WO2023232036A1 - 抗cd40抗体和抗pd-l1×cd40双特异抗体及其应用 - Google Patents

抗cd40抗体和抗pd-l1×cd40双特异抗体及其应用 Download PDF

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WO2023232036A1
WO2023232036A1 PCT/CN2023/097156 CN2023097156W WO2023232036A1 WO 2023232036 A1 WO2023232036 A1 WO 2023232036A1 CN 2023097156 W CN2023097156 W CN 2023097156W WO 2023232036 A1 WO2023232036 A1 WO 2023232036A1
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seq
amino acid
acid sequence
sequence shown
antibody
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PCT/CN2023/097156
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French (fr)
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杨亚平
赵安
张丽娜
李芸
靳照宇
霍耐凡
杨袭
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明济生物制药（北京）有限公司
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Publication of WO2023232036A1 publication Critical patent/WO2023232036A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to an anti-CD40 antibody and an anti-PD-L1 ⁇ CD40 bispecific antibody and their applications.
  • tumor immunotherapy represented by PD-L1/PD-1 inhibitors has significantly prolonged the survival of tumor patients in multiple tumor indications, and has also been positioned as first-line treatment in multiple tumor indications (Nat Rev Immunol. 2020 Nov; 20(11):651-668.). As the most likely solution to cure tumors, tumor immunotherapy has become the central focus in the field of new anti-tumor drug research and development.
  • CD40 a type I transmembrane protein
  • DC cells antigen-presenting cells
  • CD40 agonistic antibodies can achieve T cell activation and show robust anti-tumor 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 play a synergistic anti-tumor effect with immune checkpoint antibodies such as PD-L1/PD-1, which relies on the cross-action mechanism of DC cells and T cells: DCs upregulate costimulatory molecules and differentiate into IL-12 is secreted to stimulate the activation of tumor-specific T cells.
  • T cells After T cells are activated, they activate DC cells by secreting IFN- ⁇ .
  • CD40 antibodies and PD-L1/PD-1 antibodies act on different links of cross-interaction, 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.).
  • Selicrelumab a CD40 monoclonal antibody developed by Pfizer, produced an objective partial response (PR) in 4 of 15 patients with advanced melanoma in a first-in-human single-dose study.
  • PR objective partial response
  • One of the patients subsequently received repeated doses of Selicrelumab for one year.
  • Complete remission (CR) remains after 15 years.
  • CD40 monoclonal antibodies such as APX005M, SEA-CD40, etc. also only had very low objective response rates (ORR).
  • CD40 agonistic monoclonal antibodies have shown many adverse reactions in clinical practice, including cytokine release syndrome (CRS), liver damage, and thrombocytopenia.
  • the maximum tolerated doses (MTD) of Selicrelumab, APX005M, and SEA-CD40 were respectively are 0.2, 0.3 and 0.06mg/kg (J Clin Oncol.2007 Mar 1;25(7):876-83.Cancer Biol Ther.2010 Nov 15;10(10):983-93.Lancet Oncol.2021 Jan; 22(1):118-131.Oncol Lett.2020 Nov;20(5):176.Annu Rev Med.2020 Jan 27;71:47-58.). Therefore, there is an urgent need in this field for a CD40-targeting antibody with high safety and good efficacy.
  • the present invention provides an anti-CD40 antibody and an anti-PD-L1 ⁇ CD40 bispecific antibody, a new anti-CD40 agonistic antibody of the present invention It can effectively regulate the activation of DC cells, has stronger T cell activation effect, but has low toxic and side effects, and is suitable for tumor immunotherapy; the anti-PD-L1 ⁇ CD40 bispecific antibody of the present invention simultaneously activates CD40 and blocks PD-L1/PD-1 acts in the positive feedback pathway of cross-interaction between DC cells and T cells to maximize anti-tumor efficacy; it also improves the selectivity of CD40 activation and reduces CD40 through PD-L1-dependent CD40 activation. Toxic side effects of agonistic antibodies.
  • a first aspect of the present invention provides: an anti-CD40 antibody, which includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 64, so The light chain variable region includes LCDR1, LCDR2 and LCDR3 in SEQ ID NO:65.
  • CD40 includes any variant or isoform of CD40 naturally expressed by a cell.
  • the antibody of the present invention can specifically bind to human CD40 and monkey CD40 (such as cynomolgus monkey). Alternatively, the antibody may be specific for human CD40 and may not exhibit cross-reactivity with other species.
  • CD40 or any variant or isoform thereof can be derived from natural Isolated from the cells or tissues in which they are expressed, or produced by recombinant techniques using techniques common in the art and described herein.
  • the light chain variable region includes LCDR1, LCDR2, and LCDR3 in SEQ ID NO:38, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as in SEQ ID NO:40;
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3 in SEQ ID NO:38, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as in SEQ ID NO:39;
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3 as in SEQ ID NO:30, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as in SEQ ID NO:31;
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3 as in SEQ ID NO:32, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as in SEQ ID NO:33;
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3 as in SEQ ID NO:32, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as in SEQ ID NO:34;
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3 as in SEQ ID NO:35, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as in SEQ ID NO:36;
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3 as in SEQ ID NO:35, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as in SEQ ID NO:37;
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3 as in SEQ ID NO:41, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as in SEQ ID NO:42;
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3 as in SEQ ID NO:41, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as in SEQ ID NO:43.
  • the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, and in some specific embodiments, the CDRs are determined according to the Kabat numbering rule.
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 1
  • the LCDR2 includes the amino acid sequence shown in X 5 X 6 SX 7 X 8 X 9 S
  • the amino acid sequence shown wherein X 5 is Y or A; X 6 is T or A; X 7 is S, R or T; X 8 is L or R;
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:4, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:5, and the HCDR3 includes the amino acid sequence shown in SEQ ID The amino acid sequence shown in NO:6.
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO: 1
  • the amino acid sequence of LCDR2 is the amino acid sequence shown in X 5 X 6 SX 7 X 8 X 9 S, where X 5 is Y or A; X 6 is T or A; X 7 is S , R or T; X 8 is L or R; ;
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 4, and the amino acid sequence of the HCDR2
  • the acid sequence is shown in SEQ ID NO:5, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:6.
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:8, SEQ ID NO:7 or SEQ ID NO:10
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:8, SEQ ID NO:7 or SEQ ID NO:10.
  • the LCDR3 includes SEQ ID NO:19, SEQ ID NO: 17.
  • the antibody comprises the 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, such as SEQ ID NO: 14.
  • amino acid sequence of HCDR2 shown in NO:24, and the amino acid sequence of HCDR3 shown in SEQ ID NO:28, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:25 or SEQ ID NO:29 are amino acid sequence of LCDR3 shown in SEQ ID NO:16 or SEQ ID NO:20; the amino acid sequence of HCDR1 shown in SEQ ID NO:21 or SEQ ID NO:22, such as SEQ ID NO:23 or SEQ ID
  • the amino acid sequence of LCDR1 is as shown in SEQ ID NO:9, SEQ ID NO:8, SEQ ID NO:7 or SEQ ID NO:10
  • the amino acid sequence of LCDR2 is as shown in SEQ ID NO:14, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:11 or SEQ ID NO:15
  • the amino acid sequence of LCDR3 is as SEQ ID NO:19, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:16 or SEQ ID NO:20
  • the amino acid sequence of HCDR1 is shown as SEQ ID NO:21 or SEQ ID NO:22
  • the amino acid sequence of HCDR2 is shown as SEQ ID NO:23 or SEQ ID NO:24
  • the amino acid sequence of HCDR3 is as shown in SEQ ID NO:28, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:25 or SEQ ID NO:29 Show.
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:9
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:14
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO:19
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:21
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:23
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:28;
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:9
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:14
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO:19
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:21
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:24
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:28;
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:7
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:11
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO:16
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:21
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:23
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:25;
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:7
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:12
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO:17
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:22
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:24
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:26;
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:7
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:12
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO:17
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:22
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:23
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:26;
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:8, the LCDR2 includes the amino acid sequence shown in SEQ ID NO:13, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO:18,
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:22, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:24, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:27;
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:8, the LCDR2 includes the amino acid sequence shown in SEQ ID NO:13, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO:18,
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:22, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:23, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:27;
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:10
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:15
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO:20
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:21
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:24
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:29; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:10
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:15
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO:20
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:21
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:23
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:29.
  • the light chain variable region comprises SEQ ID NO:9, SEQ ID NO:14 and SEQ ID NO:19 respectively.
  • LCDR1, LCDR2 and LCDR3 are shown, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:28 respectively;
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:9, SEQ ID NO:14 and SEQ ID NO:19 respectively, and the heavy chain variable region includes as shown in SEQ ID NO:14 and SEQ ID NO:19 respectively.
  • the light chain variable region includes 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 includes as shown in SEQ ID NO:11 and SEQ ID NO:16 respectively.
  • the light chain variable region includes 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 includes as shown in SEQ ID NO:12 and SEQ ID NO:17 respectively.
  • the light chain variable region includes 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 includes as shown in SEQ ID NO:12 and SEQ ID NO:17 respectively.
