Anti-CD40 antibody and anti-PD-L1 × CD40 bispecific antibody and their use

Abstract

The present invention discloses an anti-CD40 antibody, an anti-PD-L1×CD40 bispecific antibody, and their uses. The anti-CD40 antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 64, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 65. The novel anti-CD40 agonist antibody of the present invention can effectively regulate the activation of DC cells, has a stronger T cell activation effect, has low toxicity and side effects, and is suitable for tumor immunotherapy. The bispecific antibody of the present invention can maximize the antitumor effect, improve the selectivity of CD40 activation through PD-L1-dependent CD40 activation, and reduce the toxicity and side effects of the CD40 agonist antibody.

Description

[Technical Field]

[0001] This application claims priority to Chinese patent applications 2022106137021, 2022, 2022, 2022, 2022, 2022, 2022, 2023

[0002] This invention belongs to the field of biotechnology and specifically relates to anti-CD40 antibodies, anti-PD-L1 × CD40 bispecific antibodies, and their use. [Background technology]

[0003] In recent years, the field of tumor treatment has developed rapidly, evolving from conventional therapies centered on surgery, radiotherapy, and chemotherapy to more advanced treatment protocols such as targeted therapy and immunotherapy. In particular, tumor immunotherapy, exemplified by PD-L1 / PD-1 inhibitors, has significantly extended the survival time of tumor patients in multiple tumor indications and is positioned as a first-line treatment for several tumor indications (Nat Rev Immunol. 2020 Nov;20(11):651-668). As the most promising solution for curing tumors, tumor immunotherapy has become central to the research and development of new antitumor drugs.

[0004] One of the key challenges in tumor immunology is increasing the quantity and quality of invasive T cells in cryogenic tumors. In many cancer patients, insufficient T cell priming is the reason for T cell deficiency in the tumor microenvironment, and antigen-presenting cells, particularly DC cells, loaded with tumor antigens play a crucial role in initiating the T cell response. CD40, a type I transmembrane protein, is a member of the tumor necrosis factor receptor (TNFR) superfamily and is widely expressed in antigen-presenting cells (DC cells, Mφ cells, B cells), platelets, some non-hematopoietic cells, and various types of tumor cells. It plays an important role in both innate and adaptive immunity and is crucial in the activation of DC cells (Expert Opin Biol Ther. 2021 Dec;21(12):1635-1646. Annu Rev Med. 2020 Jan 27;71:47-58. Expert Rev Anticancer Ther. 2017 Feb;17(2):175-186. Hum Vaccin Immunother. 2020;16(2):377-387.). When CD40 is activated, it activates DC cells by upregulating co-stimulatory molecules and MHC on DC cells and inducing pro-inflammatory cytokines, thereby empowering the activation of antitumor-specific T cells. This type of T cell has the potential to completely eliminate tumor cells.

[0005] In various mouse tumor models, CD40 agonist antibodies can be used to achieve T cell activation and exhibit potent antitumor effects (Science. 2011 Aug 19;333(6045):1030-4. Clin Cancer Res. 2015 Mar 1;21(5):1115-26. Int J Cancer. 2019 Sep 1;145(5):1189-1199. J Immunother Cancer. 2020 May;8(1):e000624.). CD40 agonist antibodies can exert synergistic antitumor effects with immune checkpoint antibodies such as PD-L1 / PD-1, which depends on the interaction mechanism between DC cells and T cells, in which DCs upregulate costimulatory molecules and stimulate the activation of tumor-specific T cells by secreting IL-12, and after T cells are activated, they further activate DC cells by secreting IFN-γ. CD40 antibodies and PD-L1 / PD-1 antibodies act on different interaction links to enhance positive feedback and maximize antitumor effects (Cancer Res. 2016 Nov 1;76(21):6266-6277. Immunity. 2018 Dec 18; 49(6): 1148-1161.e7.).

[0006] Currently, many biopharmaceutical companies are developing agonist monoclonal antibodies against CD40, and related patents include WO2003040170, WO2014070934A1, US20180066053, US20140348836, WO2020108611, and CN111763259, with several antibodies in clinical trials. Selicrelumab, a CD40 monoclonal antibody developed by Pfizer, showed an objective partial response (PR) in 4 out of 15 patients with advanced melanoma in the first single-dose trial in humans, and one of these patients continued to receive repeated doses of selicrelumab for one year and maintained complete remission (CR) after 15 years. However, subsequent clinical trials showed poor efficacy for sericrelumab, and other CD40 monoclonal antibodies such as APX005M and SEA-CD40 also yielded very low objective response rates (ORR). CD40-agonist monoclonal antibodies have been shown to cause numerous side effects in clinical practice, including cytokine release syndrome (CRS), liver injury, and thrombocytopenia. The maximum tolerated dose (MTD) for sericrelumab, APX005M, and SEA-CD40 is 0.2, 0.3, and 0.06 mg / kg, respectively (J Clin Oncol. 2007 Mar 1;25(7):876-83. Cancer Biol Ther. 2010 Nov 15;10(10):983-93. Lancet Oncol. 2021 Jan;22(1):118-131. Oncol Lett. 2020 Nov;20(5):176. Annu Rev Med. 2020 Jan 27;71:47-58.). Therefore, there is an urgent need in this field for CD40-targeted antibodies that offer high safety and excellent efficacy. [Overview of the project]

[0007] To address the above technical challenges, and considering the current research and development status of CD40 antibodies and / or PD-L1 antibodies, the present invention provides an anti-CD40 antibody and an anti-PD-L1 × CD40 bispecific antibody. The novel anti-CD40 agonist antibody of the present invention can effectively regulate DC cell activation and has a more potent T cell activation effect, while having low toxicity and side effects, making it suitable for tumor immunotherapy. The anti-PD-L1 × CD40 bispecific antibody of the present invention maximizes the antitumor effect by stimulating CD40 and simultaneously blocking PD-L1 / PD-1, thereby acting on the positive feedback pathway of interaction between DC cells and T cells. Furthermore, it improves the selectivity of CD40 activation through PD-L1-dependent CD40 activation, reducing the toxicity and side effects of the CD40 agonist antibody.

[0008] One aspect of the present invention provides an anti-CD40 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 64, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 65.

[0009] The term "CD40" includes all variants or isoforms of CD40 that are naturally expressed by cells. The antibodies of the present invention can specifically bind to human CD40 and monkey CD40 (e.g., cynomolgus monkeys). Alternatively, the antibodies may be specific to human CD40 and not cross-reactive with other species. CD40, or any of its variants or isoforms, can be isolated from cells or tissues that naturally express them, or can be produced by recombinant techniques common in the art and utilizing techniques described herein.

[0010] In some embodiments, the light chain variable region includes LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 38, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 40. Alternatively, the light chain variable region includes LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 38, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 39. Alternatively, the light chain variable region includes LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 30, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 31. Alternatively, the light chain variable region includes LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 32, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 33. Alternatively, the light chain variable region includes LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 32, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 34. Alternatively, the light chain variable region includes LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 35, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 36. Alternatively, the light chain variable region includes LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 35, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 37. Alternatively, the light chain variable region includes LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 41, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 42. Alternatively, the light chain variable region includes LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 41, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 43.

[0011] In some embodiments, the CDR is defined according to a Kabat, IMGT, Chothia, AbM, or Contact numbering system, and in some specific embodiments, the CDR is determined according to Kabat numbering rules.

[0012] In some embodiments, the antibody described in the first aspect of the present invention comprises the following: LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, LCDR2 comprises the amino acid sequence shown in X5X6SX7X8X9S, where X5 is Y or A, X6 is T or A, X7 is S, R or T, X8 is L or R, X9 is Q or D, LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6.

[0013] In a preferred embodiment of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of LCDR2 is the amino acid sequence shown in X5X6SX7X8X9S, where X5 is Y or A, X6 is T or A, X7 is S, R or T, X8 is L or R, and X9 is Q or D, the amino acid sequence of LCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 6.

[0014] In the antibody according to the first aspect of the present invention, LCDR1 includes the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 8, SEQ ID NO: 7, or SEQ ID NO: 10; LCDR2 includes the amino acid sequence shown in SEQ ID NO: 14, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 11, or SEQ ID NO: 15; LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 16, or SEQ ID NO: 20; HCDR1 includes the amino acid sequence shown in SEQ ID NO: 21, or SEQ ID NO: 22; HCDR2 includes the amino acid sequence shown in SEQ ID NO: 23, or SEQ ID NO: 24; and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 28, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 25, or SEQ ID NO: 29.

[0015] In a preferred embodiment of the present invention, the antibody includes the amino acid sequence of LCDR1 shown in SEQ ID NO: 9, SEQ ID NO: 8, SEQ ID NO: 7 or SEQ ID NO: 10, the amino acid sequence of LCDR2 shown in SEQ ID NO: 14, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 11 or SEQ ID NO: 15, the amino acid sequence of LCDR3 shown in SEQ ID NO: 19, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 16 or SEQ ID NO: 20, the amino acid sequence of HCDR1 shown in SEQ ID NO: 21 or SEQ ID NO: 22, the amino acid sequence of HCDR2 shown in SEQ ID NO: 23 or SEQ ID NO: 24, and the amino acid sequence of HCDR3 shown in SEQ ID NO: 28, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 25 or SEQ ID NO: 29.

[0016] In a preferred embodiment of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 9, SEQ ID NO: 8, SEQ ID NO: 7, or SEQ ID NO: 10; the amino acid sequence of LCDR2 is shown in SEQ ID NO: 14, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 11, or SEQ ID NO: 15; the amino acid sequence of LCDR3 is shown in SEQ ID NO: 19, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 16, or SEQ ID NO: 20; the amino acid sequence of HCDR1 is shown in SEQ ID NO: 21, or SEQ ID NO: 22; the amino acid sequence of HCDR2 is shown in SEQ ID NO: 23, or SEQ ID NO: 24; and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 28, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 25, or SEQ ID NO: 29.

[0017] In a preferred embodiment of the antibody described in the first aspect of the present invention: A) LCDR1 includes the amino acid sequence shown in SEQ ID NO: 9, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 14, LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19, HCDR1 includes the amino acid sequence shown in SEQ ID NO: 21, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 23, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 28.

[0018] B) LCDR1 includes the amino acid sequence shown in SEQ ID NO: 9, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 14, LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19, HCDR1 includes the amino acid sequence shown in SEQ ID NO: 21, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 24, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 28.

[0019] C) The LCDR1 includes the amino acid sequence shown in SEQ ID NO: 7, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 11, the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 16, the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 23, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 25.

[0020] D) LCDR1 includes the amino acid sequence shown in SEQ ID NO: 7, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 12, LCDR3 includes the amino acid sequence shown in SEQ ID NO: 17, HCDR1 includes the amino acid sequence shown in SEQ ID NO: 22, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 24, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 26.

[0021] E) The LCDR1 includes the amino acid sequence shown in SEQ ID NO: 7, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 12, the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 17, the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 22, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 23, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 26.

[0022] F) LCDR1 includes the amino acid sequence shown in SEQ ID NO: 8, LCDR includes the amino acid sequence shown in SEQ ID NO: 13, LCDR3 includes the amino acid sequence shown in SEQ ID NO: 18, HCDR1 includes the amino acid sequence shown in SEQ ID NO: 22, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 24, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 27.

[0023] G) The LCDR1 includes the amino acid sequence shown in SEQ ID NO: 8, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 13, the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 18, the previously described HCDR1 includes the amino acid sequence shown in SEQ ID NO: 22, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 23, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 27.

[0024] H) The LCDR1 includes the amino acid sequence shown in SEQ ID NO: 10, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 15, the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 20, the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 24, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 29, or

[0025] I) LCDR1 includes the amino acid sequence shown in SEQ ID NO: 10, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 15, LCDR3 includes the amino acid sequence shown in SEQ ID NO: 20, HCDR1 includes the amino acid sequence shown in SEQ ID NO: 21, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 23, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 29.

[0026] Further preferred embodiments of the present invention: A) The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 9, SEQ ID NO: 14, and SEQ ID NO: 19, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 28, respectively.

[0027] B) The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 9, SEQ ID NO: 14, and SEQ ID NO: 19, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 28, respectively.

[0028] C) The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 16, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25, respectively.

[0029] D) The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs. 7, 12, and 17, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs. 22, 24, and 26, respectively.

[0030] E) The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs. 7, 12, and 17, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs. 22, 23, and 26, respectively.

[0031] F) The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO: 18, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 27, respectively.

[0032] G) The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO: 18, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 27, respectively.

[0033] H) The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs. 10, 15, and 20, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs. 21, 24, and 29, respectively, or

[0034] I) The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 10, SEQ ID NO: 15, and SEQ ID NO: 20, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 29, respectively.

[0035] In a preferred embodiment of the present invention: In some embodiments, the amino acid sequence of the antibody is shown in SEQ ID NO: 9 for LCDR1, SEQ ID NO: 14 for LCDR2, SEQ ID NO: 19 for LCDR3, SEQ ID NO: 21 for HCDR1, SEQ ID NO: 23 for HCDR2, and SEQ ID NO: 28 for HCDR3.

[0036] In some embodiments, the amino acid sequence of the antibody is shown in SEQ ID NO: 9 for LCDR1, SEQ ID NO: 14 for LCDR2, SEQ ID NO: 19 for LCDR3, SEQ ID NO: 21 for HCDR1, SEQ ID NO: 24 for HCDR2, and SEQ ID NO: 28 for HCDR3.

[0037] In some embodiments, the amino acid sequence of the antibody is shown in SEQ ID NO: 7 for LCDR1, SEQ ID NO: 11 for LCDR2, SEQ ID NO: 16 for LCDR3, SEQ ID NO: 21 for HCDR1, SEQ ID NO: 23 for HCDR2, and SEQ ID NO: 25 for HCDR3.

[0038] In some embodiments, the amino acid sequence of the antibody is shown in SEQ ID NO: 7 for LCDR1, SEQ ID NO: 12 for LCDR2, SEQ ID NO: 17 for LCDR3, SEQ ID NO: 22 for HCDR1, SEQ ID NO: 24 for HCDR2, and SEQ ID NO: 26 for HCDR3.

[0039] In some embodiments, the amino acid sequence of the antibody is shown in SEQ ID NO: 7 for LCDR1, SEQ ID NO: 12 for LCDR2, SEQ ID NO: 17 for LCDR3, SEQ ID NO: 22 for HCDR1, SEQ ID NO: 23 for HCDR2, and SEQ ID NO: 26 for HCDR3.

[0040] In some embodiments, the amino acid sequence of the antibody is shown in SEQ ID NO: 8 for LCDR1, SEQ ID NO: 13 for LCDR2, SEQ ID NO: 18 for LCDR3, SEQ ID NO: 22 for HCDR1, SEQ ID NO: 24 for HCDR2, and SEQ ID NO: 27 for HCDR3.

[0041] In some embodiments, the amino acid sequence of the antibody is shown in SEQ ID NO: 8 for LCDR1, SEQ ID NO: 13 for LCDR2, SEQ ID NO: 18 for LCDR3, SEQ ID NO: 22 for HCDR1, SEQ ID NO: 23 for HCDR2, and SEQ ID NO: 27 for HCDR3.

[0042] In some embodiments, the amino acid sequence of the antibody is shown in SEQ ID NO: 10 for LCDR1, SEQ ID NO: 15 for LCDR2, SEQ ID NO: 20 for LCDR3, SEQ ID NO: 21 for HCDR1, SEQ ID NO: 24 for HCDR2, and SEQ ID NO: 29 for HCDR3.

