Anti-CD40 antibodies and anti-PD-L1×CD40 bispecific antibodies and their applications

Abstract

The present invention discloses an anti-CD40 antibody and an anti-PD-L1×CD40 bispecific antibody and their applications. The anti-CD40 antibody comprises a heavy chain variable region and a light chain variable region, 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. The new anti-CD40 agonistic antibody of the present invention can effectively regulate the activation of DC cells, has a stronger T cell activation effect, but has low toxic and side effects, and is suitable for use in tumor immunotherapy. The bispecific antibody of the present invention can maximize the anti-tumor efficacy, improve the selectivity of CD40 activation through PD-L 1-dependent CD40 activation, and reduce the toxic and side effects of CD40 agonistic antibodies.

Description

[0001] This application claims priority to Chinese Patent Application No. 2022106137021 filed on May 31, 2022, Chinese Patent Application No. 2022106162803 filed on May 31, 2022, and Chinese Patent Application No. 2023105733440 filed on May 19, 2023. This application incorporates the entirety of the aforementioned Chinese patent applications. Technical Field

[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 applications thereof. Background Art

[0003] In recent years, the field of tumor treatment has developed rapidly, evolving from classic treatments based on surgery, radiotherapy, and chemotherapy to more advanced treatment options such as targeted therapy and immunotherapy. In particular, tumor immunotherapy, represented by PD-L1 / PD-1 inhibitors, has significantly prolonged the survival of tumor patients in multiple tumor indications and has been positioned as a first-line treatment in multiple tumor indications (Nat Rev Immunol. 2020 Nov; 20(11): 651-668.). As the most likely option to cure tumors, tumor immunotherapy has become the central focus of the research and development of new anti-tumor drugs.

[0004] One of the key challenges in tumor immunology is to increase the number and quality of infiltrating T cells in cold tumors. In many cancer patients, insufficient T cell priming is due to the lack of T cells in the tumor microenvironment, and antigen-presenting cells loaded with tumor antigens, especially DCs, play a decisive role in priming T cell responses. CD40, a type I transmembrane protein, belongs to the tumor necrosis factor receptor (TNFR) superfamily. It is widely expressed in various types of cells (such as B cells), platelets, some non-hematopoietic cells, and various types of tumor cells. It plays an important role in both innate and adaptive immunity, and plays a key role in DC cell activation (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.). After CD40 is activated, it activates and authorizes DC cells to promote the activation of anti-tumor specific T cells by upregulating co-stimulatory molecules and MHC on DC cells and inducing pro-inflammatory cytokines. These T cells have the potential to completely eliminate tumor cells.

[0005] In multiple mouse tumor models, CD40 agonistic antibodies can activate T cells and demonstrate robust antitumor efficacy (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 agonistic antibodies can also synergize with immune checkpoint antibodies such as PD-L1 / PD-1 to exert synergistic antitumor effects, which rely on the cross-talk mechanism between DCs and T cells: DCs stimulate tumor-specific T cell activation by upregulating co-stimulatory molecules and secreting IL-12, and after T cell activation, they activate DCs by secreting IFN-γ. CD40 antibodies and PD-L1 / PD-1 antibodies act on different links of cross-reaction, enhancing positive feedback and maximizing anti-tumor efficacy (Cancer Res. 2016 Nov 1; 76(21): 6266-6277. Immunity. 2018 Dec 18; 49(6): 1148–1161.e7.).

[0006] Currently, several biopharmaceutical companies are developing agonist monoclonal antibodies targeting CD40, with related patents such as WO2003040170, WO2014070934A1, US20180066053, US20140348836, WO2020108611, and CN111763259, and several antibodies have entered clinical testing. In a first-in-human single-dose study, Pfizer's CD40 monoclonal antibody, Selicrelumab, produced an objective partial response (PR) in four of 15 patients with advanced melanoma. One of these patients subsequently received repeated doses of Selicrelumab for one year and maintained a complete remission (CR) 15 years later. However, Selicrelumab's efficacy has been poor in subsequent clinical trials, and other CD40 monoclonal antibodies, such as APX005M and SEA-CD40, have also had only very low objective response rates (ORRs). CD40 agonist monoclonal antibodies have shown a number of adverse reactions in clinical practice, including cytokine release syndrome (CRS), liver injury, and thrombocytopenia. The maximum tolerated doses (MTDs) 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, there is an urgent need for a CD40-targeting antibody with high safety and efficacy in this field. Summary of the Invention

[0007] In order to solve the above technical problems, in view of the current status of research and development 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 new anti-CD40 agonistic antibody of the present invention can effectively regulate the activation of DC cells, has a stronger T cell activation effect, but has low toxic side effects, and is suitable for use in tumor immunotherapy; the anti-PD-L1×CD40 bispecific antibody of the present invention acts on the positive feedback pathway of the cross-action between DC cells and T cells by simultaneously stimulating CD40 and blocking PD-L1 / PD-1, thereby maximizing the anti-tumor efficacy; it also improves the selectivity of CD40 activation through PD-L1-dependent CD40 activation and reduces the toxic side effects of CD40 agonistic antibodies.

[0008] In a first aspect, 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 may specifically bind to human CD40 and monkey CD40 (e.g., cynomolgus monkey). Alternatively, the antibodies may be specific for human CD40 and may not exhibit cross-reactivity with other species. CD40 or any variant or isoform thereof may be isolated from cells or tissues naturally expressing them, or produced by recombinant techniques using techniques commonly used in the art and those described herein.

[0010] In some embodiments, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in SEQ ID NO:38, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO:40;

[0011] or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 38, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 39;

[0012] or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as in SEQ ID NO: 30, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as in SEQ ID NO: 31;

[0013] or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as in SEQ ID NO: 32, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as in SEQ ID NO: 33;

[0014] or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as in SEQ ID NO: 32, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as in SEQ ID NO: 34;

[0015] or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as in SEQ ID NO: 35, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as in SEQ ID NO: 36;

[0016] or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as in SEQ ID NO: 35, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as in SEQ ID NO: 37;

[0017] or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as in SEQ ID NO:41, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as in SEQ ID NO:42;

[0018] Or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as in SEQ ID NO:41, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as in SEQ ID NO:43.

[0019] In some embodiments, the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. In some specific embodiments, the CDRs are defined according to the Kabat numbering convention.

[0020] In some embodiments, in the antibody of the first aspect provided by the present invention, the LCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 1, the LCDR2 comprises the amino acid sequence as shown in X5X6SX7X8X9S, wherein X5 is Y or A; X6 is T or A; X7 is S, R or T; X8 is L or R; X9 is Q or D, the LCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 3; the HCDR1 comprises the amino acid sequence as shown in SEQ ID NO: 4, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO: 5, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO: 6.

[0021] In a preferred embodiment of the present invention, the amino acid sequence of the LCDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of the LCDR2 is as shown in X5X6SX7X8X9S, wherein X5 is Y or A; X6 is T or A; X7 is S, R or T; X8 is L or R; and X9 is Q or D; the amino acid sequence of the LCDR3 is as shown in SEQ ID NO: 3; the amino acid sequence of the HCDR1 is as shown in SEQ ID NO: 4, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO: 5, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO: 6.

[0022] According to the antibody of the first aspect of the 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, and 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.

[0023] In a preferred embodiment of the present invention, the antibody comprises an amino acid sequence of LCDR1 as shown in SEQ ID NO:9, SEQ ID NO:8, SEQ ID NO:7 or SEQ ID NO:10, an amino acid sequence of LCDR2 as shown in SEQ ID NO:14, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:11 or SEQ ID NO:15, an amino acid sequence of LCDR3 as shown in SEQ ID NO:19, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:16 or SEQ ID NO:20; an amino acid sequence of HCDR1 as shown in SEQ ID NO:21 or SEQ ID NO:22, an amino acid sequence of HCDR2 as shown in SEQ ID NO:23 or SEQ ID NO:24, and an amino acid sequence of HCDR3 as shown in SEQ ID NO:28, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:25 or SEQ ID NO:29.

[0024] 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, and 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.

[0025] The antibody according to the first aspect of the present invention, in a preferred embodiment of the present invention:

[0026] A) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28;

[0027] B) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:9, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:14, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:19, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:21, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:24, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:28;

[0028] C) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 21, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 25;

[0029] D) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 17, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 26;

[0030] E) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 17, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 26;

[0031] F) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 13, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27;

[0032] G) the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 13, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27;

[0033] H) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 10, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20, 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: 29; or

[0034] 1) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 10, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20, 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: 29.

[0035] In a more preferred embodiment of the present invention:

[0036] A) the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 14, and SEQ ID NO: 19, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 28, respectively;

[0037] B) the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 14, and SEQ ID NO: 19, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 28, respectively;

[0038] C) the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 16, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25, respectively;

[0039] D) the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 12, and SEQ ID NO: 17, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26, respectively;

[0040] E) the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 12, and SEQ ID NO: 17, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 26, respectively;

[0041] F) the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO: 18, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 27, respectively;

[0042] G) the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO: 18, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 27, respectively;

[0043] H) the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 15, and SEQ ID NO: 20, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 29, respectively; or,

[0044] I) the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 15 and SEQ ID NO: 20, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO: 29, respectively.

[0045] In a preferred embodiment of the present invention:

[0046] In some embodiments, in the antibody, the amino acid sequence of the LCDR1 is shown as SEQ ID NO:9, the amino acid sequence of the LCDR2 is shown as SEQ ID NO:14, the amino acid sequence of the LCDR3 is shown as SEQ ID NO:19, the amino acid sequence of the HCDR1 is shown as SEQ ID NO:21, the amino acid sequence of the HCDR2 is shown as SEQ ID NO:23, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO:28.

[0047] In some embodiments, in the antibody, the amino acid sequence of the LCDR1 is shown as SEQ ID NO:9, the amino acid sequence of the LCDR2 is shown as SEQ ID NO:14, the amino acid sequence of the LCDR3 is shown as SEQ ID NO:19, the amino acid sequence of the HCDR1 is shown as SEQ ID NO:21, the amino acid sequence of the HCDR2 is shown as SEQ ID NO:24, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO:28.

[0048] In some embodiments, in the antibody, the amino acid sequence of the LCDR1 is shown as SEQ ID NO:7, the amino acid sequence of the LCDR2 is shown as SEQ ID NO:11, the amino acid sequence of the LCDR3 is shown as SEQ ID NO:16, the amino acid sequence of the HCDR1 is shown as SEQ ID NO:21, the amino acid sequence of the HCDR2 is shown as SEQ ID NO:23, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO:25.

[0049] In some embodiments, in the antibody, the amino acid sequence of the LCDR1 is shown as SEQ ID NO:7, the amino acid sequence of the LCDR2 is shown as SEQ ID NO:12, the amino acid sequence of the LCDR3 is shown as SEQ ID NO:17, the amino acid sequence of the HCDR1 is shown as SEQ ID NO:22, the amino acid sequence of the HCDR2 is shown as SEQ ID NO:24, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO:26.

[0050] In some embodiments, in the antibody, the amino acid sequence of the LCDR1 is shown as SEQ ID NO:7, the amino acid sequence of the LCDR2 is shown as SEQ ID NO:12, the amino acid sequence of the LCDR3 is shown as SEQ ID NO:17, the amino acid sequence of the HCDR1 is shown as SEQ ID NO:22, the amino acid sequence of the HCDR2 is shown as SEQ ID NO:23, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO:26.

[0051] In some embodiments, in the antibody, the amino acid sequence of the LCDR1 is shown as SEQ ID NO:8, the amino acid sequence of the LCDR2 is shown as SEQ ID NO:13, the amino acid sequence of the LCDR3 is shown as SEQ ID NO:18, the amino acid sequence of the HCDR1 is shown as SEQ ID NO:22, the amino acid sequence of the HCDR2 is shown as SEQ ID NO:24, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO:27.

[0052] In some embodiments, in the antibody, the amino acid sequence of the LCDR1 is shown as SEQ ID NO:8, the amino acid sequence of the LCDR2 is shown as SEQ ID NO:13, the amino acid sequence of the LCDR3 is shown as SEQ ID NO:18, the amino acid sequence of the HCDR1 is shown as SEQ ID NO:22, the amino acid sequence of the HCDR2 is shown as SEQ ID NO:23, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO:27.

[0053] In some embodiments, in the antibody, the amino acid sequence of the LCDR1 is shown as SEQ ID NO:10, the amino acid sequence of the LCDR2 is shown as SEQ ID NO:15, the amino acid sequence of the LCDR3 is shown as SEQ ID NO:20, the amino acid sequence of the HCDR1 is shown as SEQ ID NO:21, the amino acid sequence of the HCDR2 is shown as SEQ ID NO:24, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO:29.

[0054] In some embodiments, in the antibody, the amino acid sequence of the LCDR1 is shown as SEQ ID NO:10, the amino acid sequence of the LCDR2 is shown as SEQ ID NO:15, the amino acid sequence of the LCDR3 is shown as SEQ ID NO:20, the amino acid sequence of the HCDR1 is shown as SEQ ID NO:21, the amino acid sequence of the HCDR2 is shown as SEQ ID NO:23, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO:29.

