Anti-CD40 Antibody, Anti-PD-L1×CD40 Bispecific Antibody, and Use Thereof

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 the priority of Chinese Patent Application No. 2022106137021 with an application date of May 31, 2022, Chinese Patent Application No. 2022106162803 with an application date of May 31, 2022, and Chinese Patent Application No. 2023105733440 with an application date of May 19, 2023. This application incorporates the entire text of the above Chinese patent applications by reference.

[0002] The present invention belongs to the field of biotechnology, and specifically relates to an anti-CD40 antibody and an anti-PD-L1×CD40 bispecific antibody, and their uses.

Background Art

[0003] In recent years, the field of tumor treatment has developed rapidly, and more advanced treatment protocols such as targeted therapy and immunotherapy have been developed from traditional treatment methods centered on surgery, radiotherapy, and chemotherapy. In particular, tumor immunotherapy represented by PD-L1 / PD-1 inhibitors has significantly extended the survival period of tumor patients in multiple tumor indications and is also positioned as the first-choice treatment in multiple tumor indications (Nat Rev Immunol. 2020 Nov;20(11):651-668.). As the most promising solution for curing tumors, tumor immunotherapy has become the center of the research and development field of new anti-tumor drugs.

[0004] One of the important challenges in tumor immunology is to increase the quantity and quality of infiltrating T cells in cold tumors. In many cancer patients, insufficient T cell priming is the reason for the lack of T cells in the tumor microenvironment, and antigen-presenting cells loaded with tumor antigens, especially DC cells, play a decisive 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, playing important roles in both innate and adaptive immunity and playing an important role 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 upregulates co-stimulatory molecules and MHC on DC cells and induces pro-inflammatory cytokines, thereby activating DC cells and enabling them to promote the activation of anti-tumor specific T cells, and this type of T cell has the potential to completely eliminate tumor cells.

[0005] In various mouse tumor models, the use of CD40 agonist antibodies can achieve T cell activation and exhibit a potent antitumor effect (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 a synergistic antitumor effect with immune checkpoint antibodies such as PD-L1 / PD-1. This depends on the interaction mechanism between DC cells and T cells, where DCs upregulate costimulatory molecules and secrete IL-12 to stimulate the activation of tumor-specific T cells. 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 the antitumor effect (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, CN111763259, etc. Multiple antibodies have entered the clinical trial stage. Selicrelumab, a CD40 monoclonal antibody developed by Pfizer, brought objective partial response (PR) to 4 out of 15 patients with advanced melanoma in the first human single-dose trial, and 1 of the patients received repeated doses of selicrelumab for 1 year thereafter and maintained complete remission (CR) even after 15 years. However, the efficacy of selicrelumab was not good in subsequent clinical trials, and other CD40 monoclonal antibodies such as APX005M and SEA-CD40 also achieved only very low objective response rates (ORR). CD40-activating monoclonal antibodies show many side effects in the clinical setting, such as cytokine release syndrome (CRS), liver injury, thrombocytopenia, etc. The maximum tolerated doses (MTD) of selicrelumab, APX005M, and SEA-CD40 are 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, in this field, there is an urgent need for antibodies targeting CD40 with high safety and excellent efficacy.

Summary of the Invention

[0007] To solve the above technical problems, 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 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 anti-PD-L1×CD40 bispecific antibody of the present invention acts on the positive feedback pathway of the interaction between DC cells and T cells by stimulating CD40 and blocking PD-L1 / PD-1 at the same time, maximizing the anti-tumor effect. In addition, through PD-L1-dependent CD40 activation, the selectivity of CD40 activation is improved, and the toxicity and side effects of the CD40 agonist antibody are reduced.

[0008] The first 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 any variant or isoform of CD40 naturally expressed by cells. The antibodies of the present invention can specifically bind to human CD40 and monkey CD40 (e.g., cynomolgus monkey). As another option, the antibody may be specific for human CD40 and may not show cross-reactivity with other species. CD40, or any variant or isoform thereof, can be isolated from the cells or tissues in which they are naturally expressed, or can be produced by recombinant techniques that are common in the art and described herein.

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

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

[0012] In some embodiments, for the antibody according to the first aspect provided by the present invention, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, and the LCDR2 is X 5 X 6 SX 7 X 8 X 9 comprises the amino acid sequence shown by S, where X 5 is Y or A, and X 6 is T or A, and X 7 is S, R or T, and X 8 is L or R, and X 9 is Q or D. The LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and the 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 the LCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the LCDR2 is X 5 X 6 SX 7 X 8 X 9 shown by S, where X 5 is Y or A, and X 6 is T or A, and X 7 is S, R or T, and X 8 is L or R, and X 9 is Q or D. The amino acid sequence of the LCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 6.

[0014] In the antibody according to the first aspect of the present invention, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 8, SEQ ID NO: 7 or SEQ ID NO: 10, the LCDR2 comprises 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, the LCDR3 comprises 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, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24, and the HCDR3 comprises 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 comprises 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 the 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 the 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 the 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 the HCDR1 is shown in SEQ ID NO: 21 or SEQ ID NO: 22, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 23 or SEQ ID NO: 24, and the amino acid sequence of the 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 present invention, in the antibody according to the first aspect of the present invention: A) The LCDR1 includes the amino acid sequence shown in SEQ ID NO: 9, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 14, the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19, 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: 28,

[0018] B) The LCDR1 includes the amino acid sequence shown in SEQ ID NO: 9, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 14, the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19, 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: 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) 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: 24, and the 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) The LCDR1 includes the amino acid sequence shown in SEQ ID NO: 8, the LCDR 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 HCDR1 includes the amino acid sequence shown in SEQ ID NO: 22, 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: 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 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) 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: 23, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 29.

[0026] In a further preferred embodiment of the present invention: A) The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 9, 14, and 19, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 21, 23, and 28, respectively.

[0027] B) The light chain variable region contains 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 contains 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 contains 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 contains 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 contains 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 contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26, respectively.

[0030] E) The light chain variable region contains 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 contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 26, respectively.

[0031] F) The light chain variable region contains 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 contains 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 contains 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 contains 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 contains 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 contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 29, respectively, or,

[0034] I) The light chain variable region contains 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 contains 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, for the antibody, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 14, the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 19, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 21, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 23, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 28.

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

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

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

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

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

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

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

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

[0044] In some of the above embodiments, the amino acid sequences of the enumerated CDRs are determined according to the Kabat definition rules. However, it is well known to those skilled in the art that the CDRs of antibodies can be defined by various methods in the art. For example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (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)), Kabat based on the variability of the antibody sequence (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the International Immunogenetics Database (IMGT) (World Wide Web: imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures, etc. Unless otherwise specified, the terms "CDR" and "complementary determining region" of a given antibody or its region (e.g., variable region) are understood by those skilled in the art to include the complementary determining regions defined by any of the above-known schemes described by the present invention. Various numbering systems and their corresponding CDRs are well known to those skilled in the art and are as shown in Table 1.

[0045] Table 1 Definition methods of antibody CDRs

Table 1

[0046] In the antibody according to 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 according to the first aspect of the present invention, in a preferred embodiment of the present invention: a) 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: 38, 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: 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 the binding function to the same antigen (e.g., human CD40) as the original sequence.

[0053] The calculation of sequence homology between arrays is as follows. To determine the percentage of homology between two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced into 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 reference sequence to be aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. Next, the amino acid residues at the corresponding amino acid positions are compared. If the position within the first sequence is occupied by the same amino acid residue at the corresponding position within the second sequence, the molecule is the same at this position. Mathematical algorithms can be utilized to perform sequence comparison and calculation of the percentage of homology between two sequences. In one preferred embodiment, the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithms (obtained from http: / / www.gcg.com) already integrated into the GAP program of the GCG software package are used, along with the Blossum 62 matrix or the 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 to determine the percentage of homology between two amino acid sequences. A particularly preferred parameter set (and the parameter set that needs to be used unless otherwise specified) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Also, the percentage of homology between two amino acid sequences can be determined using the PAM120 weighted residue table, a gap length penalty of 12, a gap penalty of 4, and the E. Meyers and W. Miller algorithms ((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, such as to identify other family member sequences or related sequences, for example.

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

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

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

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

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

[0059] In the antibody according to the first aspect of the present invention, in an even more preferred embodiment of the present invention: In some embodiments, the antibody comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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, for the antibody, the amino acid sequence of the light chain variable region 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, for the antibody, the amino acid sequence of the light chain variable region 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, in the antibody, the amino acid sequence of the light chain variable region 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, in the antibody, the amino acid sequence of the light chain variable region 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, in the antibody, the amino acid sequence of the light chain variable region 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, in the antibody, the amino acid sequence of the light chain variable region 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, in the antibody, the amino acid sequence of the light chain variable region 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, in the antibody, the amino acid sequence of the light chain variable region 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, in the antibody, the amino acid sequence of the light chain variable region 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, in the antibody, the amino acid sequence of the light chain variable region 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, for the antibody, the amino acid sequence of the light chain variable region 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, for the antibody, the amino acid sequence of the light chain variable region 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, for the antibody, the amino acid sequence of the light chain variable region 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, for the antibody, the amino acid sequence of the light chain variable region 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] For the antibody described in the first aspect of the present invention, the antibody satisfies any one or more of the following three conditions: (1) the antibody is a full-length antibody, Fab, Fab′, F(ab′) 2 or Fv, and preferably the Fv is a scFv; (2) the antibody is a monospecific antibody or a multispecific antibody; (3) the antibody is a monoclonal antibody or a polyclonal antibody prepared by the above antibody.

[0083] The antibody of the present invention includes monoclonal antibodies (abbreviation: mAb or Ab), which refer to antibodies obtained from a single clone cell line, and the cell line is not limited to eukaryotic cell lines, prokaryotic cell lines, or phage clone cell lines.

[0084] For 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 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.