[0043] In some embodiments, the amino acid sequence of the antibody is shown in SEQ ID NO: 10 for LCDR1, SEQ ID NO: 15 for LCDR2, SEQ ID NO: 20 for LCDR3, SEQ ID NO: 21 for HCDR1, SEQ ID NO: 23 for HCDR2, and SEQ ID NO: 29 for HCDR3.

[0044] In some of the embodiments described above, the amino acid sequences of the listed CDRs are determined according to Kabat definition rules. However, it is well known to those skilled in the art that the CDR of an antibody can be defined by various methods in this art, for example, Chothia (Chothia et al., (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)) based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed, US Department of Health and Human Services, National Institutes of Health (1987)) based on the variability of the antibody sequence, AbM (University of Bath), Contact (University College London), the International Immunogen Database (IMGT) (World Wide Web: imgt.cines.fr / ), and North (affinity propagation clustering) based on numerous crystal structures. This includes the definition of CDRs. Unless otherwise specified, the terms “CDR” and “complementarity-determining region” of a given antibody or region thereof (e.g., variable region) will be understood by those skilled in the art to include the complementarity-determining region as defined by any of the known schemes described in this invention. Various numbering systems and their corresponding CDRs are well known to those skilled in the art and are shown in Table 1.

[0045] Table 1 Definition Method for Antibody CDR [Table 1] Note: In Table 1, Laa-Lbb refers to the amino acid sequence from position aa to bb, starting from the N-terminus of the antibody light chain, according to the corresponding coding rule. Haa-Hbb refers to the amino acid sequence from position aa to bb, starting from the N-terminus of the antibody heavy chain, according to the corresponding coding rule. For example, L24-L34 in the second column of the second row of Table 1 refers to the amino acid sequence starting from the N-terminus of the variable region of the antibody light chain, determined by residues at positions 24-34 according to the Kabat coding rule. Other entries are similar.

[0046] In the antibody described in the first aspect of the present invention, the framework region of the light chain variable region is a human-derived framework region, and the framework region of the heavy chain variable region is a human-derived framework region.

[0047] In the antibody described in the first aspect of the present invention, in a preferred embodiment of the present invention: a) The light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence homology with SEQ ID NO: 38, and the heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence homology with SEQ ID NO: 40 or SEQ ID NO: 39.

[0048] b) The light chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence homology with SEQ ID NO: 30, and the heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence homology with SEQ ID NO: 31.

[0049] c) The light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence homology with SEQ ID NO: 32, and the heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence homology with SEQ ID NO: 33 or SEQ ID NO: 34.

[0050] d) The light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence homology with SEQ ID NO: 35, and the heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence homology with SEQ ID NO: 36 or SEQ ID NO: 37, or,

[0051] e) The light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence homology with SEQ ID NO: 41, and the heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence homology with SEQ ID NO: 42 or SEQ ID NO: 43.

[0052] In a preferred embodiment of the present invention, the variable region having an amino acid sequence with at least 90%, at least 95%, or at least 99% sequence homology maintains binding function to the same antigen (e.g., human CD40) as the original sequence.

[0053] The calculation of sequence homology between sequences is as follows: To determine the homology percentage of two amino acid sequences, the sequences are aligned for optimal comparison purposes (for example, a gap may be introduced between the first and second amino acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In one preferred embodiment, for comparison purposes, the length of the aligned reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the reference sequence length. Next, the amino acid residues at the corresponding amino acid positions are compared. If a position in the first sequence is occupied by the same amino acid residue at the corresponding position in the second sequence, then the molecules are the same at this position. Sequence comparison and homology percentage calculation between two sequences can be performed using mathematical algorithms. In one preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available from http: / / www.gcg.com), already integrated into the GAP program of the GCG software package, is used to determine the homology percentage between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred parameter set (and, unless otherwise specified, the parameter set that should be used) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap expansion penalty of 4, and a frameshift gap penalty of 5. Additionally, the percentage of homology between two amino acid sequences can be determined using the PAM120 weighted remainder table, gap length penalty 12, gap penalty 4, and the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) incorporated into the ALIGN program (version 2.0).Furthermore, or alternatively, the protein sequences described in the present invention may be further used as “query sequences” for performing searches against public databases, for example, to identify other family member sequences or related sequences.

[0054] In the antibody described in the first aspect of the present invention, in a more preferred embodiment of the present invention: a) The light chain variable region includes the amino acid sequence shown in SEQ ID NO: 38, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 40 or SEQ ID NO: 39.

[0055] b) The light chain variable region includes the amino acid sequence shown in SEQ ID NO: 30, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 31.

[0056] c) The light chain variable region includes the amino acid sequence shown in SEQ ID NO: 32, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 33 or SEQ ID NO: 34.

[0057] d) The light chain variable region includes the amino acid sequence shown in SEQ ID NO: 35, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 36 or SEQ ID NO: 37, or

[0058] e) The light chain variable region includes the amino acid sequence shown in SEQ ID NO: 41, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 42 or SEQ ID NO: 43.

[0059] In the antibody described in the first aspect of the present invention, in a more preferred embodiment of the present invention: In some embodiments, the antibody includes a light chain variable region shown in SEQ ID NO: 38 and a heavy chain variable region shown in SEQ ID NO: 40.

[0060] In some embodiments, the antibody includes a light chain variable region shown in SEQ ID NO: 38 and a heavy chain variable region shown in SEQ ID NO: 39.

[0061] In some embodiments, the antibody includes a light chain variable region shown in SEQ ID NO: 30 and a heavy chain variable region shown in SEQ ID NO: 31.

[0062] In some embodiments, the antibody includes a light chain variable region shown in SEQ ID NO: 32 and a heavy chain variable region shown in SEQ ID NO: 33.

[0063] In some embodiments, the antibody includes a light chain variable region shown in SEQ ID NO: 32 and a heavy chain variable region shown in SEQ ID NO: 34.

[0064] In some embodiments, the antibody includes a light chain variable region shown in SEQ ID NO: 35 and a heavy chain variable region shown in SEQ ID NO: 36.

[0065] In some embodiments, the antibody includes a light chain variable region shown in SEQ ID NO: 35 and a heavy chain variable region shown in SEQ ID NO: 37.

[0066] In some embodiments, the antibody includes a light chain variable region shown in SEQ ID NO: 41 and a heavy chain variable region shown in SEQ ID NO: 42.

[0067] In some embodiments, the antibody includes a light chain variable region shown in SEQ ID NO: 41 and a heavy chain variable region shown in SEQ ID NO: 43.

[0068] In a preferred embodiment of the present invention: In some embodiments, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 38, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 40 or SEQ ID NO: 39.

[0069] In some embodiments, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 30, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31.

[0070] In some embodiments, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 32, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 33 or SEQ ID NO: 34.

[0071] In some embodiments, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36 or SEQ ID NO: 37.

[0072] In some embodiments, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 41, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 42 or SEQ ID NO: 43.

[0073] In a preferred embodiment of the present invention: In some embodiments, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 38, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 40.

[0074] In some embodiments, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 38, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 39.

[0075] In some embodiments, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 30, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31.

[0076] In some embodiments, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 32, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 33.

[0077] In some embodiments, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 32, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 34.

[0078] In some embodiments, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36.

[0079] In some embodiments, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37.

[0080] In some embodiments, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 41, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 42.

[0081] In some embodiments, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 41, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 43.

[0082] In the antibody described in the first aspect of the present invention, the antibody satisfies one or more of the following three conditions: (1) the antibody is a full-length antibody, Fab, Fab′, F(ab′)2, or Fv, and the Fv is preferably scFv; (2) the antibody is a monospecific antibody or a multispecific antibody; or (3) the antibody is a monoclonal antibody or a polyclonal antibody prepared using the above antibody.

[0083] The antibodies of the present invention include monoclonal antibodies (abbreviated as mAb or Ab) and refer to antibodies obtained from a single clonal cell line, the cell line being said to be a eukaryotic cell line, a prokaryotic cell line, or a phage clonal cell line.

[0084] In the antibody described in the first aspect of the present invention, the antibody includes a heavy chain constant region and / or a light chain constant region.

[0085] In some embodiments, the heavy chain constant region of the antibody is derived from the heavy chain constant region of the humanized antibody IgG1, IgG2, IgG3, or IgG4, and / or the light chain constant region of the antibody is derived from the κ chain of the humanized antibody.

[0086] In some embodiments, the constant region includes constant region variants that do not alter the structure and function of the antibody variable region. The prior art discloses various such constant region variants, for example, in which the Fc of the antibody heavy chain constant region has substitutions of one or more amino acids from 238, 265, 269, 270, 297, 327, and 329 (using the EU numbering system) (U.S. Patent No. 6 / 737056), or in which the Fc of the antibody heavy chain constant region has substitutions of one or more from 234, 235, 265, and 329 (using the EU numbering system). The antibody has an amino acid substitution, or the Fc in the heavy chain constant region of the antibody has one or more amino acid substitutions from 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (using the EU numbering system) (see U.S. Patent No. 7 / 371826). These mutations have been shown to give the antibody new properties, but do not alter the function of the antibody's variable region.

[0087] In a preferred embodiment of the present invention, the heavy chain constant region includes the amino acid sequence shown in SEQ ID NO: 45, and the light chain constant region includes the amino acid sequence shown in SEQ ID NO: 44.

[0088] In a more preferred embodiment of the present invention, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 94, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 95.

[0089] In a more preferred embodiment of the present invention, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 96, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 81.

[0090] In a more preferred embodiment of the present invention, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 87, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 81.

[0091] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 45, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 44.

[0092] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 94, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 95.

[0093] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 96, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 81.

[0094] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 87, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 81.

[0095] A second aspect of the present invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to human CD40 and a second antigen-binding domain that specifically binds to human PD-L1, wherein the first antigen-binding domain that specifically binds to human CD40 is as defined in the anti-CD40 antibody described in the first aspect of the present invention.

[0096] The term "antibody" as used in this invention is used in its broadest sense and includes monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv), and conventional antibodies (tetrapeptide chain antibodies composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds), and antigen-binding antibodies such as Fab, Fab', F(ab')2, Fv, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobody, peptidebody, and domain antibodies (heavy chain (VH) antibodies, light chain (VL) antibodies). Conventional antibodies (also called "full-length antibodies" or "complete antibodies") are typically heterotetrameric proteins of approximately 150,000 daltons, with a tetrapeptide chain structure consisting of two identical light chains (L) and two identical heavy chains (H) linked by interchain disulfide bonds. Each heavy chain of a full-length antibody consists of a heavy chain variable region (abbreviated as VH in this invention) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this invention) and a light chain constant region (abbreviated as CL in this invention). The light chain constant region consists of one domain, CL. Mammalian heavy chains are classified into α, δ, ε, γ, and μ heavy chains. Mammalian light chains are classified into λ or κ light chains. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are immunoglobulins (Ig) A, IgD, IgE, IgG, and IgM. A complete antibody forms a "Y" shape. The stem of the Y is formed by the joining of the second and third constant regions (or the fourth constant region in the case of IgE and IgM) of two heavy chains, with a disulfide bond (interchain) formed within the hinge. Heavy chains γ, α, and δ have a constant region composed of three tandem (arranged in a line) Ig domains and a hinge region to increase flexibility, while heavy chains μ and ε have a constant region composed of four immunoglobulin domains. The second and third constant regions are called the "CH2 domain" and the "CH3 domain," respectively.Each arm of the Y contains a single heavy chain variable region and a first constant region (CH1) attached to a single light chain. The "Fc" region consists of two heavy chain fragments containing the CH2 and CH3 domains of the antibody, which are linked by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. The prior art has disclosed various Fc constant region variants, for example, in which the Fc of the antibody's heavy chain constant region has one or more amino acid substitutions from 238, 265, 269, 270, 297, 327, and 329 (using the EU numbering system) (U.S. Patent No. 6 / 737056), or in which the Fc of the antibody's heavy chain constant region has one or more amino acid substitutions from 234, 235, 265, and 329 (using the EU numbering system) The antibody has amino acid substitutions, or the Fc in the heavy chain constant region of the antibody has one or more amino acid substitutions from among 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (using the EU numbering system) (see U.S. Patent No. 7 / 371826). These mutations have been shown to give the antibody new properties, but do not alter the function of the antibody's variable region.

[0097] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in the binding of the antibody to an antigen. VHH, VH, and VL each contain four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). Here, the term "complementarity-determining region" or "CDR" refers to a region within the variable domain that primarily contributes to antigen binding, and "framework" or "FR" refers to variable domain residues other than CDR residues. VH or VHH contains three CDR regions; for ease of distinction, the three CDRs of VH are identified by HCDR1, HCDR2, and HCDR3, the three CDRs of VHH are identified by VHH-CDR1, VHH-CDR2, and VHH-CDR3, and VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. A single VH or VL may be sufficient to confer antigen-binding specificity. As used herein, the terms "VHH" and "nano-antibody" are synonymous and interchangeable, referring to the variable region of a cloned antibody heavy chain, and a nanobody consisting of only one heavy chain variable region with complete antigen-binding function. The specific binding epitope of VHH does not require recognition by other antigen-binding domains (this differs from antibodies with conventional tetrapeptide chain structures where the epitope is recognized together by a structural pair formed by VL and VH). VHH is a small, stable, and efficient antigen-recognition unit formed by a single heavy chain variable domain. The nanobody possesses excellent biological properties, with a molecular weight of 12-15 kDa, which is one-tenth that of a complete antibody, and exhibits excellent tissue penetration, high specificity, and good water solubility. Its unique structural characteristics combine the advantages of conventional antibodies and small molecule drugs, almost completely overcoming the drawbacks of conventional antibodies such as long development cycles, low stability, and strict storage conditions. It is gradually becoming a force in the new generation of antibody therapy, showing promise for a wide range of applications in immunodiagnosis and treatment.VHHs include, but are not limited to, natural antibodies produced by camelids, and may also include antibodies produced by camelids and subsequently humanized, or may be obtained through phage display technology screening. Methods for obtaining VHHs that bind to specific antigens or epitopes are disclosed in prior literature, for example, the following: R. van der Linden et al., Journal of Immunological Methods, 240(2000)185-195; Li et al., J Biol Chem., 287(2012)13713-13721; Deffer et al., African Journal of Biotechnology Vol.8(12), pp.2645-2652, 17 June, 2009 and WO94 / 04678.

[0098] The term "PD-L1" includes all variants or isoforms of PD-L1 that are naturally expressed by cells. The antibodies of the present invention may cross-react with PD-L1 from non-human species (e.g., cynomolgus monkeys). Alternatively, the antibodies may be specific to human PD-L1 and may not exhibit cross-reactivity with other species. PD-L1 or any of its variants or isoforms can be isolated from cells or tissues that naturally express them, or can be produced by recombinant techniques common in the art and described herein.

[0099] In the bispecific antibody described in the present invention, preferably the second antigen-binding domain comprises at least one VHH, wherein the VHH comprises VHH-CDR1, VHH-CDR2, and VHH-CDR3 of SEQ ID NO: 66. In some embodiments, the VHH-CDR1 is X 41 YYX 42 X 43 The amino acid sequence is shown in C, where X 41 is D or E, and X 42 is S or T, X 43is K or Q, and VHH-CDR2 includes the amino acid sequence shown in SEQ ID NO: 76, and VHH-CDR3 includes the amino acid sequence shown in SEQ ID NO: 77.