  • the light chain variable region includes 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 includes as shown in SEQ ID NO:13 and SEQ ID NO:18 respectively.
  • the light chain variable region includes 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 includes as shown in SEQ ID NO:13 and SEQ ID NO:18 respectively.
  • the light chain variable region includes 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 includes as shown in SEQ ID NO:15 and SEQ ID NO:20 respectively.
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3 respectively as shown in SEQ ID NO:10, SEQ ID NO:15 and SEQ ID NO:20, and the heavy chain variable region includes respectively as SEQ ID NO:15 and SEQ ID NO:20.
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:9
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:14
  • the amino acid sequence of LCDR3 is shown in SEQ ID
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:21
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:23
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:28. Show.
  • the amino acid sequence of LCDR1 is as shown in SEQ ID NO:9
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO: 14
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO: 19
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO: 21
  • the amino acid sequence of HCDR2 The sequence is shown in SEQ ID NO:24
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO:28.
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:7
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:11
  • the amino acid sequence of LCDR3 is shown in SEQ ID
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:21
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:23
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:25. Show.
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:7
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:12
  • the amino acid sequence of LCDR3 is shown in SEQ ID
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:22
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:24
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:26. Show.
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:7
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:12
  • the amino acid sequence of LCDR3 is shown in SEQ ID
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:22
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:23
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:26. Show.
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is shown in SEQ ID NO:13, and the amino acid sequence of LCDR3 is shown in SEQ ID
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:22, the amino acid sequence of HCDR2 is shown in SEQ ID NO:24, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:27. Show.
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is shown in SEQ ID NO:13, and the amino acid sequence of LCDR3 is shown in SEQ ID
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:22, the amino acid sequence of HCDR2 is shown in SEQ ID NO:23, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:27. Show.
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:10
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:15
  • the amino acid sequence of LCDR3 is shown in SEQ ID
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:21
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:24
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:29. Show.
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:10
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:15
  • the amino acid sequence of LCDR3 is shown in SEQ ID
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:21
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:23
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:29. shown.
  • the amino acid sequences of the listed CDRs are determined according to Kabat's definition rules.
  • the CDRs of an antibody can be defined by a variety of methods in the art, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883 , Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, U.S.
  • CDR complementarity determining region
  • Various numbering systems and their corresponding CDRs are well known to those skilled in the art, as shown in Table 1:
  • Laa-Lbb refers to the amino acid sequence starting from the N-terminus of the antibody light chain and from position aa to position bb according to its corresponding coding rules
  • Haa-Hbb refers to the amino acid sequence starting from the N-terminus of the antibody heavy chain. Starting from the amino acid sequence from position aa to position bb according to its corresponding coding rules.
  • L24-L34 in the second column of the second row of Table 1 refers to the amino acid sequence starting from the N-terminus of the antibody light chain variable region and determined from residues 24 to 34 according to Kabat coding rules; the rest are analogous.
  • the framework region of the light chain variable region is a human framework region
  • the framework region of the heavy chain variable region is a human framework region
  • the light chain variable region comprises an amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:38, and the heavy chain variable region comprises an amino acid sequence with SEQ ID NO:40 or SEQ ID NO:39 An amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity;
  • the light chain variable region comprises an amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:30, and the heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:31 An amino acid sequence with at least 90%, at least 95% or at least 99% sequence identity;
  • the light chain variable region includes an amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:32, and the heavy chain variable region includes an amino acid sequence with SEQ ID NO:33 or SEQ ID NO:34 An amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity;
  • the light chain variable region comprises an amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:35, and the heavy chain variable region comprises an amino acid sequence with SEQ ID NO:36 or SEQ ID NO:37 has an amino acid sequence of at least 90%, at least 95%, or at least 99% sequence identity; or
  • the light chain variable region comprises an amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:41
  • the heavy chain variable region comprises an amino acid sequence with SEQ ID NO:42 or SEQ ID NO: 43 is an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity.
  • variable region of the amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity maintains the function of binding to an antigen (eg, human CD40) that is identical to the original sequence.
  • an antigen eg, human CD40
  • sequence identity between sequences is performed as follows. To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the first and second amino acid sequences for optimal alignment or may be Discard non-homologous sequences).
  • the length of the aligned reference sequences 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.
  • the amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue at the corresponding position in the second sequence, then the molecules are identical at that position.
  • Mathematical algorithms can be used to perform sequence comparison and calculation of percent identity between two sequences.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm is used which has been integrated into the GAP program of the GCG software package (available at http://www.gcg.com available), determine the distance between two amino acid sequences using the Blossum 62 matrix or the PAM250 matrix with gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6 Percent identity.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) 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 light chain variable region includes the amino acid sequence shown in SEQ ID NO:38, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:40 or SEQ ID NO:39;
  • the light chain variable region includes the amino acid sequence shown in SEQ ID NO:30, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:31;
  • the light chain variable region includes the amino acid sequence shown in SEQ ID NO:32, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:33 or SEQ ID NO:34;
  • the light chain variable region includes the amino acid sequence shown in SEQ ID NO:35, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:36 or SEQ ID NO:37; or
  • the light chain variable region includes the amino acid sequence shown in SEQ ID NO:41, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:42 or SEQ ID NO:43.
  • the antibody comprises a light chain variable region set forth in SEQ ID NO:38 and a heavy chain variable region set forth in SEQ ID NO:40.
  • the antibody comprises a light chain variable region set forth in SEQ ID NO:38 and a heavy chain variable region set forth in SEQ ID NO:39.
  • the antibody comprises a light chain variable region set forth in SEQ ID NO:30 and a heavy chain variable region set forth in SEQ ID NO:31.
  • the antibody comprises a light chain variable region set forth in SEQ ID NO:32 and a heavy chain variable region set forth in SEQ ID NO:33.
  • the antibody comprises a light chain variable region set forth in SEQ ID NO:32 and a heavy chain variable region set forth in SEQ ID NO:34.
  • the antibody comprises a light chain variable region set forth in SEQ ID NO:35 and a heavy chain variable region set forth in SEQ ID NO:36.
  • the antibody comprises a light chain variable region set forth in SEQ ID NO:35 and a heavy chain variable region set forth in SEQ ID NO:37.
  • the antibody comprises a light chain variable region set forth in SEQ ID NO:41 and a heavy chain variable region set forth in SEQ ID NO:42.
  • the antibody comprises a light chain variable region set forth in SEQ ID NO:41 and a heavy chain variable region set forth in SEQ ID NO:43.
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:38, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:40 or SEQ ID Shown in NO:39.
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:30, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:31.
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:32, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:33 or SEQ ID Shown in NO:34.
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:35, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:36 or SEQ ID Shown in NO:37.
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:41, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:42 or SEQ ID Shown in NO:43.
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:38, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:40.
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:38, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:39.
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:30, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:31.
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:32, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:33.
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:32, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:34.
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:35, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:36.
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:35, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:37.
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:41, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:42.
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:41, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:43.
  • 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 , 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 antibody.
  • the antibodies of the present invention include monoclonal antibodies (abbreviated as mAb or Ab), which refer to antibodies obtained from a single clonal cell strain.
  • mAb or Ab monoclonal antibodies
  • the cell strain is not limited to eukaryotic, prokaryotic or phage clonal cell strains.
  • said antibody comprises a heavy chain constant region and/or a light chain constant region.
  • 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 kappa of a human antibody. chain.
  • the constant region includes constant region variants that do not alter the structure and function of the antibody variable region.
  • constant region variants have been disclosed in the prior art.
  • the Fc of the heavy chain constant region of an antibody has one or more of 238, 265, 269, 270, 297, 327 and 329 (using the EU numbering system) substitution of amino acids (U.S. Patent No.
  • the Fc of the heavy chain constant region of the antibody has one or more substitutions of amino acids at 234, 235, 265, 329 (using the EU numbering system), or the heavy chain constant region of the antibody
  • the Fc of the chain constant region has 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, Substitution of one or more amino acids at 424 or 434 (using the EU numbering system) (see U.S. Patent No. 7,371,826), etc.
  • the heavy chain constant region includes the amino acid sequence shown in SEQ ID NO:45
  • the light chain constant region includes the amino acid sequence shown in SEQ ID NO:44.
  • the heavy chain of the antibody includes the amino acid sequence shown in SEQ ID NO:94, and the light chain of the antibody includes the amino acid sequence shown in SEQ ID NO:95.
  • the heavy chain of the antibody includes the amino acid sequence shown in SEQ ID NO:96, and the light chain of the antibody includes the amino acid sequence shown in SEQ ID NO:81.
  • the heavy chain of the antibody includes the amino acid sequence shown in SEQ ID NO:87, and the light chain of the antibody includes the amino acid sequence shown in SEQ ID NO:81.
  • amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:45
  • amino acid sequence of the light chain constant region is as shown in SEQ ID NO:44.
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:94
  • amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:95.
  • amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:96
  • amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:81.
  • 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.
  • a second aspect of the present invention provides: a bispecific antibody comprising a first antigen-binding domain that specifically binds human CD40 and a second antigen-binding domain that specifically binds human PD-L1; wherein, the specificity The first antigen-binding domain that binds human CD40 is as defined in the anti-CD40 antibody of the first aspect of the invention.