[0086] In some embodiments, the constant region includes constant region variants that do not change the structure and function of the antibody variable region. The prior art discloses various such constant region variants. For example, the Fc of the heavy chain constant region of an antibody has substitutions of one or more amino acids among 238, 265, 269, 270, 297, 327, and 329 (adopting the EU numbering system) (U.S. Patent No. 6,737,056), or the Fc of the heavy chain constant region of an antibody has substitutions of one or more amino acids among 234, 235, 265, 329 (adopting the EU numbering system), or the Fc of the heavy chain constant region of an antibody has substitutions of one or more amino acids 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 (adopting the EU numbering system) (see U.S. Patent No. 7,371,826). These mutations have been shown to endow the antibody with new properties, but do not change the function of the variable region of the antibody.

[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 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.

[0089] In a more preferred embodiment of the present invention, 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.

[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] The 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 the present invention is used in the broadest sense and includes monoclonal antibodies, polyclonal antibodies, single-specificity antibodies, multispecific antibodies (e.g., bispecific antibodies, diabodies, tribodies, and tetra-bodies, tandem di-scFv, tandem tri-scFv), as well as conventional antibodies (tetrapeptide chain structure antibodies composed of two identical heavy chains and two identical light chains connected by inter-chain disulfide bonds), and Fab, Fab', F(ab') having antigen-binding activity 2、Fv, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies, domain antibodies (heavy chain (VH) antibodies, light chain (VL) antibodies). Conventional antibodies (also called "full-length antibodies" or "complete antibodies") are usually heterotetrameric proteins of about 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H) connected by interchain disulfide bonds, forming a tetrapeptide chain structure. Each heavy chain of a full-length antibody consists of a heavy chain variable region (abbreviated as VH in the present invention) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in the present invention) and a light chain constant region (abbreviated as CL in the present invention). The light chain constant region is composed 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 immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. A complete antibody forms a "Y" shape. The stem of the Y is formed by the binding of the second and third constant regions of the two heavy chains (the fourth constant region in the case of IgE and IgM), and an interchain disulfide bond is formed within the hinge. Heavy chains γ, α, and δ have a constant region composed of three tandem (arranged in a row) Ig domains and a hinge region for enhancing flexibility, and 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 the first constant region (CH1) bound to a single light chain. The "Fc" region is two heavy chain fragments containing the CH2 and CH3 domains of the antibody, and the two heavy chain fragments are bound by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.The prior art discloses various Fc constant region mutants. For example, the Fc of the heavy chain constant region of an antibody has substitutions of one or more amino acids among 238, 265, 269, 270, 297, 327, and 329 (adopting the EU numbering system) (U.S. Patent No. 6,737,056), or the Fc of the heavy chain constant region of an antibody has substitutions of one or more amino acids among 234, 235, 265, 329 (adopting the EU numbering system), or the Fc of the heavy chain constant region of an antibody has substitutions of one or more amino acids 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 (adopting the EU numbering system) (see U.S. Patent No. 7,371,826). These mutations have been shown to endow the antibody with new properties, but do not change the function of the variable region of the antibody.

[0097] The term "variable region" or "variable domain" refers to the domain of an 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 the region within the variable domain that mainly contributes to antigen binding, and "framework" or "FR" refers to the variable domain residues other than the CDR residues. VH or VHH contains three CDR regions. For ease of distinction, the three CDRs of VH are identified as HCDR1, HCDR2, and HCDR3, the three CDRs of VHH are identified as VHH-CDR1, VHH-CDR2, and VHH-CDR3, and VL contains three CDR regions of LCDR1, LCDR2, and LCDR3. Each VH and VL is composed of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. To confer antigen-binding specificity, a single VH or VL may be sufficient. As used herein, the terms "VHH" and "nanobody" have the same meaning and are used interchangeably, referring to the variable region of a cloned antibody heavy chain, 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 is different from conventional tetrapeptide chain structure antibodies where the epitope is recognized together by the 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. Nanobodies have excellent biological properties, with a molecular weight of 12 - 15 kDa, which is one-tenth of that of a complete antibody, and are equipped with excellent tissue penetration, high specificity, and good water solubility. Due to its special structural properties, it combines the advantages of conventional antibodies and small molecule pharmaceuticals, almost completely overcoming the disadvantages of conventional antibodies such as long development cycles, low stability, and strict storage conditions, and gradually becoming a new emerging force in the new generation of antibody therapies, showing great potential for wide applications in immuno-diagnosis and treatment.VHHs include, but are not limited to, natural antibodies produced by camels, and may also be antibodies produced by camels and then humanized, or obtained through phage display technology screening. Methods for obtaining VHHs that bind to specific antigens or epitopes are disclosed in the prior art, and for example, the following documents can be referred to: R. van der Linden et al., Journal of Immunological Methods, 240(2000)185 - 195; Li et al., J Biol Chem., 287(2012)13713 - 13721; Deffar et al., African Journal of Biotechnology Vol.8(12), pp.2645 - 2652, 17 June, 2009 and WO94 / 04678.

[0098] The term "PD - L1" includes any variant or isoform of PD - L1 that is naturally expressed by cells. The antibodies of the present invention may cross - react with PD - L1 derived from non - human (e.g., cynomolgus monkey) species. As another option, the antibody may be specific for human PD - L1 and may not show cross - reactivity with other species. PD - L1 or any of its variants or isoforms can be isolated from the cells or tissues in which they are naturally expressed, or can be generated by recombinant techniques using techniques that are 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, and the VHH comprises VHH - CDR1, VHH - CDR2, and VHH - CDR3 of SEQ ID NO: 66. In some embodiments, the VHH - CDR1 comprises 41 YYX 42 X 43 an amino acid sequence shown in C, where X 41 is D or E, X 42 is S or T, X 43is K or Q, the VHH-CDR2 contains the amino acid sequence shown in SEQ ID NO: 76, and the VHH-CDR3 contains the amino acid sequence shown in SEQ ID NO: 77.

[0100] In some embodiments, the VHH contains VHH-CDR1, VHH-CDR2, and VHH-CDR3 of SEQ ID NO: 49, SEQ ID NO: 74, SEQ ID NO: 72, or SEQ ID NO: 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. 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 contains the amino acid sequence shown in SEQ ID NO: 46, SEQ ID NO: 67, or SEQ ID NO: 70, the VHH-CDR2 contains the amino acid sequence shown in SEQ ID NO: 47, SEQ ID NO: 68, or SEQ ID NO: 71, and the VHH-CDR3 contains the amino acid sequence shown in SEQ ID NO: 48 or SEQ ID NO: 69.

[0102] In some embodiments, the VHH contains 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, the VHH-CDR1 contains the amino acid sequence shown in SEQ ID NO: 46, the VHH-CDR2 contains the amino acid sequence shown in SEQ ID NO: 47, and the VHH-CDR3 contains the amino acid sequence shown in SEQ ID NO: 48.

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

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

[0106] In some embodiments, the VHH is: VHH-CDR1 with the sequence shown in SEQ ID NO: 46, VHH-CDR2 with the sequence shown in SEQ ID NO: 47, and VHH-CDR3 with the sequence shown in SEQ ID NO: 48, VHH-CDR1 with the sequence shown in SEQ ID NO: 67, VHH-CDR2 with the sequence shown in SEQ ID NO: 68, and VHH-CDR3 with the sequence shown in SEQ ID NO: 69, or VHH-CDR1 with the sequence shown in SEQ ID NO: 70, VHH-CDR2 with the sequence shown in SEQ ID NO: 71, and VHH-CDR3 with the sequence shown in SEQ ID NO: 48.

[0107] In some embodiments, the amino acid sequence of the 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 sequences 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 a 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 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: 83, 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: 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 the 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 with the amino acid sequences 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 the 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 comprises 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, the heavy chain constant region is preferably the Fc region of human IgG1, and the heavy chain constant region more preferably comprises 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, and the linker is preferably a linker having the amino acid sequence shown by (G4S)x, where x is independently selected from integers of 1 to 20, and more preferably is the linker shown in SEQ ID NO: 89.

[0116] Even 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 two amino acid sequences shown in SEQ ID NO: 93.

[0118] In the present invention, "Fab" is composed of one light chain and the CH1 and variable regions of one heavy chain. "Fab'" includes one light chain, the VH domain and the CH1 domain, and the region between the CH1 and CH2 domains, and to form an F(ab') 2 molecule, an interchain disulfide bond may be formed between the two heavy chains of the two Fab' fragments. F(ab') 2The fragment is composed of two Fab′ fragments linked by a disulfide bond between two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an 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 pair with each other via a 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 an scFv molecule may have a general structure: NH2-VL-linker-VH-COOH, or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repetitive G 4 S amino acid sequence or a variant thereof. For example, the amino acid sequence (G 4 S) 4 or (G 4 S) 3 can be used as the linker, and variants thereof may also be used.

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

[0121] The term "epitope" refers to the area or region on an antigen that can specifically bind to an antibody. An epitope is formed from a sequence of contiguous amino acids (linear epitope) or contains non-contiguous amino acids (conformational epitope), for example, the spatial proximity that occurs by folding of the antigen (i.e., by the tertiary folding in the protein nature of the antigen). Difference between conformational epitope and linear epitope: In the presence of a denaturing solvent, the binding of the antibody to the conformational epitope is lost. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8 to 10 amino acids in a specific 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, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), but is not limited thereto.

[0122] The term "specifically binds" refers to an antibody binding to a particular antigen or an epitope within that antigen with a higher affinity than to other antigens or epitopes. Generally, an antibody binds to an antigen or an epitope within the antigen with an equilibrium dissociation constant (KD) of about 1×10-7 M or less (e.g., about 1×10-8 M or less, about 1×10-9 M or less, about 1×10-10 M or less, about 1×10-10 M or less, about 1×10-9 M or less). 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 a BIACORE® surface plasmon resonance assay. However, an antibody that specifically binds to an antigen or an epitope within the antigen may cross-react with other related antigens, for example, with homologous antigens from other species (e.g., human or monkey such as cynomolgus (Macaca fascicularis), chimpanzee (Pan troglodytes), or common marmoset (Callithrix jacchus)).