[0100] In some embodiments, the VHH includes VHH-CDR1, VHH-CDR2, and VHH-CDR3 of sequence number 49, sequence number 74, sequence number 72, or sequence number 73.

[0101] In some embodiments, the VHH-CDR1, VHH-CDR2, and VHH-CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system, and in some specific embodiments, the VHH-CDR1, VHH-CDR2, and VHH-CDR3 are determined according to the Kabat numbering rules. In some embodiments, the VHH-CDR1 includes the amino acid sequence shown in SEQ ID NO: 46, SEQ ID NO: 67, or SEQ ID NO: 70, the VHH-CDR2 includes the amino acid sequence shown in SEQ ID NO: 47, SEQ ID NO: 68, or SEQ ID NO: 71, and the VHH-CDR3 includes the amino acid sequence shown in SEQ ID NO: 48, or SEQ ID NO: 69.

[0102] In some embodiments, the VHH includes VHH-CDR1 shown in SEQ ID NO: 46, SEQ ID NO: 67, or SEQ ID NO: 70, VHH-CDR2 shown in SEQ ID NO: 47, SEQ ID NO: 68, or SEQ ID NO: 71, and VHH-CDR3 shown in SEQ ID NO: 48, or SEQ ID NO: 69.

[0103] In a more preferred embodiment of the present invention, VHH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 46, VHH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 47, and VHH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 48.

[0104] Alternatively, VHH-CDR1 includes the amino acid sequence shown in SEQ ID NO: 67, VHH-CDR2 includes the amino acid sequence shown in SEQ ID NO: 68, and VHH-CDR3 includes the amino acid sequence shown in SEQ ID NO: 69.

[0105] Alternatively, VHH-CDR1 includes the amino acid sequence shown in SEQ ID NO: 70, VHH-CDR2 includes the amino acid sequence shown in SEQ ID NO: 71, and VHH-CDR3 includes the amino acid sequence shown in SEQ ID NO: 48.

[0106] In some embodiments, the VHH is: VHH-CDR1, whose sequence is shown in sequence number 46; VHH-CDR2, whose sequence is shown in sequence number 47; and VHH-CDR3, whose sequence is shown in sequence number 48. VHH-CDR1, whose sequence is shown in sequence number 67; VHH-CDR2, whose sequence is shown in sequence number 68; and VHH-CDR3, whose sequence is shown in sequence number 69. Alternatively, it may include VHH-CDR1, whose sequence is shown in sequence number 70; VHH-CDR2, whose sequence is shown in sequence number 71; and VHH-CDR3, whose sequence is shown in sequence number 48.

[0107] In some embodiments, the amino acid sequence of VHH is as shown in SEQ ID NO: 49, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO: 74, or has at least 90%, at least 95%, or at least 99% sequence homology with the amino acid sequence shown in SEQ ID NO: 49, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO: 74.

[0108] In some embodiments, the VHH is humanized VHH.

[0109] In some embodiments, the VHH includes framework regions FR1, FR2, FR3, and FR4, where FR1 includes the amino acid sequence shown in SEQ ID NO: 85, FR2 includes the amino acid sequence shown in SEQ ID NO: 79, FR3 includes the amino acid sequence shown in SEQ ID NO: 86, and FR4 includes the amino acid sequence shown in SEQ ID NO: 84. In some embodiments, FR1 includes the amino acid sequence shown in SEQ ID NO: 82 or SEQ ID NO: 78, FR2 includes the amino acid sequence shown in SEQ ID NO: 79, FR3 includes the amino acid sequence shown in SEQ ID NO: 83 or SEQ ID NO: 80, and FR4 includes the amino acid sequence shown in SEQ ID NO: 84.

[0110] In some embodiments, FR1 comprises the amino acid sequence shown in SEQ ID NO: 82, FR2 comprises the amino acid sequence shown in SEQ ID NO: 79, FR3 comprises the amino acid sequence shown in SEQ ID NO: 83, and FR4 comprises the amino acid sequence shown in SEQ ID NO: 84. Alternatively, FR1 includes the amino acid sequence shown in SEQ ID NO: 78, FR2 includes the amino acid sequence shown in SEQ ID NO: 79, FR3 includes the amino acid sequence shown in SEQ ID NO: 80, and FR4 includes the amino acid sequence shown in SEQ ID NO: 84. Alternatively, FR1 includes the amino acid sequence shown in SEQ ID NO: 82, FR2 includes the amino acid sequence shown in SEQ ID NO: 79, FR3 includes the amino acid sequence shown in SEQ ID NO: 80, and FR4 includes the amino acid sequence shown in SEQ ID NO: 84.

[0111] In some embodiments, the amino acid sequence of VHH is shown in SEQ ID NO: 49, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO: 74, or has at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 49, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO: 74.

[0112] In some embodiments, the amino acid sequence of VHH is shown in SEQ ID NO: 49, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO: 74.

[0113] In some embodiments, the second antigen-binding domain further includes a heavy chain constant region.

[0114] Preferably, the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4, and the heavy chain constant region is preferably the Fc region of human IgG1, and more preferably the heavy chain constant region includes the amino acid sequence shown in SEQ ID NO: 88.

[0115] More preferably, the VHH and the heavy chain constant region are linked via a linker, which is preferably a linker having the amino acid sequence shown in (G4S)x, where x is independently selected from an integer between 1 and 20, and more preferably a linker shown in Sequence ID No. 89.

[0116] More preferably, the amino acid sequence of the second antigen-binding domain is shown in SEQ ID NO: 93, SEQ ID NO: 90, SEQ ID NO: 91, or SEQ ID NO: 92, or has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 93, SEQ ID NO: 90, SEQ ID NO: 91, or SEQ ID NO: 92.

[0117] Most preferably, the second antigen-binding domain has the two amino acid sequence shown in Sequence ID No. 93.

[0118] In the present invention, "Fab" consists of one light chain and one heavy chain with a CH1 and a variable region. "Fab'" includes one light chain, a VH domain and a CH1 domain, and a region between the CH1 and CH2 domains, and interchain disulfide bonds may be formed between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule. The F(ab')2 fragment consists of two Fab' fragments linked by disulfide bonds between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody.

[0119] In the present invention, the scFv (single-chain antibody fragment) refers to a polypeptide chain formed by linking a VH domain and a VL domain via a linker (also called a linker). Here, the VL and VH domains are paired by the linker to form a monovalent molecule, enabling the production of a single polypeptide chain [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH, or NH2-VH-linker-VL-COOH. A suitable prior art linker consists of a repeating G4S amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (G4S)4 or (G4S)3 may be used, but variants thereof may also be used.

[0120] The term "multispecific antibody" is used in its broadest sense and includes antibodies that have specificity for two or more epitopes, such as bispecific antibodies. These multispecific antibodies include, but are not limited to, antibodies containing a heavy chain variable region (VH) and a light chain variable region (VL) (where the VH-VL unit has specificity for two or more epitopes), antibodies containing two or more VL and VH regions (each VH-VL unit binds to a different target or a different epitope of the same target), and antibodies containing two or more single variable domains (e.g., VHH) (each single variable domain binds to a different target or a different epitope of the same target).

[0121] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody. Epitopes are formed from a sequence of amino acids (linear epitopes) or contain discontinuous amino acids (structural epitopes), and are spatial proximitys that arise, for example, from the folding of an antigen (i.e., from tertiary folding in the protein properties of the antigen). The difference between structural epitopes and linear epitopes is that antibody binding to structural epitopes is lost in the presence of a denaturing solvent. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial structure. Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind to the same epitope) can be performed using routine methods in the art, such as alanine scanning and peptide blotting (see Meth.Mol.Biol.248(2004)443-463), but are not limited to these.

[0122] The term "specifically binding" refers to an antibody binding to a specific antigen or an epitope within that antigen with higher affinity than other antigens or epitopes. Generally, antibodies bind to an antigen or an epitope within an antigen with an equilibrium dissociation constant (KD) of approximately 1 × 10⁻⁷ M or less (e.g., approximately 1 × 10⁻⁸ M, approximately 1 × 10⁻⁹ M, approximately 1 × 10⁻¹⁰ M, approximately 1 × 10⁻¹⁰ M, approximately 1 × 10⁻⁹ M). In some embodiments, the KD of an antibody that binds to an antigen is 10% or 1% of the KD of an antibody that binds to a non-specific antigen (e.g., BSA, casein). The KD can be measured using standard procedures such as the BIACORE® surface plasmon resonance assay. However, antibodies that specifically bind to an antigen or an epitope within an antigen may cross-react with other related antigens, for example, with homologous antigens of other species (homologous species) such as humans or monkeys, including cynomolgus monkeys (Macaca fascicularis) (cynomolgus, cyno), chimpanzees (Pan troglodytes) (chimpanzee, chimp), or common marmosets (Callithrix jacchus) (commonmarmoset, marmoset).

[0123] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, "affinity" as used herein refers to internal binding affinity, which reflects the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y is generally expressed by the dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. The term "kassoc" or "ka" refers to the binding rate of a particular antibody-antigen interaction, while the term "kdis" or "kd" as used herein refers to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" refers to the dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and expressed as molar concentration (M). The KD value of an antibody can be determined using methods well established in the art. Methods for measuring the KD of antibodies include measuring surface plasmon resonance using biosensing systems such as ion systems, or measuring affinity in solution by solution equilibrium titration (SET).

[0124] The terms "anti-CD40 antibody" and "antibody that specifically binds to CD40" refer to antibodies that can bind to CD40 with sufficient affinity to be used as diagnostic and / or therapeutic agents targeting CD40. In some embodiments, antibodies that bind to CD40 have a dissociation constant (KD) of < about 1 μM, < about 100 nM, < about 10 nM, < about 1 nM, < about 0.1 nM, < about 0.01 nM, or < about 0.001 nM (e.g., less than or equal to 10⁻⁸ M, e.g., 10⁻⁸ M to 10⁻¹² M, e.g., 10⁻⁹ M to 10⁻¹⁰ M). In some embodiments, anti-CD40 antibodies bind to antigenic epitopes conserved in CD40 of various species.

[0125] The terms "anti-PD-L1 antibody" and "antibody that specifically binds to PD-L1" refer to antibodies that can bind to PD-L1 with sufficient affinity to be used as diagnostic and / or therapeutic agents targeting PD-L1. In some embodiments, antibodies that bind to PD-L1 have a dissociation constant (KD) of < about 1 μM, < about 100 nM, < about 10 nM, < about 1 nM, < about 0.1 nM, < about 0.01 nM, or < about 0.001 nM (e.g., less than or equal to 10⁻⁸ M, e.g., 10⁻⁸ M to 10⁻¹² M, e.g., 10⁻⁹ M to 10⁻¹⁰ M). In some embodiments, anti-PD-L1 antibodies bind to antigenic epitopes conserved in various species of PD-L1.

[0126] In some preferred embodiments, the first antigen-binding domain and the second antigen-binding domain are operably connected directly or via a linker.

[0127] Preferably, the second antigen-binding domain is ligated to the N-terminus of the light chain variable region or heavy chain variable region of the first antigen-binding domain, or to the C-terminus of the light chain constant region, or to the C-terminus of IgG.

[0128] The linker is preferably a peptide sequence, and more preferably (G4S) n Includes G or (G4S) n It consists of G, where n is an integer from 1 to 10, for example, n is 3.

[0129] The bispecific antibody described in the present invention comprises two primary polypeptide chains and two secondary polypeptide chains.

[0130] In some preferred embodiments, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 50 or has at least 99%, at least 95%, or at least 90% sequence homology with SEQ ID NO: 50, and / or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 51 or has at least 99%, at least 95%, or at least 90% sequence homology with SEQ ID NO: 51.

[0131] In some preferred embodiments, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 52 or has at least 99%, at least 95%, or at least 90% sequence homology with SEQ ID NO: 52, and / or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 53 or SEQ ID NO: 60, or has at least 99%, at least 95%, or at least 90% sequence homology with SEQ ID NO: 53 or SEQ ID NO: 60.

[0132] In some preferred embodiments, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 54 or has at least 99%, at least 95%, or at least 90% sequence homology with SEQ ID NO: 54, and / or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 55 or SEQ ID NO: 61, or has at least 99%, at least 95%, or at least 90% sequence homology with SEQ ID NO: 55 or SEQ ID NO: 61.

[0133] In some preferred embodiments, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 56 or has at least 99%, at least 95%, or at least 90% sequence homology with SEQ ID NO: 56, and / or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 57 or SEQ ID NO: 62 or has at least 99%, at least 95%, or at least 90% sequence homology with SEQ ID NO: 62.

[0134] In some preferred embodiments, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 58 or has at least 99%, at least 95%, or at least 90% sequence homology with SEQ ID NO: 58, and / or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 59 or SEQ ID NO: 63, or has at least 99%, at least 95%, or at least 90% sequence homology with SEQ ID NO: 59 or SEQ ID NO: 63.

[0135] The third aspect of the present invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to human CD40 and a second antigen-binding domain that specifically binds to human PD-L1, wherein the second antigen-binding domain comprises at least one VHH, and the sequence of the VHH is as defined in the bispecific antibody described in the second aspect of the present invention.

[0136] In some preferred embodiments, the first antigen-binding domain is as defined in the anti-CD40 antibody described in the first aspect of the present invention.

[0137] In some preferred embodiments, the first antigen-binding domain is operably linked to the second antigen-binding domain directly or via a linker.

[0138] Preferably, the second antigen-binding domain is linked to the N-terminus of the variable region of the light chain or heavy chain of the first antigen-binding domain, or the C-terminus of the light chain constant region, or the C-terminus of IgG.

[0139] The linker is preferably a peptide sequence, more preferably (G4S) n containing G, or (G4S) n consisting of G, where n is an integer from 1 to 10, for example, n is 3.

[0140] The bispecific antibody described in the present invention comprises two first polypeptide chains and two second polypeptide chains.

[0141] In some preferred embodiments, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 50, and / or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 51.

[0142] In some preferred embodiments, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 52, and / or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 53 or SEQ ID NO: 60.

[0143] In some preferred embodiments, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 54, and / or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 55 or SEQ ID NO: 61.

[0144] In some preferred embodiments, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 56, and / or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 57 or SEQ ID NO: 62.

[0145] In some preferred embodiments, the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 58, and / or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 59 or SEQ ID NO: 63.

[0146] A fourth aspect of the present invention provides isolated nucleic acids encoding the anti-CD40 antibody described in the first aspect of the present invention, or the bispecific antibody described in the second or third aspect.

[0147] As is known in the art, “nucleic acid” in the present invention refers to a nucleotide chain of any length and includes DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and / or analogs thereof, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0148] A fifth aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid described in the fourth aspect of the present invention.

[0149] Preferably, the recombinant expression vector is a plasmid, cosmid, phage, or viral vector.

[0150] For example, the plasmid backbone is pcDNA3.4.

[0151] The term “recombinant expression vector” refers to a genetically modified oligonucleotide or polynucleotide construct, the construct comprising a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the construct enabling the expression of mRNA, protein, polypeptide, or peptide by a host cell when the vector comes into contact with a cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the cell. The vectors of this disclosure are generally not found in nature. However, some of the vectors may be found in nature. The recombinant expression vectors of the present invention include, but are not limited to, single-stranded or double-stranded, synthetic, or partially derived from natural sources DNA and RNA, which may contain natural, unnatural, or modified nucleotides. The recombinant expression vectors may contain naturally occurring or unnatural nucleotide interlinks, or both types of interlinks. In exemplary aspects, modified nucleotides or unnatural nucleotide interlinks do not inhibit the transcription or replication of the vector.