  • antibody used in the present invention is used in the broadest sense, which encompasses monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (such as bispecific antibodies, diabodies, triabodies and tetrabodies, tandem two- scFv, tandem tri-scFv), also covers traditional antibodies (tetrapeptide chain structure antibodies composed of two identical heavy chains and two identical light chains connected by inter-chain disulfide bonds), as well as antibodies with antigen-binding activity Fab, Fab', F(ab')2, Fv, linear antibody, single chain antibody, scFv, sdAb, sdFv, Nanobody, peptide antibody peptibody, domain antibody (heavy chain (VH) antibody, light chain (VL) Antibody).
  • Each heavy chain of a full-length antibody consists of a heavy chain variable region (abbreviated as VH in the present invention) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated as VL in the present invention) and a light chain constant region (abbreviated as CL in the present invention).
  • the light chain constant region consists of one domain, CL.
  • Mammalian heavy chains are divided into alpha, delta, epsilon, gamma and mu heavy chains.
  • Mammalian light chains are classified as lambda or kappa light chains.
  • Immunoglobulins containing alpha, delta, epsilon, gamma and mu heavy chains are immunoglobulin (Ig)A, IgD, IgE, IgG and IgM.
  • IgA, IgD, IgE, IgG and IgM immunoglobulin
  • Complete antibodies form a "Y" shape.
  • the stem of Y consists of the second and third constant regions (and for IgE and IgM, the fourth constant region) of the two heavy chains held together, with disulfide bonds (interchain) formed in the hinge.
  • Heavy chains ⁇ , ⁇ , and ⁇ have a constant region composed of three tandem (in a row) Ig domains, and a hinge region for increased flexibility; heavy chains ⁇ and ⁇ have a constant region composed of four immunoglobulin domains. district.
  • the second and third constant regions are called "CH2 domain” and "CH3 domain” respectively.
  • Each arm of 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 the two heavy chain fragments containing the CH2 and CH3 domains of the antibody, which are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.
  • Fc constant region variants have been disclosed in the prior art.
  • the Fc of the heavy chain constant region of an antibody has one or more of 238, 265, 269, 270, 297, 327 and 329 (using the EU numbering system) Substitution of amino acids (U.S. Patent No. 6,737,056), or the Fc of the heavy chain constant region of the antibody has a substitution of one or more amino acids at 234, 235, 265, 329 (using the EU numbering system), or the heavy chain of the antibody
  • the Fc of the constant region has 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or substitution of one or more amino acids at 434 (using the EU numbering system) (see U.S. Patent No. 7,371,826), etc.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in the binding of the antibody to an antigen.
  • VHH, VH and VL each contain four conserved framework regions (FR) and three complementarity determining regions (CDR).
  • FR conserved framework regions
  • CDR complementarity determining region
  • CDR refers to the region within the variable domain that mainly contributes to binding to the antigen
  • framework or "FR” refers to the variable domain residues other than CDR residues.
  • VH or VHH contains 3 CDR regions: for easy differentiation, the 3 CDRs of VH are identified by HCDR1, HCDR2 and HCDR3, and the 3 CDRs of VHH are identified by VHH-CDR1, VHH-CDR2 and VHH-CDR3 identification; VL contains 3 CDR areas: LCDR1, LCDR2 and LCDR3.
  • Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • a single VH or VL may be sufficient to confer antigen binding specificity.
  • VHH and “Nanobody” have the same meaning and are used interchangeably and refer to the variable region of a cloned antibody heavy chain, a Nanobody consisting of only one heavy chain variable region, which has a complete Antigen binding function.
  • the specific binding epitope of VHH does not require recognition by other antigen-binding domains (this is different from conventional tetrapeptide chain structure antibodies, where the epitope of conventional tetrapeptide chain structure antibodies is recognized together by the structural pair formed by VL and VH).
  • VHH is a small, stable and efficient antigen recognition unit formed by a single heavy chain variable domain.
  • Nanobodies have excellent biological properties, with a molecular weight of 12-15kDa, one-tenth that of intact antibodies, good tissue penetration, high specificity, and good water solubility. Because of its special structural properties, it combines the advantages of traditional antibodies and small molecule drugs, almost perfectly overcoming the shortcomings of traditional antibodies such as long development cycles, low stability, and harsh storage conditions, and has gradually become an emerging new generation of antibody therapy. power, showing broad application prospects in immunodiagnosis and therapy.
  • VHH includes but is not limited to natural antibodies produced by camelids. They can also be antibodies produced by camelids and then humanized, or they can be obtained through phage display technology screening.
  • VHHs that bind specific antigens or epitopes have been previously disclosed, for example, in the following documents: 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, 17June, 2009 and WO94/04678.
  • PD-L1 includes any variant or isoform of PD-L1 naturally expressed by a cell.
  • Antibodies of the invention may cross-react with PD-L1 from 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 can be isolated from the cells or tissues in which they are naturally expressed, or produced by recombinant techniques using techniques common in the art and described herein.
  • the second antigen-binding domain includes at least one VHH
  • the VHH includes VHH-CDR1, VHH-CDR2 and VHH-CDR3 in SEQ ID NO: 66.
  • the VHH-CDR1 comprises an amino acid sequence represented by X 41 YYX 42 X 43 C, wherein is D or E, is S or T, is K or Q
  • the VHH-CDR2 includes the amino acid sequence shown in SEQ ID NO:76
  • the VHH-CDR3 includes the amino acid sequence shown in SEQ ID NO:77.
  • the VHH comprises VHH-CDR1, VHH-CDR2, and VHH-CDR3 of SEQ ID NO:49, SEQ ID NO:74, SEQ ID NO:72, or SEQ ID NO:73.
  • the VHH-CDR1, VHH-CDR2 and VHH-CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. In some specific embodiments, the VHH-CDR1, VHH-CDR2 and VHH-CDR3 is determined according to the Kabat numbering rule.
  • the VHH- CDR1 includes the amino acid sequence shown in SEQ ID NO:46, SEQ ID NO:67 or SEQ ID NO:70
  • the VHH-CDR2 includes the amino acid sequence shown in SEQ ID NO:47, SEQ ID NO:68 or SEQ ID NO:71
  • the amino acid sequence shown is
  • the VHH-CDR3 includes the amino acid sequence shown in SEQ ID NO:48 or SEQ ID NO:69.
  • the VHH comprises a VHH-CDR1 as set forth in SEQ ID NO:46, SEQ ID NO:67 or SEQ ID NO:70, such as SEQ ID NO:47, SEQ ID NO:68 or SEQ ID VHH-CDR2 as shown in NO:71, and VHH-CDR3 as shown in SEQ ID NO:48 or SEQ ID NO:69.
  • the VHH-CDR1 includes the amino acid sequence shown in SEQ ID NO:46
  • the VHH-CDR2 includes the amino acid sequence shown in SEQ ID NO:47
  • the VHH-CDR3 includes the amino acid sequence shown in SEQ ID NO:47.
  • the VHH-CDR1 includes the amino acid sequence shown in SEQ ID NO:67
  • the VHH-CDR2 includes the amino acid sequence shown in SEQ ID NO:68
  • the VHH-CDR3 includes the amino acid sequence shown in SEQ ID NO:69 sequence;
  • the VHH-CDR1 includes the amino acid sequence shown in SEQ ID NO:70
  • the VHH-CDR2 includes the amino acid sequence shown in SEQ ID NO:71
  • the VHH-CDR3 includes the amino acid sequence shown in SEQ ID NO:48 sequence.
  • the VHH includes:
  • VHH-CDR1 whose sequence is shown in SEQ ID NO:46
  • VHH-CDR2 whose sequence is shown in SEQ ID NO:47
  • VHH-CDR3 whose sequence is shown in SEQ ID NO:48;
  • VHH-CDR1 with the sequence shown in SEQ ID NO:67
  • VHH-CDR2 with the sequence shown in SEQ ID NO:68
  • VHH-CDR3 with the sequence shown in SEQ ID NO:69
  • VHH-CDR1 with the sequence shown in SEQ ID NO:70
  • VHH-CDR2 with the sequence shown in SEQ ID NO:71
  • VHH-CDR3 with the sequence shown in SEQ ID NO:48.
  • 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 is the same as SEQ ID NO:49, SEQ ID
  • the amino acid sequence shown in NO:72, SEQ ID NO:73 or SEQ ID NO:74 has at least 90%, at least 95% or at least 99% sequence identity.
  • the VHH is a humanized VHH.
  • 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 and the FR2 comprises the amino acid sequence set forth in SEQ ID NO:79
  • the FR1 comprises the amino acid sequence set forth in SEQ ID NO:82 or SEQ ID NO:78
  • 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:79.
  • the FR4 includes the amino acid sequence shown in SEQ ID NO:84.