[0123] The term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, the "affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y is generally represented by the dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including the methods described herein. The term "kassoc" or "ka" refers to the association 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 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 an antibody include methods for measuring surface plasmon resonance using a biosensing system such as a system, or methods for measuring the affinity in solution by solution equilibrium titration (SET).

[0124] The terms "anti-CD40 antibody", "antibody that specifically binds to CD40" refer to an antibody that can bind to CD40 with sufficient affinity to be used as a diagnostic and / or therapeutic agent targeting CD40. In some embodiments, the antibody that binds to CD40 has 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 10-8 M, e.g., between 10-8 M and 10-12 M, e.g., between 10-9 M and 10-10 M). In some embodiments, the anti-CD40 antibody binds to antigen epitopes conserved in CD40 of various species.

[0125] The terms "anti-PD-L1 antibody" and "antibody that specifically binds to PD-L1" refer to an antibody that can bind to PD-L1 with sufficient affinity to be used as a diagnostic agent and / or therapeutic agent targeting PD-L1. In some embodiments, the antibody that binds to PD-L1 has 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., 10-8 M or less, e.g., 10-8 M to 10-12 M, e.g., 10-9 M to 10-10 M). In some embodiments, the anti-PD-L1 antibody binds to antigenic epitopes conserved in PD-L1 of various species.

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

[0128] The linker is preferably a peptide sequence, more preferably (G 4 S) n contains G, or consists of (G 4 S) n 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 first polypeptide chains and two second 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 light chain or variable heavy chain of the first antigen-binding domain, or the C-terminus of the constant light chain, or the C-terminus of IgG.

[0139] The linker is preferably a peptide sequence, more preferably, it contains (G 4 S) n G, or consists of (G 4 S) n 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] The fourth aspect of the present invention provides an isolated nucleic acid encoding the anti-CD40 antibody according to the first aspect of the present invention or the bispecific antibody according to the second or third aspect of the present invention.

[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. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0148] The 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 backbone of the plasmid is pcDNA3.4.

[0151] The term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and when the vector contacts a cell under conditions sufficient for the expression of the mRNA, protein, polypeptide, or peptide intracellularly, the construct enables the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors of the present disclosure are generally not naturally occurring. However, a portion of the vector may be a naturally occurring substance. The recombinant expression vectors of the present invention include, but are not limited to, DNA and RNA that may be single-stranded or double-stranded, synthetic, or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vector may contain naturally occurring, non-naturally occurring, or both types of nucleotide linkages. In an exemplary aspect, the modified nucleotides or non-natural nucleotide linkages do not inhibit transcription or replication of the vector.

[0152] The recombinant expression vector of the present invention can be any suitable recombinant expression vector for delivering one or more target genes or sequences to any suitable host cell and preferably for use in transformation or transfection to express said gene or sequence in the host cell. Suitable vectors include vectors designed for amplification and propagation, or expression, or both. Examples of vectors include viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, including, but not limited to, certain eukaryotic cells such as production cells.

[0153] The 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 transformant 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 cell is, for example, an EXPI-293 cell or a CHO cell.

[0157] As used herein, the term "host cell" refers to any type of cell that may contain a nucleic acid or vector described herein. The host cell may be a eukaryotic cell such as a plant, animal, fungus, or alga, or may be a prokaryotic cell such as a bacterium or protozoan. As described herein, the host cell may be a cell derived from or obtained from an individual. The host cell may be derived from or obtainable from a mammal. As used herein, the term "mammal" refers to any mammal including, but not limited to, mammals of the order Rodentia such as mice and hamsters, and mammals of the order Lagomorpha such as rabbits. Preferably, the mammal is derived from the order Carnivora including the families Felidae (cats) and Canidae (dogs). More preferably, the mammal is derived from the order Artiodactyla including the families Bovidae (cattle) and Suidae (pigs), or the order Perissodactyla including the family Equidae (horses). Most preferably, the mammal belongs to the order Primate, the New World monkeys (Ceboids) or the Simoid order (monkeys), or the order Anthropoids (humans and apes). Particularly preferably, the mammal is a human.

[0158] The expression vector can be transfected or introduced into a suitable host cell. To achieve this purpose, various techniques can be used, such as 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 gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, the cells are cultured in a medium and screened for the appropriateness of their activity. The methods and conditions for culturing the generated 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 the methods known from this specification and the prior art. Also, cells with stably integrated DNA into the chromosome can be selected by introducing one or more markers that enable the selection of transfected host cells. The markers can provide, for example, prototrophy for auxotrophic hosts, biocidal resistance (e.g., antibiotics), or heavy metal (e.g., copper) resistance. The selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional components may be required for optimal synthesis of mRNA. These components may include splicing signals, and transcriptional promoters, enhancers, and termination signals.

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

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

[0161] Preferably, the pharmaceutical composition further comprises other agents, and in some embodiments, the other agents are selected from one or more of the group consisting of hormonal preparations, target 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] "Pharmaceutically acceptable carrier" refers to any of the conventionally used carriers, limited only by physicochemical considerations (such as solubility and lack of reactivity with the CD40-targeted antibody) and subject to limitations of the route of administration. Pharmaceutically acceptable carriers such as solvents, adjuvants, excipients, and diluents described herein are well known to those skilled in the art and are generally readily available. In one aspect, a pharmaceutically acceptable carrier is one that is chemically inert to the active ingredient of the pharmaceutical composition and has no harmful side effects or toxicity under the conditions of use. In some embodiments, when administered to an animal or a human, the carrier does not cause an adverse reaction, an allergic reaction, or other inappropriate reaction. In some embodiments, the pharmaceutical composition does not contain pyrogens or other impurities that may be harmful to humans or animals. Pharmaceutically acceptable carriers include any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., and their uses are well known in the art.

[0163] Therapeutic formulations of the compositions suitable for practicing 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 A. R. Gennaro, Mack Publishing Company (1990)). The pharmaceutical compositions can be prepared by mixing with one or more appropriate carriers or adjuvants such as water, mineral oil, polyethylene glycol, starch, talc, lactose, thickening agents, stabilizers, suspending agents, and the like. Such compositions may be in the form of solutions, suspensions, tablets, capsules, creams, ointments, pastes, or other conventional forms.

[0164] Compositions for in vivo administration should be sterile. This can be readily accomplished by filtration through sterile filtration membranes either before or after lyophilization and reconstitution. Therapeutic compositions generally can be placed in a container having a sterile access port, for example, a sealed vial or bag with a stopper into which a hypodermic injection needle can be inserted. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In some circumstances, the form must be sterile and fluid to the extent that easy injection is possible. These are stable under the conditions of preparation and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. Compositions for parenteral administration are generally stored in a lyophilized form or in solution form.

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

[0166] The pharmaceutical composition of the present invention may contain any pharmaceutically acceptable components such as acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anti-caking agents, anti-freezing agents, antibacterial preservatives, antioxidants, antiseptics, matrices, binders, buffers, chelating agents, coating agents, coloring agents, desiccants, detergents, diluents, disinfectants, disintegrants, dispersants, solubilizing agents, dyes, emollients, emulsifiers, emulsion stabilizers, fillers, film-forming agents, fragrances, flavorings, flow promoters, gelling agents, granulating agents, heat-insulating agents, lubricants, mucoadhesives, ointment bases, ointments, oily solvents, organic bases, tablet bases, pigments, plasticizers, abrasives, preservatives, sequestering agents, skin penetration enhancers, solubilizers, solvents, stabilizers, suppository bases, surface active agents, surfactants, suspending agents, sweetening agents, therapeutic agents, thickening agents, isotonic agents, toxic agents, tackifiers, water absorbents, water-miscible co-solvents, water softeners or wetting agents.

[0167] In some embodiments, the pharmaceutical composition comprising the bispecific antibody described herein is formulated for parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intrathecal administration or intraperitoneal administration. In other embodiments, the pharmaceutical composition is administered by nasal administration, spray administration, oral administration, aerosol administration, rectal administration, or vaginal administration. The composition can be administered via infusion, bolus injection, or 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 herein, parenteral administration includes, but is not limited to, injection or infusion into the vein, artery, muscle, brain, ventricle of the brain, heart, subcutaneous, intraosseous, intradermal, subarachnoid space, intraperitoneal, retrobulbar, intrapulmonary, intravesical, and intracavernous penis. Administration by surgical implantation at a specific site is also contemplated.

[0170] Injectable formulations are contemplated by the present invention. Those skilled in the art are well aware of the requirements for an effective pharmaceutical carrier for an injectable composition (see, e.g., "Pharmaceutics and Pharmacy Practice", J.B. Lippincott Company, Philadelphia, edited by Banker and Chalmers, pages 238-250 (1982), and "ASHP Handbook on Injectable Drugs", Toissel, 4th ed., pages 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 inclusion complexes such as cyclodextrin inclusion complexes or liposomes.

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

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

[0174] In a preferred embodiment of the present invention, the tumor is, but not limited to, lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric 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 the anti-CD40 antibody described in the first aspect of the present invention, the bispecific antibody described in the second or third aspect, or the pharmaceutical composition described in the eighth aspect of the present invention.

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

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

[0178] the medicine box A contains the anti-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,

[0179] the medicine box B contains another anti-tumor antibody or a pharmaceutical composition containing the other anti-tumor antibody, and / or one or more of the group consisting of hormone preparations, target small molecule preparations, proteasome inhibitors, contrast agents, diagnostic agents, chemotherapeutic agents, tumor lysing agents, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0180] The twelfth aspect of the present invention provides a method for immunoassay or measuring CD40 and / or PD-L1, which includes 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 only suitable for scientific research purposes.

[0182] The 13th aspect of the present invention provides a method for preventing and / or treating tumors, which comprises administering to a patient in need thereof a therapeutically effective amount of the CD40 antibody described in the 1st aspect of the present invention, the bispecific antibody described in the 2nd aspect or the 3rd aspect, and / or the pharmaceutical composition described in the 8th aspect of the present invention, or the medicine box set described in the 11th 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, gastric cancer, colorectal cancer, bladder cancer, rhabdomyosarcoma, esophageal cancer, cervical cancer, multiple myeloma, leukemia, gallbladder cancer, glioblastoma or melanoma.