[0152] The recombinant expression vector of the present invention may be any suitable recombinant expression vector that can be used to deliver one or more target genes or sequences to any suitable host cell and, preferably, to transform or transfect the host cell to express the gene or sequence. Suitable vectors include vectors designed for expansion and amplification, or expression, or both, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, and liposome-encapsulated DNA or RNA expression vectors, including certain eukaryotic cells such as production cells.

[0153] A sixth aspect of the present invention provides a transformant comprising the recombinant expression vector described in the fifth aspect of the present invention.

[0154] Preferably, the host cell of the transformed organism is a prokaryotic cell or a eukaryotic cell.

[0155] More preferably, the eukaryotic cell is a yeast cell or a mammalian cell.

[0156] The mammalian cells mentioned above are, for example, EXPI-293 cells or CHO cells.

[0157] As used herein, the term “host cell” means any type of cell which may contain the nucleic acids or vectors described herein. A host cell may be a eukaryotic cell, such as a plant, animal, fungus, or algae, or it may be a prokaryotic cell, such as a bacterium or protist. As described herein, a host cell may be derived from or obtained from an individual. A host cell may be derived from or obtained from a mammal. As used herein, the term “mammal” means any mammal, including but not limited to rodents such as mice and hamsters, and mammals of the order Lagomorpha such as rabbits. Preferably, mammals are derived from the order Carnivora, which includes the families Felidae (cats) and Canidae (dogs). More preferably, mammals are derived from the order Artiodactyla, which includes the families Bovidae (cats) and Suidae (pigs), or from the order Perissodactyla, which includes the family Equidae (horses). Most preferably, the mammal belongs to the order Primates, New World monkeys (Ceboids), or Simoids (monkeys), or the order Anthropoids (humans and apes). Particularly preferably, the mammal is human.

[0158] Expression vectors can be transfected or introduced into suitable host cells. To achieve this objective, various techniques can be used, including protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene editing (CRISPR-Cas system, ZFN system, or TALEN system), transposon (Sleeping Beauty or PiggyBAC) gene guns, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, cells are cultured in a culture medium and screened for the appropriateness of their activity. Methods and conditions for culturing the resulting transfected cells and recovering the generated antibody molecules are known to those skilled in the art and can be modified or optimized according to the specific expression vector and mammalian host cell used, based on methods known from this specification and the prior art. Furthermore, cells with stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that enable the selection of transfected host cells. These markers can provide, for example, prototrophicity, biocidal resistance (e.g., antibiotics), or heavy metal (e.g., copper) resistance to a trophic host. Selectable marker genes can be directly ligated to the expressed DNA sequence or introduced into the same cell by co-transformation. Additional components may be required for optimal mRNA synthesis. These components may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0159] A seventh aspect of the present invention provides a method for preparing an anti-CD40 antibody or a bispecific antibody, comprising the step of culturing the transformant described in the sixth aspect of the present invention and obtaining an anti-CD40 antibody or a bispecific antibody from the culture.

[0160] The eighth aspect of the present invention provides a pharmaceutical composition comprising an anti-CD40 antibody as described in the first aspect of the present invention, a bispecific antibody as described in the second or third aspect, and a pharmaceutically acceptable carrier.

[0161] Preferably, the pharmaceutical composition further comprises other agents, in some embodiments, the other agents being selected from 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.

[0162] A "pharmaceutically acceptable carrier" is any conventionally used carrier, limited only by physicochemical considerations (e.g., solubility and lack of reactivity with CD40-targeted antibodies) and subject to restrictions on the route of administration. The pharmaceutically acceptable carriers described herein, such as solvents, adjuvants, excipients, and diluents, are well known to those skilled in the art and are generally readily available. In one respect, a pharmaceutically acceptable carrier is one that is chemically inert to the active ingredient of the pharmaceutical composition and does not have adverse side effects or toxicity under the conditions of use. In some embodiments, when administered to animals or humans, the carrier does not cause adverse reactions, allergic reactions, or other inappropriate reactions. In some embodiments, the pharmaceutical composition does not contain pyrogens or other impurities that may be harmful to humans or animals. Pharmacochemically acceptable carriers include all solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., whose uses are well known in the art.

[0163] Therapeutic formulations of compositions suitable for carrying out the methods disclosed herein, such as polypeptides, polynucleotides, or antibodies, can be prepared for storage by mixing a selected composition having the desired purity in the form of a lyophilized cake or aqueous solution with any physiologically and pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 18th edition, edited by AR Gennaro, Mack Publishing Company (1990)). Pharmaceutical compositions can be prepared by mixing with one or more suitable carriers or adjuvants such as water, mineral oil, polyethylene glycol, starch, talc, lactose, thickeners, stabilizers, and suspending agents. Such compositions may be in the form of solutions, suspensions, tablets, capsules, creams, ointments, or other conventional forms.

[0164] Compositions for in vivo administration should be sterile. This can be easily achieved by filtering through a sterile filter membrane before or after lyophilization and reconstitution. Therapeutic compositions can generally be placed in containers with sterile access ports, such as infusion bags or vials with stoppers into which a subcutaneous needle can be inserted. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In some situations, these forms must be sterile and fluid enough to be easily injected. They need to be stable under preparation and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. Compositions for parenteral administration are generally stored in lyophilized or solution form.

[0165] The carrier may be, for example, a solvent or dispersion medium containing water or a suitable mixture thereof and a vegetable oil. Appropriate flow properties can be maintained, for example, by the use of a coating such as lecithin, the maintenance of a desired particle size in the case of a dispersion, and the use of a surfactant. The choice of carrier is determined in part by the specific type of pharmaceutical composition and the route of administration of the pharmaceutical composition. Accordingly, a variety of suitable pharmaceutical composition formulations can be prepared.

[0166] The pharmaceutical compositions of the present invention include acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anti-coagulation agents, antimicrobial preservatives, antioxidants, antiseptics, matrices, binders, buffers, chelating agents, coating agents, colorants, drying agents, detergents, diluents, disinfectants, disintegrants, dispersants, dissolution accelerators, dyes, emollients, emulsifiers, emulsifying stabilizers, fillers, film-forming agents, fragrances, fragrances, flow accelerators, gelling agents, granulators, heat-retaining agents, lubricants, mucosal adhesives, ointment bases, ointments, oily solvents, organic bases, tablet bases, pigments, plasticizers, abrasives, preservatives, metal ion sequestering agents, skin penetration agents, solubilizers, solvents, stabilizers, suppository bases, and surfactants (surface active). It may contain any pharmaceutically acceptable components such as an agent, surfactant, suspension agent, sweetener, therapeutic agent, thickener, isotonic agent, toxic agent, tackifier, water absorbent, water-miscible cosolvent, water softener, or wetting agent.

[0167] In some embodiments, the pharmaceutical composition comprising the bispecific antibodies described herein is formulated for parenteral, subcutaneous, intravenous, intramuscular, intra-arterial, subarachnoid, or intraperitoneal administration. In other embodiments, the pharmaceutical composition is administered by nasal, spray, oral, aerosol, rectal, or vaginal administration. The composition may be administered by infusion, bolus injection, or via an implantable device.

[0168] Topical formulations are well known to those skilled in the art. Such formulations are particularly suitable for use on the skin in the context of the present invention.

[0169] In some embodiments, the pharmaceutical compositions described herein are formulated for parenteral administration. For the purposes of this specification, parenteral administration includes, but is not limited to, injection or infusion into the vein, artery, muscle, brain, ventricle, heart, subcutaneous, bone, skin, subarachnoid space, abdominal cavity, retroocular, lung, bladder, and corpus cavernosum of the penis. Administration by surgical implantation to specific sites is also included.

[0170] An injectable formulation is according to the present invention. Those skilled in the art are well aware of the requirements for effective pharmaceutical carriers for injectable compositions (see, for example, *Pharmaceutics and Pharmacy Practice*, JBLippincott Company, Philadelphia, edited by Banker and Chalmers, pp. 238-250 (1982), and *ASHP Handbook on Injectable Drugs*, Toissel, 4th ed., pp. 622-630 (1986)).

[0171] Those skilled in the art will understand that, in addition to the pharmaceutical compositions described above, the compositions of the present invention may be formulated as cyclodextrin inclusion complexes or liposomes or other inclusion complexes.

[0172] The ninth aspect of the present invention provides the use of the anti-CD40 antibody described in the first aspect of the present invention, the bispecific antibody described in the second or third aspect of the present invention, and / or the pharmaceutical composition described in the eighth aspect of the present invention in the preparation of a pharmaceutical for the prevention and / or treatment of tumors.

[0173] The tumor is preferably a PD-L1-positive and / or CD40-positive tumor.

[0174] In preferred embodiments of the present invention, the tumor is, but is not limited to, lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, rhabdomyosarcoma, esophageal cancer, cervical cancer, multiple myeloma, leukemia, gallbladder cancer, glioblastoma, or melanoma.

[0175] The tenth aspect of the present invention provides a kit comprising an anti-CD40 antibody described in the first aspect of the present invention, a bispecific antibody described in the second or third aspect, or a pharmaceutical composition described in the eighth aspect of the present invention.

[0176] Preferably, the kit further includes (i) a device for administering an antibody or pharmaceutical composition, and / or (ii) instructions for use.

[0177] The eleventh aspect of the present invention provides a medicine box set including medicine box A and medicine box B, wherein,

[0178] The aforementioned medicine box A contains an anti-CD40 antibody according to the first aspect of the present invention, a bispecific antibody according to the second or third aspect of the present invention, and / or a pharmaceutical composition according to the eighth aspect of the present invention.

[0179] The medicine box B contains other antitumor antibodies or pharmaceutical compositions containing 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.

[0180] A twelfth aspect of the present invention provides an immunodetection method for measuring CD40 and / or PD-L1, comprising using the CD40 antibody described in the first aspect of the present invention, the bispecific antibody described in the second or third aspect, and / or the pharmaceutical composition described in the eighth aspect of the present invention.

[0181] In a preferred embodiment of the present invention, the detection is for non-diagnostic purposes and is suitable only for scientific research purposes.

[0182] A thirteenth aspect of the present invention provides a method for preventing and / or treating tumors, comprising administering to a patient in need a therapeutically effective amount of the CD40 antibody described in the first aspect of the present invention, the bispecific antibody described in the second or third aspect, and / or the pharmaceutical composition described in the eighth aspect of the present invention, or the medicine box set described in the eleventh aspect of the present invention.

[0183] For example, the tumors include, but are not limited to, lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, rhabdomyosarcoma, esophageal cancer, cervical cancer, multiple myeloma, leukemia, gallbladder cancer, glioblastoma, or melanoma.

[0184] As used in this invention, the term “effective dose” refers to the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as determined by a researcher or clinician. Furthermore, the term “therapeutic effective dose” refers to the amount that, compared to a corresponding subject not administered that amount, results in an improvement in the treatment, cure, prevention, or reduction of a disease, condition, or side effect, or reduces the rate of progression of the disease or condition. The term also includes amounts that are effective in enhancing normal physiological function.

[0185] A fourteenth aspect of the present invention provides a combination therapy comprising administering the CD40 antibody described in the first aspect of the present invention, the bispecific antibody described in the second or third aspect, and / or the pharmaceutical composition described in the eighth aspect of the present invention to a patient in need of a second therapeutic agent.

[0186] The second therapeutic agent preferably comprises another antitumor antibody or a pharmaceutical composition containing the other antitumor antibody, 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.

[0187] A fifteenth aspect of the present invention provides an anti-CD40 antibody as described in the first aspect of the present invention, a bispecific antibody as described in the second or third aspect, and / or a pharmaceutical composition as described in the eighth aspect of the present invention, which in some technical solutions is used for the prevention and / or treatment of tumors.

[0188] Preferably, the tumor is PD-L1 positive and / or CD40 positive.

[0189] For example, the tumors mentioned above include lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, rhabdomyosarcoma, esophageal cancer, cervical cancer, multiple myeloma, leukemia, gallbladder cancer, glioblastoma, or melanoma.

[0190] Preferably, the tumor is colon cancer.

[0191] The present invention also provides antibody-drug conjugates comprising an anti-CD40 antibody according to the first aspect of the present invention, or a bispecific antibody according to the second or third aspect, conjugated with one or more therapeutic agents or radioisotopes.

[0192] Preferably, the therapeutic agent is a cytotoxic agent, a chemotherapeutic agent, a drug, a proliferation inhibitor, and / or a toxin, and / or the conjugation is achieved by using a linker to conjugate the antibody with the therapeutic agent or radioisotope.

[0193] Based on common sense in this field, the above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0194] The novel anti-CD40 agonist antibody of the present invention can effectively control the activation of DC cells and has a more potent T cell activation effect, while having low toxicity and side effects, making it suitable for tumor immunotherapy. The bispecific antibody of the present invention stimulates CD40 and simultaneously blocks PD-L1 / PD-1, thereby acting on the positive feedback pathway of interaction between DC cells and T cells to maximize the antitumor effect. Furthermore, PD-L1-dependent CD40 activation improves the selectivity of CD40 activation and reduces the toxicity and side effects of the CD40 agonist antibody. [Brief explanation of the drawing]