[0055] In some of the above embodiments, the amino acid sequences of the listed CDRs are determined according to the Kabat definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in the art by a variety of methods, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (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 antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US 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. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions defined by any of the above-mentioned known schemes described herein. Various numbering systems corresponding to CDRs are well known to those skilled in the art, as shown in Table 1:

[0056] Table 1 Antibody CDR definition method

[0057]

[0058] Note: In Table 1, Laa-Lbb refers to the amino acid sequence starting from the N-terminus of the antibody light chain and following the corresponding numbering rules, from positions aa to bb; Haa-Hbb refers to the amino acid sequence starting from the N-terminus of the antibody heavy chain and following the corresponding numbering rules, from positions aa to bb. For example, L24-L34 in the second row, second column of Table 1 refers to the amino acid sequence starting from the N-terminus of the antibody light chain variable region and following the Kabat numbering rules, from residues 24 to 34; and so on.

[0059] As for the antibody according to the first aspect of the present invention, the framework region of the light chain variable region is a human framework region, and the framework region of the heavy chain variable region is a human framework region.

[0060] The antibody according to the first aspect of the present invention, in a preferred embodiment of the present invention:

[0061] a) the light chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 38, and the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 40 or SEQ ID NO: 39;

[0062] b) the light chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 30, and the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 31;

[0063] c) the light chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 32, and the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 33 or SEQ ID NO: 34;

[0064] d) the light chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 35, and the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 36 or SEQ ID NO: 37; or

[0065] e) the light chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:41, and the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:42 or SEQ ID NO:43.

[0066] 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 identity maintains the same antigen (eg, human CD40) binding function as the original sequence.

[0067] The calculation of sequence identity between sequences is as follows. For determining the percent identity of two amino acid sequences, the sequences are compared for optimal comparison purposes (for example, rooms can be introduced in the first and second amino acid sequences for optimal comparison or non-homologous sequences can be abandoned for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the compared reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. Subsequently, the amino acid residues at corresponding amino acid positions are compared. When the position in the first sequence is occupied by the same amino acid residue at the corresponding position in the second sequence, the molecules are identical at this position. A mathematical algorithm can be utilized to realize the sequence comparison and the calculation of percent identity between two sequences. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred parameter set (and the one that should be used unless otherwise specified) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4. Additionally or alternatively, a protein sequence described in the present invention can be used as a "query sequence" to perform a search against public databases to, for example, identify other family member sequences or related sequences.

[0068] The antibody according to the first aspect of the present invention, in a more preferred embodiment of the present invention:

[0069] 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;

[0070] 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;

[0071] 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;

[0072] 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

[0073] 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.

[0074] The antibody according to the first aspect of the present invention, in a further preferred embodiment of the present invention:

[0075] In some embodiments, the antibody comprises a light chain variable region as set forth in SEQ ID NO:38 and a heavy chain variable region as set forth in SEQ ID NO:40.

[0076] In some embodiments, the antibody comprises a light chain variable region as set forth in SEQ ID NO:38 and a heavy chain variable region as set forth in SEQ ID NO:39.

[0077] In some embodiments, the antibody comprises a light chain variable region as set forth in SEQ ID NO:30 and a heavy chain variable region as set forth in SEQ ID NO:31.

[0078] In some embodiments, the antibody comprises a light chain variable region as set forth in SEQ ID NO:32 and a heavy chain variable region as set forth in SEQ ID NO:33.

[0079] In some embodiments, the antibody comprises a light chain variable region as set forth in SEQ ID NO:32 and a heavy chain variable region as set forth in SEQ ID NO:34.

[0080] In some embodiments, the antibody comprises a light chain variable region as set forth in SEQ ID NO:35 and a heavy chain variable region as set forth in SEQ ID NO:36.

[0081] In some embodiments, the antibody comprises a light chain variable region as set forth in SEQ ID NO:35 and a heavy chain variable region as set forth in SEQ ID NO:37.

[0082] In some embodiments, the antibody comprises a light chain variable region as set forth in SEQ ID NO:41 and a heavy chain variable region as set forth in SEQ ID NO:42.

[0083] In some embodiments, the antibody comprises a light chain variable region as set forth in SEQ ID NO:41 and a heavy chain variable region as set forth in SEQ ID NO:43.

[0084] In a preferred embodiment of the present invention:

[0085] 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 or SEQ ID NO:39.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] In a more preferred embodiment of the present invention:

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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:37.

[0098] 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.

[0099] 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:43.

[0100] As described in the first aspect of the present invention, the antibody satisfies one or more of the following three items: (1) the antibody is a full-length antibody, Fab, Fab', F(ab')2 or Fv, and the Fv is preferably scFv; (2) the antibody is a monospecific antibody or a multispecific antibody; (3) the antibody is a monoclonal antibody or a polyclonal antibody prepared from the above antibodies.

[0101] The antibodies of the present invention include monoclonal antibodies (abbreviated as mAb or Ab), which refer to antibodies obtained from a single clonal cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage clonal cell lines.

[0102] The antibody according to the first aspect of the present invention comprises a heavy chain constant region and / or a light chain constant region.

[0103] In some embodiments, the heavy chain constant region of the antibody is derived from the heavy chain constant region of a human antibody IgG1, IgG2, IgG3, or IgG4, and / or the light chain constant region of the antibody is derived from the κ chain of a human antibody.

[0104] In some embodiments, the constant region includes a constant region variant that does not change the structure and function of the antibody variable region. A variety of such constant region variants have been disclosed in the prior art, for example, the Fc of the heavy chain constant region of an antibody has one or more amino acids substituted at 238, 265, 269, 270, 297, 327 and 329 (using the EU numbering system) (U.S. Patent No. 6,737,056), or the Fc of the heavy chain constant region of an antibody has one or more amino acids substituted at 234, 235, 265, 329 (using the EU numbering system) , or the Fc of the heavy chain constant region of the antibody has one or more amino acid substitutions at positions 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434 (using the EU numbering system) (see U.S. Patent No. 7,371,826), etc. These mutations have been shown to confer new properties on antibodies without changing the function of the antibody variable region.

[0105] In a preferred embodiment of the present invention, 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.

[0106] In a more preferred embodiment of the present invention, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:94, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:95.

[0107] In a more preferred embodiment of the present invention, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:96, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:81.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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 according to the first aspect of the present invention.

[0114] The term "antibody" as used herein is used in the broadest sense, encompassing monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, and tandem tri-scFv), as well as traditional antibodies (antibodies with a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds), as well as Fab, Fab', F(ab')2, Fv, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies, and domain antibodies (heavy chain (VH) antibodies, light chain (VL) antibodies) with antigen-binding activity. Traditional antibodies (also called "full-length antibodies" or "complete antibodies") are typically heterotetrameric glycoproteins of approximately 150,000 daltons, which are composed of a tetrapeptide chain structure composed of two identical light chains (L) and two identical heavy chains (H) linked by interchain disulfide bonds. Each heavy chain of a full-length antibody is composed of a heavy chain variable region (abbreviated as VH in this invention) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL in this invention) and a light chain constant region (abbreviated as CL in this invention). The light chain constant region is composed of one domain, CL. Mammalian heavy chains are divided into α, δ, ε, γ, and μ heavy chains. Mammalian light chains are divided into λ or κ light chains. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are immunoglobulins (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of the Y is composed of the second and third constant regions of the two heavy chains (and the fourth constant region for IgE and IgM) bound together, and disulfide bonds (between chains) are formed in the hinge. The heavy chains γ, α, and δ have a constant region consisting of three tandem (in a row) Ig domains and a hinge region for increased flexibility; the heavy chains μ and ε have a constant region consisting of four immunoglobulin domains. The second and third constant regions are called "CH2 domains" and "CH3 domains," respectively. Each arm of the Y includes the variable region of a single heavy chain bound to a single light chain and the first constant region (CH1). The "Fc" region is two heavy chain fragments comprising the CH2 and CH3 domains of an antibody, held together by two or more disulfide bonds and the hydrophobic interaction of the CH3 domain.Various Fc constant region variants have been disclosed in the prior art, such as an antibody heavy chain constant region Fc having one or more amino acid substitutions at positions 238, 265, 269, 270, 297, 327, and 329 (using the EU numbering system) ( U.S. Patent No. 6,737,056 ), or an antibody heavy chain constant region Fc having one or more amino acid substitutions at positions 234, 235, 265, and 329 (using the EU numbering system). , or the Fc of the heavy chain constant region of the antibody has one or more amino acid substitutions at positions 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434 (using the EU numbering system) (see U.S. Patent No. 7,371,826), etc. These mutations have been shown to confer new properties on antibodies without changing the function of the antibody variable region.

[0115] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain involved in antigen binding. VHH, VH, and VL each contain four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region of the variable domain that primarily contributes to antigen binding; "framework" or "FR" refers to the variable domain residues excluding the CDR residues. A VH or VHH contains three CDR regions: HCDR1, HCDR2, and HCDR3 for VHH, and VHH-CDR1, VHH-CDR2, and VHH-CDR3 for VHH. The VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen binding specificity. As used herein, the terms "VHH" and "nanoantibody" have the same meaning and are used interchangeably, referring to the variable region of the heavy chain of a cloned antibody, a nanoantibody consisting of only one heavy chain variable region, which has complete antigen binding function. VHH specific binding epitopes do not require other antigen binding domains to recognize together (this is different from conventional tetrapeptide chain structure antibodies, in which the epitopes of conventional tetrapeptide chain structure antibodies are 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. Nanoantibodies have excellent biological properties, with a molecular weight of 12-15kDa, which is one-tenth of that of a complete antibody, and have good tissue penetration, high specificity and good water solubility. Due to its special structural properties, it combines the advantages of traditional antibodies and small molecule drugs, almost perfectly overcoming the defects of traditional antibodies such as long development cycle, low stability and harsh storage conditions, and gradually becoming an emerging force in the new generation of antibody therapy, showing broad application prospects in immune diagnosis and treatment. VHHs include, but are not limited to, natural antibodies produced by camelids, antibodies produced by camelids that have been humanized, or antibodies obtained through phage display technology. Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in, for example, the following literature: 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.

[0116] The term "PD-L1" includes any variant or isoform of PD-L1 naturally expressed by cells. The antibodies of the present invention may cross-react with PD-L1 of non-human species (such as cynomolgus monkeys). Alternatively, the antibody may be specific for human PD-L1 and may not exhibit cross-reactivity with other species. PD-L1 or any variant or isoform thereof may be isolated from cells or tissues that naturally express them, or produced by recombinant techniques using techniques commonly used in the art and those described herein.

[0117] In the bispecific antibody of the present invention, preferably, the second antigen-binding domain comprises at least one VHH, wherein the VHH comprises VHH-CDR1, VHH-CDR2 and VHH-CDR3 in SEQ ID NO: 66. In some embodiments, the VHH-CDR1 comprises 41 YYX 42 X 43 C, wherein is D or E, is S or T, is K or Q, the VHH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 76, and the VHH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 77.

[0118] In some embodiments, the VHH comprises the 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.

[0119] In some embodiments, the VHH-CDR1, VHH-CDR2, and VHH-CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. In some specific embodiments, the VHH-CDR1, VHH-CDR2, and VHH-CDR3 are determined according to the Kabat numbering convention. In some embodiments, the VHH-CDR1 comprises the amino acid sequence of SEQ ID NO: 46, SEQ ID NO: 67, or SEQ ID NO: 70, the VHH-CDR2 comprises the amino acid sequence of SEQ ID NO: 47, SEQ ID NO: 68, or SEQ ID NO: 71, and the VHH-CDR3 comprises the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 69.

[0120] In some embodiments, the VHH comprises a VHH-CDR1 as shown in SEQ ID NO:46, SEQ ID NO:67 or SEQ ID NO:70, a VHH-CDR2 as shown in SEQ ID NO:47, SEQ ID NO:68 or SEQ ID NO:71, and a VHH-CDR3 as shown in SEQ ID NO:48 or SEQ ID NO:69.

[0121] In a more preferred embodiment of the present invention, the VHH-CDR1 comprises the amino acid sequence shown in SEQ ID NO:46, the VHH-CDR2 comprises the amino acid sequence shown in SEQ ID NO:47, and the VHH-CDR3 comprises the amino acid sequence shown in SEQ ID NO:48;

[0122] Or, 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;

[0123] 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.

[0124] In some embodiments, the VHH comprises:

[0125] The VHH-CDR1 sequence is shown in SEQ ID NO:46, the VHH-CDR2 sequence is shown in SEQ ID NO:47, and the VHH-CDR3 sequence is shown in SEQ ID NO:48;

[0126] The VHH-CDR1 sequence is shown in SEQ ID NO: 67, the VHH-CDR2 sequence is shown in SEQ ID NO: 68, and the VHH-CDR3 sequence is shown in SEQ ID NO: 69,

[0127] Or, the VHH-CDR1 sequence is shown in SEQ ID NO:70, the VHH-CDR2 sequence is shown in SEQ ID NO:71 and the VHH-CDR3 sequence is shown in SEQ ID NO:48.

[0128] 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 identity with the amino acid sequence shown in SEQ ID NO:49, SEQ ID NO:72, SEQ ID NO:73 or SEQ ID NO:74.

[0129] In some embodiments, the VHH is a humanized VHH.