[0184] As used in the present invention, the term "effective amount" 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. Further, the term "therapeutically effective amount" refers to an amount that provides an improvement in the treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or reduces the rate of progression of a disease or condition, as compared to a corresponding subject not administered such amount. The term also includes amounts effective to enhance normal physiological functions.

[0185] The 14th aspect of the present invention provides a combination therapy which comprises administering to a patient in need thereof the CD40 antibody described in the 1st aspect of the present invention, the bispecific antibody described in the 2nd aspect or the 3rd aspect, and / or the pharmaceutical composition described in the 8th aspect of the present invention and a second therapeutic agent.

[0186] The second therapeutic agent preferably comprises one or more of the group consisting of other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and / or hormonal preparations, target small molecule preparations, proteasome inhibitors, contrast agents, diagnostic agents, chemotherapeutic agents, tumor lysing agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0187] The 15th aspect of the present invention provides the anti-CD40 antibody described in the 1st aspect of the present invention as a medicament, the bispecific antibody described in the 2nd or 3rd aspect, and / or the pharmaceutical composition described in the 8th aspect of the present invention. In some technical solutions, the medicament is used for the prevention and / or treatment of tumors.

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

[0189] For example, the tumor is lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric 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 an antibody-drug conjugate comprising the anti-CD40 antibody described in the 1st aspect of the present invention, the bispecific antibody described in the 2nd or 3rd aspect, which is conjugated with one or more therapeutic agents or radioisotopes.

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

[0193] Based on the common general knowledge 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, 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 acts on the positive feedback pathway of the interaction between DC cells and T cells by stimulating CD40 and simultaneously blocking PD-L1 / PD-1, maximizing the anti-tumor effect. In addition, PD-L1-dependent CD40 activation improves the selectivity of CD40 activation and reduces the toxicity and side effects of the CD40 agonist antibody.

Brief Description of Drawings

[0195]

Figure 1

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Figure 4A

Figure 4B

Figure 4C

Figure 5

Figure 6

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Figure 8

Figure 9

Figure 10A

Figure 10B

Figure 10C

Figure 10D

Figure 11A

Figure 11B

Figure 11C

Figure 12A

Figure 12B

Figure 13

Figure 14

Modes for Carrying Out the Invention

[0196] The present invention will be further described by the following examples, but the present invention is not limited to the scope of these examples. For the experimental methods without specific conditions shown in the following examples, the conventional methods and conditions or the product specifications should be selected. The reagents without specific sources indicated are conventional reagents that can be purchased on the market.

[0197] Example 1 Construction of CD40 antibody and PD-L1 antibody phage libraries Based on the crystal structure of human CD40 protein, the variable region sequence of human CD40 antibody was designed, and the single-chain Fv sequence (single chain Fv) (VL-G4S linker-VH) sequence was obtained by gene synthesis technology. It 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 SS320 E. coli (purchased from Lucigen) grew to the logarithmic growth phase, helper phage (M13K07, purchased from NEB) was added for infection, cultured overnight, and phages were extracted from the culture supernatant to obtain a CD40 antibody phage library.

[0198] Based on the crystal structure of human PD-L1 protein, the variable region sequence of human PD-L1 nanobody was designed, and the VHH sequence was obtained by gene synthesis technology. It was 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 SS320 grew to the logarithmic growth phase, helper phage was added for infection, cultured overnight, and 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] Using protein-based phage antibody panning technology, three rounds of panning of the phage display library were performed. Screening method for one round: 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 added to the same volume of 2% skim milk premix for culturing, the premix was added to the coated wells for reaction, and after washing with sterile PBST to remove the premix, the phage adsorbed to the well plate was eluted using 75 mM sodium citrate buffer. After neutralizing the phage library, the screened phage library was amplified 100-fold using M13K07 helper phage. Subsequently, the second and third rounds of screening were performed in the same manner as the first round. After three rounds of screening, a concentrated phage library was obtained. The concentration of phage was monitored by the initial phage dosage at the start of each round of screening and the phage titer collected after screening.

[0201] Using the concentrated CD40 phage library and the PD-L1 phage library, SS320 Escherichia coli was infected with each of them, and then spread on an agarose plate and cultured. Monoclonal colonies were selected, transferred to a 96-well deep well plate, and cultured with shaking at 37 °C in 2YT medium containing ampicillin and kanamycin to obtain a supernatant containing monoclonal phage. The monoclonal phage supernatant was cultured for 1 hour in an ELISA plate coated with human CD40-mFc antigen or human PD-L1-mFc antigen, then washed with sterile PBST, and then anti-M13-HRP (purchased from Sino biological) was added and cultured at 4 °C for 30 minutes. Next, a microplate reader was used to detect the binding of the phage to the antigen, and CD40 monoclonal phages and PD-L1 monoclonal phages with high binding affinity to the antigen were screened.

[0202] Example 3 Preparation of Recombinant CD40 Monoclonal Antibody and Preparation of Recombinant PD-L1 Monoclonal Antibody The cDNA sequences of the heavy and light chain variable regions of the screened CD40 monoclonal phages were cloned into the pcDNA3.4 vector (Invitrogen) that already contains the antibody constant region, respectively, that is, a plurality of anti-CD40 monoclonal antibodies were obtained and named 1605CD, 1606CD, 1607CD, 1608CD, 1609CD, 1652CD, 1653CD, 1654CD, 1655CD, respectively. The recombinant plasmids of the heavy and light chains were co-transfected into EXPI-293 cells (Invitrogen) using the PEI method, transiently transfected for 7 to 10 days, centrifuged, and the supernatant was collected. The supernatant was purified by protein A to obtain a purified anti-CD40 monoclonal antibody. The CDR sequences of the CD40 antibody were referred to Table 7 (determined by the Kabat CDR system), and the amino acid sequences of the heavy chain variable region and the light chain variable region were referred to Table 8.

[0203] As an example, the full-length amino acid sequence of 1654CD is as follows.

[0204] Heavy chain amino acid sequence of 1654CD (SEQ ID NO: 94): EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVGFIRNKANAYTTEYAASVKGRFTISRDNSKSTLYLQMNRLRAEDTAVYYCARYGGLKVGWYFDLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

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

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

[0207] Heavy chain amino acid sequence of 1606CD (SEQ ID NO: 96): EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVGFIRNKANGYTTEYAASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYGGLRQGWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0208] The light chain amino acid sequence of 1606CD (SEQ ID NO: 81): DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRRDSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGKTLPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0209] The full-length amino acid sequence of 1652CD is as follows.

[0210] The heavy chain amino acid sequence of 1652CD (SEQ ID NO: 87): EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVGFIRNKANAYTTEYAASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYGGLRQGWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0211] Amino acid sequence of the light chain of 1652CD (SEQ ID NO: 81): DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRRDSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGKTLPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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

[0213] Table 2 CDR Sequences of PD-L1 Monoclonal Antibodies

Table 2

[0214] Table 3 FR Sequences of PD-L1 Monoclonal Antibodies

Table 3-1

Table 3-2

[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] Here, X 41 is either D or E, X 42 is either S or T, X 43 is either K or Q, X 44 is either G or S, X 45 is either S or T, X 46 is either D or E, X 47 is either K or N, X 48 is either L or F, X 49is A or S, X 50 is K or R, X 51 is A or T.

[0218] Table 5 Amino acid sequences of the linker and constant region of the PD-L1 monoclonal antibody [Table 5]

[0219] Table 6 Full-length amino acid sequences of the PD-L1 monoclonal antibody [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0220] Example 4 Preparation of recombinant anti-PD-L1 / CD40 bispecific antibody The recombinant anti-PD-L1 / CD40 bispecific antibody used the PD-L1 monoclonal antibody 1541 and the CD40 monoclonal antibodies 1605CD, 1606CD, 1607CD, 1608CD, 1609CD, 1652CD, 1653CD, 1654CD, 1655CD.

[0221] The cDNA sequences of the heavy and light chain variable regions of the screened CD40 monoclonal phages were cloned into the pcDNA3.4 vector (Invitrogen) that already contains the antibody constant region. The cDNA sequence (VHH) of the variable region of the screened PD-L1 monoclonal phage was cloned into the pcDNA3.4 vector (Invitrogen) that already contains CD40-VH and the antibody constant region. The VHH sequence was incorporated into the C-terminus of the antibody constant region, and a (G4S) linker was introduced between the constant region and VHH. That is, multiple anti-PD-L1 / CD40 bispecific antibodies (the structure is as shown in Figure 7) were obtained and named 1605, 1606, 1607, 1608, 1609, 1652, 1653, 1654, 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 the light chain variable region are shown in Table 8, and the amino acid sequences of the heavy and light chains of the bispecific antibodies are shown in Table 9. The recombinant plasmids of the heavy and light chains were co-transfected into EXPI-293 cells (Invitrogen) using the PEI method, transiently transfected for 7 to 10 days, centrifuged, and the supernatant was collected. The supernatant was purified by Protein A to obtain the purified anti-PD-L1 / CD40 bispecific antibody.

[0222] Table 7 CDR Sequences of Bispecific Antibodies

Table 7-1

Table 7-2

Table 7-3

Table 7-4

[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 8-3

Table 8-4

Table 8-5

Table 8-6

[0224] Here, the consensus sequence of the heavy chain variable region of Anti-CD40: EVQLVESGGGLVQPGGSLRLSCAASGFTFSX 19 YYMSWVRQAPGKGLEWVX 20 FIRNKANX 21 YTTEYAASVKGRFTISRDNSKX 22 TLYLQMNX 23 LRAEDTAVYYCARYGGX 24 X 25 X 26 GWYFDX 27 WGQGTLVTVSS (SEQ ID NO: 64): X 19 is N or D, X 20 is G or A, X 21 is A or G, X 22 is N or S, X 23 is R or S, X 24 is L or I, X 25 is K or R, X 26 is V, K, I or Q, X 27 is L or V.