[0195] [Figure 1] This is the CD40 agonist activity of the monoclonal antibody measured by the reporter gene method in Example 7 of the present invention. [Figure 2] This describes the DC cell regulatory activity of the monoclonal antibody in Example 8 of the present invention, using CD83 as a DC activation indicator. [Figure 3] This describes the T cell regulatory activity of the monoclonal antibody in Example 9 of the present invention, using IFN-γ as a T cell activation indicator. [Figure 4A] This describes the safety of the monoclonal antibody in human CD40 gene knock-in mice in Example 10 of the present invention. Here, Figure 4A shows the change in body weight of the mice, Figure 4B shows the changes in liver function and hematology of the mice, and Figure 4C shows the changes in organ coefficients of the mice. [Figure 4B] This describes the safety of the monoclonal antibody in human CD40 gene knock-in mice in Example 10 of the present invention. Here, Figure 4A shows the change in body weight of the mice, Figure 4B shows the changes in liver function and hematology of the mice, and Figure 4C shows the changes in organ coefficients of the mice. [Figure 4C] This describes the safety of the monoclonal antibody in human CD40 gene knock-in mice in Example 10 of the present invention. Here, Figure 4A shows the change in body weight of the mice, Figure 4B shows the changes in liver function and hematology of the mice, and Figure 4C shows the changes in organ coefficients of the mice. [Figure 5]This is the PD-L1 / PD-1 inhibitory activity of the monoclonal antibody measured by the reporter gene method of Example 12 of the present invention. [Figure 6] This is the T cell regulatory activity of the monoclonal antibody in Example 13 of the present invention. [Figure 7] This is the structure of the bispecific antibody in Example 4 of the present invention. [Figure 8] In Example 15 of the present invention, a bispecific antibody is used that simultaneously binds to PD-L1 and CD40. [Figure 9] In Example 16 of the present invention, the PD-L1 / PD-1 inhibitory activity of the bispecific antibody was measured by the reporter gene method. [Figure 10A] In Example 17 of the present invention, the CD40 agonist activity of the bispecific antibodies was measured by the reporter gene method. Figure 10A: CD40 agonist activity of 1605, 1606, and 1607; Figure 10B: CD40 agonist activity of 1608 and 1609; Figure 10C: CD40 agonist activity of 1652 and 1653; Figure 10D: CD40 agonist activity of 1654, 1655, and CP-870893. [Figure 10B] In Example 17 of the present invention, the CD40 agonist activity of the bispecific antibodies was measured by the reporter gene method. Figure 10A: CD40 agonist activity of 1605, 1606, and 1607; Figure 10B: CD40 agonist activity of 1608 and 1609; Figure 10C: CD40 agonist activity of 1652 and 1653; Figure 10D: CD40 agonist activity of 1654, 1655, and CP-870893. [Figure 10C] In Example 17 of the present invention, the CD40 agonist activity of the bispecific antibodies was measured by the reporter gene method. Figure 10A: CD40 agonist activity of 1605, 1606, and 1607; Figure 10B: CD40 agonist activity of 1608 and 1609; Figure 10C: CD40 agonist activity of 1652 and 1653; Figure 10D: CD40 agonist activity of 1654, 1655, and CP-870893. [Figure 10D]In Example 17 of the present invention, the CD40 agonist activity of the bispecific antibodies was measured by the reporter gene method. Figure 10A: CD40 agonist activity of 1605, 1606, and 1607; Figure 10B: CD40 agonist activity of 1608 and 1609; Figure 10C: CD40 agonist activity of 1652 and 1653; Figure 10D: CD40 agonist activity of 1654, 1655, and CP-870893. [Figure 11A] This shows the DC regulatory activity of the bispecific antibody in Example 19 of the present invention. Figure 11A compares the activity of different bispecific antibodies using IL-12p40 as an indicator, and Figures 11B and 11C compare the activity of the parent monoclonal antibody and the bispecific antibody, with Figure 11B showing CD83 and Figure 11C showing IL-12p40. [Figure 11B] This shows the DC regulatory activity of the bispecific antibody in Example 19 of the present invention. Figure 11A compares the activity of different bispecific antibodies using IL-12p40 as an indicator, and Figures 11B and 11C compare the activity of the parent monoclonal antibody and the bispecific antibody, with Figure 11B showing CD83 and Figure 11C showing IL-12p40. [Figure 11C] This shows the DC regulatory activity of the bispecific antibody in Example 19 of the present invention. Figure 11A compares the activity of different bispecific antibodies using IL-12p40 as an indicator, and Figures 11B and 11C compare the activity of the parent monoclonal antibody and the bispecific antibody, with Figure 11B showing CD83 and Figure 11C showing IL-12p40. [Figure 12A] This is the T cell regulatory activity of the bispecific antibody in Example 20 of the present invention. [Figure 12B] This is the T cell regulatory activity of the bispecific antibody in Example 20 of the present invention. [Figure 13] This describes the efficacy of the bispecific antibody against MC38 tumors in mice in Example 21 of the present invention. [Figure 14] This describes the efficacy of an anti-PD-L1 / CD40 bispecific antibody against in vivo anti-MC38 / hPD-L1 colon cancer. [Modes for carrying out the invention]

[0196] The present invention will be further described below with reference to examples, but the present invention is not limited to the scope of these examples. Experimental methods for which specific conditions are not given in the following examples should be selected according to conventional methods and conditions, or according to the product instructions. Reagents for which specific sources are not given are conventional reagents available on the market.

[0197] Example 1: Construction of CD40 antibody and PD-L1 antibody phage libraries Based on the crystal structure of the human CD40 protein, the variable region sequence of the human CD40 antibody was designed, and the single-chain Fv sequence (VL-G4S linker-VH) was obtained using gene synthesis technology. This sequence was loaded into a phage vector (pComb3XSS, purchased from Beijing Zoman Biotechnology Co., Ltd.) using restriction endonuclease to obtain a recombinant plasmid library. The phage plasmid incorporating the scFv gene was electroporated into SS320 E. coli competent cells. After the SS320 E. coli (purchased from Lucigen) grew to the logarithmic growth phase, a helper phage (M13K07, purchased from NEB) was added for infection, and the cells were cultured overnight. The phages were extracted from the culture supernatant to obtain a CD40 antibody phage library.

[0198] Based on the crystal structure of the human PD-L1 protein, a variable region sequence of a human PD-L1 nanoantibody was designed. The VHH sequence was obtained using gene synthesis technology and loaded into a phage vector using restriction endonuclease to obtain a recombinant plasmid library. The phage plasmid incorporating the VHH gene was electroporated into SS320 E. coli competent cells. After the SS320 cells grew to the logarithmic growth phase, helper phages were added for infection, and the cells were cultured overnight. The phages were extracted from the culture supernatant to obtain a PD-L1 antibody phage library.

[0199] Example 2 Screening of CD40 and PD-L1 antibodies from an antibody phage library CD40 antibodies were screened from a CD40 antibody phage library, and PD-L1 antibodies were screened from a PD-L1 antibody phage library.

[0200] Three rounds of panning were performed on a phage display library using a protein-based phage antibody panning technique. Round 1 screening method: ELISA plates were coated with human CD40-mFc antigen or human PD-L1-mFc antigen (purchased from ACROBiosystems). The corresponding phage library was taken and cultured in the same volume of 2% skim milk premix. After reacting with the premix in the coated wells, the premix was removed by washing with sterile PBST. The phages adsorbed to the well plates were eluted using 75 mM sodium citrate buffer, and the phage library was neutralized. The screened phage library was then amplified 100-fold using the M13K07 helper phage. Rounds 2 and 3 screening were then performed in the same manner as round 1. After three rounds of screening, a concentrated phage library was obtained. Phage concentration was monitored by the starting phage dose for each round of screening and the phage titer collected after screening.

[0201] Concentrated CD40 phage libraries and PD-L1 phage libraries were used to infect SS320 E. coli, respectively, and then cultured on agarose plates. Monoclonal colonies were selected, transferred to 96-well deep-well plates, and cultured with shaking at 37°C in 2YT medium containing ampicillin and kanamycin to obtain a supernatant containing monoclonal phages. The monoclonal phage supernatant was cultured for 1 hour on ELISA plates coated with human CD40-mFc antigen or human PD-L1-mFc antigen, then washed with sterile PBST, and further inoculated with anti-M13-HRP (purchased from Sino Biological), and cultured at 4°C for 30 minutes. Next, phage-antigen binding was detected using a microplate reader, and CD40 monoclonal phages and PD-L1 monoclonal phages with high antigen binding affinity were screened.

[0202] Example 3: Preparation of recombinant CD40 monoclonal antibody and recombinant PD-L1 monoclonal antibody The cDNA sequences of the heavy and light chain variable regions of screened CD40 monoclonal phages were cloned into pcDNA3.4 vectors (Invitrogen) that already contained the antibody constant region, thereby obtaining multiple anti-CD40 monoclonal antibodies, which were named 1605CD, 1606CD, 1607CD, 1608CD, 1609CD, 1652CD, 1653CD, 1654CD, and 1655CD, respectively. The recombinant heavy and light chain plasmids were co-transfected into EXPI-293 cells (Invitrogen) using the PEI method, transfected transiently for 7-10 days, centrifuged, and the supernatant was collected. The supernatant was purified with protein A to obtain purified anti-CD40 monoclonal antibodies. The CDR sequences of the CD40 antibodies are shown in Table 7 (determined by the Kabat CDR system), and the amino acid sequences of the heavy and light chain variable regions are shown in Table 8.

[0203] For example, the full-length amino acid sequence of 1654CD is as follows:

[0204] Heavy chain amino acid sequence of 1654CD (SEQ ID NO: 94): EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVGFIRNKANAYTTEYAASVKGRFTISRDNSKSTLYLQMNRLRAEDTAVYYCARYGGLKVGWYFDL WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERK CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0205] Light chain amino acid sequence of 1654CD (SEQ ID NO: 95): DIQMTQSPSSLSASVGDRVTITCRASQSISNYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGSSYPWTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0206] The full-length amino acid sequence of 1606CD is as follows:

[0207] Heavy chain amino acid sequence of 1606CD (SEQ ID NO: 96): EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVGFIRNKANGYTTEYAASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYGGLRQGWYFDV WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERK CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0208] Light chain amino acid sequence of 1606CD (SEQ ID NO: 81): DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRRDSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGKTLPWTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0209] The full amino acid sequence of 1652CD is as follows:

[0210] Heavy chain amino acid sequence of 1652CD (SEQ ID NO: 87): EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVGFIRNKANAYTTEYAASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYGGLRQGWYFDV WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERK CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0211] Light chain amino acid sequence of 1652CD (SEQ ID NO: 81): DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRRDSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGKTLPWTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0212] The cDNA sequences (VHH) of the variable regions of screened PD-L1 monoclonal phages were cloned into a pcDNA3.4 vector (Invitrogen) that already contained the antibody constant region, thereby obtaining several anti-PD-L1 monoclonal antibodies, which were named 1029, 1031, 1102, and 1541, respectively. The complementarity-determining region (CDR) sequences of the PD-L1 monoclonal antibodies are shown in Table 2 (determined by the Kabat CDR system), the framework region (FR) sequences of the monoclonal antibodies are shown in Table 3, the amino acid sequences of the variable region (VHH) are shown in Table 4, the amino acid sequences of the linker and constant region of the monoclonal antibodies are shown in Table 5, and the full-length amino acid sequences of the monoclonal antibodies are shown in Table 6. Plasmids were transfected into EXPI-293 cells (Invitrogen) using the PEI method, transfected transiently for 7-10 days, centrifuged, and the supernatant was collected. The supernatant was purified with protein A to obtain purified anti-PD-L1 monoclonal antibody.

[0213] Table 2 CDR sequences of PD-L1 monoclonal antibodies [Table 2] Note: The bold and underlined parts of the table represent amino acids that are different from 1029. Here, X 41 is D or E, and X 42 is S or T, X 43 is K or Q, and X 44 is G or S, X 45 is S or T, X 46 is D or E, and X 47 It is either K or N.

[0214] Table 3 FR sequences of PD-L1 monoclonal antibodies [Table 3-1] [Table 3-2] Note: The bold and underlined parts in the table represent amino acids different from those in 1029. Here, X 48 is L or F, X 49 is A or S, X 50 is K or R, X 51 is A or T.

[0215] Table 4 Amino acid sequences of VHHs of PD-L1 monoclonal antibodies

Table 4

[0216] Amino acid sequence of the general formula of VHH (SEQ ID NO: 66): EVQLVESGGGLVQPGGSLRLSCAASGFTX 48 X 41 YYX 42 X 43 CWFRQAPGKEREWVSCIX 44 X 45 SX 46 GSTYYADSVKGRFTISRDNX 49 KNTVYLQMNSLX 50 X 51 EDTAVYYCAARX 47 GGPLTIENFFDYWGQGTQVTVSS

[0217] ​​​​​​​​​​​​​​​​​​​is A or S, X 50 is K or R, X 51 is A or T.

[0218] Table 5 Amino Acid Sequences of Linkers and Constant Regions of PD-L1 Monoclonal Antibodies [Table 5]

[0219] Table 6 Full-Length Amino Acid Sequences of PD-L1 Monoclonal Antibodies [Table 6-1] [Table 6-2] [Table​​​​​​​​​​​​​​​​​​The cDNA sequences of the heavy and light chain variable regions of screened CD40 monoclonal phages were cloned into pcDNA3.4 vectors (Invitrogen) that already contained the antibody constant region. The cDNA sequences (VHH) of the variable region of screened PD-L1 monoclonal phages were cloned into pcDNA3.4 vectors (Invitrogen) that already contained CD40-VH and the antibody constant region. The VHH sequences were incorporated into the C-terminus of the antibody constant region, and a (G4S) linker was introduced between the constant region and the VHH. This resulted in obtaining several anti-PD-L1 / CD40 bispecific antibodies (the structures of which are shown in Figure 7), which were named 1605, 1606, 1607, 1608, 1609, 1652, 1653, 1654, and 1655, respectively. The CDR sequences of the bispecific antibodies are shown in Table 7 (determined by the Kabat CDR system), the amino acid sequences of the heavy chain variable region and light chain variable region are shown in Table 8, and the amino acid sequences of the heavy chain and light chain of the bispecific antibodies are shown in Table 9. Recombinant plasmids of the heavy chain and light chain were co-transfected into EXPI-293 cells (Invitrogen) using the PEI method, transiently transfected for 7-10 days, centrifuged, and the supernatant was collected. The supernatant was purified with protein A to obtain purified anti-PD-L1 / CD40 bispecific antibodies.

[0222] Table 7. CDR sequences of bispecific antibodies [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] Note: In RASQX1IX2X3YLX4 (SEQ ID NO: 1), X1 is D, G, S or T, X2 is R or S, X3 is N or S, and X4 is N or A. In X5X6SX7X8X9S, X5 is Y or A, X6 is T or A, X7 is S, R or T, X8 is L or R, and X9 is Q or D. QQG X 10 X 11 X 12 In PW (SEQ ID NO: 3), X 10 is K, I, N, Q or S, and X 11 is S, A, N or T, and X 12 is L, Y or F. SX 13 In YYMS (SEQ ID NO: 4), X 13 is N or D. FIRNKAN X 14 In YT (SEQ ID NO: 5), X 14 is A or G. YGG X 15 X 16 X 17 GWYFD X 18 (SEQ ID NO: 6), X 15 is L or I, and X 16 is K or R, and X 17 is V, K, I or Q, and X 18 is L or V.

[0223] Table 8 Amino Acid Sequences of Heavy Chain Variable Region and Light Chain Variable Region of Bispecific Antibody [Table 8-1] [Table 8-2] [Table 8-�?] 4][Table 8-�?] [Table 8-5] [Table 8-6]

[0224] Here, the shared sequence of the heavy chain variable region of Anti-CD40: EVQLVESGGGLVQPGGSLRLSCAASGTFSX 19 YYMSWVRQAPGKGLEWVX 20 FIRNKANX 21 YTTEYAASVKGRFTISRDNSKX 22 TLYLQMNX 23 LRAEDTAVYYCARYGGX 24 X 25 X 26 GWYFDX 27 WGQGTLVTVSS(Sequence ID 64):X 19 is N or D, and X 20 is G or A, and X 21 is A or G, and X 22 is N or S, X 23 is R or S, and X 24 is L or I, and X 25 is K or R, and X 26 is V, K, I, or Q, and X 27 It is either L or V.

[0225] Common sequence in the light chain variable region of Anti-CD40 (SEQ ID NO: 65): DIQMTQSPSSLSASVGDRVTITCRASQX 28 IX 29 X 30 YLX 31 WYQQKPGKAPKLLIYX 32 X 33 SX 34 X 35 X 36 SGVPSRFSGSGSGTDYTLTISSLQPEDFATYX37 CQQGX 38 X 39 X 40 PWTFGGGTKVEIK:X 28 is D, G, S or T, X 29 is R or S, X 30 is N or S, X 31 is N or A, X 32 is Y or A, X 33 is T or A, X 34 is S, R or T, X 35 is L or R, X 36 is Q or D, X 37 is Y or F, X 38 is K, I, N, Q or S, X 39 is S, A, N or T, X 40 is L, Y or F.