[0130] In some embodiments, the VHH comprises framework regions FR1, FR2, FR3, and FR4, wherein the FR1 comprises the amino acid sequence set forth in SEQ ID NO: 85, the FR2 comprises the amino acid sequence set forth in SEQ ID NO: 79, the FR3 comprises the amino acid sequence set forth in SEQ ID NO: 86, and the FR4 comprises the amino acid sequence set forth in SEQ ID NO: 84;

[0131] In some embodiments, the FR1 comprises the amino acid sequence shown in SEQ ID NO:82 or SEQ ID NO:78, the FR2 comprises the amino acid sequence shown in SEQ ID NO:79, the FR3 comprises the amino acid sequence shown in SEQ ID NO:83 or SEQ ID NO:80, and the FR4 comprises the amino acid sequence shown in SEQ ID NO:84.

[0132] In some embodiments, the FR1 comprises the amino acid sequence set forth in SEQ ID NO: 82, the FR2 comprises the amino acid sequence set forth in SEQ ID NO: 79, the FR3 comprises the amino acid sequence set forth in SEQ ID NO: 83, and the FR4 comprises the amino acid sequence set forth in SEQ ID NO: 84;

[0133] Or, the FR1 comprises the amino acid sequence shown in SEQ ID NO: 78, the FR2 comprises the amino acid sequence shown in SEQ ID NO: 79, the FR3 comprises the amino acid sequence shown in SEQ ID NO: 80, and the FR4 comprises the amino acid sequence shown in SEQ ID NO: 84;

[0134] Or, the FR1 comprises the amino acid sequence shown in SEQ ID NO: 82, the FR2 comprises the amino acid sequence shown in SEQ ID NO: 79, the FR3 comprises the amino acid sequence shown in SEQ ID NO: 80, and the FR4 comprises the amino acid sequence shown in SEQ ID NO: 84.

[0135] 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% sequence identity with the amino acid sequence shown in SEQ ID NO:49, SEQ ID NO:72, SEQ ID NO:73 or SEQ ID NO:74.

[0136] 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.

[0137] In some embodiments, the second antigen binding domain further comprises a heavy chain constant region.

[0138] Preferably, the heavy chain constant region is selected from the heavy chain constant region 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.

[0139] More preferably, the VHH is connected to the heavy chain constant region via a linker; the linker is preferably a linker having an amino acid sequence as shown in (G4S)x, wherein x is independently selected from an integer of 1-20, more preferably a linker as shown in SEQ ID NO: 89.

[0140] Even more preferably, the amino acid sequence of the second antigen-binding domain is as 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 identity with the amino acid sequence shown in SEQ ID NO: 93, SEQ ID NO: 90, SEQ ID NO: 91 or SEQ ID NO: 92;

[0141] Most preferably, the second antigen-binding domain has two amino acid sequences as shown in SEQ ID NO:93.

[0142] In the present invention, "Fab" is composed of one light chain and the CH1 and variable regions of one heavy chain. "Fab'" contains one light chain and a portion encompassing the VH domain, CH1 domain, and the region between the CH1 and CH2 domains. The two heavy chains of two Fab' fragments can form an interchain disulfide bond to form a F(ab')2 molecule. A F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.

[0143] In the present invention, the scFv (single chain antibody fragment) refers to a polypeptide chain formed by connecting a VH domain and a VL domain via a linker (also known as a linker). The VL and VH domains are paired to form a monovalent molecule via a linker that enables them to be produced as a single polypeptide chain [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers are composed of repeated G4S amino acid sequences or variants thereof. For example, linkers having the amino acid sequence (G4S)4 or (G4S)3 may be used, but variants thereof may also be used.

[0144] The term "multispecific antibody" is used in its broadest sense to encompass antibodies with two or more epitope specificities, such as bispecific antibodies. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has two or more epitope specificities; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable domains (e.g., VHH), each single variable domain binding to a different target or a different epitope of the same target.

[0145] The term "epitope" refers to an area (area or region) on an antigen that is capable of specific binding to an antibody. An epitope can be formed by a continuous string of amino acids (linear epitope) or contain non-continuous amino acids (conformational epitope), for example, brought into spatial proximity due to the folding of the antigen (i.e., by the tertiary folding of the antigen in a proteinaceous manner). The difference between a conformational epitope and a linear epitope is that the binding of the antibody to the conformational epitope is lost in the presence of a denaturing solvent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed using routine methods in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463),

[0146] The term "specific binding" refers to an antibody that binds to an antigen or an epitope within the antigen with a higher affinity than for other antigens or epitopes. Typically, an antibody binds to an antigen or an epitope within an antigen with an equilibrium dissociation constant (KD) of about 1 x 10-7 M or less (e.g., about 1 x 10-8 M or less, about 1 x 10-9 M or less, about 1 x 10-10 M or less, about 1 x 10-11 M or less, or about 1 x 10-12 M or less). In some embodiments, the KD for antibody binding to an antigen is 10% or 1% of the KD for the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using standard procedures, for example, by However, an antibody that specifically binds to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, for example, to the same antigen from other species (homologous), such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), chimpanzees (Pantroglodytes (chimpanzee, chimp), or marmosets (Callithrix jacchus (common marmoset, marmoset).

[0147] The term "affinity" refers to the overall intensity of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "affinity" refers to internal binding affinity, which reflects 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X to its part Y can generally be represented by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art (including those described herein). The terms "kassoc" or "ka" refer to the on-rate of specific antibody-antigen interactions, and the terms "kdis" or "kd" as used herein refer to the off-rate of specific antibody-antigen interactions. As used herein, the term "KD" refers to a dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and is expressed as molar concentration (M). The KD value of an antibody can be measured using the method well established in this area. Methods for determining antibody KD include measuring surface plasmon resonance using a biosensing system, such as a BIOMEDIC® system, or measuring affinity in solution by solution equilibrium titration (SET).

[0148] The terms "anti-CD40 antibody" and "antibody that specifically binds to CD40" refer to an antibody that binds to CD40 with sufficient affinity to allow the antibody to be used as a diagnostic and / or therapeutic agent targeting CD40. In certain embodiments, the antibody binds to CD40 with 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 certain embodiments, the anti-CD40 antibody binds to an epitope that is conserved among CD40 from different species.

[0149] 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 so that the antibody can be used as a diagnostic and / or therapeutic agent targeting PD-L1. In certain 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 certain embodiments, the anti-PD-L1 antibody binds to an epitope that is conserved in PD-L1 from different species.

[0150] In certain preferred embodiments, the first antigen-binding domain and the second antigen-binding domain are operably linked directly or through a linker.

[0151] Preferably, the second antigen-binding domain is linked to the N-terminus of the light chain variable region or the 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 the IgG.

[0152] The linker is preferably a peptide sequence, more preferably comprises or consists of (G4S) n G, wherein n=1-10 and is an integer, for example, n is 3.

[0153] The bispecific antibody of the present invention contains two first polypeptide chains and two second polypeptide chains.

[0154] In certain preferred embodiments, the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO:50 or has at least 99%, at least 95%, or at least 90% sequence identity with SEQ ID NO:50, and / or the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO:51 or has at least 99%, at least 95%, or at least 90% sequence identity with SEQ ID NO:51.

[0155] In certain preferred embodiments, the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO:52 or has at least 99%, at least 95%, or at least 90% sequence identity with SEQ ID NO:52, and / or the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO:53 or SEQ ID NO:60 or has at least 99%, at least 95%, or at least 90% sequence identity with SEQ ID NO:53 or SEQ ID NO:60.

[0156] In certain preferred embodiments, the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO:54, or has at least 99%, at least 95%, or at least 90% sequence identity with SEQ ID NO:54, and / or the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO:55 or SEQ ID NO:61, or has at least 99%, at least 95%, or at least 90% sequence identity with SEQ ID NO:55 or SEQ ID NO:61.

[0157] In certain preferred embodiments, the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO:56, or has at least 99%, at least 95%, or at least 90% sequence identity with SEQ ID NO:56, and / or the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO:57 or SEQ ID NO:62, or has at least 99%, at least 95%, or at least 90% sequence identity with SEQ ID NO:62.

[0158] In certain preferred embodiments, the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO:58 or has at least 99%, at least 95%, or at least 90% sequence identity with SEQ ID NO:58, and / or the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO:59 or SEQ ID NO:63 or has at least 99%, at least 95%, or at least 90% sequence identity with SEQ ID NO:59 or SEQ ID NO:63.

[0159] 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, the sequence of the VHH being as defined in the bispecific antibody according to the second aspect of the present invention.

[0160] In certain preferred embodiments, the first antigen-binding domain is as defined in the anti-CD40 antibody according to the first aspect of the present invention.

[0161] In certain preferred embodiments, the first antigen-binding domain and the second antigen-binding domain are operably linked directly or through a linker.

[0162] Preferably, the second antigen-binding domain is linked to the N-terminus of the light chain variable region or the 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 the IgG.

[0163] The linker is preferably a peptide sequence, more preferably comprises or consists of (G4S) n G, wherein n=1-10 and is an integer, for example, n is 3.

[0164] The bispecific antibody of the present invention contains two first polypeptide chains and two second polypeptide chains.

[0165] In certain preferred embodiments, the amino acid sequence of the first polypeptide chain is shown as SEQ ID NO: 50, and / or the amino acid sequence of the second polypeptide chain is shown as SEQ ID NO: 51.

[0166] In certain preferred embodiments, the amino acid sequence of the first polypeptide chain is shown as SEQ ID NO: 52, and / or the amino acid sequence of the second polypeptide chain is shown as SEQ ID NO: 53 or SEQ ID NO: 60.

[0167] In certain preferred embodiments, the amino acid sequence of the first polypeptide chain is shown as SEQ ID NO: 54, and / or the amino acid sequence of the second polypeptide chain is shown as SEQ ID NO: 55 or SEQ ID NO: 61.

[0168] In certain preferred embodiments, the amino acid sequence of the first polypeptide chain is shown as SEQ ID NO: 56, and / or the amino acid sequence of the second polypeptide chain is shown as SEQ ID NO: 57 or SEQ ID NO: 62.

[0169] In certain preferred embodiments, the amino acid sequence of the first polypeptide chain is shown as SEQ ID NO: 58, and / or the amino acid sequence of the second polypeptide chain is shown as SEQ ID NO: 59 or SEQ ID NO: 63.

[0170] The fourth aspect of the present invention provides: an isolated nucleic acid encoding the anti-CD40 antibody of the first aspect, or the bispecific antibody of the second aspect or the third aspect of the present invention.

[0171] As known in the art, "nucleic acid" in the present invention refers to a nucleotide chain of any length, and includes DNA and RNA. The nucleotides can 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.

[0172] 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.

[0173] Preferably, the recombinant expression vector is a plasmid, cosmid, phage or viral vector.

[0174] For example, the backbone of the plasmid is pcDNA3.4.

[0175] The term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that permits expression of an mRNA, protein, polypeptide, or peptide by a host cell when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell. The vectors disclosed herein are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the present invention may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthesized or partially obtained from natural sources, and which may contain natural, non-natural, or altered nucleotides. The recombinant expression vector may comprise naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. In exemplary aspects, the altered nucleotides or non-naturally occurring internucleotide linkages do not hinder transcription or replication of the vector.

[0176] The recombinant expression vector of the present invention can be any suitable recombinant expression vector that can be used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably express the genes or sequences in the host cell. Suitable vectors include those designed for expansion and amplification or for expression or both, examples of vectors include but are not limited to 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, and certain eukaryotic cells, such as production cells.

[0177] The sixth aspect of the present invention provides: a transformant comprising the recombinant expression vector described in the fifth aspect of the present invention.

[0178] Preferably, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

[0179] More preferably, the eukaryotic cell is a yeast cell or a mammalian cell.

[0180] The mammalian cells are, for example, EXPI-293 cells or CHO cells.

[0181] As used herein, the term "host cell" refers to any type of cell that can contain a nucleic acid or vector described herein. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or algae; or the host cell can be a prokaryotic cell, such as a bacterium or a protozoan. As described herein, the host cell can be a cell originating from or obtained from an individual. The host cell can be derived from or obtained from a mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to rodents (order Rodentia) mammals, such as mice and hamsters; and lagomorphs (order Lagomorpha) mammals, such as rabbits. Preferably, the mammal is from the order Carnivora, including felines (cats) and canines (dogs). More preferably, the mammal is from the order Artiodactyla, including bovines (cows) and suids (pigs), or belongs to the order Perssodactyla, including equines (horses). Most preferably, the mammal is of the order Primate, Ceboids or Simoids (monkeys) or of the order Anthropoids (humans and apes). A particularly preferred mammal is a human.

[0182] Expression vector can be transfected or introduced into suitable host cells.Multiple techniques can be used to achieve this purpose, for example, 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, cells are cultivated in culture medium and screened for suitable activity. The method and conditions for cultivating the transfected cells produced and for recovering the antibody molecules produced are well known to those skilled in the art and can be based on this specification and prior art known methods, according to the specific expression vector used and mammalian host cell changes or optimization. In addition, one or more markers of the host cell transfected can be selected by introducing, and the cell stably incorporated into its chromosome by DNA can be selected. Markers can, for example, provide prototrophy, biocidal resistance (for example, antibiotics) or heavy metal (such as copper) resistance to auxotrophic hosts. Selectable marker genes can be directly connected to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.

[0183] The seventh aspect of the present invention provides: a method for preparing an anti-CD40 antibody or a bispecific antibody, comprising the following steps: culturing the transformant according to the sixth aspect of the present invention, and obtaining the anti-CD40 antibody or bispecific antibody from the culture.

[0184] 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 aspect or the third aspect, and a pharmaceutically acceptable carrier.