  • 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
  • the amino acid sequence, and the FR4 includes the amino acid sequence shown in SEQ ID NO: 84;
  • the FR1 includes the amino acid sequence shown in SEQ ID NO:78
  • the FR2 includes the amino acid sequence shown in SEQ ID NO:79
  • the FR3 includes the amino acid sequence shown in SEQ ID NO:80
  • the FR4 includes the amino acid sequence shown in SEQ ID NO:84;
  • the FR1 includes the amino acid sequence shown in SEQ ID NO:82
  • the FR2 includes the amino acid sequence shown in SEQ ID NO:79
  • the FR3 includes the amino acid sequence shown in SEQ ID NO:80
  • the FR4 includes the amino acid sequence shown in SEQ ID NO:84.
  • 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 is the same as SEQ ID NO:49, SEQ ID NO :72.
  • the amino acid sequence shown in SEQ ID NO:73 or SEQ ID NO:74 has at least 90% sequence identity.
  • 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.
  • the second antigen binding domain further comprises a heavy chain constant region.
  • 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; the heavy chain constant region is more preferably a protein containing, for example, SEQ ID The amino acid sequence shown in NO:88.
  • the VHH and the heavy chain constant region are connected through a linker;
  • the linker is preferably a linker having an amino acid sequence shown as (G4S)x, wherein x is independently selected from an integer of 1-20 , more preferably the linker shown in SEQ ID NO:89.
  • 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 is the same as SEQ ID NO:93 , the amino acid sequence shown in SEQ ID NO:90, SEQ ID NO:91 or SEQ ID NO:92 has at least 90% sequence identity;
  • the second antigen-binding domain has 2 amino acid sequences as shown in SEQ ID NO: 93.
  • Fab consists of the CH1 and variable regions of a light chain and a heavy chain.
  • Fab' contains a light chain and a portion containing the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, two Fab' An interchain disulfide bond can be formed between the two heavy chains of the fragment to form an F(ab') 2 molecule.
  • the F(ab') 2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • Fv refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.
  • the scFv single chain antibody fragment, single chain antibody refers to a polypeptide chain formed by connecting a VH domain and a VL domain through a linker (also called a linker). wherein the VL and VH domains are paired to form a monovalent molecule via a linker that enables production of a single polypeptide chain [see, e.g., 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 consist of repeated G4S amino acid sequences or variants thereof.
  • linkers having the amino acid sequence (G 4 S) 4 or (G 4 S) 3 can be used, but variants thereof can also be used.
  • multispecific antibody is used in its broadest sense and encompasses antibodies with specificity for two or more epitopes, such as bispecific antibodies.
  • These multispecific antibodies include, but are not limited to: antibodies containing a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has two or more epitope specificities; Antibodies with one or more VL and VH regions, each VH-VL unit binds to a different target or different epitopes of the same target; antibodies with two or more single variable regions (such as VHH) , each single variable region binds to a different target or a different epitope of the same target.
  • VHH light chain variable region
  • epitope refers to an area or region on an antigen that can specifically bind to an antibody.
  • An epitope may be formed from a contiguous string of amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope) brought into spatial proximity, for example by folding of the antigen (ie by tertiary folding of the antigen in its proteinaceous nature).
  • the difference between conformational epitopes and linear epitopes is that in the presence of denaturing solvents, the antibody's binding to the conformational epitope is lost.
  • An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.
  • Screening for antibodies that bind a specific epitope can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443 -463) ⁇
  • antibodies are present in about 1 ⁇ 10-7M or less (e.g., about 1 ⁇ 10-8M or less, about 1 ⁇ 10-9M or less, about 1 ⁇ 10-10M or less, about 1 ⁇ 10- Binds the antigen or an epitope within the antigen with an equilibrium dissociation constant (KD) of 11 M or less, or about 1 x 10-12 M or less).
  • KD equilibrium dissociation constant
  • the KD of the antibody binding to the antigen is 10%, or 1% of the KD of the antibody binding to a non-specific antigen (eg, BSA, casein).
  • KD can be measured using standard procedures, e.g. by Measured by surface plasmon resonance assay.
  • antibodies that specifically bind to an antigen or an epitope within the antigen may be cross-reactive to other related antigens, e.g., to antibodies from other species (homologous) such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or marmoset (Callithrix jacchus) (commonmarmoset, marmoset) have cross-reactivity with the same antigen.
  • affinity refers to the overall strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise specified, as used herein, “affinity” refers to the internal binding affinity that reflects a 1:1 interaction between the members of a binding pair (eg, antibody and antigen).
  • affinity of a molecule X for its ligand Y can often be expressed by the dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.
  • kassoc or “ka” refers to the association rate of a particular antibody-antigen interaction
  • kdis or “kd” as used herein refers to the dissociation rate of a particular antibody-antigen interaction
  • KD refers to the dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd/ka) and is expressed as molar concentration (M).
  • M molar concentration
  • the KD value of an antibody can be determined using methods well established in the art. Methods for determining antibody KD include measuring surface plasmon resonance using biosensing systems such as systems, or measuring affinity in solution by solution equilibrium titration (SET).
  • anti-CD40 antibody and "antibody that specifically binds CD40” refer to an antibody that is capable of binding to CD40 with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent targeting CD40.
  • the antibody that binds to CD40 has a dissociation constant (KD) of ⁇ about 1 ⁇ M, ⁇ about 100 nM, ⁇ about 10 nM, ⁇ about 1 nM, ⁇ about 0.1 nM, ⁇ about 0.01 nM, or ⁇ about 0.001 nM. (For example, 10-8M or less, such as 10-8M to 10-12M, such as 10-9M to 10-10M).
  • anti-CD40 antibodies bind to epitopes conserved in CD40 from different species.
  • anti-PD-L1 antibody and "antibody that specifically binds to PD-L1” refer to an antibody that is capable of binding to PD-L1 with sufficient affinity such that the antibody can be used as a diagnostic agent and/or therapy targeting PD-L1 agent.
  • the antibody that binds to PD-L1 has a dissociation constant (KD) of ⁇ about 1 ⁇ M, ⁇ about 100 nM, ⁇ about 10 nM, ⁇ about 1 nM, ⁇ about 0.1 nM, ⁇ about 0.01 nM, or ⁇ about 0.001 nM (eg 10-8M or less, eg 10-8M to 10-12M, eg 10-9M to 10-10M).
  • anti-PD-L1 antibodies bind to epitopes conserved in PD-L1 from different species.
  • the first antigen binding domain and the second antigen binding domain are operably connected directly or through a linker.
  • the second antigen-binding domain is connected 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 IgG C-side.
  • the bispecific antibody of the present invention contains two first polypeptide chains and two second polypeptide chains.
  • 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
  • the second polypeptide The amino acid sequence of the chain is as set forth in SEQ ID NO:51 or has at least 99%, at least 95%, or at least 90% sequence identity with SEQ ID NO:51.
  • 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
  • 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% or at least 95% similarity with SEQ ID NO:53 or SEQ ID NO:60. , or at least 90% sequence identity.
  • 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
  • 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% or at least 95% similarity with SEQ ID NO:55 or SEQ ID NO:61 , or at least 90% sequence identity.
  • 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
  • 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 with SEQ ID NO:62 Identity.
  • 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
  • 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% or at least 95% similarity with SEQ ID NO:59 or SEQ ID NO:63. , or at least 90% sequence identity.
  • a third aspect of the present invention provides: a bispecific antibody comprising a first antigen-binding domain that specifically binds human CD40 and a second antigen-binding domain that specifically binds human PD-L1, wherein: the second The antigen-binding domain includes at least one VHH, the sequence of which is as defined in the bispecific antibody of the second aspect of the invention.
  • the first antigen binding domain is as defined in the anti-CD40 antibody of the first aspect of the invention.
  • the first antigen binding domain and the second antigen binding domain are operably connected directly or through a linker.
  • the second antigen-binding domain is connected 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 IgG C-side.
  • the bispecific antibody of the present invention contains two first polypeptide chains and two second polypeptide chains.
  • amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 50, and/or, The amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 51.
  • amino acid sequence of the first polypeptide chain is as shown in 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 Shown as ID NO:60.
  • amino acid sequence of the first polypeptide chain is as shown in 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 Shown as ID NO:61.
  • amino acid sequence of the first polypeptide chain is as shown in 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 Shown as ID NO:62.
  • the amino acid sequence of the first polypeptide chain is as shown in 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 Shown as ID NO:63.
  • the fourth aspect of the present invention provides: an isolated nucleic acid encoding the anti-CD40 antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect or the third aspect.
  • nucleic acid in the present invention refers to a chain of nucleotides of any length, and includes DNA and RNA.
  • the nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate capable of being incorporated into the strand by DNA or RNA polymerase.
  • a fifth aspect of the present invention provides: a recombinant expression vector comprising the isolated nucleic acid described in the fourth aspect of the present invention.
  • the recombinant expression vector is a plasmid, cosmid, phage or viral vector.
  • the backbone of the plasmid is pcDNA3.4.
  • recombinant expression vector means a genetically modified oligonucleotide or polynucleotide construct when the construct contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and the vector is in a state sufficient to allow the mRNA, protein, or The construct permits expression of the mRNA, protein, polypeptide or peptide by the host cell when the polypeptide, or peptide is contacted with the cell under conditions for intracellular expression.
  • the vectors of the present disclosure are generally not naturally occurring. However, portions of the carrier may be naturally occurring.