[0226] Table 9 Amino Acid Sequences of Heavy and Light Chains of Bispecific Antibodies

Table 9-1

Table 9-2

Table 9-3

Table 9-4

Table 9-5

Table 9-6

Table 9-7

Table 9-8

[0227] Example 5: Detection of binding of recombinant CD40 monoclonal antibody to CD40. The binding affinity and species specificity of monoclonal antibodies to human CD40 were detected using enzyme-linked immunosorbent assay (ELISA). Specific method: Human CD40, cynomolgus monkey CD40, rat CD40, and mouse CD40 (all purchased from ACROBiosystems) were coated in ELISA plates at 1 μg / mL and 100 μL / well, respectively, using a pH=9.6 carbonate buffer, and incubated overnight at 4°C. Washed five times with PBST. Blocked with 300 μL / well of PBST containing 1% BSA and incubated at room temperature for 1 hour. Washed five times with PBST. Monoclonal antibodies gradient-diluted with PBST containing 1% BSA were added, and 100 μL / well of the control CD40 monoclonal antibody CP-870893 (IMGT database ID 10523) was added and incubated at room temperature for 1 hour. Washed five times with PBST. HRP-labeled anti-human IgG antibody (Jackson ImmunoResearch, catalog number: 109-035-088), diluted in PBST containing 1% BSA, was added at 100 μL / well and incubated at room temperature for 1 hour. The mixture was washed 5 times with PBST. 100 μL / well of colorimetric substrate TMB was added and allowed to develop at room temperature for 10 minutes, after which 1 M sulfuric acid was added to stop the reaction. OD was measured using a microplate reader. 450nm Read the data, analyze the results, and use the EC four-parameter fitting combined curve. 50 I calculated it.

[0228] The results are shown in Table 10. Monoclonal antibodies 1605CD, 1606CD, 1607CD, 1608CD, 1609CD, 1652CD, 1653CD, 1654CD, and 1655CD were all able to bind to human CD40 and cynomolgus monkey CD40, and their binding affinity to human and cynomolgus monkey antigens was equivalent. They could not bind to rat CD40 or mouse CD40.

[0229] Table 10 Binding of monoclonal antibodies to antigens [Table 10]

[0230] Example 6: Effect of anti-CD40 monoclonal antibody on CD40 / CD40L binding The inhibitory effect of a monoclonal antibody on CD40 / CD40L binding was tested using HEK-Blue CD40L cells (purchased from InvivoGen) that highly express CD40. Specific method: HEK-Blue CD40L cells were collected and placed in pre-cooled PBS containing 2% FBS (1 × 10⁶ cells). 7 After resuspending at a density of / mL, 50 μL / well, i.e., 5 × 10⁶, is placed in a 96-well plate. 5 After adding cells / well, biotin-CD40L (FutureGen Biopharmaceutical) and gradient-diluted CD40 monoclonal antibody were added at 50 μL / well, with a final concentration of 10 nM of biotin-CD40L. The cells were incubated at 4°C for 1 hour. They were washed twice with pre-cooled PBS. Streptoavidin-PE (BioLegend, catalog number: 405203), diluted in pre-cooled PBS containing 2% FBS, was added, and the cells were incubated at 4°C for 30 minutes. They were washed twice with pre-cooled PBS. Next, the cells were resuspended in pre-cooled PBS containing 2% FBS and detected by flow analyzer.

[0231] The results are shown in Table 11. Monoclonal antibodies 1605CD, 1606CD, 1607CD, 1608CD, 1609CD, 1652CD, 1653CD, 1654CD, and 1655CD inhibited the CD40 / CD40L interaction, while the control CD40 monoclonal antibody CP-870893 did not affect the CD40 / CD40L interaction.

[0232] Table 11 Effect of monoclonal antibodies on CD40 / CD40L binding [Table 11]

[0233] Example 7: Detection of CD40 agonist activity of anti-CD40 monoclonal antibody using reporter gene method The CD40 agonist activity of a monoclonal antibody was detected using HEK-Blue CD40L. HEK-Blue CD40L cells were purchased from InvivoGen and highly expressed CD40 and a SEAP reporter gene under the control of the NF-κB response element. Activation of CD40 on HEK-Blue CD40L cells induced activation of the downstream signaling pathway NF-κB, which in turn induced SEAP production. The activation status of CD40 can be monitored by detecting the amount of secreted SEAP using QUANTI-Blue reagent (InvivoGen). Specific method: HEK-Blue CD40L cells were collected and placed in complete culture medium (RPMI 1640 containing 10% FBS) in 3 × 10⁶ cells. 5 Resuspend in / mL and pour 100μL / well into a 96-well plate, i.e., 3×10 4 Cells were uniformly spread in each well, and 100 μL / well of anti-CD40 monoclonal antibody sample and control antibody (CP-870893), gradient diluted with complete medium, were added. The cells were incubated at 37°C in a 5% CO2 incubator for 20-24 hours. After incubation, the 96-well plate was removed, centrifuged at 300 g for 5 minutes, and 40 μL / well of supernatant was transferred to a new 96-well plate. 160 μL / well of QUANTI-Blue reagent was added to the supernatant, and the cells were incubated at 37°C in a 5% CO2 incubator for 20-30 minutes. The cells were then OD-filtered in a microplate. 655nm We read the data and analyzed the results.

[0234] The results are shown in Figure 1. The CD40 agonist activity of the monoclonal antibodies can be broadly divided into two categories. The CD40 agonist activity of antibodies 1608CD, 1609CD, 1654CD, and 1655CD was weaker than that of the control CD40 monoclonal antibody CP-870893, while the activity of antibodies 1605CD, 1606CD, 1607CD, 1652CD, and 1653CD was stronger than that of the control CD40 monoclonal antibody CP-870893.

[0235] Example 8: DC-modulating activity of anti-CD40 monoclonal antibody The dendritic cell (DC) regulatory activity of CD40 monoclonal antibodies was tested.

[0236] The provided human PBMC cells were resuspended in complete medium (RPMI 1640 containing 10% FBS), seeded in 10 cm cell culture dishes, and cultured in a 37°C carbon dioxide incubator for 2 hours. The culture supernatant and suspension cells were discarded, and the adherent cells were mononuclear. The mononuclear cells were cultured for 6 days in complete medium containing 100 ng / mL GM-CSF (PeproTech, catalog number: 300-03) and 100 ng / mL IL-4 (PeproTech, catalog number: 200-04), with the liquid changed every 2 days to obtain imDC cells. The imDC cells were collected, resuspended in complete medium, seeded in 24-well plates, and 100 nM antibody sample and control antibody (CP-870893) were added. The culture plates were placed in a 37°C carbon dioxide incubator and cultured for 2 days. After culturing, the supernatant was removed from the wells, and cytokine IL-12 / IL-23 p40 (R&D, catalog number: DY1240) was detected according to the kit manual. Simultaneously, cells from the well plate were collected, and after culturing the test antibody (APC anti-human CD83 antibody, Biolegend, 305312), CD83 expression on DC cells was detected by flow cytometry.

[0237] Figure 2 compares the DC regulatory activity of the anti-CD40 monoclonal antibodies of the present invention, showing that all different monoclonal antibodies were able to significantly upregulate CD83 on DC cells.

[0238] Example 9: T cell regulatory activity of anti-CD40 monoclonal antibody The most important antitumor biological effect of CD40 agonist antibodies is the activation of T cells after activating DC cells. The T cell regulatory activity of CD40 monoclonal antibodies was tested using an MLR (Multiple Cell Regeneration) system cultured with allogeneic DC cells and T cells.

[0239] DC cells were obtained using the same method as in Example 8. Allogeneic T cells were isolated from human PBMC cells, and the specific isolation method was referred to in the instructions for the Pan T cell isolation kit (Miltenyi Biotech, catalog number: 130-096-535). Briefly, first, the PBMCs were washed once with PBS, and then resuspended in isolation buffer (PBS containing 2 mm EDTA and 0.5% BSA, pH=7.2) at a rate of 1E7 cells / 40 μL (the amounts used below are counted based on 1E7 cells). 10 μL of Pan T cell biotin antibody cocktail was added, and the cells were cultured at 4°C for 5 minutes. Next, 30 μL of isolation buffer and 20 μL of Pan T cell biotin antibody cocktail were added, and the cells were cultured at 4°C for 10 minutes. T cells were obtained by passing the cells through a MACS isolation column.

[0240] The obtained human DC cells and human T cells were collected, resuspended in complete medium (RPMI 1640 containing 10% FBS), and seeded in 96-well plates. DC cells and T cells were seeded at 1E4 / well and 1E5 / well, respectively, and mixed culture was performed. Monoclonal antibody samples and control antibodies, gradient diluted in complete medium, were added. The culture plates were placed in a 37°C carbon dioxide incubator and cultured for 5 days. After the culture period, the supernatant was taken from the wells, and the cytokine IFN-γ (Biolegend, catalog number: 430101) was detected according to the kit manual.

[0241] The results are shown in Figure 3. Both the potent CD40 agonist monoclonal antibody 1606CD and the weak CD40 agonist monoclonal antibody 1654CD of the present invention were able to enhance T cell activation under conditions of a low DC ratio, while the control monoclonal antibody CP-870893 had virtually no effect.

[0242] Example 10: Preliminary safety evaluation of an anti-CD40 monoclonal antibody. A preliminary safety evaluation of anti-CD40 monoclonal antibodies was performed in human CD40 gene knock-in mice. Experimental animals were purchased from Biocytogen and randomly divided into four groups (5 animals / group). Each group received either a solvent control, 21 mg / kg of monoclonal antibody 1606CD, 21 mg / kg of monoclonal antibody 1654CD, or 21 mg / kg of control CD40 monoclonal antibody CP-870893. The antibodies were administered intraperitoneally twice a week for two consecutive weeks, for a total of four doses. During the study, the animals' clinical symptoms, body weight, liver function biochemistry, and blood cell counts were monitored. After the end of the administration period, all animals were euthanized as planned the day after the final dose, dissected, examined for abnormalities, and their organ weights were measured.

[0243] As shown in Figures 4A to 4C, mice repeatedly injected with the potent CD40 agonist monoclonal antibody 1606CD and the weak CD40 agonist monoclonal antibody 1654CD showed no significant abnormalities, but blood tests revealed a decrease in lymphocytes and granulocytes. On the other hand, mice repeatedly injected with the monoclonal antibody CP-870893 showed weight loss, increased ALT, and a significant decrease in RBC, HGB, and PLT, and significant organ necrosis and organ enlargement were observed during autopsy. The results suggest that monoclonal antibodies 1606CD and 1654CD are safer than monoclonal antibody CP-870893.

[0244] Example 11: Detection of binding of recombinant PD-L1 monoclonal antibody to PD-L1 The binding affinity and species specificity of monoclonal antibodies to human PD-L1 were detected using enzyme-linked immunosorbent assay (ELISA). Specific method: Using a pH=9.6 carbonate buffer, human PD-L1, cynomolgus PD-L1, rat PD-L1, and mouse PD-L1 antigens (all purchased from ACROBiosystems) were coated at 1 μg / mL and 100 μL / well in ELISA plates, and incubated overnight at 4°C. Washed five times with PBST. Blocked with PBST containing 1% BSA in 300 μL / well and incubated at room temperature for 1 hour. Washed five times with PBST. Monoclonal antibodies gradient-diluted with PBST containing 1% BSA were added, and 100 μL / well of the control PD-L1 monoclonal antibody durvalumab (IMGT database ID 10010) was added, and incubated at room temperature for 1 hour. Washed five times with PBST. 100 μL / well of HRP-labeled anti-human IgG antibody (Jackson ImmunoResearch, catalog number: 109-035-088), diluted in PBST containing 1% BSA, was added and incubated at room temperature for 1 hour. Washed five times with PBST. 100 μL / well of chromometric substrate TMB was added and allowed to develop color at room temperature for 10 minutes, then 1 M sulfuric acid was added to stop the reaction. OD was measured using a microplate reader. 450nm Read the data, analyze the results, and use the EC four-parameter fitting combined curve. 50 I calculated it.

[0245] The results are shown in Table 12. Monoclonal antibodies 1029, 1031, 1102, and 1541 were all able to bind to human PD-L1 and cynomolgus monkey PD-L1, and their binding affinity to human and cynomolgus monkey antigens was the same as that of the control antibody. They were unable to bind to rat PD-L1 or mouse PD-L1.

[0246] Table 12 Binding of monoclonal antibodies to antigens [Table 12] NB: Do not combine.

[0247] The interaction between monoclonal antibodies and antigens was detected using Gator, a label-free biomolecular interaction analyzer based on the principle of biolayer interferometry (BLI). Specific method: Using a PA probe, a monoclonal antibody was diluted to 50 nM and added to a probe plate, where the monoclonal antibody was captured by the PA probe. Next, the antigen, human PD-L1, was added, gradient-diluted starting at 200 nM. The antigen interacted with the bispecific antibody captured by the PA probe, and the interaction was analyzed by detecting the signal change in the reflection interference spectrum on the probe surface. Finally, the antibody binding rate constant was calculated.

[0248] The results are shown in Table 13, and the binding rate constants of monoclonal antibodies 1029, 1031, 1102, and 1541 to human PD-L1 were in the range of 4.37 to 8.30 nM.

[0249] Table 13 Binding kinetics of monoclonal antibodies and antigens [Table 13]

[0250] Example 12: Detection of PD-L1 / PD-1 inhibitory activity of PD-L1 monoclonal antibody using reporter gene method A method for detecting reporter genes with PD-L1 / PD-1 inhibitory activity was established using Jurkat / PD-1-NFAT-luciferase cells (highly expressing luciferase reporter genes under the control of PD-1 and NFAT response elements) and WIL2S / PD-L1 cells (highly expressing PD-L1), constructed by FutureGen Biopharmaceutical, and an anti-CD20 / CD3 bispecific antibody. Specific method: WIL2S / PD-L1 cells were collected, and 4 × 10⁶ cells were used. 6 50 μL / well in complete medium (RPMI 1640 containing 10% FBS) at a cell density of 2 × 10⁶ / mL, i.e., 2 × 10⁶ / mL. 5Resuspend in 10 cells / well, spread uniformly in a 96-well plate, collect Jurrkat-PD-1-NFAT-luciferase cells, 4 × 10 6 50 μL / well in complete medium (RPMI 1640 containing 10% FBS) at a cell density of 2 × 10⁶ / mL, i.e., 2 × 10⁶ / mL. 5 The cells were resuspended in individual cells / well and spread uniformly in a 96-well plate. The anti-CD20 / CD3 bispecific antibody was diluted in complete medium and added to the 96-well plate at 25 μL / well. The anti-PD-L1 monoclonal antibody sample and control antibody were gradient diluted in complete medium and added to the 96-well plate at 25 μL / well. The cells were then incubated at 37°C in a 5% CO2 incubator for 6 hours. After incubation, 50 μL / well of one-glo reagent (Promega, catalog number: E6120) was added to the 96-well plate, and the plate was shaken in a plate shaker for 5 minutes and then allowed to stand for 10 minutes. The relative chemiluminescence unit values ​​(RLU) were then read using a chemiluminescence module on a microplate reader (MD, SpectraMax iD3) and the results were analyzed.

[0251] The results are shown in Figure 5. Monoclonal antibodies 1029, 1031, 1102, and 1541 were all able to block the negative signal transmitted from PD-L1 to PD-1. The antibody activities were similar, but slightly stronger than those of the PD-L1 controlled monoclonal antibody durvalumab.

[0252] Example 13: T cell regulatory activity of PD-L1 monoclonal antibody The T cell regulatory activity of monoclonal antibodies was tested using an MLR experimental system cultured with allogeneic DC cells and T cells.

