 targeting antigen-binding proteins, and / or the C-terminuses of the two VHs are linked to the N-terminuses of two heavy chains of the immunoglobulin via a linker shown in GGS or SEQ ID NO: 101, respectively.

8. The antigen-binding protein targeting PD-L1 and CD40 according to claim 1, characterized in that the first protein functional region comprises two light chains containing the amino acid sequence shown in any one of SEQ ID NO: 88 to 92 and two heavy chains containing the amino acid sequence shown in SEQ ID NO: 84, and the second protein functional region comprises two VHs containing the amino acid sequence shown in any one of SEQ ID NO: 79 to 83, and the C-terminuses of the two VHs are linked to the N-terminuses of the two heavy chains of the first protein functional region, respectively, via a GGS or a linker shown in SEQ ID NO:

101.

9. The antigen-binding protein that targets PD-L1 and CD40 is The amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 89, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 93, or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 88, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 94, or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 91, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 94, or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 90, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 93, or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 92, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 93, or the amino acid sequence is shown in SEQ ID NO: The second polypeptide chain shown in 88, the amino acid sequence of the first polypeptide chain shown in 95, or the amino acid sequence of the first polypeptide chain shown in 88, the amino acid sequence of the first polypeptide chain shown in 96, or the amino acid sequence of the first polypeptide chain shown in 88, the amino acid sequence of the first polypeptide chain shown in 97, or the amino acid sequence of the first polypeptide chain shown in 91, the amino acid sequence of the first polypeptide chain shown in 95, or the amino acid sequence of the first polypeptide chain shown in 91, the amino acid sequence of the first polypeptide chain shown in 93, or the amino acid sequence of the second polypeptide chain shown in 91, the amino acid sequence of the first polypeptide chain shown in 95 The first polypeptide chain shown in 96, or the amino acid sequence is SEQ ID NO: The second polypeptide chain shown in 89, the amino acid sequence is SEQ ID NO: The first polypeptide chain shown in 97, or the amino acid sequence is SEQ ID NO: The second polypeptide chain shown in 90, the amino acid sequence is SEQ ID NO: The first polypeptide chain shown in 97, or the amino acid sequence is SEQ ID NO: The second polypeptide chain shown in 88, the amino acid sequence is SEQ ID NO:The antigen-binding protein targeting PD-L1 and CD40 according to claim 8, characterized in that it comprises a first polypeptide chain shown in 98, or a second polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 88, a first polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 99, or a second polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 91, a first polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 98, or a second polypeptide chain whose amino acid sequence is shown in SEQ ID NO: 91, and a first polypeptide chain whose amino acid sequence is shown in SEQ ID NO:

99.

10. A chimeric antigen receptor characterized by comprising an antigen-binding protein that targets PD-L1 and CD40 as described in Claim 1.

11. An antibody-drug conjugate comprising the antigen-binding protein that targets PD-L1 and CD40 as described in Claim 1, and a cytotoxic agent or label.

12. Genetically modified cells expressing the chimeric antigen receptor described in Claim 10.

13. An isolated nucleic acid characterized by encoding an antigen-binding protein that targets PD-L1 and CD40 as described in Claim 1.

14. A recombinant expression vector comprising the isolated nucleic acid described in Claim 13.

15. A transformant comprising the isolated nucleic acid described in Claim 14 or the recombinant expression vector.

16. A method for producing antigen-binding proteins targeting PD-L1 and CD40 according to any one of claims 1 to 9, wherein the method comprises the step of culturing the transformant according to claim 15 and obtaining antigen-binding proteins targeting PD-L1 and CD40 from the culture.

17. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises one or more of the following: an antigen-binding protein targeting PD-L1 and CD40 as described in any one of claims 1 to 9, a chimeric antigen receptor as described in claim 10, and an antibody-drug conjugate as described in claim 11, and / or a pharmaceutically acceptable carrier.

18. A detection reagent, characterized in that the detection reagent comprises an antigen-binding protein that targets PD-L1 and CD40 as described in any one of claims 1 to 9 and / or an antibody-drug conjugate as described in claim 11.

19. A parts kit comprising Kit A, which comprises one or more of the following: an antigen-binding protein targeting PD-L1 and CD40 as described in any one of claims 1 to 9, a chimeric antigen receptor as described in claim 10, an antibody-drug conjugate as described in claim 11, and a pharmaceutical composition as described in claim 17. The parts kit further comprises Kit B, wherein Kit B contains one or more of the following: other antitumor antibodies or pharmaceutical compositions comprising the other antitumor antibodies, and / or hormone preparations, targeted small molecule preparations, proteasome inhibitors, contrast agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

20. One or more of the following for use in diagnosing, preventing and / or treating diseases related to PD-L1 and / or CD40: an antigen-binding protein targeting PD-L1 and CD40 according to any one of claims 1 to 9, a chimeric antigen receptor according to claim 10, an antibody-drug conjugate according to claim 11, and a pharmaceutical composition according to claim 17, wherein the diseases related to PD-L1 and / or CD40 are PD-L1-related tumors, CD40-related tumors, or PD-L1 × CD40-related tumors, the CD40-related tumors include solid tumors and hematological tumors, and the PD-L1-related tumors include solid tumors and hematological tumors.

21. The CD40-related tumors include B-series 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-related 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), and primary mediastinal large myeloma. B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), large B-cell lymphoma rich in T cells / histiocytes, multiple myeloma, myelocellular leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), gastrointestinal cancer, kidney 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 tract cancer Antigen-binding proteins targeting PD-L1 and CD40, chimeric antigen receptors, antibody-drug conjugates, and pharmaceutical compositions for use in claim 20, including 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.

22. A method for detecting PD-L1 and / or CD40 in a sample, the method comprising the step of contacting the sample with a detection reagent, wherein the sample is a blood sample and a reagent containing PD-L1 and / or CD40, and the method is not intended for therapeutic / diagnostic purposes. The method is characterized in that the detection reagent comprises the antigen-binding protein that targets PD-L1 and CD40 according to any one of claims 1 to 9 and / or the antibody-drug conjugate according to claim 11.

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