[0185] Preferably, the pharmaceutical composition further contains other agents. In some embodiments, the other agents are selected from one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

[0186] A "pharmaceutically acceptable carrier" is any of those conventionally used and is limited only by physico-chemical considerations (such as solubility and lack of reactivity with the CD40-targeting antibody) and by the route of administration. Pharmaceutically acceptable carriers, such as vehicles, adjuvants, excipients, and diluents described herein are well known to those skilled in the art and are readily available to the public. In one aspect, a pharmaceutically acceptable carrier is a carrier that is chemically inert to the active ingredients of the pharmaceutical composition and does not have adverse side effects or toxicity under the conditions of use. In some embodiments, the carrier does not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. In some aspects, the pharmaceutical composition is free of pyrogens and other impurities that may be harmful to humans or animals. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like; their uses are well known in the art.

[0187] Therapeutic formulations of compositions suitable for practicing the methods disclosed herein, such as polypeptides, polynucleotides, or antibodies, can be prepared for storage by mixing the selected composition having the desired purity with optional physiologically pharmaceutically acceptable carriers, excipients, or stabilizers in the form of a lyophilized cake or aqueous solution (Remington's Pharmaceutical Sciences, 18th edition, edited by A.R. Gennaro, Mack Publishing Company (1990)). Pharmaceutical compositions can be prepared by admixing with one or more suitable carriers or adjuvants, such as water, mineral oil, polyethylene glycol, starch, talc, lactose, thickeners, stabilizers, suspending agents, and the like. Such compositions can be in the form of solutions, suspensions, tablets, capsules, creams, ointments, ointments, or other conventional forms.

[0188] The composition used for in vivo administration should be sterile. This is easily achieved by filtering through a sterile filtration membrane before or after freeze-drying and reconstitution. The therapeutic composition is generally placed in a container with a sterile access port, for example, an intravenous solution bag or a bottle with a stopper that can be pierced by a hypodermic needle. The medical form suitable for injectable purposes includes sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. In some cases, the form should be sterile and should be fluid, to the extent that it can be easily injected. It should be stable under the conditions of manufacture and storage and should be preserved to avoid the contamination of microorganisms such as bacteria and fungi. Compositions for parenteral administration will usually be stored in lyophilized form or in solution form.

[0189] The carrier can be a solvent or dispersion medium containing, for example, water or a suitable mixture thereof and a vegetable oil. Suitable fluidity can be determined, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant to maintain the choice of carrier, which will be determined in part by the particular type of pharmaceutical composition and the route of administration of the pharmaceutical composition. Accordingly, various formulations of suitable pharmaceutical compositions can be prepared.

[0190] The pharmaceutical compositions of the present invention may contain any pharmaceutically acceptable ingredient, including, for example, acidifiers, additives, adsorbents, aerosol propellants, air displacers, alkalizers, anti-caking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffers, chelating agents, coatings, colorants, desiccants, detergents, diluents, disinfectants, disintegrants, dispersants, solubility enhancers, dyes, emollients, emulsifiers, emulsion stabilizers, fillers, film formers, fragrance enhancers, flavorings, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oily vehicles, organic bases, tablet bases, pigments, plasticizers, polishing agents, preservatives, sequestrants, skin penetrants, solubilizers, solvents, stabilizers, suppository bases, surface active agents, agents), surfactants, suspending agents, sweeteners, therapeutic agents, thickeners, tonicity agents, toxicants, viscosity-increasing agents, water-absorbing agents, water-miscible co-solvents, water softeners, or wetting agents.

[0191] In some embodiments, the pharmaceutical compositions comprising the bispecific antibodies described herein are formulated for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, or intraperitoneal administration. In other embodiments, the pharmaceutical compositions are administered nasally, by spray, orally, by aerosol, rectally, or vaginally. The compositions can be administered by infusion, bolus injection, or by implantable device.

[0192] Topical formulations are well known to those skilled in the art. Such formulations are particularly suitable for application to the skin in the context of the present invention.

[0193] In some embodiments, the pharmaceutical compositions described herein are formulated for parenteral administration. For purposes herein, parenteral administration includes, but is not limited to, intravenous, intraarterial, intramuscular, intracerebral, intracerebroventricular, intracardiac, subcutaneous, intraosseous, intradermal, intrathecal, intraperitoneal, retrobulbar, intrapulmonary, intravesical, and intracavernous injection or infusion. Administration by surgical implantation at a specific site is also contemplated.

[0194] Injectable formulations are according to the present invention. Those of ordinary skill in the art are familiar with the requirements for effective pharmaceutical carriers for injectable compositions (see, for example, Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, Pennsylvania, eds. Banker and Chalmers, pp. 238-250 (1982); and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pp. 622-630 (1986)).

[0195] Those skilled in the art will appreciate that, in addition to the above-mentioned pharmaceutical compositions, the compositions of the present invention can be formulated into inclusion compounds, such as cyclodextrin inclusion compounds, or liposomes.

[0196] The ninth aspect of the present invention provides: use of the anti-CD40 antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect or the 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 preventing and / or treating tumors.

[0197] The tumor is preferably a PD-L1 positive and / or CD40 positive tumor.

[0198] In a preferred embodiment of the present invention, 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, but is not limited thereto.

[0199] The tenth aspect of the present invention provides: a kit comprising the anti-CD40 antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect or the third aspect, or the pharmaceutical composition according to the eighth aspect of the present invention.

[0200] Preferably, the kit further comprises (i) a device for administering the antibody or pharmaceutical composition; and / or (ii) instructions for use.

[0201] The eleventh aspect of the present invention provides: a set of medicine kits, comprising medicine kit A and medicine kit B, wherein:

[0202] The drug kit A contains the anti-CD40 antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect or the third aspect, and / or the pharmaceutical composition according to the eighth aspect of the present invention;

[0203] The drug kit B contains other anti-tumor antibodies or pharmaceutical compositions comprising the other anti-tumor antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

[0204] The twelfth aspect of the present invention provides: a method for immunodetection or determination of CD40 and / or PD-L1, comprising using the anti-CD40 antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect or the third aspect and / or the pharmaceutical composition according to the eighth aspect of the present invention.

[0205] In a preferred embodiment of the present invention, the detection is a non-diagnostic detection and is only suitable for scientific research purposes.

[0206] The thirteenth aspect of the present invention provides: a method for preventing and / or treating tumors, comprising administering to a patient in need thereof a therapeutically effective amount of the anti-CD40 antibody as described in the first aspect of the present invention, the bispecific antibody as described in the second aspect or the third aspect of the present invention, and / or the pharmaceutical composition as described in the eighth aspect of the present invention, or the kit as described in the eleventh aspect of the present invention.

[0207] 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, but is not limited thereto.

[0208] As used herein, the term "effective amount" refers to an amount of a drug or pharmaceutical agent that elicits the biological or pharmaceutical response of a tissue, system, animal, or human that is being sought, for example, by a researcher or clinician. Additionally, the term "therapeutically effective amount" refers to an amount that results in improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or that reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received that amount. The term also includes within its scope amounts that are effective to enhance normal physiological function.

[0209] The fourteenth aspect of the present invention provides: a combination therapy comprising administering the anti-CD40 antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect or the third aspect of the present invention, and / or the pharmaceutical composition according to the eighth aspect of the present invention, and a second therapeutic agent, respectively, to a patient in need thereof;

[0210] The second therapeutic agent preferably comprises other anti-tumor antibodies or pharmaceutical compositions comprising such other anti-tumor antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0211] The fifteenth aspect of the present invention provides: the anti-CD40 antibody as described in the first aspect of the present invention, the bispecific antibody as described in the second aspect or the third aspect of the present invention and / or the pharmaceutical composition as described in the eighth aspect of the present invention for use as a medicine; in some technical solutions, the medicine is used to prevent and / or treat tumors.

[0212] Preferably, the tumor is PD-L1 positive and / or CD40 positive.

[0213] 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.

[0214] Preferably, the tumor is colon cancer.

[0215] The present invention also provides an antibody-drug conjugate comprising the anti-CD40 antibody of the first aspect, or the bispecific antibody of the second aspect or the third aspect of the present invention conjugated to one or more therapeutic agents or radioisotopes.

[0216] Preferably, the therapeutic agent is a cytotoxic agent, a chemotherapeutic agent, a drug, a growth inhibitory agent and / or a toxin; and / or the conjugation is to link the antibody to the therapeutic agent or a radioisotope using a linker.

[0217] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0218] The novel anti-CD40 agonistic antibodies of the present invention can effectively regulate DC activation, exhibiting stronger T cell activation with minimal toxic side effects, making them suitable for tumor immunotherapy. The bispecific antibodies of the present invention simultaneously activate CD40 and block PD-L1 / PD-1, acting in the positive feedback loop between DC and T cells to maximize anti-tumor efficacy. They also enhance the selectivity of CD40 activation through PD-L1-dependent CD40 activation, thereby reducing the toxic side effects of CD40 agonistic antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0219] Figure 1This is the reporter gene method used in Example 7 of the present invention to determine the CD40 agonist activity of the monoclonal antibody.

[0220] Figure 2 The DC cell regulatory activity of the monoclonal antibody in Example 8 of the present invention is shown, with CD83 being used as a DC activation indicator.

[0221] Figure 3 The T cell regulatory activity of the monoclonal antibody in Example 9 of the present invention is shown, with IFN-γ being used as the T cell activation indicator.

[0222] Figures 4A to 4C The safety of the monoclonal antibody in Example 10 of the present invention in human CD40 gene knock-in mice. Figure 4A is the weight change of mice; Figure 4B for the liver function and hematological changes in mice; Figure 4C The changes in organ coefficients of mice.

[0223] Figure 5 This is the PD-L1 / PD-1 blocking activity of the monoclonal antibody determined by the reporter gene method in Example 12 of the present invention.

[0224] Figure 6 This is the T cell regulatory activity of the monoclonal antibody in Example 13 of the present invention.

[0225] Figure 7 This is the structure of the bispecific antibody in Example 4 of the present invention.

[0226] Figure 8 The bispecific antibody in Example 15 of the present invention simultaneously binds to PD-L1 and CD40.

[0227] Figure 9 This is the PD-L1 / PD-1 blocking activity of the bispecific antibody determined by the reporter gene method in Example 16 of the present invention.

[0228] FIG10 shows the CD40 agonistic activity of the bispecific antibody measured by the reporter gene method in Example 17 of the present invention: Figure 10A : CD40 agonist activity of 1605, 1606, and 1607; Figure 10B : CD40 agonistic activity of 1608 and 1609; Figure 10C :CD40 agonist activity of 1652, 1653; Figure 10D : CD40 agonistic activity of 1654, 1655, and CP-870893.

[0229] FIG11 shows the DC regulatory activity of the bispecific antibody in Example 19 of the present invention: Figure 11A Comparison of the activities of different bispecific antibodies using IL-12p40 as an indicator; Figure 11B and Figure 11CComparison of the activities of the parental mAb and the bispecific antibody, Figure 11B The one shown is CD83, Figure 11C Shown is IL-12p40.

[0230] Figure 12A and Figure 12B This is the T cell regulatory activity of the bispecific antibody in Example 20 of the present invention.

[0231] Figure 13 This is the efficacy of the bispecific antibody in Example 21 of the present invention against MC38 tumors in mice.

[0232] Figure 14 In vivo anti-MC38 / hPD-L1 colon cancer tumor efficacy of an anti-PD-L1 / CD40 bispecific antibody. DETAILED DESCRIPTION

[0233] The present invention is further illustrated by the following examples, but the invention is not limited to the scope of the examples. Experimental methods in the following examples, where specific conditions are not specified, were performed according to conventional methods and conditions, or according to the product specifications. Reagents whose specific sources are not specified were commercially available.

[0234] Example 1 Establishment of CD40 Antibody and PD-L1 Antibody Phage Libraries

[0235] Based on the crystal structure of the human CD40 protein, the variable region sequence of the human CD40 antibody was designed. A single-chain Fv sequence (VL-G4S linker-VH) was generated by gene synthesis. This sequence was then inserted into a phage vector (pComb3XSS, purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.) using restriction enzymes to generate a recombinant plasmid library. The phage plasmid carrying the scFv gene was transformed into SS320 competent E. coli cells (purchased from Lucigen) by electroporation. After the SS320 E. coli cells (purchased from Lucigen) reached logarithmic phase, helper phage (M13K07, purchased from New England Biolabs) was added for infection. The cells were incubated overnight, and phage were extracted from the culture supernatant to generate the CD40 antibody phage library.

[0236] Based on the crystal structure of the human PD-L1 protein, the variable region sequence of the human PD-L1 nanobody was designed. The VHH sequence was synthesized and incorporated into a phage vector using restriction enzymes to generate a recombinant plasmid library. The phage plasmid carrying the VHH gene was transformed into SS320 competent Escherichia coli cells using electroporation. After the SS320 cells reached logarithmic phase, helper phage was added for infection, and the cells were cultured overnight. Phage were then extracted from the culture supernatant to generate the PD-L1 antibody phage library.

[0237] Example 2 Screening of CD40 Antibodies and PD-L1 Antibodies from Antibody Phage Libraries

[0238] CD40 antibodies were screened from a CD40 antibody phage library, and PD-L1 antibodies were screened from a PD-L1 antibody phage library.