[0253] The provided human PBMC cells were resuspended in complete medium (RPMI 1640 containing 10% FBS), seeded in a 10 cm cell culture dish, and cultured in a 37°C carbon dioxide incubator for 2 hours. The culture supernatant and suspension cells were discarded, and the adherent cells were mononuclear. The mononuclear cells were cultured for 6 days in complete medium containing 100 ng / mL GM-CSF (PeproTech, catalog number: 300-03) and 100 ng / mL IL-4 (PeproTech, catalog number: 200-04), with the medium changed every 2 days. TNFα and IL-1β (both purchased from PeproTech) were then added, and the cells were cultured for 2 days to obtain DC cells.

[0254] Allogeneic T cells were obtained by isolating them from the provided human PBMC cells. For specific isolation methods, refer to the instructions for the Pan T cell isolation kit (Miltenyi Biotech, catalog number: 130-096-535). Briefly, the PBMCs were first washed once with PBS, and then 1 × 10⁶ PBMCs were separated. 7 Resuspend cells in 40 μL of separation buffer (PBS containing 2 mM EDTA and 0.5% BSA, pH=7.2) (the amount used below is 1 × 10 7 (Based on cell counting), 10 μL of Pan T cell biotin antibody cocktail was added and the cells were cultured at 4°C for 5 minutes. Further 30 μL of separation buffer and 20 μL of Pan T cell biotin antibody cocktail were added and the cells were cultured at 4°C for 10 minutes. T cells were obtained by passing the cells through a MACS separation column.

[0255] Human DC cells and human T cells were collected, resuspended in complete medium (RPMI 1640 containing 10% FBS), seeded in 96-well plates, and the seeded DC and T cells were each 1 × 10⁶ cells. 4 / well, and 1×10 5 Seeds were seeded in one well and mixed culture was performed. Antibody samples, gradient diluted with complete medium, were added. The culture plates were placed in a 37°C carbon dioxide incubator and cultured for 5 days. After the culture period, the supernatant was taken from the wells and the cytokine IFN-γ (Biolegend, catalog number: 430101) was detected according to the kit manual.

[0256] The results are shown in Figure 6, and the monoclonal antibody significantly enhanced T cell activation.

[0257] Example 14: Detection of binding of recombinant anti-PD-L1 / CD40 bispecific antibody to PD-L1 and CD40. The binding affinity and species specificity of bispecific antibodies against human PD-L1 and human CD40 were detected using enzyme-linked immunosorbent assay (ELISA). Specific method: Using a pH=9.6 carbonate buffer, ELISA plates were coated with 1 μg / mL of the following antigens: human PD-L1, cynomolgus monkey PD-L1, rat PD-L1, mouse PD-L1, human CD40, cynomolgus monkey CD40, rat CD40, and mouse CD40 (all purchased from SINOBiological). The plates were incubated overnight at 4°C. Washed five times with PBST. Blocked with PBST containing 300 μL / well of 1% BSA and incubated at room temperature for 1 hour. Washed five times with PBST. Bispecific antibodies, gradient diluted with PBST containing 1% BSA, were added. Control PD-L1 monoclonal antibody durvalumab (IMGT database ID 10010) and CD40 monoclonal antibody CP-870893 (IMGT database ID 10523) were added at 100 μL / well, and the mixture was incubated at room temperature for 1 hour. The mixture was washed 5 times with PBST. HRP-labeled anti-human IgG antibody (Jackson ImmunoResearch, catalog number: 109-035-088), diluted with PBST containing 1% BSA, was added at 100 μL / well, and the mixture was incubated at room temperature for 1 hour. The mixture was washed 5 times with PBST. 100 μL / well of colorimetric substrate TMB was added, and the mixture was allowed to develop color at room temperature for 10 minutes. The reaction was then stopped by adding 1 M sulfuric acid. The mixture was then OD-read using a microplate reader. 450nm Read the data, analyze the results, and use the EC four-parameter fitting combined curve. 50 I calculated it.

[0258] The results are shown in Table 14. Bispecific antibodies 1605, 1606, 1607, 1608, 1609, 1652, 1653, 1654, and 1655 were all able to bind to human PD-L1 and CD40, and cynomolgus monkey PD-L1 and CD40, and their binding affinity to human and cynomolgus monkey antigens was the same. They were not able to bind to rat PD-L1 and CD40, or mouse PD-L1 and CD40.

[0259] Table 14 Binding of bispecific antibodies to antigens [Table 14] NB: Do not combine.

[0260] Example 15: Detection of simultaneous binding of recombinant anti-PD-L1 / CD40 bispecific antibody to PD-L1 and CD40. Using a pH=9.6 carbonate buffer, human CD40 antigen (purchased from SINOBiological) was coated at 1 μg / mL, 100 μL / well, in an ELISA plate and incubated overnight at 4°C. Washed 5 times with PBST. Blocked with PBST containing 300 μL / well of 1% BSA and incubated at room temperature for 1 hour. Washed 5 times with PBST. Bispecific antibodies, gradient diluted with PBST containing 1% BSA, were added, along with 100 μL / well of control PD-L1 monoclonal antibody durvalumab and CD40 monoclonal antibody CP-870893, and incubated at room temperature for 1 hour. Washed 5 times with PBST. Biotin-labeled human PD-L1 (FutureGen Biopharmaceutical), diluted with PBST containing 1% BSA, was added at 100 μL / well and incubated at room temperature for 1 hour. Washed 5 times with PBST. Streptoavidin-HRP (BioLegend, catalog number: 405210), diluted in PBST containing 1% BSA, was added at 100 μL / well and incubated at room temperature for 30 minutes. 100 μL / well of the colorimetric substrate TMB was added and allowed to develop color at room temperature for 10 minutes, after which 1 M sulfuric acid was added to stop the reaction. The OD was measured using a microplate reader. 450nmRead the data, analyze the results, and use the EC four-parameter fitting combined curve. 50 I calculated it.

[0261] The results are shown in Figure 8. Bispecific antibodies 1605, 1606, 1607, 1608, 1609, 1652, 1653, 1654, and 1655 were all able to simultaneously bind to human PD-L1 and human CD40, but the control monoclonal antibody showed no signal and was unable to bind to both antigens simultaneously.

[0262] Example 16: Detection of PD-L1 / PD-1 inhibitory activity of anti-PD-L1 / CD40 bispecific antibody using reporter gene method. Jurkat / PD-1-NFAT-luciferase cells (highly expressing luciferase reporter genes under the control of PD-1 and NFAT response elements) and WIL2S / PD-L1 cells (highly expressing PD-L1) were constructed by lentiviral transfection, referring to methods described in the literature (Xiaoyin Wang, et al. J Vis Exp. 2009; (32): 1499. Jonathan Elegheert, et al. Nat Protoc. 2018 Dec; 13(12): 2991-3017. Andreas Rinne, et al. J Physiol. 2010 Sep 1; 588(Pt 17): 3211-3216.). A method for detecting reporter genes with PD-L1 / PD-1 blocking activity was established using Jurkat and WIL2S cells and anti-CD20 / CD3 bispecific antibodies. Specific method: WIL2S / PD-L1 cells were collected and resuspended in complete medium (RPMI 1640 containing 10% FBS) at a cell density of 4E6 / mL at 50 μL / well, i.e., 2E5 cells / well, and spread uniformly in a 96-well plate. Jurkat-PD-1-NFAT-luciferase cells were collected and resuspended in complete medium (RPMI 1640 containing 10% FBS) at a cell density of 4E6 / mL at 50 μL / well, i.e., 2E5 cells / well, and spread uniformly in the above 96-well plate. Anti-CD20 / CD3 bispecific antibody was diluted in complete medium and added to the above 96-well plate at 25 μL / well. Anti-PD-L1 / CD40 bispecific antibody sample and control antibody were gradient diluted in complete medium and added to the above 96-well plate at 25 μL / well, and cultured for 6 hours at 37°C in a 5% CO2 incubator. After culturing was complete, 50 μl / well of one-glo reagent (Promega, catalog number: E6120) was added to the 96-well plate described above, and the plate was shaken in a plate shaker for 5 minutes, followed by standing for 10 minutes. Next, relative chemiluminescence unit values ​​(RLU) were read using a chemiluminescence module on a microplate reader (MD, SpectraMax iD3), and the results were analyzed.

[0263] The results are shown in Figure 9. The bispecific antibodies 1605, 1606, 1607, 1608, 1609, 1652, 1653, 1654, and 1655 were able to block the negative signal transmitted to PD-1 by PD-L1. The activity of the different bispecific antibodies was similar, as was the activity of the PD-L1 controlled monoclonal antibody durvalumab.

[0264] Example 17: Detection of CD40 agonist activity of anti-PD-L1 / CD40 bispecific antibody using reporter gene method The CD40 agonist activity of bispecific antibodies was measured using HEK-Blue CD40L. HEK-Blue CD40L cells were purchased from InvivoGen and highly expressed CD40 and a SEAP reporter gene under the control of the NF-κB response element. Activation of CD40 on HEK-Blue CD40L cells induced activation of the downstream signaling pathway NF-κB, which in turn induced SEAP production. The activation status of CD40 was monitored by detecting the amount of secreted SEAP using QUANTI-Blue reagent (InvivoGen). The CD40 agonist activity of bispecific antibodies was tested by culturing CHO / PD-L1 cells with HEK-Blue CD40L cells in the presence of PD-L1. Specific method: HEK-Blue CD40L cells were collected, resuspended in complete medium (RPMI 1640 containing 10% FBS) at 3E5 / mL, and uniformly spread in a 96-well plate at 100 μL / well, i.e., 3E4 cells / well. CHO / PD-L1 cells were collected, resuspended in complete medium (RPMI 1640 containing 10% FBS) at 6E5 / mL at 50 μL / well, i.e., 3E4 cells / well, and added to a 96-well plate. If CHO / PD-L1 cells were not needed, 50 μL / well of complete culture medium was added, and a gradient-diluted anti-PD-L1 / CD40 bispecific antibody sample and control antibody were added at 50 μL / well. The cells were then cultured for 20-24 hours in a 37°C, 5% CO2 incubator. After culturing is complete, remove the 96-well plate, centrifuge at 300g for 5 minutes, aspirate 40μL / well supernatant and transfer to a new 96-well plate, add 160μL / well of QUANTI-Blue reagent to the supernatant, and culture in a 37°C, 5% CO2 incubator for 20-30 minutes, then perform OD on a microplate. 655nm We read the data and analyzed the results.

[0265] The results are shown in Figures 10A to 10D and Table 15. The CD40 agonist activity of bispecific antibodies 1608, 1609, 1654, and 1655 was significantly enhanced by the PD-L1 crosslinking signal provided by CHO / PD-L1. This indicates that the activity of these bispecific antibodies is selective (low activity in environments with low PD-L1 expression (e.g., blood) and high activity in environments with high PD-L1 expression (e.g., tumor environment)), resulting in a therapeutic effect that enhances drug efficacy and suppresses side effects. However, bispecific antibodies 1605, 1606, 1607, 1652, and 1653 were not significantly enhanced by CHO / PD-L1, and the control CD40 monoclonal antibody CP-870893 was not enhanced by CHO / PD-L1.

[0266] Table 15 CD40 agonist activity of bispecific antibodies [Table 15]

[0267] Example 18: Effect of anti-PD-L1 / CD40 bispecific antibody on CD40 / CD40L binding The blocking effect of a bispecific antibody against CD40 / CD40L binding was tested using HEK-Blue CD40L cells that highly express CD40. Specific methods: HEK-Blue CD40L cells were collected, resuspended in pre-cooled PBS containing 2% FBS at a density of 1E7 / mL, and then added to a 96-well plate at 50 μL / well (i.e., 5E5 cells / well). Biotin-CD40L (FutureGen Biopharmaceutical) and gradient-diluted bispecific antibody were then added at 50 μL / well, with a final concentration of 10 nM for biotin-CD40L. The cells were cultured at 4°C for 1 hour. They were washed twice with pre-cooled PBS. Streptoavidin-PE (BioLegend, catalog number: 405203), diluted in pre-cooled PBS containing 2% FBS, was added, and the cells were cultured at 4°C for 30 minutes. They were washed twice with pre-cooled PBS. Next, the cells were resuspended in pre-cooled PBS containing 2% FBS and detected using a flow analyzer.

[0268] The results are shown in Table 16. The bispecific antibodies 1605, 1606, 1607, 1608, 1609, 1652, 1653, 1654, and 1655 blocked the CD40 / CD40L interaction, while the control CD40 monoclonal antibody CP-870893 did not affect the CD40 / CD40L interaction.

[0269] Table 16 Effect of bispecific antibodies on CD40 / CD40L binding [Table 16]

[0270] Example 19: DC regulatory activity of anti-PD-L1 / CD40 bispecific antibody The dendritic cell (DC) regulatory activity of bispecific antibodies was tested.

[0271] Human PBMC cells were resuspended in complete medium (RPMI 1640 containing 10% FBS), seeded in 10 cm cell culture dishes, and cultured in a 37°C carbon dioxide incubator for 2 hours. The culture supernatant and suspension cells were discarded, and the adherent cells were mononuclear. The mononuclear cells were cultured for 6 days in complete medium containing 100 ng / mL GM-CSF (PeproTech, catalog number: 300-03) and 100 ng / mL IL-4 (PeproTech, catalog number: 200-04), with the liquid changed every 2 days to obtain imDC cells. The imDC cells were collected, resuspended in complete medium, seeded in 24-well plates, and gradient-diluted bispecific antibody samples and control antibodies were added. The culture plates were placed in a 37°C carbon dioxide incubator and cultured for 2 days. After culturing was complete, the supernatant was removed from the wells, and cytokine IL-12 / IL-23 p40 (R&D, catalog number: DY1240) was detected according to the kit manual. In some experiments, cells in the well plate were simultaneously collected and cultured with detection antibodies (APC anti-human CD83 antibody, Biolegend, 305312, PE / Cyanine7 anti-human CD86 antibody, Biolegend, 374210), and then the expression of CD83 and CD86 on DC cells was detected by flow cytometry.

[0272] Figure 11A compares the DC regulatory activity of different bispecific antibodies, using IL-12 / IL-23 p40 as an indicator. The bispecific antibodies stimulated DCs to secrete IL-12 p40 in a concentration-dependent manner, and the activity of the different bispecific antibodies differed significantly. Figures 11B and 11C compare the DC regulatory activity of parental PD-L1 monoclonal antibody, parental CD40 monoclonal antibody, bispecific antibody (1609), and control CD40 monoclonal antibody CP-870893. The detection indicators were CD83 and IL-12 / IL-23 p40. The data showed that the parental PD-L1 monoclonal antibody did not regulate DCs, the parental CD40 monoclonal antibody had weak activity, and after constructing the biantibody, its DC activity was significantly enhanced.

[0273] Example 20: T cell regulatory activity of anti-PD-L1 / CD40 bispecific antibody The T cell regulatory activity of bispecific antibodies was tested using an MLR experimental system cultured with allogeneic DC cells and T cells.

[0274] DC cells were obtained using the same method as in Example 19. Allogeneic T cells were isolated from human PBMC cells, and the specific isolation method was referred to in the instructions for the Pan T cell isolation kit (Miltenyi Biotech, catalog number: 130-096-535). Briefly, the PBMCs were first washed once with PBS, then resuspended in 1E7 cells / 40 μL of isolation buffer (PBS containing 2 mM EDTA and 0.5% BSA, pH=7.2) (the amounts used below are counted based on 1E7 cells), 10 μL of Pan T cell biotin antibody cocktail was added, and the cells were cultured at 4°C for 5 minutes. Furthermore, 30 μL of isolation buffer and 20 μL of Pan T cell biotin antibody cocktail were added, and the cells were cultured at 4°C for 10 minutes. T cells were obtained by passing the cells through a MACS isolation column.