  • the recombinant expression vector of the present invention may contain any type of nucleotide, including but not limited to DNA and RNA: it may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and it may contain natural , unnatural or altered nucleotides.
  • Recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages.
  • the altered nucleotides or non-naturally occurring inter-nucleotide linkages do not hinder transcription or replication of the vector.
  • the recombinant expression vector of the present invention can be any suitable recombinant expression vector that can be used for transformation or transfection to deliver one or more genes or sequences of interest into any suitable host cell and preferably expresses the gene or sequence in the host cell. gene or sequence.
  • 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, and DNA or RNA expression vector related to cationic coagulant, DNA encapsulated in liposomes or RNA expression vectors and certain eukaryotic cells, such as production cells.
  • a sixth aspect of the present invention provides: a transformant comprising the recombinant expression vector described in the fifth aspect of the present invention.
  • the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.
  • the eukaryotic cell is a yeast cell or a mammalian cell.
  • the mammalian cells are, for example, EXPI-293 cells or CHO cells.
  • the term "host cell” refers to any type of cell that can contain the nucleic acids or vectors 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 protozoa.
  • a host cell may be a cell originating from or obtained from an individual.
  • Host cells can be derived from or obtained from mammals.
  • the term "mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters; and mammals of the order Lagomorpha, such as rabbits.
  • the mammal is from the order Carnivora, including felines (cats) and canidae (dogs). More preferably, the mammal is from the order Artiodactyla, including Bovids (cows) and Suids (pig), or of the order Perssodactyla, including Equidae (horses). Most preferably, the mammal belongs to the order Primates, New World Ceboids or Simoids (monkeys) or to the order Anthropoids (humans and apes). A particularly preferred mammal is human.
  • the expression vector can be transfected or introduced into a suitable host cell.
  • a variety of techniques can be used to achieve this purpose, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene editing (CRISPR-Cas system, ZFN system or TALEN system), transfection Transposon (Sleeping Beauty or PiggyBAC), gene gun, lipid-based transfection or other conventional techniques.
  • protoplast fusion cells are grown in culture medium and screened for appropriate activity.
  • cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers may, for example, provide prototrophy, biocidal resistance (eg, antibiotics), or heavy metal (eg, copper) resistance to an auxotrophic host, etc.
  • the selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional components may also be required for optimal synthesis of mRNA. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.
  • the seventh aspect of the present invention provides: a method for preparing an anti-CD40 antibody or a bispecific antibody, which includes the following steps: cultivating the transformant as described in the sixth aspect of the present invention, and obtaining an anti-CD40 antibody or bispecific antibody from the culture Bispecific antibodies.
  • 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 pharmaceutical composition also contains other pharmaceutical agents.
  • the other pharmaceutical agents are selected from the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, solvents, etc.
  • 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 CD40-targeting antibodies, and is subject to Drug route restrictions.
  • 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.
  • a pharmaceutically acceptable carrier is one that is chemically inert toward 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 adverse, allergic, or other inappropriate reactions when administered to animals or humans.
  • compositions are 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.
  • compositions suitable for practicing the methods disclosed herein can be prepared by combining a selected composition of desired purity in the form of a lyophilized cake or aqueous solution with optional Mixed with physiologically and pharmaceutically acceptable carriers, excipients or stabilizers ("Remington's Pharmaceutical Sciences", 18th edition, edited by A.R. Gennaro, Mack Publishing Company (1990) ) to prepare for storage.
  • Pharmaceutical compositions may be manufactured by admixing with one or more suitable carriers or adjuvants such as water, mineral oil, polyethylene glycol, starch, talc, lactose, thickeners, stabilizers , suspending agent, etc.
  • suitable carriers or adjuvants such as water, mineral oil, polyethylene glycol, starch, talc, lactose, thickeners, stabilizers , suspending agent, etc.
  • Such compositions may be in the form of solutions, suspensions, tablets, capsules, creams, salves, ointments or other conventional forms.
  • compositions for in vivo administration should be sterile. This is easily achieved by filtration through sterile filtration membranes before or after lyophilization and reconstitution. Therapeutic compositions are typically placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be pierced by a hypodermic needle. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In some cases, the form should be sterile and fluid to the extent that it can be easily injected. They should be stable under the conditions of manufacture and storage and should be preserved against the contaminating effects of microorganisms such as bacteria and fungi. Compositions for parenteral administration will generally be stored in lyophilized form or in solution.
  • the carrier may be a solvent or dispersion medium containing, for example, water or suitable mixtures thereof and vegetable oil. Appropriate flow properties can be maintained, for example, by the use of coatings such as lecithin, in the case of dispersions by maintaining the desired particle size, and by the use of surfactants.
  • the choice of carrier will depend in part on the particular type of pharmaceutical composition as well as on the pharmaceutical composition. application diameter to determine. Accordingly, a variety of suitable pharmaceutical composition formulations may be formulated.
  • the pharmaceutical composition of the present invention may contain any pharmaceutically acceptable ingredients, including, for example, acidulants, additives, adsorbents, aerosol propellants, air displacing agents, alkalinizing agents, anti-caking agents, anti-caking agents, etc.
  • compositions comprising bispecific antibodies described herein are formulated for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal administration. drug or intraperitoneally.
  • the pharmaceutical compositions are administered by nasal, spray, oral, aerosol, rectal, or vaginal administration.
  • the composition can be administered by infusion, bolus injection, or via an implanted device.
  • 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.
  • parenteral administration includes, but is not limited to, intravenous, intraarterial, intramuscular, intracerebral, intracerebroventricular, intracardiac, subcutaneous, intraosseous, intradermal, intrathecal, intraperitoneal, retrobulbar, pulmonary Intravesical, intravesical, and intracavernosal injection or infusion. Administration by surgical implantation at a specific site is also covered.
  • Injectable formulations are according to the invention.
  • Those of ordinary skill in the art are familiar with the requirements for effective pharmaceutical carriers for injectable compositions (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA ( Philadelphia), Banker and Chalmers, eds., pp. 238-250 (1982); and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pp. 622-630 (1986)).
  • compositions of the present invention may be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.
  • a ninth 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 or third aspect, and/or the pharmaceutical composition as described in the eighth aspect of the present invention, in Application in the preparation of drugs for the prevention and/or treatment of tumors.
  • the tumor is preferably a PD-L1 positive and/or CD40 positive tumor.
  • 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 not limited thereto.
  • the tenth aspect of the present invention provides: a kit, which includes the anti-CD40 antibody described in the first aspect of the present invention, the bispecific antibody described in the second or third aspect, or the anti-CD40 antibody described in the eighth aspect of the present invention.
  • Pharmaceutical compositions which includes the anti-CD40 antibody described in the first aspect of the present invention, the bispecific antibody described in the second or third aspect, or the anti-CD40 antibody described in the eighth aspect of the present invention.
  • the kit further includes (i) a device for administering the antibody or pharmaceutical composition; and/or (ii) instructions for use.
  • An eleventh aspect of the present invention provides: a medicine box set, which includes medicine box A and medicine box B, wherein:
  • kit A contains the anti-CD40 antibody as described in the first aspect of the present invention, the bispecific antibody as described in the second or third aspect, and/or the pharmaceutical composition as described in the eighth aspect of the present invention;
  • the kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and/or consists of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapy agents, One or more from the group consisting of oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
  • a twelfth aspect of the present invention provides: an immune detection, or a method for measuring CD40 and/or PD-L1, which includes using the anti-CD40 antibody as described in the first aspect of the present invention, the second aspect or the third aspect.
  • the bispecific antibody and/or the pharmaceutical composition according to the eighth aspect of the present invention is not limited to: an immune detection, or a method for measuring CD40 and/or PD-L1, which includes using the anti-CD40 antibody as described in the first aspect of the present invention, the second aspect or the third aspect.
  • the detection is a non-diagnostic detection and is only suitable for scientific research purposes.
  • a thirteenth aspect of the present invention provides: a method for preventing and/or treating tumors, which includes administering to a patient in need a therapeutically effective amount of an anti-CD40 antibody as described in the first aspect of the present invention, the second aspect or the third aspect.
  • 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 are not limited thereto.
  • the term "effective amount” means an amount of a drug or agent that elicits the biological or pharmaceutical response in a tissue, system, animal, or human, for example, that is sought by a researcher or clinician.
  • therapeutically effective amount means an amount that results in improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or a reduced rate of progression of a disease or condition, as compared to a corresponding subject who does not receive such amount. amount.
  • the term also includes within its scope amounts effective to enhance normal physiological functions.
  • a fourteenth aspect of the present invention provides: a combination therapy, which includes administering to a patient in need an anti-CD40 antibody as described in the first aspect of the present invention, a bispecific antibody as described in the second or third aspect, and /or the pharmaceutical composition according to the eighth aspect of the present invention, and the second therapeutic agent;
  • the second therapeutic agent preferably contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and/or consists of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents , one or more from the group consisting of chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
  • a fifteenth aspect of the present invention provides: an anti-CD40 antibody as described in the first aspect of the present invention, a bispecific antibody as described in the second or third aspect, and/or as described in the eighth aspect of the present invention for use as a medicine Pharmaceutical compositions; in some technical solutions, the medicine is used to prevent and/or treat tumors.