[0239] Three rounds of panning of phage display libraries were performed using a protein-based phage antibody panning technique. The first round of screening was as follows: an ELISA plate was coated with either the human CD40-mFc antigen or the human PD-L1-mFc antigen (purchased from Biopsies). The corresponding phage library was premixed with an equal volume of 2% skim milk and incubated. The premix was added to the coated wells for reaction, and then washed with sterile PBST to remove the premix. Adsorbed phage from the plate were eluted with 75 mM sodium citrate buffer. After neutralization, the phage library was amplified 100-fold using M13K07 helper phage. Second and third rounds of screening were then performed using methods similar to the first round. After three rounds of screening, an enriched phage library was obtained. Phage enrichment was monitored by the initial phage dosage and the titer of phage collected after each round of screening.

[0240] The enriched CD40 phage library and PD-L1 phage library were used to infect SS320 Escherichia coli, respectively, and then smeared on agarose plates for culture. Monoclonal colonies were picked and placed in a 96-well deep-well plate and cultured with 2YT culture medium containing ampicillin and kanamycin at 37°C to obtain the supernatant containing monoclonal phage. The monoclonal phage supernatant was incubated with an ELISA plate coated with human CD40-mFc antigen or human PD-L1-mFc antigen for 1 hour, then washed with sterile PBST, and then Anti-M13-HRP (purchased from Yiqiao Shenzhou) was added and incubated at 4°C for 30 minutes. The binding of phage and antigen was then detected using a microplate reader to screen out CD40 monoclonal phage and PD-L1 monoclonal phage with high antigen binding.

[0241] Example 3 Preparation of recombinant CD40 monoclonal antibody and recombinant PD-L1 monoclonal antibody

[0242] The cDNA sequences of the heavy and light chain variable regions of the selected CD40 monoclonal phage were cloned into the pcDNA3.4 vector (Invitrogen), which already contains the antibody constant region. This yielded several anti-CD40 monoclonal antibodies, designated 1605CD, 1606CD, 1607CD, 1608CD, 1609CD, 1652CD, 1653CD, 1654CD, and 1655CD. The heavy and light chain recombinant plasmids were co-transfected into EXPI-293 cells (Invitrogen) using the PEI method. Transient transfection was continued for 7-10 days, followed by centrifugation and collection of the supernatant. The supernatant was purified using protein A to obtain purified anti-CD40 monoclonal antibodies. The CDR sequences of the CD40 antibodies are shown in Table 7 (determined using the Kabat CDR system); the amino acid sequences of the heavy and light chain variable regions are shown in Table 8.

[0243] Illustratively, the full-length amino acid sequence of 1654CD is as follows:

[0244] The amino acid sequence of the heavy chain of 1654CD (SEQ ID NO: 94):

[0245]

[0246]

[0247] The amino acid sequence of the light chain of 1654CD (SEQ ID NO: 95):

[0248]

[0249] The full-length amino acid sequence of 1606CD is as follows:

[0250] The amino acid sequence of the heavy chain of 1606CD (SEQ ID NO: 96):

[0251]

[0252] The amino acid sequence of the light chain of 1606CD (SEQ ID NO: 81):

[0253]

[0254] The full-length amino acid sequence of 1652CD is as follows:

[0255] The amino acid sequence of the heavy chain of 1652CD (SEQ ID NO: 87):

[0256]

[0257]

[0258] The amino acid sequence of the light chain of 1652CD (SEQ ID NO: 81):

[0259]

[0260] The cDNA sequences of the variable regions (VHH) of the selected PD-L1 monoclonal phages were cloned into the pcDNA3.4 vector (Invitrogen) containing the antibody constant regions, resulting in multiple anti-PD-L1 monoclonal antibodies designated 1029, 1031, 1102, and 1541. The complementarity-determining region (CDR) sequences of the PD-L1 monoclonal antibodies are shown in Table 2 (determined using the Kabat CDR system), the framework region (FR) sequences of the monoclonal antibodies are shown in Table 3, the amino acid sequences of the variable regions (VHH) are shown in Table 4, the amino acid sequences of the linker and constant regions of the monoclonal antibodies are shown in Table 5, and the full-length amino acid sequences of the monoclonal antibodies are shown in Table 6. Plasmids were transfected into EXPI-293 cells (Invitrogen) using the PEI method for transient transfection 7-10 days after which the cells were centrifuged and the supernatant collected. The supernatant was purified with protein A to obtain purified anti-PD-L1 monoclonal antibodies.

[0261] Table 2 CDR sequences of PD-L1 monoclonal antibodies

[0262]

[0263] Note: The bold and underlined amino acids in the table are different from those in 1029. is D or E; is S or T; is K or Q; is G or S; is S or T; is D or E; K or N.

[0264] Table 3 FR sequences of PD-L1 monoclonal antibodies

[0265]

[0266] Note: The bold and underlined amino acids in the table are different from those in 1029. 48 L or F; X 49 A or S; X 50 K or R; X 51 A or T.

[0267] Table 4 Amino acid sequences of VHHs of PD-L1 monoclonal antibodies

[0268]

[0269] Note: The italicized and underlined parts in the table are the above CDR sequences; the bold and underlined parts are the different amino acids compared with 1029.

[0270] The amino acid sequence of the general formula of VHH (SEQ ID NO: 66):

[0271]

[0272]

[0273] in is D or E; is S or T; is K or Q; is G or S; is S or T; is D or E; K or N, X 48 L or F; X 49 A or S; X 50 K or R; X 51 A or T.

[0274] Table 5 Amino acid sequences of the linker and constant regions of PD-L1 monoclonal antibodies

[0275]

[0276] Table 6 Full-length amino acid sequences of PD-L1 monoclonal antibodies

[0277]

[0278]

[0279] Example 4 Preparation of recombinant anti-PD-L1 / CD40 bispecific antibody

[0280] The recombinant anti-PD-L1 / CD40 bispecific antibody uses PD-L1 monoclonal antibody 1541 and CD40 monoclonal antibodies 1605CD, 1606CD, 1607CD, 1608CD, 1609CD, 1652CD, 1653CD, 1654CD, and 1655CD.

[0281] The cDNA sequences of the heavy and light chain variable regions of the screened CD40 monoclonal phage were cloned into the pcDNA3.4 vector (Invitrogen) containing the antibody constant region. The cDNA sequence of the variable region (VHH) of the screened PD-L1 monoclonal phage was cloned into the pcDNA3.4 vector (Invitrogen) containing CD40-VH and the antibody constant region. The VHH sequence was inserted into the C-terminus of the antibody constant region, and a (G4S) linker was introduced between the constant region and the VHH. Thus, multiple anti-PD-L1 / CD40 bispecific antibodies (structures such as Figure 7 The CDR sequences of the bispecific antibody are shown in Table 7 (determined by the Kabat CDR system); the amino acid sequences of the heavy and light chain variable regions are shown in Table 8; and the amino acid sequences of the heavy and light chains of the bispecific antibody are shown in Table 9. The heavy and light chain recombinant plasmids were co-transfected into EXPI-293 cells (Invitrogen) using the PEI method. After transient transfection for 7-10 days, the cells were centrifuged and the supernatant was collected. The supernatant was purified with protein A to obtain the purified anti-PD-L1 / CD40 bispecific antibody.

[0282] Table 7 CDR sequences of bispecific antibodies

[0283]

[0284]

[0285] Note: In RASQX1IX2X3YLX4 (SEQ ID NO: 1), X1 is D, G, S, or T; X2 is R or S, X3 is N or S; X4 is N or A. In X5X6SX7X8X9S, X5 is Y or A; X6 is T or A; X7 is S, R, or T; X8 is L or R; X9 is Q or D. QQGX 10 X 11 X 12 In PW (SEQ ID NO: 3), X 10 K, I, N, Q or S; X 11 S, A, N or T; X 12 L, Y or F. SX 13 In YYMS (SEQ ID NO: 4), X 13 N or D. FIRNKANX 14 In YT(SEQ ID NO:5), X 14 A or G. YGGX 15 X 16 X 17GWYFDX 18 (SEQ ID NO:6), X 15 L or I; X 16 K or R; X 17 V, K, I or Q; X 18 It is L or V.

[0286] Table 8 Amino acid sequences of the heavy chain variable region and light chain variable region of bispecific antibodies

[0287]

[0288]

[0289] Among them, the common sequence of the heavy chain variable region of Anti-CD40 is:

[0290] 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 N or D; X 20 G or A; X 21 A or G; X 22 N or S; X 23 R or S; X 24 L or I; X 25 K or R; X 26 V, K, I or Q; X 27 It is L or V.

[0291] The consensus sequence of the light chain variable region of Anti-CD40 is (SEQ ID NO: 65):

[0292] DIQMTQSPSSLSASVGDRVTITCRASQX 28 IX 29 X 30 YLX 31 WYQQKPGKAPKLLIYX 32 X 33 SX 34 X 35 X36 SGVPSRFSGSGSGTDYTLTISSLQPEDFATYX 37 CQQGX 38 X 39 X 40 PWTFGGGTKVEIK:X 28 D, G, S or T; X 29 R or S; X 30 N or S; X 31 N or A; X 32 is Y or A; X 33 T or A; X 34 S, R or T; X 35 L or R; X 36 Q or D; X 37 Y or F; X 38 K, I, N, Q or S; X 39 S, A, N or T; X 40 It is L, Y or F.

[0293] Table 9 Amino acid sequences of heavy and light chains of bispecific antibodies

[0294]

[0295]

[0296]

[0297]

[0298] Example 5 Detection of binding of recombinant CD40 monoclonal antibody to CD40

[0299] The binding ability and species specificity of the monoclonal antibodies to human CD40 were determined using an enzyme-linked immunosorbent assay (ELISA). The following method was used: 100 μL of each of the 1 μg / mL human CD40, cynomolgus CD40, rat CD40, and mouse CD40 antigens (all purchased from Biopsies) were coated onto an ELISA plate using carbonate buffer (pH 9.6). The plates were incubated overnight at 4°C. The plates were washed five times with PBST. The plates were blocked with 300 μL / well of 1% BSA in PBST and incubated for 1 hour at room temperature. The plates were washed five times with PBST. The monoclonal antibodies were serially diluted in PBST containing 1% BSA, and the control CD40 monoclonal antibody CP-870893 (IMGT database ID 10523) was added at 100 μL / well. The plates were incubated for 1 hour at room temperature. The plates were washed five times with PBST. Add 100 μL of HRP-labeled anti-human IgG antibody (Jackson ImmunoResearch, Cat. No. 109-035-088) diluted in PBST containing 1% BSA to each well and incubate at room temperature for 1 hour. Wash five times with PBST. Add 100 μL of the colorimetric substrate TMB to each well and develop at room temperature for 10 minutes. Then, add 1 M sulfuric acid to terminate the reaction. Read the OD value on a microplate reader. 450nm , analyze the results and calculate the EC using a 4-parameter binding curve fit 50 .

[0300] The results are shown in Table 10. Monoclonal antibodies 1605CD, 1606CD, 1607CD, 1608CD, 1609CD, 1652CD, 1653CD, 1654CD, and 1655CD all bind to human CD40 and cynomolgus CD40, and their binding affinity to human and cynomolgus macaque antigens is comparable. They do not bind to rat CD40 or mouse CD40.

[0301] Table 10 Binding of monoclonal antibodies to antigens

[0302]

[0303] Example 6 Effect of anti-CD40 monoclonal antibodies on CD40 / CD40L binding

[0304] The blocking effect of monoclonal antibodies on CD40 / CD40L binding was tested using HEK-Blue CD40L cells (purchased from InvivoGen) that highly express CD40. The specific method is as follows: HEK-Blue CD40L cells were collected and resuspended in pre-cooled PBS containing 2% FBS at a density of 1×10 7 / mL, and then added to a 96-well plate, 50 μL per well, i.e. 5×10 5Cells were then plated at 50 μL per well of Biotin-CD40L (Mingji Bio) and serially diluted CD40 monoclonal antibodies, with a final concentration of 10 nM Biotin-CD40L. The cells were incubated at 4°C for 1 hour. The cells were washed twice with pre-chilled PBS. Streptavidin-PE (BioLegend, Cat. No. 405203) diluted in pre-chilled PBS containing 2% FBS was added and the cells were incubated at 4°C for 30 minutes. The cells were washed twice with pre-chilled PBS. The cells were then resuspended in pre-chilled PBS containing 2% FBS and analyzed on a flow cytometer.

[0305] The results are shown in Table 11. Monoclonal antibodies 1605CD, 1606CD, 1607CD, 1608CD, 1609CD, 1652CD, 1653CD, 1654CD, and 1655CD inhibited the CD40 / CD40L interaction, while the control CD40 monoclonal antibody CP-870893 had no effect on the CD40 / CD40L interaction.