[0225] The consensus sequence of the light chain variable region of Anti-CD40 (SEQ ID NO: 65): DIQMTQSPSSLSASVGDRVTITCRASQX 28 IX29 X 30 YLX 31 WYQQKPGKAPKLLIYX 32 X 33 SX 34 X 35 X 36 SGVPSRFSGSGSGTDYTLTISSLQPEDFATYX 37 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 the heavy and light chains of the bispecific antibody

Table 9-1

Table 9-2

Table 9-3

Table 9-4

Table 9-5

Table 9-6

Table 9-7

Table 9-8

Table 9-9

Table 9-10

Table 9-11

Table 9-12

Table 9-13

Table 9-14

Table 9-15

Table 9-16

Table 9-17

Table 9-18

[0227] Example 5 Detection of the Binding between Recombinant CD40 Monoclonal Antibody and CD40 The binding affinity and species specificity of monoclonal antibodies against human CD40 were detected using enzyme-linked immunosorbent assay (ELISA). Specific method: Using carbonate buffer at pH = 9.6, antigens human CD40, cynomolgus monkey CD40, rat CD40, mouse CD40 (all purchased from ACROBiosystems) were each coated at 1 μg / mL, 100 μL / well in an ELISA plate and cultured overnight at 4°C. Washed 5 times with PBST. Blocked with PBST containing 1% BSA at 300 μL / well and cultured at room temperature for 1 hour. Washed 5 times with PBST. Added monoclonal antibodies serially diluted with PBST containing 1% BSA, and added control CD40 monoclonal antibody CP-870893 (IMGT database ID 10523) at 100 μL / well and cultured at room temperature for 1 hour. Washed 5 times with PBST. Added HRP-labeled anti-human IgG antibody (Jackson ImmunoResearch, catalog number: 109-035-088) diluted with PBST containing 1% BSA at 100 μL / well and cultured at room temperature for 1 hour. Washed 5 times with PBST. Added colorimetric substrate TMB at 100 μL / well, allowed to develop color at room temperature for 10 minutes, and then added 1 M sulfuric acid to stop the reaction. Read OD 450nm using a microplate reader, analyzed the results, and calculated EC 50 using a four-parameter fitting binding curve.

[0228] The results are as shown in Table 10. Monoclonal antibodies 1605CD, 1606CD, 1607CD, 1608CD, 1609CD, 1652CD, 1653CD, 1654CD, 1655CD could all bind to human CD40 and cynomolgus monkey CD40, and the binding affinities for human antigen and cynomolgus monkey antigen were 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 Influence of anti-CD40 monoclonal antibody on CD40 / CD40L binding Using HEK-Blue CD40L cells (purchased from InvivoGen) that highly express CD40, the inhibitory effect of monoclonal antibody on CD40 / CD40L binding was tested. Specific method: HEK-Blue CD40L cells were collected and resuspended in pre-cooled PBS containing 2% FBS at a density of 1×10 7 / mL. After that, 50 μL / well, that is, 5×10 5 cells / well were added to a 96-well plate. Then, biotin-CD40L (FutureGen Biopharmaceutical) and serially diluted CD40 monoclonal antibody were added at 50 μL / well. The final concentration of biotin-CD40L was 10 nM, and the cells were cultured at 4°C for 1 hour. They were washed twice with pre-cooled PBS. Streptavidin-PE (BioLegend, catalog number: 405203) diluted with 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 with a flow analyzer.

[0231] The results are as shown in Table 11. Monoclonal antibodies 1605CD, 1606CD, 1607CD, 1608CD, 1609CD, 1652CD, 1653CD, 1654CD, 1655CD inhibited the interaction of CD40 / CD40L, while the control CD40 monoclonal antibody CP-870893 had no effect on the interaction of CD40 / CD40L.

[0232] Table 11 Influence of monoclonal antibody on CD40 / CD40L binding

Table 11

[0233] Example 7 Detection of CD40 agonist activity of anti-CD40 monoclonal antibody by reporter gene method The CD40 agonist activity of monoclonal antibodies was detected using HEK-Blue CD40L. HEK-Blue CD40L cells were purchased from InvivoGen, highly expressed CD40, and highly expressed the SEAP reporter gene under the control of the NF-κB response element. When CD40 on HEK-Blue CD40L cells was activated, the activation of downstream signal transduction NF-κB was induced, thereby inducing the production of SEAP. The activation status of CD40 can be monitored by detecting the amount of secreted SEAP using the QUANTI-Blue reagent (InvivoGen). Specific method: HEK-Blue CD40L cells were collected, resuspended in complete culture medium (RPMI 1640 containing 10% FBS) at 3×10 5 / mL, and spread evenly in a 96-well plate at 100 μL / well, that is, 3×10 4 cells / well. Anti-CD40 monoclonal antibody samples and control antibody (CP-870893) diluted in gradient with complete medium were added at 100 μL / well, and cultured in an incubator at 37°C and 5% CO 2 for 20 - 24 hours. After culturing, the 96-well plate was taken out, centrifuged at 300 g for 5 minutes, 40 μL / well of the supernatant was transferred to a new 96-well plate, 160 μL / well of the QUANTI-Blue reagent was added to the supernatant, and cultured in an incubator at 37°C and 5% CO 2 for 20 - 30 minutes, and OD 655nm was read on a microplate and the results were analyzed.

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

[0235] Example 8 DC Regulatory Activity of Anti-CD40 Monoclonal Antibodies The dendritic cell (DC) regulatory activity of the CD40 monoclonal antibody was tested.

[0236] 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 carbon dioxide incubator at 37 °C for 2 hours. The culture supernatant and floating cells were discarded, and the adherent cells were monocytes. The monocytes were cultured in complete medium containing 100 ng / mL of GM-CSF (PeproTech, catalog number: 300 - 03) and 100 ng / mL of IL-4 (PeproTech, catalog number: 200 - 04) for 6 days, with the liquid being changed every 2 days to obtain imDC cells. The imDC cells were collected, resuspended in complete medium, seeded in a 24-well plate, and 100 nM of the antibody sample and a control antibody (CP-870893) were added. The culture plate was placed in a carbon dioxide incubator at 37 °C and cultured for 2 days. After culturing, the supernatant was taken out from the well, and cytokine IL-12 / IL-23 p40 (R&D, catalog number: DY1240) was detected according to the kit manual. At the same time, the cells in the well plate were collected, and after culturing with the test antibody (APC anti-human CD83 antibody, Biolegend, 305312), the CD83 expression on DC cells was detected by flow cytometry.

[0237] Figure 2 compares the DC regulatory activity of the anti-CD40 monoclonal antibody of the present invention, and 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 After activating DC cells approved by the CD40 agonist antibody, activating T cells is the most important anti-tumor biological effect. The T cell regulatory activity of the CD40 monoclonal antibody was tested using an MLR experimental system cultured with allogeneic DC cells and T cells.

[0239] DC cells were obtained in the same manner as in Example 8. Allogeneic T cells were obtained by separating from human PBMC cells. For the specific separation method, refer to the instruction manual of the Pan T cell isolation kit (Miltenyi Biotech, catalog number: 130-096-535). Briefly, first, PBMC was washed once with PBS, and PBMC was resuspended with a separation buffer (PBS containing 2 mM EDTA and 0.5% BSA, pH = 7.2) at 1E7 cells / 40 μL (the following usage amounts are counted based on 1E7 cells), 10 μL of Pan T cell Biotin Antibody Cocktail was added, and cultured at 4°C for 5 minutes. Next, 30 μL of separation buffer and 20 μL of Pan T cell Biotin Antibody Cocktail were added, and cultured at 4°C for 10 minutes. T cells were obtained by passing through a MACS separation column.

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

[0241] The results are as shown in Figure 3. Both the potent CD40 agonist monoclonal antibody 1606CD and the weak CD40 agonist monoclonal antibody 1654CD of the present invention can enhance T cell activation under conditions where the proportion of DC is low, while the control monoclonal antibody CP-870893 basically had no effect.

[0242] Example 10 Preliminary Safety Evaluation of Anti-CD40 Monoclonal Antibody The preliminary safety evaluation of the anti-CD40 monoclonal antibody was conducted in human CD40 gene knock-in mice. The experimental animals were purchased from Biocytogen and randomly divided into four groups (5 animals / group), and were administered with vehicle control, 21 mg / kg monoclonal antibody 1606CD, 21 mg / kg monoclonal antibody 1654CD, and 21 mg / kg control CD40 monoclonal antibody CP-870893, respectively. They were injected intraperitoneally twice a week for 2 consecutive weeks, for a total of 4 injections. During the test, the clinical symptoms, body weight, liver function biochemistry, and blood cell count of the animals were monitored. After the end of the administration period, all animals were euthanized as planned on the day after the last administration, dissected, the presence of abnormalities was observed, and the organ weights were measured.

[0243] As shown in FIGS. 4A to 4C, no significant abnormalities were observed in the mice repeatedly injected with the strong CD40 agonist monoclonal antibody 1606CD and the weak CD40 agonist monoclonal antibody 1654CD, but a decrease in lymphocytes and granulocytes was observed in the blood test. On the other hand, in the mice repeatedly injected with the monoclonal antibody CP-870893, a decrease in body weight, an increase in ALT, and significant decreases in RBC, HGB, and PLT were observed, and significant organ necrosis and organ hypertrophy were observed during dissection. The results suggested that the monoclonal antibodies 1606CD and 1654CD were safer than the monoclonal antibody CP-870893.