  • the tumor is a PD-L1 positive and/or CD40 positive tumor.
  • 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.
  • the tumor is colon cancer.
  • the present invention also provides an antibody-drug conjugate, which contains the anti-CD40 antibody as described in the first, second or third aspect of the present invention conjugated to one or more therapeutic agents or radioactive isotopes.
  • the bispecific antibody contains the anti-CD40 antibody as described in the first, second or third aspect of the present invention conjugated to one or more therapeutic agents or radioactive isotopes.
  • the bispecific antibody contains the anti-CD40 antibody as described in the first, second or third aspect of the present invention conjugated to one or more therapeutic agents or radioactive isotopes.
  • the bispecific antibody which contains the anti-CD40 antibody as described in the first, second or third aspect of the present invention conjugated to one or more therapeutic agents or radioactive isotopes.
  • the therapeutic agent is a cytotoxic agent, a chemotherapeutic agent, a drug, a growth inhibitor and/or a toxin; and/or the conjugation is using a linker to connect the antibody to the therapeutic agent or a radioactive isotope. .
  • the new anti-CD40 agonistic antibody of the present invention can effectively regulate the activation of DC cells, has 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 by stimulating CD40 and blocking PD-L1/PD-1 at the same time, acts on the positive feedback pathway of the cross-interaction of DC cells and T cells to maximize the anti-tumor efficacy; it also acts on PD- L1-dependent CD40 activation improves the selectivity of CD40 activation and reduces the toxic side effects of CD40 agonistic antibodies.
  • Figure 1 shows the reporter gene method in Example 7 of the present invention to measure the CD40 agonistic activity of monoclonal antibodies.
  • Figure 2 shows the DC cell regulatory activity of the monoclonal antibody in Example 8 of the present invention, using CD83 as the DC activation indicator.
  • Figure 3 shows the T cell regulatory activity of monoclonal antibodies in Example 9 of the present invention, using IFN- ⁇ as a T cell activation indicator.
  • Figures 4A to 4C show the safety of monoclonal antibodies in human CD40 gene knock-in mice in Example 10 of the present invention.
  • Figure 4A shows the changes in body weight of mice;
  • Figure 4B shows changes in liver function and hematology of mice;
  • Figure 4C shows changes in organ coefficients of mice.
  • Figure 5 shows the PD-L1/PD-1 blocking activity of monoclonal antibodies measured by the reporter gene method in Example 12 of the present invention.
  • Figure 6 shows the T cell regulatory activity of monoclonal antibodies in Example 13 of the present invention.
  • Figure 7 shows the structure of the bispecific antibody in Example 4 of the present invention.
  • Figure 8 shows the bispecific antibody binding to PD-L1 and CD40 simultaneously in Example 15 of the present invention.
  • Figure 9 shows the PD-L1/PD-1 blocking activity of the bispecific antibody measured by the reporter gene method in Example 16 of the present invention.
  • Figure 10 shows the CD40 agonistic activity of bispecific antibodies measured by the reporter gene method in Example 17 of the present invention:
  • Figure 10A CD40 agonistic activity of 1605, 1606, and 1607;
  • Figure 10B CD40 agonistic activity of 1608 and 1609;
  • Figure 10C CD40 agonistic activity of 1652 and 1653;
  • Figure 10D CD40 agonistic activity of 1654, 1655 and CP-870893.
  • Figure 11 shows the DC regulatory activity of bispecific antibodies in Example 19 of the present invention:
  • Figure 11A compares the activities of different bispecific antibodies, using IL-12p40 as an indicator;
  • Figure 11B and Figure 11C compares the activities of parent monoclonal antibodies and bispecific antibodies,
  • Figure 11B shows CD83 and
  • Figure 11C shows IL-12p40.
  • FIG. 12A and Figure 12B show the T cell regulatory activity of the bispecific antibody in Example 20 of the present invention.
  • Figure 13 shows the efficacy of the bispecific antibody against MC38 tumors in mice in Example 21 of the present invention.
  • Figure 14 shows the in vivo anti-MC38/hPD-L1 colon cancer tumor efficacy of the anti-PD-L1/CD40 bispecific antibody.
  • the variable region sequence of human CD40 antibody was designed, and the single chain Fv sequence (VL-G4S linker-VH) was obtained through gene synthesis technology, and loaded into the phage vector (pComb3XSS, purchased from (Beijing Zhuangmeng International Biogene Technology Co., Ltd.), a recombinant plasmid library was obtained.
  • the phage plasmid carrying the scFv gene was electroporated into SS320 E. coli competent cells. After SS320 E. coli (purchased from Lucigen) has grown to the logarithmic phase, helper phage (M13K07, purchased from NEB) is added for infection and cultured overnight. The phage are extracted from the culture supernatant to obtain a CD40 antibody phage library.
  • variable region sequence of the human PD-L1 nanobody was designed, and the VHH sequence was obtained through gene synthesis technology, and then loaded into a phage vector using restriction endonucleases to obtain a recombinant plasmid library.
  • the phage plasmid carrying the VHH gene was electroporated into SS320 E. coli competent cells. After SS320 has proliferated to the logarithmic phase, helper phage is added for infection and cultured overnight. The phage are extracted from the culture supernatant to obtain a PD-L1 antibody phage library.
  • the first round of screening method is as follows: Coat the enzyme plate with human CD40-mFc antigen or human PD-L1-mFc antigen (purchased from Biopsis), take the corresponding phage library and add an equal volume of 2% skim milk pre-prepared. Mix and incubate, add the premix to the coated wells for reaction, then wash with sterile PBST to remove the premix. Use 75mM sodium citrate buffer to elute the adsorbed phage in the well plate; after neutralizing the phage library, use M13K07 to assist phage to amplify the screened phage library 100 times.
  • CD40 phage library and PD-L1 phage library to infect SS320 E. coli respectively, and then spread them on agarose plates for culture. Pick single clone colonies, place them in a 96-well deep well plate, and culture them with 2YT containing ampicillin and kanamycin based on shaking at 37°C to obtain a supernatant containing monoclonal phage.
  • the monoclonal phage supernatant was incubated with an enzyme plate coated with human CD40-mFc antigen or human PD-L1-mFc antigen for 1 hour, then washed with sterile PBST, and then added Anti-M13-HRP (purchased from Yiqiao Shenzhou) and incubated at 4°C for 30 minutes. Then, a microplate reader is used to detect the binding of phages and antigens, and CD40 monoclonal phages and PD-L1 monoclonal phages with high binding to antigens are screened out.
  • the cDNA sequences of the heavy chain and light chain variable regions of the selected CD40 monoclonal phage were cloned into the pcDNA3.4 vector (Invitrogen) that already contains the antibody constant region, that is, multiple anti-CD40 monoclonal antibodies were obtained and named respectively.
  • the heavy chain and light chain recombinant plasmids were co-transfected into EXPI-293 cells (Invitrogen) using the PEI method, transiently transfected for 7-10 days, centrifuged and the supernatant was collected.
  • the supernatant is purified by protein A to obtain purified anti-CD40 monoclonal antibody.
  • the CDR sequence of the CD40 antibody is referred to Table 7 (determined by Kabat CDR system); the amino acid sequence of the heavy chain variable region and light chain variable region is referred to Table 8.
  • the full-length amino acid sequence of 1654CD is as follows:
  • Amino acid sequence of the heavy chain of 1654CD (SEQ ID NO:94):
  • the full-length amino acid sequence of 1606CD is as follows:
  • Amino acid sequence of the heavy chain of 1606CD (SEQ ID NO:96):
  • the full-length amino acid sequence of 1652CD is as follows:
  • Amino acid sequence of the heavy chain of 1652CD (SEQ ID NO:87):
  • the cDNA sequence (VHH) of the variable region of the selected PD-L1 monoclonal phage was cloned into the pcDNA3.4 vector (Invitrogen) that already contains the antibody constant region, to obtain multiple anti-PD-L1 monoclonal antibodies, respectively. Named, 1029, 1031, 1102, 1541.
  • the complementarity determining region (CDR) sequence of the PD-L1 monoclonal antibody is shown in Table 2 (determined by the Kabat CDR system), the framework region (FR) sequence of the monoclonal antibody is shown in Table 3, and the variable region (VHH) sequence is shown in Table 2.
  • the amino acid sequence is shown in Table 4, the amino acid sequence of the linker and constant region of the monoclonal antibody is shown in Table 5, and the full-length amino acid sequence of the monoclonal antibody is shown in Table 6.
  • the plasmid was transfected into EXPI-293 cells (Invitrogen) using the PEI method, transiently transfected for 7-10 days, centrifuged and the supernatant was collected. The supernatant is purified by protein A to obtain purified anti-PD-L1 monoclonal antibody.
  • X 48 is L or F
  • X 49 is A or S
  • X 50 is K or R
  • X 51 is A or T.