[0275] The obtained human DC cells and human T cells were harvested, resuspended in complete medium (RPMI 1640 containing 10% FBS), and seeded in 96-well plates. The seeded DC cells and T cells were 1E4 / well and 1E5 / well, respectively, and were cultured together. Bispecific antibody samples and control antibodies, gradient diluted in complete medium, were added. The culture plates were placed in a 37°C carbon dioxide incubator and cultured for 5 days. After the culture period, the supernatant was taken from the wells and the cytokine IFN-γ (Biolegend, catalog number: 430101) was detected according to the kit manual.

[0276] The results are shown in Figures 12A and 12B. The bispecific antibody enhanced T cell activation, and its T cell regulatory activity was significantly stronger than that of the parental PD-L1 monoclonal antibody, the parental CD40 monoclonal antibody, and PD-L1 controlled monoclonal durvalumab.

[0277] Example 21: In vivo antitumor effect of anti-mouse PD-L1 / mouse CD40 bispecific antibody In this example, the antitumor effect of a bispecific antibody in mice was detected. To more easily evaluate the in vivo efficacy of the anti-PD-L1 / CD40 bispecific antibody, an anti-mouse PD-L1 / mouse CD40 bispecific antibody 1058 was constructed as a substitute for expressing the bispecific antibody (the anti-PD-L1 sequence was taken from IMGT database ID 9814, and the anti-CD40 sequences were No.33_VH and No.34_VL of WO2018185045A1). The purpose of using this substitute is to evaluate the in vivo effect of a bispecific antibody formed by an antibody targeting PD-L1 and an antibody targeting CD40 in wild-type mice, and it is used solely to verify the efficacy of such a bispecific antibody.

[0278] C57BL / 6 female mice, 6-8 weeks old, were selected and purchased from Beijing Vital River. After the mice had adapted to the environment for one week, each mouse was seeded with 3E5 MC38 mouse colon cancer cells (purchased from Basic Medical Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences). The tumor volume was approximately 100 mm². 3 When the tumors grew, they were divided into groups according to their tumor volume, with 8 mice in each group. These groups were designated as a solvent control group, an anti-mouse PD-L1 monoclonal antibody group, an anti-mouse CD40 monoclonal antibody group, and an anti-mouse PD-L1 / mouse CD40 bispecific antibody group. The antibody was administered intraperitoneally once a week at a dose of 35 nmol / kg for two consecutive weeks. From the day of administration, the tumor volume was measured three times a week, and its longest diameter a and shortest diameter b were measured. 3 ) = (a × b 2 I calculated ) / 2.

[0279] The results are shown in Figure 13. Anti-mouse PD-L1 / mouse CD40 bispecific antibody 1058, a substitute for the bispecific antibody, significantly inhibited the proliferation of MC38 colon cancer xenograft tumors in mice, demonstrating a good antitumor effect. This effect was stronger than that of the PD-L1 monoclonal antibody and stronger than that of the CD40 monoclonal antibody.

[0280] Example 22: In vivo antitumor effect of anti-PD-L1 / CD40 bispecific antibody In this study, the antitumor effect of an anti-PD-L1 / CD40 bispecific antibody in PD-L1 / CD40 humanized mice was evaluated. The experiment was completed in cooperation with Beijing Agricultural University. Female PD-L1 / CD40 humanized mice, 6-8 weeks old, were purchased from Biocytogen. After the mice had adapted to the environment for one week, 5E6 MC38 / hPD-L1 mouse colon cancer cells were seeded into each mouse. The tumor volume was approximately 100 mm². 3 When the tumors grew, they were divided into groups according to tumor volume. Six mice in each group received intraperitoneal injections of a solvent, anti-PD-L1 monoclonal antibody, anti-CD40 monoclonal antibody, and anti-PD-L1 / anti-CD40 bispecific antibody 1654 twice a week for two consecutive weeks. From the day of administration, the clinical expression of the mice, their body weight, and tumor volume were monitored. The longest diameter a and shortest diameter b of the tumor were measured, and the tumor volume (mm²) was measured. 3 ) = (a × b 2 I calculated ) / 2.

[0281] The results are shown in Figure 14. 1654 significantly inhibited the growth of MC38 / hPD-L1 transplanted tumors in PD-L1 / CD40 humanized mice. At a low dose of 7 nmol / kg, the tumor growth inhibition rate (TGI) reached 72%, which was stronger than that of PD-L1 monoclonal antibodies or CD40 monoclonal antibodies at doses of 20 nmol / kg, both of which had a TGI of 28%. During the experiment, one mouse in the solvent control group died, but no abnormalities were observed in the other mice. The mice's body weight was normal.

[0282] Example 23: Preliminary safety evaluation of an anti-PD-L1 / CD40 bispecific antibody. A preliminary safety evaluation of the anti-PD-L1 / CD40 bispecific antibody was commissioned to JOINN (Suzhou) New Drug Research Center Co., Ltd.

[0283] The experimental animals used were male cynomolgus monkeys, randomly divided into five groups (2 animals / group). Each group received either a solvent control, 12 mg / kg of bispecific antibodies 1607, 1608, and 1609, or 10 mg / kg of control CD40 monoclonal CP-870893. The drugs were administered subcutaneously via intravenous infusion at a dose of 10 mL / kg and a rate of 0.5 mL / kg / min into the forelimb or hindlimb of the animals using a syringe pump. The drugs were administered once a week for two consecutive weeks, for a total of three doses. During the study, the animals' clinical symptoms, body weight, food intake, body temperature, blood cell count, coagulation function, blood biochemistry, urinalysis, immunocytophenotype, cytokines, blood drug concentration, and anti-drug antibodies were regularly monitored. After the end of the administration period, all surviving animals in groups 2-5 were euthanized as planned the day after the final dose. Gross dissection was performed to observe for abnormalities and measure the weight of the organs.

[0284] In animals receiving 12 mg / kg doses of bispecific antibodies 1607, 1608, and 1609, no abnormal changes related to the test products were observed in clinical observation, body weight, body weight gain, food intake, body temperature, blood cell count, coagulation function, blood biochemistry, urinalysis, organ weight, or pathological gross dissection. Only increases in immune cell phenotype and some cytokines were observed and were related to the pharmacological mechanism of action. However, in the group receiving a 10 m / kg dose of the CD40 monoclonal antibody CP-870893, animals exhibited small amounts of loose stools on days D7-D8, D10-D11, and D7-D13, respectively. Body weight gain on day D14 was slightly less than before drug administration, food intake significantly decreased from day D5 onward, RBC, HGB, HCT, and PLT showed a decreasing trend, the phenotype of immune cells changed, some cytokines increased, and during dissection of cynomolgus monkeys, significant changes were observed, with a decrease in thymus organ weight, organ-to-body weight ratio, and organ-to-brain ratio, and an increase in spleen organ weight, organ-to-body weight ratio, and organ-to-brain ratio.

[0285] The results suggested that anti-PD-L1 / CD40 bispecific antibodies were safer than CD40 monoclonal antibodies.

Claims

1. It includes a heavy chain variable region and a light chain variable region, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 14, and SEQ ID NO: 19, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 28, respectively. The light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 14, and SEQ ID NO: 19, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 28, respectively. The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 16, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25, respectively. The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 7, SEQ ID NO: 12, and SEQ ID NO: 17, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO:

26. The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 7, SEQ ID NO: 12, and SEQ ID NO: 17, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 26, respectively. The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO: 18, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HC shown in SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 27, respectively. Including DR3, The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO: 18, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 27, respectively. The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 10, SEQ ID NO: 15, and SEQ ID NO: 20, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 29, respectively, or An anti-CD40 antibody wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 15, and SEQ ID NO: 20, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 29, respectively.

2. The framework region of the light chain variable region is a humanized framework region, and the framework region of the heavy chain variable region is a humanized framework region. The light chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 38, and the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 40 or SEQ ID NO:

39. The light chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 30, and the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

31. The light chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 32, and the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 33 or SEQ ID NO:

34. The light chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 35, and the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 36 or SEQ ID NO:

37. Or, The light chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 41, and the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 42 or SEQ ID NO:

43. The antibody according to claim 1.

3. The light chain variable region includes the amino acid sequence shown in SEQ ID NO: 38, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 40 or SEQ ID NO:

39. The light chain variable region includes the amino acid sequence shown in SEQ ID NO: 30, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:

31. The light chain variable region includes the amino acid sequence shown in SEQ ID NO: 32, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 33 or SEQ ID NO:

34. The light chain variable region includes the amino acid sequence shown in SEQ ID NO: 35, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 36 or SEQ ID NO: 37, or The light chain variable region includes the amino acid sequence shown in Sequence ID No. 41, and the heavy chain variable The variant region includes the amino acid sequence shown in SEQ ID NO: 42 or SEQ ID NO:

43. The antibody according to claim 2.

4. The aforementioned antibody is (1) The antibody is a full-length antibody, Fab, Fab', F(ab') ２ , Fv, or scFv, (2) The antibody is a single-specific antibody or a multispecific antibody, (3) The antibody is a monoclonal antibody prepared from the above antibody. A characteristic that satisfies one or more of the following: The antibody according to claim 2.

5. The antibody according to claim 4, wherein the antibody comprises a heavy chain constant region and / or a light chain constant region.

6. The antibody according to claim 5, wherein the heavy chain constant region of the antibody is derived from the heavy chain constant region of a humanized antibody IgG1, IgG2, IgG3, or IgG4, and / or the light chain constant region of the antibody is derived from the κ chain of a humanized antibody.

7. The antibody according to claim 6, characterized in that the heavy chain of the antibody includes the amino acid sequence shown in SEQ ID NO: 94, and the light chain of the antibody includes the amino acid sequence shown in SEQ ID NO: 95, or the heavy chain of the antibody includes the amino acid sequence shown in SEQ ID NO: 96, and the light chain of the antibody includes the amino acid sequence shown in SEQ ID NO: 81, or the heavy chain of the antibody includes the amino acid sequence shown in SEQ ID NO: 87, and the light chain of the antibody includes the amino acid sequence shown in SEQ ID NO:

81.

8. A bispecific antibody comprising a first antigen-binding domain that specifically binds to human CD40 and a second antigen-binding domain that specifically binds to human PD-L1, wherein the first antigen-binding domain is the anti-CD40 antibody described in claim 1, The second antigen-binding domain comprises at least one VHH, The aforementioned VHH is, The sequence is VHH-CDR1, shown in sequence number 46; the sequence is VHH-CDR2, shown in sequence number 47; and the sequence is VHH-CDR3, shown in sequence number 48. VHH-CDR1, whose sequence is shown in sequence number 67; VHH-CDR2, whose sequence is shown in sequence number 68; and VHH-CDR3, whose sequence is shown in sequence number 69. Alternatively, VHH-CDR1 whose sequence is shown in sequence number 70, VHH-CDR2 whose sequence is shown in sequence number 71, and VHH-CDR3 whose sequence is shown in sequence number 48, A bispecific antibody containing [specific antibody].

9. The bispecific antibody according to claim 8, characterized in that the amino acid sequence of VHH is as shown in SEQ ID NO: 49, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO: 74, or has at least 90% sequence identity with SEQ ID NO: 49, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO:

74.

10. The first antigen-binding domain and the second antigen-binding domain are operably linked directly or via a linker. The second antigen-binding domain is linked to the N-terminus of the light chain variable region or heavy chain variable region of the first antigen-binding domain, or to the C-terminus of the light chain constant region, or to the C-terminus of IgG, and the linker is a peptide sequence, (G ４ S) ｎ Includes G, or (G ４ S) ｎ The bispecific antibody according to claim 8, characterized in that it consists of G, where n is an integer from 1 to 10.

11. It comprises two first polypeptide chains and two second polypeptide chains, where, The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 56, or has at least 90% sequence identity with SEQ ID NO: 56, and / or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 62 or SEQ ID NO: 57, or has at least 90% sequence identity with SEQ ID NO: 62 or SEQ ID NO:

57. The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 50, or has at least 90% sequence identity with SEQ ID NO: 50, and / or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 51, or has at least 90% sequence identity with SEQ ID NO:

51. The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 52, or has at least 90% sequence identity with SEQ ID NO: 52, and / or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 53 or SEQ ID NO: 60, or has at least 90% sequence identity with SEQ ID NO: 53 or SEQ ID NO:

60. The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 54, or has at least 90% sequence identity with SEQ ID NO: 54, and / or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 55 or SEQ ID NO: 61, or has at least 90% sequence identity with SEQ ID NO: 55 or SEQ ID NO: 61, or The bispecific antibody according to claim 8, characterized in that the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 58 or has at least 90% sequence identity with SEQ ID NO: 58, and / or the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 59 or SEQ ID NO: 63, or has at least 90% sequence identity with SEQ ID NO: 59 or SEQ ID NO:

63.

12. An isolated nucleic acid characterized by encoding an anti-CD40 antibody according to any one of claims 1 to 7, or a bispecific antibody according to any one of claims 8 to 11.

13. A recombinant expression vector comprising the isolated nucleic acid described in claim 12.

14. A transformant comprising the recombinant expression vector described in claim 13.

15. A method for preparing an anti-CD40 antibody or a bispecific antibody, comprising the step of culturing the transformant described in claim 14 to obtain an anti-CD40 antibody or a bispecific antibody from the culture.

16. A pharmaceutical composition comprising an anti-CD40 antibody according to any one of claims 1 to 7 or a bispecific antibody according to any one of claims 8 to 11, and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition according to claim 16, further comprising an additional agent, the additional agent being selected from 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.

18. A kit comprising an anti-CD40 antibody according to any one of claims 1 to 7, a bispecific antibody according to any one of claims 8 to 11, or a pharmaceutical composition comprising the anti-CD40 antibody or the bispecific antibody and a pharmaceutically acceptable carrier.

19. Further comprising (i) an apparatus for administering an antibody or pharmaceutical composition, and / or (ii) instructions for use, The kit according to claim 18, wherein the pharmaceutical composition further comprises an additional agent, the additional agent being selected from 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.

20. A medicine box set including medicine box A and medicine box B, The medicine box A contains an anti-CD40 antibody according to any one of claims 1 to 7, a bispecific antibody according to any one of claims 8 to 11, and / or a pharmaceutical composition comprising the anti-CD40 antibody or the bispecific antibody and a pharmaceutically acceptable carrier. The aforementioned medicine box B contains one or more of the following: other antitumor antibodies or pharmaceutical compositions containing 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. Medicine box set.

21. An in vitro method for immunodetection or measurement of CD40 and / or PD-L1, using an anti-CD40 antibody according to any one of claims 1 to 7, or a bispecific antibody according to any one of claims 8 to 11.

22. A pharmaceutical composition for use as a pharmaceutical, comprising an anti-CD40 antibody according to any one of claims 1 to 7 or a bispecific antibody according to any one of claims 8 to 11, and a pharmaceutically acceptable carrier.

23. The pharmaceutical agent is used to prevent and / or treat tumors, The pharmaceutical composition according to claim 22, further comprising an additional agent, the additional agent being selected from 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.

24. The pharmaceutical composition according to claim 23, wherein the tumor is lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, rhabdomyosarcoma, esophageal cancer, cervical cancer, multiple myeloma, leukemia, gallbladder cancer, glioblastoma, or melanoma.

Images