[0244] Example 11: Detection of the binding between the recombinant PD-L1 monoclonal antibody and PD-L1 The binding affinity and species specificity of monoclonal antibodies against human PD-L1 were detected using enzyme-linked immunosorbent assay (ELISA). Specific method: Using carbonate buffer at pH = 9.6, antigens human PD-L1, cynomolgus PD-L1, rat PD-L1, and mouse PD-L1 (all purchased from ACROBiosystems) were each coated at 1 μg / mL, 100 μL / well in an ELISA plate and cultured overnight at 4°C. Washed 5 times with PBST. Blocked with PBST containing 1% BSA at 300 μL / well and cultured at room temperature for 1 hour. Washed 5 times with PBST. Monoclonal antibodies serially diluted with PBST containing 1% BSA were added, and the control PD-L1 monoclonal antibody durvalumab (IMGT database ID 10010) was added at 100 μL / well and cultured at room temperature for 1 hour. 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 cultured at room temperature for 1 hour. Washed 5 times with PBST. Colorimetric substrate TMB was added at 100 μL / well, allowed to develop color at room temperature for 10 minutes, and then 1 M sulfuric acid was added to stop the reaction. The OD 450nm was read using a microplate reader, the results were analyzed, and the EC 50 was calculated using a four-parameter fitting binding curve.

[0245] The results are as shown in Table 12. Monoclonal antibodies 1029, 1031, 1102, and 1541 were all able to bind to human PD-L1 and cynomolgus PD-L1, and the binding affinities for human and cynomolgus antigens were the same as those of the control antibody. They could not bind to rat PD-L1 and mouse PD-L1.

[0246] Table 12 Binding of Monoclonal Antibodies to Antigens

Table 12

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

[0248] The results are as shown in Table 13. The binding rate constants of monoclonal antibodies 1029, 1031, 1102, and 1541 against human PD-L1 were in the range of 4.37 - 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 Antibodies by Reporter Gene Assay Jurkat / PD-1-NFAT-luciferase cells (highly expressing the luciferase reporter gene under the control of PD-1 and NFAT response elements) and WIL2S / PD-L1 (highly expressing PD-L1) cells constructed by FutureGen Biopharmaceutical, and an anti-CD20 / CD3 bispecific antibody were used to establish a reporter gene detection method for PD-L1 / PD-1 inhibitory activity. Specific method: WIL2S / PD-L1 cells were collected and seeded at a cell density of 4×10 6 / mL in complete medium (RPMI 1640 containing 10% FBS) at 50 μL / well, that is, 2×10 5Resuspend in cells / well, spread evenly on a 96-well plate, collect Jurrkat-PD-1-NFAT-luciferase cells, and 6 resuspend in complete medium (RPMI 1640 containing 10% FBS) at a cell density of 4×10 5 / mL, i.e., 50 μL / well, that is, 2×10 2 resuspend in cells / well, spread evenly on a 96-well plate, dilute the anti-CD20 / CD3 bispecific antibody in complete medium, add 25 μL / well to the above 96-well plate, serially dilute the anti-PD-L1 monoclonal antibody sample and the control antibody in complete medium, add 25 μL / well to the above 96-well plate, and then incubate in a 37 °C, 5% CO

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

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

[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 floating cells were discarded, and the adherent cells were monocytes. The monocytes were cultured 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) for 6 days, with the medium changed every 2 days. Furthermore, TNFα and IL-1β (both purchased from PeproTech) were added and cultured for 2 days to obtain DC cells.

[0254] Allogeneic T cells were isolated from the provided human PBMC cells. For the specific isolation method, refer to the instruction manual of the Pan T cell isolation kit (Miltenyi Biotech, catalog number: 130-096-535). Briefly, first, the PBMCs were washed once with PBS, and then the PBMCs were resuspended in 1×10 7 cells / 40 μL of separation buffer (PBS containing 2 mM EDTA and 0.5% BSA, pH = 7.2) (the following usage amount is based on the count of 1×10 7 cells), 10 μL of Pan T cell biotin antibody cocktail was added, and cultured at 4°C for 5 minutes. Furthermore, 30 μL of separation buffer and 20 μL of Pan T cell biotin antibody cocktail were added, and cultured at 4°C for 10 minutes. The T cells were obtained by passing through a MACS separation column.

[0255] The human DC cells and human T cells were collected, resuspended in complete medium (RPMI 1640 containing 10% FBS), seeded in a 96-well plate, and the seeded DC cells and T cells were seeded at 1×10 4 / well and 1×10 5 / well, respectively, and co-cultured. Antibody samples serially diluted with complete medium were added. The culture plate was placed in a 37°C carbon dioxide incubator and cultured for 5 days. After the culture was completed, the supernatant of the well was taken out, and the cytokine IFN-γ (Biolegend, catalog number: 430101) was detected according to the kit manual.

[0256] The results were as shown in Figure 6, and the monoclonal antibody significantly enhanced the activation of T cells.

[0257] Example 14 Detection of the Binding of Recombinant Anti-PD-L1 / CD40 Bispecific Antibody to PD-L1 and CD40 Enzyme-linked immunosorbent assay (ELISA) was used to detect the binding affinity and species specificity of the bispecific antibody for human PD-L1 and human CD40. Specific method: Using carbonate buffer at pH = 9.6, the antigens human PD-L1, cynomolgus monkey PD-L1, rat PD-L1, mouse PD-L1, human CD40, cynomolgus monkey CD40, rat CD40, mouse CD40 (all purchased from SINOBiological) were each coated at 1 μg / mL, 100 μL / well in an ELISA plate and cultured overnight at 4°C. Washed 5 times with PBST. Blocked with PBST containing 1% BSA at 300 μL / well and cultured at room temperature for 1 hour. Washed 5 times with PBST. The bispecific antibody serially diluted with PBST containing 1% BSA was added, and the 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 cultured at room temperature for 1 hour. 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 cultured at room temperature for 1 hour. Washed 5 times with PBST. Colorimetric substrate TMB was added at 100 μL / well, allowed to develop color at room temperature for 10 minutes, and then 1 M sulfuric acid was added to stop the reaction. OD 450nm was read using a microplate reader, the results were analyzed, and EC 50 was calculated using a four-parameter fitting binding curve.

[0258] The results are as 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, cynomolgus monkey PD-L1 and CD40, and had the same binding affinity for human and cynomolgus monkey antigens. They were unable to bind to rat PD-L1 and CD40, and mouse PD-L1 and CD40.

[0259] Table 14 Binding of Bispecific Antibodies to Antigens

Table 14

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

[0261] The results are as shown in Figure 8. Bispecific antibodies 1605, 1606, 1607, 1608, 1609, 1652, 1653, 1654, and 1655 can all bind to human PD-L1 and human CD40 simultaneously, but there was no signal in the control monoclonal antibody, and it could not bind to two antigens simultaneously.

[0262] Example 16 Detection of PD-L1 / PD-1 Inhibitory Activity of Anti-PD-L1 / CD40 Bispecific Antibody by Reporter Gene Assay Jurkat / PD-1-NFAT-luciferase cells (highly expressing the luciferase reporter gene under the control of PD-1 and NFAT response elements) and WIL2S / PD-L1 cells (highly expressing PD-L1) were constructed by lentiviral transfection with reference to the methods 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.). Using Jurkat and WIL2S cells and anti-CD20 / CD3 bispecific antibodies, a reporter gene detection method for PD-L1 / PD-1 blocking activity was established. Specific method: Collect WIL2S / PD-L1 cells, resuspend them in complete medium (RPMI 1640 containing 10% FBS) at a cell density of 4E6 / mL at 50 μL / well, that is, 2E5 cells / well, spread them evenly on a 96-well plate. Collect Jurkat-PD-1-NFAT-luciferase cells, resuspend them in complete medium (RPMI 1640 containing 10% FBS) at a cell density of 4E6 / mL at 50 μL / well, that is, 2E5 cells / well, and spread them evenly on the above 96-well plate. Dilute the anti-CD20 / CD3 bispecific antibody with complete medium and add it to the above 96-well plate at 25 μL / well. Gradient dilute the anti-PD-L1 / CD40 bispecific antibody sample and the control antibody with complete medium and add them to the above 96-well plate at 25 μL / well. Incubate in a 37 °C, 5% CO 2 incubator for 6 hours. After the culture, add 50 μl / well of one-glo reagent (Promega, catalog number: E6120) to the above 96-well plate, place it on a plate shaker and shake for 5 minutes, then leave it for 10 minutes. Next, use a microplate reader (MD, SpectraMax iD3) to read the relative chemiluminescence unit value (RLU) using the chemiluminescence module and analyze the results.

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

[0264] Example 17 Detection of CD40 agonist activity of anti-PD-L1 / CD40 bispecific antibody by reporter gene method HEK-Blue CD40L was used to measure the CD40 agonist activity of the bispecific antibody. HEK-Blue CD40L cells were purchased from InvivoGen and highly expressed the CD40 and SEAP reporter gene under the control of the NF-κB response element. When CD40 on HEK-Blue CD40L cells was activated, the activation of downstream signaling NF-κB was induced, thereby inducing the production of SEAP. The activation status of CD40 was monitored by detecting the amount of SEAP secreted using the QUANTI-Blue reagent (InvivoGen). In the presence of PD-L1, the CD40 agonist activity of the bispecific antibody was tested by culturing CHO / PD-L1 cells with HEK-Blue CD40L cells. Specific method: HEK-Blue CD40L cells were collected and resuspended in complete medium (RPMI 1640 containing 10% FBS) at 3E5 / mL, evenly spread at 100 μL / well in a 96-well plate, that is, 3E4 cells / well. CHO / PD-L1 cells were collected and resuspended in complete medium (RPMI 1640 containing 10% FBS) at 6E5 / mL at 50 μL / well, that is, 3E4 cells / well, and added to a 96-well plate. If CHO / PD-L1 cells were not required, 50 μL / well of complete culture medium was added. Anti-PD-L1 / CD40 bispecific antibody samples and control antibodies serially diluted with complete medium were added at 50 μL / well, and incubated at 37 °C, 5% CO 2It was cultured in an incubator for 20 to 24 hours. After the culture was completed, the 96-well plate was taken out, centrifuged at 300 g for 5 minutes, 40 μL / well of the supernatant was aspirated and transferred to a new 96-well plate, and 160 μL / well of QUANTI-Blue reagent was added to the supernatant, and then incubated at 37 °C in 5% CO 2 It was cultured in an incubator for 20 to 30 minutes, and OD was measured with a microplate 655nm and the results were analyzed.