[0306] Table 11 Effects of monoclonal antibodies on CD40 / CD40L binding

[0307]

[0308] Example 7 Detection of CD40 Agonistic Activity of Anti-CD40 Monoclonal Antibodies by Reporter Gene Method

[0309] HEK-Blue CD40L was used to test the CD40 agonist activity of the monoclonal antibody. HEK-Blue CD40L cells were purchased from InvivoGen and highly express CD40 and the SEAP reporter gene under the control of the NF-κB response element. When CD40 on HEK-BlueCD40L cells is activated, it induces the activation of the downstream signal NF-κB, which in turn induces the production of SEAP. The amount of secreted SEAP can be detected by QUANTI-Blue reagent (InvivoGen) to monitor CD40 activation. The specific method is as follows: Collect HEK-Blue CD40L cells, 3×10 5 / mL was resuspended in complete culture medium (RPMI 1640 containing 10% FBS) and evenly plated in a 96-well plate at 100 μL / well, i.e. 3×10 4cells; 100 μL / well of anti-CD40 monoclonal antibody samples and control antibodies (CP-870893) serially diluted in complete medium were added and incubated in a 37°C, 5% CO2 incubator for 20-24 hours. After the incubation, the 96-well plate was removed and centrifuged at 300g for 5 minutes. 40 μL / well of supernatant was transferred to a new 96-well plate, and 160 μL / well of QUANTI-Blue reagent was added to the supernatant. The plate was incubated in a 37°C, 5% CO2 incubator for 20-30 minutes, and the OD was read on a microplate reader. 655nm , analyze the results.

[0310] The results are as follows Figure 1 As shown in the figure, the CD40 agonistic activity of monoclonal antibodies can be roughly divided into two categories. The CD40 agonistic activity of one category of antibodies 1608CD, 1609CD, 1654CD, and 1655CD is weaker, weaker than that of the control CD40 monoclonal antibody CP-870893; the activity of the other category of antibodies 1605CD, 1606CD, 1607CD, 1652CD, and 1653CD is stronger, stronger than that of the control CD40 monoclonal antibody CP-870893.

[0311] Example 8 DC regulatory activity of anti-CD40 monoclonal antibodies

[0312] CD40 monoclonal antibodies were tested for their dendritic cell (DC) modulating activity.

[0313] Donated human PBMCs were resuspended in complete culture medium (RPMI 1640 with 10% FBS) and seeded into 10 cm cell culture dishes. The cells were incubated at 37°C in a CO2 incubator for 2 hours. The culture supernatant and suspended cells were discarded, and adherent cells were identified as monocytes. Immunofluorescent DCs were obtained by incubating the monocytes in complete culture medium containing 100 ng / mL GM-CSF (PeproTech, Catalog No. 300-03) and 100 ng / mL IL-4 (PeproTech, Catalog No. 200-04) for 6 days, with the medium changed every 2 days. The imDCs were harvested, resuspended in complete culture medium, and seeded into 24-well plates. Antibody samples and a control antibody (CP-870893) were added at 100 nM. The plates were incubated at 37°C in a CO2 incubator for 2 days. After incubation, the supernatant was removed from the wells and the cytokine IL-12 / IL-23 p40 (R&D, Catalog No. DY1240) was detected according to the kit manual. At the same time, the cells in the well plate were collected and incubated with the detection antibody (APC anti-human CD83 Antibody, Biolegend, 305312). The expression of CD83 on DC cells was detected by flow cytometer.

[0314] Figure 2The DC regulatory activities of the anti-CD40 monoclonal antibodies of the present invention were compared, and different monoclonal antibodies were able to significantly upregulate CD83 on DC cells.

[0315] Example 9 T cell regulatory activity of anti-CD40 monoclonal antibodies

[0316] The most important anti-tumor biological effect of CD40 agonistic antibodies is the activation of authorized DCs, which in turn activate T cells. The T cell regulatory activity of CD40 monoclonal antibodies was tested using an MLR assay system incubated with allogeneic DCs and T cells.

[0317] DCs were obtained using the same method as in Example 8. Allogeneic T cells were isolated from human PBMCs. The specific isolation method is described in the instructions for the Pan T Cell Isolation Kit (Miltenyi Biotech, Cat. No. 130-096-535). Briefly, PBMCs were washed once with PBS. The PBMCs were then resuspended in 40 μL of separation buffer (PBS containing 2 mM EDTA, 0.5% BSA, pH 7.2) at a ratio of 1E7 cells per 40 μL (the following amounts are based on 1E7 cells). 10 μL of Pan T cell Biotin Antibody Cocktail was added, and the cells were incubated at 4°C for 5 minutes. 30 μL of separation buffer and 20 μL of Pan T cell MicroBead Cocktail were then added, and the cells were incubated at 4°C for 10 minutes. T cells were obtained by passing the cells through a MACS column.

[0318] Human DCs and T cells were harvested, resuspended in complete culture medium (RPMI 1640 with 10% FBS), and seeded into 96-well plates at a ratio of 1E4 DCs and 1E5 T cells per well, respectively, for mixed culture. Monoclonal antibody samples and control antibodies serially diluted in complete culture medium were then added. The plates were incubated in a 37°C CO2 incubator for 5 days. After incubation, the supernatants were removed and assayed for the cytokine IFN-γ (Biolegend, Cat. No. 430101) according to the kit manual.

[0319] The results are as follows Figure 3 As shown, the strong CD40 agonist mAb 1606CD and the weak CD40 agonist mAb 1654CD of the present invention can enhance T cell activation under the condition of low DC ratio, while the control mAb CP-870893 has little effect.

[0320] Example 10 Preliminary Safety Evaluation of Anti-CD40 Monoclonal Antibodies

[0321] A preliminary safety evaluation of the anti-CD40 monoclonal antibody was conducted in human CD40 knock-in mice. The experimental animals were purchased from Biocytogen and randomly divided into four groups (5 animals / group) to receive vehicle control, 21 mg / kg of monoclonal antibody 1606CD, 21 mg / kg of monoclonal antibody 1654CD, and 21 mg / kg of the control CD40 monoclonal antibody CP-870893. The drugs were administered intraperitoneally twice weekly for two consecutive weeks, for a total of four doses. During the trial, the animals were monitored for clinical symptoms, body weight, liver function biochemistry, and blood cell counts. After the dosing period, all animals were euthanized as planned the day after the last dose. Autopsies were performed to observe any abnormalities and weigh their organs.

[0322] like Figures 4A to 4C As shown, mice repeatedly injected with the strong CD40 agonist mAb 1606CD or the weak CD40 agonist mAb 1654CD showed no significant abnormalities, with only a decrease in lymphocytes and granulocytes observed in hematological tests. In contrast, mice repeatedly injected with mAb CP-870893 showed weight loss, elevated ALT, and significant decreases in RBC, HGB, and PLT counts. Autopsies revealed significant organ necrosis and enlargement. These results suggest that mAbs 1606CD and 1654CD have a better safety profile than mAb CP-870893.

[0323] Example 11: Binding Detection of Recombinant PD-L1 Monoclonal Antibody to PD-L1

[0324] The binding ability and species specificity of the monoclonal antibodies to human PD-L1 were tested using an enzyme-linked immunosorbent assay (ELISA). The specific method was as follows: 1 μg / mL of human PD-L1, cynomolgus monkey PD-L1, rat PD-L1, and mouse PD-L1 (all purchased from Biopsies) were coated onto an ELISA plate using carbonate buffer (pH 9.6). The plate was incubated overnight at 4°C with 100 μL per well of 1 μg / mL of each antigen. The plate was washed five times with PBST. The plate was blocked with 300 μL / well of PBST containing 1% BSA and incubated at room temperature for 1 hour. The plate was washed five times with PBST. A serial dilution of the monoclonal antibodies in PBST containing 1% BSA was added, along with 100 μL per well of the control PD-L1 monoclonal antibody, Durvalumab (IMGT database ID 10010), and the plate was incubated at room temperature for 1 hour. The plate was washed five times with PBST. Add 100 μL of HRP-labeled anti-human IgG antibody (Jackson ImmunoResearch, Cat. No. 109-035-088) diluted in PBST containing 1% BSA to each well and incubate at room temperature for 1 hour. Wash five times with PBST. Add 100 μL of the colorimetric substrate TMB to each well and develop at room temperature for 10 minutes. Then, add 1 M sulfuric acid to terminate the reaction. Read the OD value on a microplate reader. 450nm, analyze the results and calculate the EC using a 4-parameter binding curve fit 50 .

[0325] The results are shown in Table 12. Monoclonal antibodies 1029, 1031, 1102, and 1541 all bind to human PD-L1 and cynomolgus monkey PD-L1, and their binding affinity to human and cynomolgus monkey antigens is comparable to that of the control antibody. They do not bind to rat PD-L1 or mouse PD-L1.

[0326] Table 12 Binding of monoclonal antibodies to antigens

[0327]

[0328] NB: Not combined

[0329] The interaction between monoclonal antibodies and antigens was detected using the Gator, a label-free biomolecular interaction analyzer based on the biolayer interferometry (BLI) principle. The specific method is as follows: Using a PA probe, the monoclonal antibody is diluted to 50 nM and added to the probe plate, where it is captured by the PA probe. Then, the human PD-L1 antigen is added in a serial dilution starting from 200 nM. The antigen interacts with the bispecific antibody captured by the PA probe. The interaction is analyzed by detecting changes in the interference spectrum reflected from the probe surface, ultimately calculating the binding kinetic constant of the antibody.

[0330] The results are shown in Table 13. The binding kinetic constants of monoclonal antibodies 1029, 1031, 1102, and 1541 to human PD-L1 ranged from 4.37 to 8.30 nM.

[0331] Table 13 Binding kinetics of monoclonal antibodies to antigens

[0332]

[0333] Example 12: Detection of PD-L1 / PD-1 Blocking Activity of PD-L1 Monoclonal Antibodies by Reporter Gene Assay

[0334] A reporter gene assay for PD-L1 / PD-1 blocking activity was established using Jurkat / PD-1-NFAT-luciferase cells (highly expressing PD-1 and a luciferase reporter gene under the control of the NFAT response element) and WIL2S / PD-L1 (highly expressing PD-L1) cells constructed by Mingji Bio, along with an anti-CD20 / CD3 bispecific antibody. The specific method is as follows: WIL2S / PD-L1 cells were collected and plated at a cell density of 4×10 6 / mL was resuspended in complete culture medium (RPMI 1640 containing 10% FBS), 50 μL / well, i.e. 2×10 5cells were evenly plated in 96-well plates; Jurrkat-PD-1-NFAT-luciferase cells were collected and plated at a cell density of 4×10 6 / mL was resuspended in complete culture medium (RPMI 1640 containing 10% FBS), 50 μL / well, i.e. 2×10 5 cells were evenly added to the above 96-well plate; anti-CD20 / CD3 bispecific antibody was diluted in complete medium and added to the above 96-well plate at 25 μL / well; anti-PD-L1 monoclonal antibody samples and control antibody were serially diluted in complete medium and added to the above 96-well plate at 25 μL / well; then incubated in a 37°C, 5% CO2 incubator for 6 hours. After incubation, 50 μL / well of one-glo reagent (Promega, Cat. No. E6120) was added to the above 96-well plate, placed on a shaker for 5 minutes, and allowed to stand for 10 minutes. The relative chemiluminescence unit value (RLU) was then read using a chemiluminescence module on a microplate reader (MD, SpectraMax iD3) and the results were analyzed.

[0335] The results are as follows Figure 5 As shown in the results, monoclonal antibodies 1029, 1031, 1102, and 1541 can all block the negative signal transmitted by PD-L1 to PD-1. The activities of the antibodies are similar, but slightly stronger than that of the PD-L1 control monoclonal antibody Durvalumab.

[0336] Example 13: T cell regulatory activity of PD-L1 monoclonal antibody

[0337] The T cell regulatory activity of monoclonal antibodies was tested using the MLR experimental system incubated with allogeneic DC cells and T cells.

[0338] Donated human PBMCs were resuspended in complete culture medium (RPMI 1640 with 10% FBS) and seeded into 10 cm cell culture dishes. The cells were incubated at 37°C in a CO2 incubator for 2 hours. The culture supernatant and suspended cells were discarded, and adherent cells were identified as monocytes. Monocytes were cultured in complete culture medium containing 100 ng / mL GM-CSF (PeproTech, Cat. No. 300-03) and 100 ng / mL IL-4 (PeproTech, Cat. No. 200-04) for 6 days, with the medium changed every 2 days. TNFα and IL-1β (both purchased from PeproTech) were then added and incubated for 2 days to obtain DCs.

[0339] Allogeneic T cells were isolated from donated human PBMCs. The specific isolation method was described in the instructions for the Pan T cell isolation kit (Miltenyi Biotech, Cat. No. 130-096-535). Briefly, PBMCs were washed once with PBS and then plated at 1×10 7 Resuspend the cells in 40 μL of separation buffer (PBS containing 2 mM EDTA, 0.5% BSA, pH = 7.2) (the following usage is based on 1×10 7 cytometer), add 10 μL of Pan T cell Biotin Antibody Cocktail, and incubate at 4°C for 5 minutes. Then add 30 μL of separation buffer and 20 μL of Pan T cell MicroBead Cocktail, and incubate at 4°C for 10 minutes. Pass the cells through a MACS separation column to obtain T cells.

[0340] The obtained human DC cells and human T cells were collected, resuspended in complete culture medium (RPMI 1640 containing 10% FBS), and inoculated into 96-well plates. The inoculated DC cells and T cells were 1×10 4 / hole and 1×10 5 Each plate was plated and mixed. Antibody samples serially diluted in complete culture medium were added. The plates were incubated in a 37°C CO2 incubator for 5 days. After incubation, the supernatant was removed and the cytokine IFN-γ (Biolegend, Cat. No. 430101) was assayed according to the kit manual.

[0341] The results are as follows Figure 6 As shown, the monoclonal antibody significantly enhanced T cell activation.