  • the recombinant anti-PD-L1/CD40 bispecific antibody uses PD-L1 monoclonal antibody 1541 and CD40 monoclonal antibody 1605CD, 1606CD, 1607CD, 1608CD, 1609CD, 1652CD, 1653CD, 1654CD, 1655CD.
  • the cDNA sequences of the heavy chain and light chain variable regions of the selected CD40 monoclonal phage were cloned into the pcDNA3.4 vector (Invitrogen) that already contained the antibody constant region; the cDNA sequences of the selected PD-L1 monoclonal phage were The cDNA sequence of the variable region (VHH) was cloned into the pcDNA3.4 vector (Invitrogen) that already contained CD40-VH and antibody constant region.
  • the VHH sequence was loaded into the C-terminus of the antibody constant region and introduced between the constant region and VHH ( G4S) linker; that is, multiple anti-PD-L1/CD40 bispecific antibodies (the structure is shown in Figure 7) were obtained, named 1605, 1606, 1607, 1608, 1609, 1652, 1653, 1654, and 1655 respectively.
  • the CDR sequence of the bispecific antibody is shown in Table 7 (determined by the Kabat CDR system); the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in Table 8; the amino acids of the heavy chain and light chain of the bispecific antibody The sequence is shown in Table 9.
  • the heavy chain and light chain recombinant plasmids were co-transfected into EXPI-293 cells (Invitrogen) using the PEI method, transiently transfected for 7-10 days, centrifuged and the supernatant was collected. The supernatant is purified by protein A to obtain purified anti-PD-L1/CD40 bispecific antibody.
  • RASQX 1 IX 2 X 3 YLX 4 (SEQ ID NO:1), X 1 is D, G, S or T; X 2 is R or S, X 3 is N or S; X 4 is N or A .
  • X 5 X 6 SX 7 X 8 X 9 S X 5 is Y or A; X 6 is T or A; X 7 is S, R or T; X 8 is L or R;
  • QQGX 10 X 11 X 12 PW (SEQ ID NO : 3), X 10 is K, I, N, Q or S;
  • SX 13 YYMS (SEQ ID NO: 4), X 13 is N or D.
  • FIRNKANX 14 YT (SEQ ID NO: 5), X 14 is A or G.
  • YGGX 15 X 16 X 17 GWYFDX 18 (SEQ ID NO: 6 ), X 15 is L or I;
  • X 16 is K or R;
  • the common sequence of the heavy chain variable region of Anti-CD40 is:
  • X 20 is G or A ;
  • X 21 is A or G ;
  • X 22 is N or S;
  • X 23 is R or S;
  • X 24 is L or I;
  • X 25 is K or R;
  • X 26 is V, K, I or Q;
  • the common sequence of the light chain variable region of Anti-CD40 is (SEQ ID NO:65):
  • X 29 is R or S;
  • X 30 is N or S;
  • X 31 is N or A;
  • X 32 is Y or A ;
  • X 33 is T or A;
  • X 34 is S, R or T;
  • X 38 is K, I, N, Q or S;
  • X 39 is S, A, N or T;
  • X 40 is L, Y or F.
  • Enzyme-linked immunosorbent assay was used to detect the binding ability and species specificity of monoclonal antibodies to human CD40.
  • Monoclonal antibodies 1605CD, 1606CD, 1607CD, 1608CD, 1609CD, 1652CD, 1653CD, 1654CD, and 1655CD can all bind to human CD40 and cynomolgus CD40, and their binding ability to human and cynomolgus monkey antigens is comparable. Cannot bind to rat CD40 or mouse CD40.
  • HEK-Blue CD40L cells purchased from InvivoGen
  • the specific method is as follows: collect HEK-Blue CD40L cells, resuspend them in pre-cooled PBS containing 2% FBS, with a density of 1 ⁇ 10 7 /mL, and then add them to a 96-well plate, 50 ⁇ L per well, that is, 5 ⁇ 10 5 cells /well, then add Biotin-CD40L (Mingji Biotechnology) and serially diluted CD40 monoclonal antibody, 50 ⁇ L per well, the final concentration of Biotin-CD40L is 10 nM, and incubate at 4°C for 1 hour.
  • Example 7 Reporter gene method to detect CD40 agonistic activity of anti-CD40 monoclonal antibodies
  • HEK-Blue CD40L was used to test the CD40 agonistic activity of monoclonal antibodies.
  • HEK-Blue CD40L cells were purchased from InvivoGen and highly express CD40, as well as the SEAP reporter gene under the control of NF- ⁇ B response element.
  • CD40 on HEK-Blue CD40L cells is activated, it induces the activation of downstream signaling NF- ⁇ B, which in turn induces the production of SEAP.
  • CD40 activation can be monitored by detecting the amount of secreted SEAP using QUANTI-Blue reagent (InvivoGen).
  • the specific method is as follows: collect HEK-Blue CD40L cells, 3 ⁇ 10 5 /mL resuspended in complete culture medium (RPMI 1640 containing 10% FBS), spread evenly into a 96-well plate, 100 ⁇ L/well, that is, 3 ⁇ 10 per well 4 cells; add 100 ⁇ L/well of anti-CD40 monoclonal antibody sample serially diluted in complete culture medium and control antibody (CP-870893), and incubate in a 37°C, 5% CO2 incubator for 20-24 hours. After the incubation, take out the 96-well plate and centrifuge at 300 g for 5 minutes. Transfer 40 ⁇ L/well supernatant to a new 96-well plate.
  • the results are shown in Figure 1.
  • the CD40 agonistic activity of monoclonal antibodies can be roughly divided into two categories.
  • the CD40 agonistic activity of antibodies 1608CD, 1609CD, 1654CD, and 1655CD is weak and weaker than the control CD40 monoclonal antibody CP-870893; the other Antibodies 1605CD, 1606CD, 1607CD, 1652CD, and 1653CD have stronger activities than the control CD40 monoclonal antibody CP-870893.
  • CD40 monoclonal antibodies were tested for their dendritic cell (DC) regulatory activity.
  • the donated human PBMC cells were resuspended in complete culture medium (RPMI 1640 containing 10% FBS) and seeded in a 10cm cell culture dish, and incubated in a 37°C carbon dioxide incubator for 2 hours. Discard the culture supernatant and suspended cells. The adherent cells are monocytes. Mononuclear cells were cultured in complete medium containing 100ng/mL GM-CSF (PeproTech, Cat. No. 300-03) and 100ng/mL IL-4 (PeproTech, Cat. No. 200-04) for 6 days and replaced every 2 days. liquid to obtain imDC cells.
  • complete culture medium RPMI 1640 containing 10% FBS
  • Figure 2 compares the DC regulatory activities of the anti-CD40 monoclonal antibodies of the present invention. Different monoclonal antibodies can significantly up-regulate CD83 on DC cells.
  • CD40 agonistic antibodies activate authorized DC cells and then activate T cells, which is its most important anti-tumor biological effect.
  • the MLR experimental system incubated with allogeneic DC cells and T cells was used to test the T cell regulatory activity of CD40 monoclonal antibodies.
  • Allogeneic T cells are isolated from human PBMC cells.
  • Allogeneic T cells are isolated from human PBMC cells.
  • the results are shown in Figure 3.
  • the strong CD40 agonistic monoclonal antibody 1606CD and the weak CD40 agonistic monoclonal antibody 1654CD of the present invention can enhance T cell activation under the condition of low proportion of DC, while the control monoclonal antibody CP-870893 has basically no effect. .
  • mice repeatedly injected with the strong CD40 agonistic monoclonal antibody 1606CD and the weak CD40 agonistic monoclonal antibody 1654CD showed no obvious abnormalities, and only a decrease in lymphocytes and granulocytes was seen in hematology.
  • Mice that were repeatedly injected with monoclonal antibody CP-870893 showed weight loss, increased ALT, and significant decreases in RBC, HGB, and PLT. During dissection, obvious organ necrosis and organ enlargement were seen. The results suggest that monoclonal antibodies 1606CD and 1654CD have better safety than monoclonal antibody CP-870893.
  • Example 11 Binding detection of recombinant PD-L1 monoclonal antibody and PD-L1
  • Enzyme-linked immunosorbent assay was used to detect the binding ability and species specificity of monoclonal antibodies to human PD-L1.
  • Mouse (mouse) PD-L1 (all purchased from Biopsis), 1 ⁇ g/mL, 100 ⁇ L per well, incubated overnight at 4°C. Wash 5 times with PBST. Block with 300 ⁇ L/well of PBST containing 1% BSA and incubate at room temperature for 1 hour. Wash 5 times with PBST.
  • Monoclonal antibodies 1029, 1031, 1102, and 1541 can all bind to human PD-L1 and cynomolgus monkey PD-L1, and the binding ability to human and cynomolgus monkey antigens is equivalent to that of the control antibody. Cannot bind to rat PD-L1 or mouse PD-L1.
  • the interaction between monoclonal antibodies and antigens is detected using Gator, a label-free biomolecule interaction analyzer based on the principle of biolayer interference (BLI).