[0265] The results are as shown in FIGS. 10A to 10D and Table 15. The CD40 agonist activities of the bispecific antibodies 1608, 1609, 1654, and 1655 were significantly enhanced by the PD-L1 cross-linking signal provided by CHO / PD-L1, indicating that the activities of these bispecific antibodies have selectivity (low activity in an environment with low PD-L1 expression (e.g., blood), and high activity in an environment with high PD-L1 expression (e.g., tumor environment)), showing a therapeutic effect of enhancing drug efficacy and suppressing side effects. However, the 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 activities of bispecific antibodies

Table 15

[0267] Example 18 Influence of anti-PD-L1 / CD40 bispecific antibodies on CD40 / CD40L binding Using HEK-Blue CD40L cells that highly express CD40, the blocking effect of bispecific antibodies on CD40 / CD40L binding was tested. Specific method: 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, that is, 5E5 cells / well. Then, biotin-CD40L (FutureGen Biopharmaceutical) and serially diluted bispecific antibodies were added at 50 μL / well. The final concentration of biotin-CD40L was 10 nM, and the cells were cultured at 4°C for 1 hour. They were washed twice with pre-cooled PBS. Streptavidin-PE (BioLegend, catalog number: 405203) diluted with 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 with a flow analyzer.

[0268] The results are as shown in Table 16. Bispecific antibodies 1605, 1606, 1607, 1608, 1609, 1652, 1653, 1654, 1655 blocked the CD40 / CD40L interaction, while the control CD40 monoclonal antibody CP-870893 had no effect on the CD40 / CD40L interaction.

[0269] Table 16 Influence 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 into 10-cm cell culture dishes, and cultured in a carbon dioxide incubator at 37°C for 2 hours. The culture supernatant and floating cells were discarded, and the adherent cells were monocytes. The monocytes were cultured 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) for 6 days, and the liquid was changed every 2 days to obtain imDC cells. The imDC cells were collected, resuspended in complete medium, seeded into 24-well plates, and gradient-diluted bispecific antibody samples and control antibodies were added. The culture plates were placed in a carbon dioxide incubator at 37°C and cultured for 2 days. After the culture, the supernatant was taken out 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, the cells in the well plates were collected simultaneously, cultured with detection antibodies (APC anti-human CD83 antibody, Biolegend, 305312, PE / Cyanine7 anti-human CD86 antibody, Biolegend, 374210), and the expressions of CD83 and CD86 on DC cells were detected by flow cytometry.

[0272] Figure 11A compares the DC regulatory activities of different bispecific antibodies, with IL-12 / IL-23 p40 as the index. The bispecific antibodies stimulated DCs to secrete IL-12 p40 in a concentration-dependent manner, and the activities of different bispecific antibodies were significantly different. Figures 11B and 11C compare the DC regulatory activities of the parental PD-L1 monoclonal antibody, the parental CD40 monoclonal antibody, the bispecific antibody (1609), and the control CD40 monoclonal antibody CP-870893. The detection indices are CD83 and IL-12 / IL-23 p40. The data show that the parental PD-L1 monoclonal antibody has no control over DCs, the activity of the parental CD40 monoclonal antibody is weak, and after constructing the bispecific antibody, its DC activity is significantly enhanced.

[0273] Example 20 T Cell Regulatory Activity of Anti-PD-L1 / CD40 Bispecific Antibody Using an MLR experimental system cultured with allogeneic DC cells and T cells, the T cell regulatory activity of the bispecific antibody was tested.

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

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

[0276] The results are as 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 the PD - L1 control monoclonal durvalumab.

[0277] Example 21 In vivo antitumor effect of anti - mouse PD - L1 / mouse CD40 bispecific antibody In this example, the anti-tumor effect of the bispecific antibody in the mouse body was detected. To more simply evaluate the in vivo efficacy of the anti-PD-L1 / CD40 bispecific antibody, an anti-mouse PD-L1 / mouse CD40 bispecific antibody 1058, which is an alternative expressing the bispecific antibody, was constructed (the anti-PD-L1 sequence is from IMGT database ID 9814, and the anti-CD40 sequences are No.33_VH and No.34_VL of WO2018185045A1). The purpose of using this alternative is to evaluate the in vivo effect of the bispecific antibody formed by the antibody targeting PD-L1 and the antibody targeting CD40 in wild-type mice, and it is only used to verify the efficacy of such bispecific antibodies.

[0278] C57BL / 6 mice, female, 6 - 8 weeks old, were selected and purchased from Beijing Vital River. After the mice adapted to the environment for 1 week, 3E5 MC38 mouse colon cancer cells (purchased from Basic Medical Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were seeded into each mouse. When the tumor volume grew to about 100 mm 3 , the mice were grouped according to the tumor volume, with 8 mice in each group, and were set as the solvent control group, the anti-mouse PD-L1 monoclonal antibody administration group, the anti-mouse CD40 monoclonal antibody administration group, and the anti-mouse PD-L1 / mouse CD40 bispecific antibody group, respectively. They were intraperitoneally injected at a dose of 35 nmol / kg once a week for 2 consecutive weeks. From the administration day, the tumor volume was measured three times a week, and its major axis a and minor axis b were measured, and the tumor volume (mm 3 ) = (a × b 2 ) / 2 was calculated.

[0279] The results are as shown in Figure 13. The anti-mouse PD-L1 / mouse CD40 bispecific antibody 1058, which is a substitute for the bispecific antibody, significantly inhibited the growth of MC38 colon cancer xenograft tumors in the mouse body, showed a good anti-tumor effect, and its 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 anti-tumor effect of anti-PD-L1 / CD40 bispecific antibody In this example, the anti-tumor effect of the anti-PD-L1 / CD40 bispecific antibody in PD-L1 / CD40 humanized mice was evaluated. The experiment was completed with the cooperation of China Agricultural University. PD-L1 / CD40 humanized mice, female, 6-8 weeks old, were purchased from Biocytogen. After the mice adapted to the environment for 1 week, 5×10^6 MC38 / hPD-L1 mouse colon cancer cells were seeded into each mouse. When the tumor volume grew to about 100 mm 3 , the mice were grouped according to the tumor volume, with 6 mice in each group. The solvent, anti-PD-L1 monoclonal antibody, anti-CD40 monoclonal antibody, and anti-PD-L1 / anti-CD40 bispecific antibody 1654 were intraperitoneally injected twice a week for 2 consecutive weeks. From the administration day, the clinical manifestations of the mice, the body weight of the mice, and the tumor volume were monitored. The tumor was measured for its major axis a and minor axis b, and the tumor volume (mm 3 ) = (a × b 2 ) / 2 was calculated.

[0281] The results are as shown in Figure 14. 1654 significantly inhibited the growth of MC38 / hPD-L1 transplanted tumors in the PD-L1 / CD40 humanized mouse body. The tumor growth inhibition rate (TGI) reached 72% at a low dose of 7 nmol / kg, which was stronger than that of the PD-L1 monoclonal antibody or CD40 monoclonal antibody at a dose of 20 nmol / kg, and the TGI of both was 28%. During the experiment, 1 mouse in the solvent control group died, but no abnormalities were found in the other mice. The body weight of the mice was normal.

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

[0283] The experimental animals used were male cynomolgus monkeys, which were randomly divided into 5 groups (2 animals / group), and were administered with solvent control, 12 mg / kg of bispecific antibodies 1607, 1608, 1609, and 10 mg / kg of control CD40 monoclonal CP-870893, respectively. A syringe pump was used to perform subcutaneous intravenous injection into the forelimbs or hindlimbs of the animals at a dose of 10 mL / kg and a dosing rate of 0.5 mL / kg / min. Administration was carried out once a week for 2 consecutive weeks, for a total of 3 times. During the test, the clinical symptoms, body weight, food intake, body temperature, blood cell count, coagulation function, blood biochemistry and urine tests, immune cell phenotype, cytokines, blood drug concentration and anti-drug antibodies of the animals were detected regularly. After the end of the administration period, on the day after the final administration, all surviving animals in groups 2-5 were euthanized as planned, macroscopic dissection was performed to observe the presence of abnormalities, and the weights of the organs were measured.

[0284] In the 12 mg / kg dose groups of bispecific antibodies 1607, 1608, and 1609, no abnormal changes related to the test product were observed in the clinical observation, body weight, weight gain, food intake, body temperature, blood cell count, coagulation function, blood biochemistry, urine test, organ weight and macroscopic pathological dissection of the animals. Only an increase in the phenotype of immune cells and some cytokines was observed, which was related to the pharmacological mechanism of action. However, in the 10 m / kg dose group of the CD40 monoclonal antibody CP-870893, a small amount of loose stools / soft stools were observed in the animals on D7-D8, D10-D11, and D7-D13, respectively. The weight gain on D14 was slightly decreased compared with that before drug administration. The food intake was significantly decreased after D5. RBC, HGB, HCT, and PLT showed a decreasing trend. The phenotype of immune cells changed, some cytokines increased. During the dissection of cynomolgus monkeys, the organ weight, organ-body weight ratio, and organ-brain ratio of the thymus decreased, while the organ weight, organ-body weight ratio, and organ-brain ratio of the spleen increased, showing significant changes.

[0285] The results suggested that the anti-PD-L1 / CD40 bispecific antibody was safer than the CD40 monoclonal antibody.

Claims

1. comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 at SEQ ID NO: 64, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 at SEQ ID NO: 65, Preferably, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, and the LCDR2 is X ５ X ６ SX ７ X ８ X ９ comprises the amino acid sequence shown in S, where X ５ is Y or A, and X ６ is T or A, and X ７ is S, R or T, and X ８ is L or R, and X ９ is Q or D. The LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

6. An anti-CD40 antibody.

2. the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 8, SEQ ID NO: 7 or SEQ ID NO: 10, the LCDR2 comprises 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, the LCDR3 comprises 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, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24, and the HCDR3 comprises 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, preferably, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 14, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 23, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 28, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 14, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 24, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 28, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 7, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 23, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 25, The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 7, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 12, the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 17, the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 22, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 24, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 26, The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 7, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 12, the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 17, the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 22, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 23, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 26, The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 8, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 13, the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 18, the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 22, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 24, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 27, The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 8, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 13, the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 18, the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 22, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 23, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 27, The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 10, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 15, the LCDR3 contains the amino acid sequence shown in SEQ ID NO: 20, the HCDR1 contains the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 24, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 29, or, 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: 23, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:

29. More preferably, the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 9, 14, and 19, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 21, 23, and 28, respectively. The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 9, 14, and 19, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 21, 24, and 28, respectively. The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 7, 11, and 16, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 21, 23, and 25, respectively. 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. 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. The light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 8, 13, and 18, respectively, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 22, 24, and 27, respectively. The light chain variable region contains 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 contains 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 contains 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 contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 29 respectively, or The antibody according to claim 1, wherein the light chain variable region contains 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 contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 29 respectively.