[0342] Example 14 Detection of Binding of Recombinant Anti-PD-L1 / CD40 Bispecific Antibody to PD-L1 and CD40

[0343] The binding ability and species specificity of the bispecific antibodies to human PD-L1 and human CD40 were determined using an enzyme-linked immunosorbent assay (ELISA). The following method was used: 100 μL of each of the following antigens (human PD-L1, cynomolgus PD-L1, rat PD-L1, mouse PD-L1, human CD40, cynomolgus CD40, rat CD40, and mouse CD40, all purchased from Biopsies) were coated onto an ELISA plate using carbonate buffer (pH 9.6) at 1 μg / mL. The plates were incubated overnight at 4°C. The plates were washed five times with PBST. The plates were blocked with 300 μL / well of 1% BSA in PBST and incubated for 1 hour at room temperature. The plates were then washed five times with PBST. Add bispecific antibodies diluted in PBST containing 1% BSA, and add control PD-L1 monoclonal antibody Durvalumab (IMGT database ID 10010) and CD40 monoclonal antibody CP-870893 (IMGT database ID 10523), 100 μL per well, and incubate at room temperature for 1 hour. Wash 5 times with PBST. Add HRP-labeled anti-human IgG antibody (Jackson ImmunoResearch, product number 109-035-088) diluted in PBST containing 1% BSA, 100 μL per well, and incubate at room temperature for 1 hour. Wash 5 times with PBST. Add the colorimetric substrate TMB, 100 μL per well, develop color at room temperature for 10 minutes, and then add 1M sulfuric acid to terminate the reaction. Read OD on a microplate reader 450nm , analyze the results and calculate the EC using a 4-parameter binding curve fit 50 .

[0344] The results are shown in Table 14. Bispecific antibodies 1605, 1606, 1607, 1608, 1609, 1652, 1653, 1654, and 1655 all bind to human PD-L1 and CD40, as well as cynomolgus PD-L1 and CD40, with comparable binding affinity to human and cynomolgus macaque antigens. They do not bind to rat PD-L1 and CD40, or mouse PD-L1 and CD40.

[0345] Table 14 Binding of bispecific antibodies to antigens

[0346]

[0347] NB: Not combined

[0348] Example 15 Simultaneous Binding Detection of Recombinant Anti-PD-L1 / CD40 Bispecific Antibody to PD-L1 and CD40

[0349] The plate was coated with the antigen human CD40 (purchased from Biopsies) at 1 μg / mL in carbonate buffer (pH 9.6) at 100 μL per well and incubated overnight at 4°C. Washed 5 times with PBST. Blocked with 300 μL / well of PBST containing 1% BSA and incubated at room temperature for 1 hour. Washed 5 times with PBST. Added bispecific antibodies serially diluted in PBST containing 1% BSA, along with control PD-L1 monoclonal antibody Durvalumab and CD40 monoclonal antibody CP-870893, at 100 μL per well, and incubated at room temperature for 1 hour. Washed 5 times with PBST. Added Biotin-labeled human PD-L1 (Mingji Biopharmaceuticals) diluted in PBST containing 1% BSA at 100 μL per well, and incubated at room temperature for 1 hour. Washed 5 times with PBST. Add 100 μL of Streptavidin-HRP (BioLegend, Cat. No. 405210) diluted in PBST containing 1% BSA to each well and incubate at room temperature for 30 minutes. Add 100 μL of the colorimetric substrate TMB to each well and develop color at room temperature for 10 minutes. Then add 1 M sulfuric acid to terminate the reaction. Read the OD value on a microplate reader. 450nm , analyze the results and calculate the EC using a 4-parameter binding curve fit 50 .

[0350] The results are as follows Figure 8 As shown, bispecific antibodies 1605, 1606, 1607, 1608, 1609, 1652, 1653, 1654, and 1655 can simultaneously bind to human PD-L1 and human CD40, while the control monoclonal antibody has no signal and cannot bind to the two antigens simultaneously.

[0351] Example 16 Detection of PD-L1 / PD-1 Blocking Activity of Anti-PD-L1 / CD40 Bispecific Antibody Using Reporter Gene Assay

[0352] Jurkat / PD-1-NFAT-luciferase cells (overexpressing PD-1 and a luciferase reporter gene under the control of an NFAT response element) and WIL2S / PD-L1 cells (overexpressing PD-L1) were constructed by lentiviral transfection according to 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 these Jurkat and WIL2S cells and an anti-CD20 / CD3 bispecific antibody, a reporter gene assay for PD-L1 / PD-1 blocking activity was established. The specific method is as follows: WIL2S / PD-L1 cells were collected and resuspended in complete medium (RPMI 1640 with 10% FBS) at a cell density of 4E6 / mL, 50 μL / well, i.e., 2E5 cells per well, and evenly plated into a 96-well plate; Jurrkat-PD-1-NFAT-luciferase cells were collected and resuspended in complete medium (RPMI 1640 with 10% FBS) at a cell density of 4E6 / mL, 50 μL / well, i.e., 2E5 cells per well, and evenly added to the above 96-well plate; anti-CD20 / CD3 bispecific antibody was diluted in complete medium, 25 μL / well was added to the above 96-well plate; anti-PD-L1 / CD40 bispecific antibody sample and control antibody were serially diluted in complete medium, 25 μL / well was added to the above 96-well plate; and then incubated in a 37°C, 5% CO2 incubator for 6 hours. After incubation, add 50 μl / well of One-Glo reagent (Promega, Cat. No. E6120) to the 96-well plate. Place the plate on a shaker for 5 minutes and let it rest for 10 minutes. Then, analyze the relative chemiluminescence units (RLU) using a chemiluminescence module on a microplate reader (MD, SpectraMax iD3).

[0353] The results are as follows Figure 9 As shown, bispecific antibodies 1605, 1606, 1607, 1608, 1609, 1652, 1653, 1654, and 1655 can block the negative signal transmitted by PD-L1 to PD-1. The activities of different bispecific antibodies are similar, and are also similar to the activity of the PD-L1 control monoclonal antibody Durvalumab.

[0354] Example 17 Detection of CD40 Agonistic Activity of Anti-PD-L1 / CD40 Bispecific Antibody Using Reporter Gene Assay

[0355] HEK-Blue CD40L was used to test the CD40 agonist activity of the bispecific antibody. HEK-Blue CD40L cells were purchased from InvivoGen and highly express CD40 and the SEAP reporter gene under the control of the NF-κB response element. When CD40 on HEK-BlueCD40L cells is activated, it induces the activation of the downstream signal NF-κB, which in turn induces the production of SEAP. The amount of secreted SEAP can be detected by QUANTI-Blue reagent (InvivoGen) to monitor CD40 activation. The CD40 agonist activity of the bispecific antibody in the presence of PD-L1 was tested by co-incubating CHO / PD-L1 cells with HEK-Blue CD40L cells. The specific method is as follows: HEK-Blue CD40L cells were collected, resuspended in complete culture medium (RPMI 1640 containing 10% FBS) at 3E5 / mL, and evenly plated into a 96-well plate at 100 μL / well, i.e., 3E4 cells per well; CHO / PD-L1 cells were collected, resuspended in complete culture medium (RPMI 1640 containing 10% FBS) at 6E5 / mL, 50 μL / well, i.e., 3E4 cells per well were added into a 96-well plate. If CHO / PD-L1 cells were not required, 50 μL / well of complete culture medium was added; 50 μL / well of anti-PD-L1 / CD40 bispecific antibody samples and control antibodies serially diluted in complete culture medium were added and incubated in a 37°C, 5% CO2 incubator for 20-24 hours. After the incubation, remove the 96-well plate and centrifuge at 300g for 5 minutes. Pipette 40μL / well supernatant and transfer it to a new 96-well plate. Add 160μL / well QUANTI-Blue reagent to the supernatant and incubate in a 37°C, 5% CO2 incubator for 20-30 minutes. Read the OD value on a microplate reader. 655nm , analyze the results.

[0356] The results are as follows 10A to 10D As shown in Table 15, the CD40 agonist activity of bispecific antibodies 1608, 1609, 1654, and 1655 was significantly enhanced by the PD-L1 cross-linking signal provided by CHO / PD-L1. This indicates that these bispecific antibodies exhibit selective activity: lower activity in environments with low PD-L1 expression (such as blood) and higher activity in environments with high PD-L1 expression (such as tumors), resulting in enhanced efficacy and controlled side effects. However, the enhancement of CHO / PD-L1 expression by bispecific antibodies 1605, 1606, 1607, 1652, and 1653 was minimal; the control CD40 monoclonal antibody CP-870893 was not enhanced by CHO / PD-L1.

[0357] Table 15 CD40 agonistic activity of bispecific antibodies

[0358]

[0359] Example 18 Effect of Anti-PD-L1 / CD40 Bispecific Antibody on CD40 / CD40L Binding

[0360] The blocking effect of the bispecific antibody on CD40 / CD40L binding was tested using HEK-Blue CD40L cells, which overexpress CD40. The specific method was as follows: HEK-Blue CD40L cells were harvested and resuspended in ice-cold PBS containing 2% FBS to a density of 1E7 / mL. Then, 50 μL of cells were added to a 96-well plate at a density of 5E5 cells / well. Biotin-CD40L (Mingji Bio) and serially diluted bispecific antibodies were then added at a concentration of 50 μL per well to a final concentration of 10 nM. The cells were incubated at 4°C for 1 hour. The cells were washed twice with ice-cold PBS. Streptavidin-PE (BioLegend, Cat. No. 405203) diluted in ice-cold PBS containing 2% FBS was added and the cells were incubated at 4°C for 30 minutes. The cells were washed twice with ice-cold PBS. The cells were then resuspended in ice-cold PBS containing 2% FBS and analyzed on a flow cytometer.

[0361] The results are shown in Table 16. Bispecific antibodies 1605, 1606, 1607, 1608, 1609, 1652, 1653, 1654, and 1655 inhibited the CD40 / CD40L interaction, while the control CD40 monoclonal antibody CP-870893 had no effect on the CD40 / CD40L interaction.

[0362] Table 16 Effect of bispecific antibodies on CD40 / CD40L binding

[0363]

[0364] Example 19 DC regulatory activity of anti-PD-L1 / CD40 bispecific antibody

[0365] The bispecific antibodies were tested for their dendritic cell (DC) modulating activity.

[0366] Human PBMCs were resuspended in complete culture medium (RPMI 1640 with 10% FBS) and seeded into 10 cm cell culture dishes. Incubated in a 37°C CO2 incubator for 2 hours. The culture supernatant and suspended cells were discarded; adherent cells were identified as monocytes. Immobilized DCs were obtained by incubating the monocytes in complete culture medium supplemented with 100 ng / mL GM-CSF (PeproTech, Cat. No. 300-03) and 100 ng / mL IL-4 (PeproTech, Cat. No. 200-04) for 6 days, with the medium changed every 2 days. The immobilized DCs were harvested, resuspended in complete culture medium, and seeded into 24-well plates. Serial dilutions of the bispecific antibody sample and control antibody were added. The plates were incubated in a 37°C CO2 incubator for 2 days. After incubation, the supernatant was removed from the wells and the cytokine IL-12 / IL-23 p40 (R&D, Catalog No. DY1240) was detected according to the kit manual. In some experiments, cells were collected from the wells and incubated with detection antibodies (APC anti-human CD83 Antibody, Biolegend, 305312; PE / Cyanine7 anti-human CD86 Antibody, Biolegend, 374210). The expression of CD83 and CD86 on DC cells was detected by flow cytometer.

[0367] Figure 11A The DC regulatory activities of different bispecific antibodies were compared. Using IL-12 / IL-23 p40 as an indicator, the bispecific antibodies stimulated DCs to secrete IL-12 p40 in a concentration-dependent manner, and the activities of different bispecific antibodies varied greatly. Figure 11B and Figure 11C The DC regulatory activities of the parental PD-L1 monoclonal antibody, parental CD40 monoclonal antibody, bispecific antibody (1609) and control CD40 monoclonal antibody CP-870893 were compared. The detection indicators were CD83 and IL-12 / IL-23 p40. The data showed that the parental PD-L1 monoclonal antibody had no regulation on DCs, and the parental CD40 monoclonal antibody had weak activity. After the bispecific antibody was constructed, its DC activity was significantly enhanced.

[0368] Example 20 T cell regulatory activity of anti-PD-L1 / CD40 bispecific antibody

[0369] The T cell regulatory activity of bispecific antibodies was tested using an MLR experimental system incubated with allogeneic DC cells and T cells.

[0370] DCs were obtained using the same method as in Example 19. Allogeneic T cells were isolated from human PBMCs. The specific isolation method is described in the instructions for the Pan T Cell Isolation Kit (Miltenyi Biotech, Cat. No. 130-096-535). Briefly, PBMCs were washed once with PBS. The PBMCs were then resuspended in 40 μL of separation buffer (PBS containing 2 mM EDTA, 0.5% BSA, pH 7.2) at a ratio of 1E7 cells per 40 μL (the following amounts are based on 1E7 cells). 10 μL of Pan T cell Biotin Antibody Cocktail was added, and the cells were incubated at 4°C for 5 minutes. 30 μL of separation buffer and 20 μL of Pan T cell MicroBead Cocktail were then added, and the cells were incubated at 4°C for 10 minutes. T cells were obtained by passing the cells through a MACS separation column.