3. 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. Preferably, the light chain variable region contains 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 contains 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. The light chain variable region contains 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 contains an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence homology with SEQ ID NO:

31. The light chain variable region contains 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 contains 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. 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: 35, 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: 36 or SEQ ID NO:

37. Or 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: 41, 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: 42 or SEQ ID NO:

43. More preferably, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 40 or SEQ ID NO:

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

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

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

43. Even more preferably, the antibody comprises the light chain variable region shown in SEQ ID NO: 38 and the heavy chain variable region shown in SEQ ID NO:

40. Preferably, the variable region of the amino acid sequence having at least 90%, at least 95% or at least 99% sequence homology maintains the same antigen-binding function as the original sequence. The antibody according to claim 2.

4. The antibody is (1) The antibody is a full-length antibody, Fab, Fab′, F(ab′) ２ or Fv, and the Fv is preferably a condition of scFv, (2) The condition that the antibody is a monospecific antibody or a multispecific antibody, (3) The condition that the antibody is a monoclonal antibody or a polyclonal antibody prepared from the above antibody, The antibody according to claim 3, characterized by satisfying one or more of the three.

5. The antibody comprises a heavy chain constant region and / or a light chain constant region. Preferably, 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. More preferably, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 45, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

44. Most preferably, 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, or 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, or 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, the antibody according to claim 4.

6. 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 as defined in the anti-CD40 antibody according to any one of claims 1 to 5.

7. The second antigen-binding domain comprises at least one VHH, and the VHH comprises VHH-CDR1, VHH-CDR2 and VHH-CDR3 of SEQ ID NO:

66. Preferably, the VH H-CDR1 is X ４１ YYX ４２ X ４３ contains the amino acid sequence shown in C, where X ４１ is D or E, X ４２ is S or T, X ４３ is K or Q, the VH H-CDR2 contains the amino acid sequence shown in SEQ ID NO: 76, and the VH H-CDR3 contains the amino acid sequence shown in SEQ ID NO: 77 More preferably, the VHH comprises VHH-CDR1, VHH-CDR2 and VHH-CDR3 of SEQ ID NO: 49, SEQ ID NO: 74, SEQ ID NO: 72 or SEQ ID NO:

73. Even more preferably, the VHH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 46, SEQ ID NO: 67 or SEQ ID NO: 70, the VHH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 47, SEQ ID NO: 68 or SEQ ID NO: 71, and the VHH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 48 or SEQ ID NO: 69, the bispecific antibody according to claim 6.

8. The 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. Alternatively, the VH H-CDR1 contains the amino acid sequence shown in SEQ ID NO: 67, VH H-CDR2 contains the amino acid sequence shown in SEQ ID NO: 68, and VH H-CDR3 contains the amino acid sequence shown in SEQ ID NO: 69, Alternatively, the VH H-CDR1 contains the amino acid sequence shown in SEQ ID NO: 70, VH H-CDR2 contains the amino acid sequence shown in SEQ ID NO: 71, and VH H-CDR3 contains the amino acid sequence shown in SEQ ID NO: 48, Preferably, the VH H is VH H-CDR1 whose sequence is shown in SEQ ID NO: 46, VH H-CDR2 whose sequence is shown in SEQ ID NO: 47, and VH H-CDR3 whose sequence is shown in SEQ ID NO: 48, VH H-CDR1 whose sequence is shown in SEQ ID NO: 67, VH H-CDR2 whose sequence is shown in SEQ ID NO: 68, and VH H-CDR3 whose sequence is shown in SEQ ID NO: 69, or VH H-CDR1 whose sequence is shown in SEQ ID NO: 70, VH H-CDR2 whose sequence is shown in SEQ ID NO: 71, and VH H-CDR3 whose sequence is shown in SEQ ID NO: 48, contains More preferably, the amino acid sequence of the VH H 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 homology with SEQ ID NO: 49, SEQ ID NO: 72, SEQ ID NO: 73 or SEQ ID NO:

74. The bispecific antibody according to claim 7.

9. 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 VH H, and the sequence of the VH H is as defined in the bispecific antibody according to claim 7 or claim 8. Preferably, the first antigen-binding domain is as defined in the anti-CD40 antibody according to any one of claims 1 to 5.

10. The first antigen-binding domain and the second antigen-binding domain are operably linked directly or via a linker, Preferably, 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 the C-terminus of the light chain constant region, or the C-terminus of IgG, and the linker is preferably a peptide sequence, more preferably, (G ４ S) ｎ G-containing, or consisting of (G ４ S) ｎ G, where n = 1 to 10, is an integer, for example, n = 3, and the bispecific antibody according to any one of claims 6 to 9, characterized in that

11. Comprising two first polypeptide chains and two second polypeptide chains, wherein The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 56, or has 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: 62 or SEQ ID NO: 57, or has at least 90% sequence homology 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 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 90% sequence homology 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 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 90% sequence homology 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 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 90% sequence homology with SEQ ID NO: 55 or SEQ ID NO: 61, or The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 58, or has 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 90% sequence homology with SEQ ID NO: 59 or SEQ ID NO:

63. The bispecific antibody according to any one of claims 6 to 10, characterized in that it has the above characteristics.

12. An isolated nucleic acid encoding an anti-CD40 antibody according to any one of claims 1 to 5, or a bispecific antibody according to any one of claims 6 to 11.

13. Comprising the isolated nucleic acid according to claim 12, Preferably a plasmid, cosmid, phage or viral vector, More preferably, the backbone of the plasmid is pcDNA3.

4. A recombinant expression vector.

14. A transformant comprising the recombinant expression vector according to claim 13, preferably, the host cell is a prokaryotic cell or a eukaryotic cell, more preferably, the eukaryotic cell is a yeast cell or a mammalian cell, wherein the mammalian cell is, for example, an EXPI-293 cell or a CHO cell.

15. A method for preparing an anti-CD40 antibody or a bispecific antibody, comprising culturing the transformant according to claim 14 and obtaining the anti-CD40 antibody or the bispecific antibody from the culture.

16. Comprising the anti-CD40 antibody according to any one of claims 1 to 5 or the bispecific antibody according to any one of claims 6 to 11, and a pharmaceutically acceptable carrier. Preferably, further comprising other agents, and the other agents are preferably selected from one or more of the group consisting of hormonal preparations, target 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. A pharmaceutical composition.

17. Use of the anti-CD40 antibody according to any one of claims 1 to 5, the bispecific antibody according to any one of claims 6 to 11, and / or the pharmaceutical composition according to claim 16 in the preparation of a medicament for preventing and / or treating tumors. Preferably, the tumor is lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, bladder cancer, rhabdomyosarcoma, esophageal cancer, cervical cancer, multiple myeloma, leukemia, gallbladder cancer, glioblastoma or melanoma.

18. Comprising the anti-CD40 antibody according to any one of claims 1 to 5, the bispecific antibody according to any one of claims 6 to 11, or the pharmaceutical composition according to claim 16. Preferably, further comprising (i) a device for administering the antibody or the pharmaceutical composition, and / or (ii) an instruction manual. A kit.

19. A medicine box set comprising a medicine box A and a medicine box B. The medicine box A contains the anti-CD40 antibody according to any one of claims 1 to 5, the bispecific antibody according to any one of claims 6 to 11, and / or the pharmaceutical composition according to claim 16. The medicine box set B includes one or more of the group consisting of other anti-tumor antibodies or pharmaceutical compositions containing other anti-tumor antibodies, and / or hormonal preparations, target small molecule preparations, proteasome inhibitors, contrast agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

20. A method for immunoassay or measuring CD40 and / or PD-L1, comprising using the anti-CD40 antibody according to any one of Claims 1 to 5, the bispecific antibody according to any one of Claims 6 to 11, and / or the pharmaceutical composition according to Claim 16, and preferably, the detection is for non-diagnostic purposes, a method for immunoassay or measuring CD40 and / or PD-L1.

21. comprising administering a therapeutically effective amount of the anti-CD40 antibody according to any one of Claims 1 to 5, the bispecific antibody according to any one of Claims 6 to 11, and / or the pharmaceutical composition according to Claim 16, or the medicine box set according to Claim 19 to a patient in need thereof, preferably, a method for preventing and / or treating a tumor, characterized in that it is a PD-L1 positive and / or CD40 positive tumor.

22. comprising administering to a patient in need thereof the anti-CD40 antibody according to any one of Claims 1 to 5, the bispecific antibody according to any one of Claims 6 to 11, and / or the pharmaceutical composition according to Claim 16 and a second therapeutic agent, wherein the second therapeutic agent preferably includes one or more of the group consisting of other anti-tumor antibodies or pharmaceutical compositions containing other anti-tumor antibodies, and / or hormonal preparations, target small molecule preparations, proteasome inhibitors, contrast agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines, a combination therapy.

23. The anti-CD40 antibody according to any one of Claims 1 to 5, the bispecific antibody according to any one of Claims 6 to 11, and / or the pharmaceutical composition according to Claim 16 as a medicine. (Preferably, the medicament is used for preventing and / or treating tumors, and more preferably, the tumors are lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, bladder cancer, rhabdomyosarcoma, esophageal cancer, cervical cancer, multiple myeloma, leukemia, gallbladder cancer, glioblastoma or melanoma.)

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