[0371] The obtained human DCs and T cells were collected, resuspended in complete culture medium (RPMI 1640 with 10% FBS), and seeded into 96-well plates at a ratio of 1E4 DCs and 1E5 T cells per well, respectively, for mixed culture. Bispecific antibody samples and control antibodies serially diluted in complete culture medium were then added. The plates were incubated in a 37°C CO2 incubator for 5 days. After incubation, the supernatants were removed and assayed for the cytokine IFN-γ (Biolegend, Cat. No. 430101) according to the kit manual.

[0372] The results are as follows Figure 12A and Figure 12B As shown, 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 antibody Durvalumab.

[0373] Example 21 In vivo anti-tumor efficacy of anti-mouse PD-L1 / mouse CD40 bispecific antibody

[0374] This example detects the anti-tumor efficacy of bispecific antibodies in mice. In order to more conveniently evaluate the in vivo efficacy of anti-PD-L1 / CD40 bispecific antibodies, a substitute anti-mouse PD-L1 / mouse CD40 bispecific antibody 1058 expressing a bispecific antibody was constructed (the anti-PD-L1 sequence is from the IMGT database ID 9814, and the anti-CD40 sequence is No.33_VH and No.34_VL in WO2018185045A1). The purpose of using this substitute is to evaluate the in vivo effect of a bispecific antibody formed by an antibody targeting PD-L1 and an antibody targeting CD40 in wild-type mice, and is only used to verify the efficacy of such bispecific antibodies.

[0375] Mice were C57BL / 6 female mice, 6-8 weeks old, purchased from Beijing Weitonglihua. After one week of acclimatization, each mouse was inoculated with 3E5 MC38 mouse colon cancer cells (purchased from the Basic Medical Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences). 3 At the same time, the mice were divided into groups according to the tumor volume, with 8 mice in each group, which were set as the vehicle 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. The dosage was 35 nmol / kg, intraperitoneal injection, once a week for 2 consecutive weeks. The tumor volume was measured 3 times a week from the date of administration, and its long diameter a and short diameter b were measured to calculate the tumor volume (mm 3 )=(axb 2 ) / 2.

[0376] The results are as follows Figure 13 As shown, the anti-mouse PD-L1 / mouse CD40 bispecific antibody 1058, an alternative to bispecific antibodies, significantly inhibited the growth of MC38 colon cancer transplanted tumors in mice, showing good anti-tumor efficacy, which was stronger than PD-L1 monoclonal antibody and CD40 monoclonal antibody.

[0377] Example 22 In vivo anti-tumor efficacy of anti-PD-L1 / CD40 bispecific antibody

[0378] This example evaluates the anti-tumor efficacy of an anti-PD-L1 / CD40 bispecific antibody in PD-L1 / CD40 humanized mice. This experiment was conducted in collaboration with Beijing Agricultural College. Female PD-L1 / CD40 humanized mice, 6-8 weeks old, were purchased from Biocytogen. After one week of acclimatization, each mouse was inoculated with 5E6 MC38 / hPD-L1 mouse colon cancer cells. The tumors were grown to approximately 100 mm in size. 3 At the same time, the mice were divided into groups according to tumor volume, with 6 mice in each group, and were given vehicle, anti-PD-L1 monoclonal antibody, anti-CD40 monoclonal antibody, and anti-PD-L1 / anti-CD40 bispecific antibody 1654 by intraperitoneal injection, twice a week for 2 consecutive weeks. From the day of administration, the clinical manifestations of the mice, the body weight of the mice, and the tumor volume were monitored. The long diameter a and short diameter b of the tumor were measured, and the tumor volume (mm 3 )=(axb 2 ) / 2.

[0379] The results are as follows Figure 14As shown, 1654 significantly inhibited the growth of MC38 / hPD-L1 xenografts in PD-L1 / CD40 humanized mice, achieving a tumor growth inhibition rate (TGI) of 72% at a dose as low as 7 nmol / kg. This was more potent than a 20 nmol / kg dose of either PD-L1 or CD40 monoclonal antibody, both of which achieved a TGI of 28%. During the experiment, one mouse died in the vehicle control group; the remaining mice were normal. Mouse weights were normal.

[0380] Example 23 Preliminary Safety Evaluation of Anti-PD-L1 / CD40 Bispecific Antibody

[0381] The preliminary safety evaluation of the anti-PD-L1 / CD40 bispecific antibody was entrusted to Zhaoyan (Suzhou) New Drug Research Center Co., Ltd.

[0382] The experimental animals used were male cynomolgus macaques, which were randomly divided into 5 groups (2 per group) and given a vehicle control, 12 mg / kg of bispecific antibodies 1607, 1608, and 1609, and 10 mg / kg of the control CD40 monoclonal antibody CP-870893. The drug was administered subcutaneously in the forelimb or hindlimb of the animal using a syringe pump, with a dosing volume of 10 mL / kg and a dosing rate of 0.5 mL / kg / min. The drug was administered once a week for 2 consecutive weeks, for a total of 3 doses. During the trial, the animals were regularly monitored for clinical symptoms, body weight, food intake, body temperature, blood cell count, coagulation function, blood biochemistry and urine analysis, immune cell phenotype, cytokines, and blood drug concentration and anti-drug antibodies. After the end of the dosing period, all surviving animals in Groups 2-5 were euthanized as planned the day after the last dose, and gross autopsies were performed to observe for abnormalities and weigh organs.

[0383] Animals in the 12 mg / kg dose group of bispecific antibodies 1607, 1608, and 1609 showed no test article-related abnormalities in clinical observations, body weight, weight gain, food intake, body temperature, blood cell count, coagulation function, blood biochemistry, urinalysis, organ weights, and gross pathological examinations. Only increases in immune cell phenotype and some cytokines were observed, consistent with the pharmacological mechanism of action. In contrast, animals in the 10 mg / kg dose group of CD40 monoclonal antibody CP-870893 showed small amounts of loose / diaperfluous stools on Days 7-8, Days 10-11, and Days 7-13, respectively. Weight gain on Day 14 was slightly lower than pre-drug intake. Food intake decreased significantly starting on Day 5. RBC counts, HGB, HCT, and PLT counts showed decreasing trends. Changes in immune cell phenotype and increases in some cytokines were also observed. Autopsies in cynomolgus monkeys revealed decreased thymus weight, organ-to-body ratio, and organ-to-brain ratio, while increased spleen weight, organ-to-body ratio, and organ-to-brain ratio, with significant changes.

[0384] The results suggest that anti-PD-L1 / CD40 bispecific antibody has better safety than CD40 monoclonal antibody.

Claims

1. 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, characterized in that: The first antigen-binding domain comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 14, and SEQ ID NO: 19, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 28, respectively; The light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 14, and SEQ ID NO: 19, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 28, respectively; The light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 16, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25, respectively; The light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 12, and SEQ ID NO: 17, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26, respectively; The light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 12, and SEQ ID NO: 17, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 26, respectively; The light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO: 18, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 27, respectively; The light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 8, SEQ ID NO: 13, and SEQ ID NO: 18, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 27, respectively; The light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 15 and SEQ ID NO: 20, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 24 and SEQ ID NO: 29, respectively; or, The light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 15, and SEQ ID NO: 20, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 29, respectively; The second antigen-binding domain comprises at least one VHH, wherein the VHH comprises VHH-CDR1, VHH-CDR2 and VHH-CDR3; The VHH comprises a VHH-CDR1 sequence as shown in SEQ ID NO: 46, a VHH-CDR2 sequence as shown in SEQ ID NO: 47, and a VHH-CDR3 sequence as shown in SEQ ID NO: 48, or, a VHH-CDR1 sequence as shown in SEQ ID NO: 67, a VHH-CDR2 sequence as shown in SEQ ID NO: 68, and a VHH-CDR3 sequence as shown in SEQ ID NO: 69, or, a VHH-CDR1 having a sequence as shown in SEQ ID NO: 70, a VHH-CDR2 having a sequence as shown in SEQ ID NO: 71, and a VHH-CDR3 having a sequence as shown in SEQ ID NO: 48; The second antigen-binding domain is linked to the IgG C-terminus of the first antigen-binding domain; The amino acid sequence of the CDR is determined according to the Kabat definition rules.

2. The bispecific antibody according to claim 1, wherein In the first antigen binding domain: 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.

3. The bispecific antibody according to claim 2, wherein The first antigen-binding domain comprises a light chain variable region as shown in SEQ ID NO: 38 and a heavy chain variable region as shown in SEQ ID NO:

40.

4. The bispecific antibody according to claim 2, wherein The first antigen binding domain comprises a heavy chain constant region and / or a light chain constant region; The heavy chain of the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:94, and the light chain of the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:95; or, the heavy chain of the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:96, and the light chain of the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:81; or, the heavy chain of the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:87, and the light chain of the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:

81. 5 . The bispecific antibody of claim 1 , wherein the amino acid sequence of the VHH has at least 90% sequence identity with SEQ ID NO: 49, SEQ ID NO: 72, SEQ ID NO: 73 or SEQ ID NO:

74.

6. The bispecific antibody according to claim 5, wherein 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.

7. The bispecific antibody according to any one of claims 1 to 6, wherein The first antigen-binding domain and the second antigen-binding domain are operably linked via a linker. The bispecific antibody according to claim 7 , wherein the linker is a peptide sequence.

9. The bispecific antibody of claim 8, wherein the linker comprises (G4S) n G, wherein n=1-10 and is an integer.

10. The bispecific antibody according to claim 9, wherein the linker is composed of (G4S) n G, wherein n=1-10 and is an integer. The bispecific antibody according to claim 9 or 10, wherein n=3.

12. The bispecific antibody according to any one of claims 1 to 6, comprising two first polypeptide chains and two second polypeptide chains, wherein: the amino acid sequence of the first polypeptide chain has at least 90% sequence identity to SEQ ID NO:56, and the amino acid sequence of the second polypeptide chain has at least 90% sequence identity to SEQ ID NO:62 or SEQ ID NO:57; the amino acid sequence of the first polypeptide chain has at least 90% sequence identity to SEQ ID NO: 50, and the amino acid sequence of the second polypeptide chain has at least 90% sequence identity to SEQ ID NO: 51; the amino acid sequence of the first polypeptide chain has at least 90% sequence identity to SEQ ID NO:52, and the amino acid sequence of the second polypeptide chain has at least 90% sequence identity to SEQ ID NO:53 or SEQ ID NO:60; The amino acid sequence of the first polypeptide chain has at least 90% sequence identity with SEQ ID NO: 54, and the amino acid sequence of the second polypeptide chain has at least 90% sequence identity with SEQ ID NO: 55 or SEQ ID NO: 61; or The amino acid sequence of the first polypeptide chain has at least 90% sequence identity with SEQ ID NO:58, and the amino acid sequence of the second polypeptide chain has at least 90% sequence identity with SEQ ID NO:59 or SEQ ID NO:

63.

13. The bispecific antibody according to claim 12, wherein: The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO:56, and the amino acid sequence of the second polypeptide chain is shown in 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, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 51; The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO:52, and the amino acid sequence of the second polypeptide chain is shown in 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, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO:55 or SEQ ID NO:61; The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 58, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 59 or SEQ ID NO:

63.

14. An isolated nucleic acid, characterized in that It encodes the bispecific antibody according to any one of claims 1 to 13.

15. A recombinant expression vector comprising the isolated nucleic acid of claim 14. The recombinant expression vector according to claim 15 , which is a plasmid or a viral vector. The recombinant expression vector according to claim 15 , wherein the recombinant expression vector is a cosmid or a phage. The recombinant expression vector according to claim 16 , wherein the backbone of the plasmid is pcDNA3.

4.

19. A transformant comprising the recombinant expression vector according to claim 15; in, The transformant is a non-animal or plant species. The transformant according to claim 19 , wherein the host cell of the transformant is a prokaryotic cell or a eukaryotic cell. The transformant according to claim 20 , wherein the eukaryotic cell is a yeast cell or a mammalian cell. The transformant according to claim 21 , wherein the mammalian cell is an EXPI-293 cell or a CHO cell.

23. A method for preparing a bispecific antibody, comprising the following steps: The transformant according to claim 19 is cultured, and the bispecific antibody is obtained from the culture. 24 . A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of claim 24, further comprising other pharmaceutical agents.

26. The pharmaceutical composition of claim 25, wherein the other agent is selected from one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

27. Use of the bispecific antibody according to any one of claims 1 to 13, and / or the pharmaceutical composition according to any one of claims 24 to 26, in the preparation of a medicament for preventing and / or treating tumors.

28. The use according to claim 27, wherein 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.

29. A kit comprising the bispecific antibody according to any one of claims 1 to 13 or the pharmaceutical composition according to any one of claims 24 to 26.

30. The kit of claim 29, further comprising (i) a device for administering the antibody or pharmaceutical composition; and / or (ii) instructions for use.

31. A kit comprising kit A and kit B, wherein: The drug kit A contains the bispecific antibody according to any one of claims 1 to 13 and / or the pharmaceutical composition according to any one of claims 24 to 26; The drug kit B contains other anti-tumor antibodies or pharmaceutical compositions containing other anti-tumor antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

32. A method for immunoassay or determination of CD40 and / or PD-L1, characterized in that: It comprises using the bispecific antibody according to any one of claims 1 to 13 and / or the pharmaceutical composition according to claim 24; The detection is for non-diagnostic purposes.

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