  • the specific method is as follows: use PA probe, dilute the monoclonal antibody to 50nM and add it to the probe plate, so that the monoclonal antibody is captured by the PA probe. Then, the antigen human PD-L1 was added in a gradient dilution starting from 200nM. The antigen interacted with the bispecific antibody captured by the PA probe. The interaction was analyzed by detecting the signal change of the reflection interference spectrum on the probe surface, and the final calculation was performed. Find the binding kinetic constants of the antibody.
  • Example 12 Reporter gene method to detect PD-L1/PD-1 blocking activity of PD-L1 monoclonal antibody
  • Jurkat/PD-1-NFAT-luciferase cells constructed using Mingji Biotechnology (high expression of PD-1 and luciferase reporter gene under the control of NFAT response element) and WIL2S/PD-L1 (high expression of PD-L1) cells, and Anti-CD20/CD3 bispecific antibody to establish a reporter gene detection method for PD-L1/PD-1 blocking activity.
  • the specific method is as follows: collect WIL2S/PD-L1 cells and resuspend them in complete culture medium (RPMI 1640 containing 10% FBS) at a cell density of 4 ⁇ 10 6 /mL, 50 ⁇ L/well, that is, 2 ⁇ 10 5 cells per well , spread evenly into a 96-well plate; collect Jurrkat-PD-1-NFAT-luciferase cells and resuspend them in complete culture medium (RPMI 1640 containing 10% FBS) at a cell density of 4 ⁇ 10 6 /mL, 50 ⁇ L/well, That is, 2 ⁇ 10 5 cells per well are evenly added to the above-mentioned 96-well plate; dilute the anti-CD20/CD3 bispecific antibody in complete culture medium, and add 25 ⁇ L/well to the above-mentioned 96-well plate; serially dilute the anti-PD- L1 monoclonal antibody sample and control antibody were added to the above-mentioned 96-well plate at 25 ⁇ L/
  • Monoclonal antibodies 1029, 1031, 1102, and 1541 can all block the negative signal transmitted by PD-L1 to PD-1.
  • the activity of the antibodies is similar, but slightly less active than the PD-L1 control monoclonal antibody Durvalumab. powerful.
  • Example 13 T cell regulatory activity of PD-L1 monoclonal antibody
  • the MLR experimental system incubated with allogeneic DC cells and T cells was used to test the T cell regulatory activity of monoclonal antibodies.
  • the donated human PBMC cells were resuspended in complete culture medium (RPMI 1640 containing 10% FBS) and seeded in a 10cm cell culture dish, and incubated in a 37°C carbon dioxide incubator for 2 hours. Discard the culture supernatant and suspended cells. The adherent cells are monocytes. Mononuclear cells were cultured in complete medium containing 100ng/mL GM-CSF (PeproTech, Cat. No. 300-03) and 100ng/mL IL-4 (PeproTech, Cat. No. 200-04) for 6 days and replaced every 2 days. solution, then add TNF ⁇ and IL-1 ⁇ (both purchased from PeproTech) and incubate for 2 days to obtain DC cells.
  • complete culture medium RPMI 1640 containing 10% FBS
  • Allogeneic T cells are isolated from donated human PBMC cells.
  • the obtained human DC cells and human T cells were collected, resuspended in complete culture medium (RPMI 1640 containing 10% FBS), and seeded in a 96-well plate.
  • the DC cells and T cells seeded were 1 ⁇ 10 4 /well and 1 ⁇ 10 5 /well, mixed culture. And add antibody samples serially diluted in complete medium.
  • the culture plate was placed in a 37°C carbon dioxide incubator and incubated for 5 days. After the incubation, remove the supernatant from the well and detect the cytokine IFN- ⁇ (Biolegend, Cat. No. 430101) according to the kit instruction manual.
  • Enzyme-linked immunosorbent assay was used to detect the binding ability and species specificity of bispecific antibodies to human PD-L1 and human CD40.
  • CD40, mouse CD40 both purchased from Biopsis
  • 1 ⁇ g/mL 100 ⁇ L per well, incubated overnight at 4°C. Wash 5 times with PBST.
  • 109-035-088 diluted in PBST containing 1% BSA, 100 ⁇ L per well, and incubate at room temperature for 1 hour. Wash 5 times with PBST. Add colorimetric substrate TMB, 100 ⁇ L per well, develop color at room temperature for 10 minutes, and then add 1M sulfuric acid to terminate the reaction. Read the OD 450nm on a microplate reader, analyze the results and calculate the EC 50 using a 4-parameter fitting binding curve.
  • Bispecific antibodies 1605, 1606, 1607, 1608, 1609, 1652, 1653, 1654, and 1655 can all bind to human PD-L1 and CD40, cynomolgus PD-L1 and CD40, and bind to human and crab-eating crabs.
  • the binding capacity of monkey antigens was comparable.
  • mouse PD-L1 and CD40 cannot bind.
  • Biotin-labeled human PD-L1 (Mingji Biopharmaceutical) diluted in PBST containing 1% BSA, 100 ⁇ L per well, and incubate at room temperature for 1 hour. Wash 5 times with PBST.
  • Add Streptavidin-HRP (BioLegend, Cat. No. 405210) diluted in PBST containing 1% BSA, 100 ⁇ L per well, and incubate at room temperature for 30 minutes.
  • Add colorimetric substrate TMB 100 ⁇ L per well, develop color at room temperature for 10 minutes, and then add 1M sulfuric acid to terminate the reaction. Read the OD 450nm on a microplate reader, analyze the results and calculate the EC 50 using a 4-parameter fitting binding curve.
  • Bispecific antibodies 1605, 1606, 1607, 1608, 1609, 1652, 1653, 1654, and 1655 can all bind to human PD-L1 and human CD40 at the same time, while the control monoclonal antibody has no signal and cannot bind to both at the same time. species antigen.
  • Example 16 Reporter gene method to detect PD-L1/PD-1 blocking activity of anti-PD-L1/CD40 bispecific antibodies
  • Jurkat/PD-1-NFAT-luciferase cells high expression of PD-1 and luciferase reporter gene under the control of NFAT response element
  • WIL2S/PD-L1 cells high expression of PD-L1
  • a reporter gene detection method for PD-L1/PD-1 blocking activity was established.
  • the specific method is as follows: collect WIL2S/PD-L1 cells and resuspend them in complete culture medium (RPMI 1640 containing 10% FBS) at a cell density of 4E6/mL, 50 ⁇ L/well, that is, 2E5 cells per well, and spread evenly into 96 wells.
  • 96-well plate collect Jurrkat-PD-1-NFAT-luciferase cells and resuspend them in complete culture medium (RPMI 1640 containing 10% FBS) at a cell density of 4E6/mL, 50 ⁇ L/well, that is, 2E5 cells per well, and add evenly to the above-mentioned 96-well plate; dilute the anti-CD20/CD3 bispecific antibody in complete medium, 25 ⁇ L/well, and add to the above-mentioned 96-well plate; serially dilute the anti-PD-L1/CD40 bispecific antibody sample and control antibody in the complete medium, 25 ⁇ L /well was added to the above-mentioned 96-well plate; then incubated in a 37°C, 5% CO2 incubator for 6 hours.
  • complete culture medium RPMI 1640 containing 10% FBS
  • 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.
  • the activity of the PD-L1 control monoclonal antibody Durvalumab is also similar.
  • Example 17 Reporter gene method to detect CD40 agonistic activity of anti-PD-L1/CD40 bispecific antibodies
  • HEK-Blue CD40L was used to test the CD40 agonistic activity of bispecific antibodies.
  • HEK-Blue CD40L cells Purchased from InvivoGen, it has high expression of CD40 and SEAP reporter gene under the control of NF- ⁇ B response element.
  • CD40 on HEK-Blue CD40L cells is activated, it induces the activation of downstream signaling NF- ⁇ B, which in turn induces the production of SEAP.
  • CD40 activation can be monitored by detecting the amount of secreted SEAP using QUANTI-Blue reagent (InvivoGen).
  • the CD40 agonistic activity of the bispecific antibody in the presence of PD-L1 was tested by incubating CHO/PD-L1 cells with HEK-Blue CD40L cells.
  • the specific method is as follows: collect HEK-Blue CD40L cells, 3E5/mL resuspended in complete culture medium (RPMI 1640 containing 10% FBS), spread evenly into a 96-well plate, 100 ⁇ L/well, that is, 3E4 cells per well; collect CHO /PD-L1 cells, 6E5/mL resuspended in complete culture medium (RPMI 1640 containing 10% FBS), 50 ⁇ L/well, that is, 3E4 cells per well, was added to a 96-well plate.
  • CHO/PD-L1 cells are not required, Add 50 ⁇ L/well of complete culture medium; add 50 ⁇ L/well of anti-PD-L1/CD40 bispecific antibody samples serially diluted with complete culture medium and control antibodies, and incubate in a 37°C, 5% CO2 incubator for 20-24 hours. After the incubation, take out the 96-well plate and centrifuge at 300 g for 5 minutes. Transfer 40 ⁇ L/well supernatant to a new 96-well plate. Add 160 ⁇ L/well QUANTI-Blue reagent to the supernatant and incubate at 37°C and 5% CO2. Incubate in the box for 20-30 minutes, read OD 655nm on a microplate reader, and analyze the results.
  • the results are shown in Figure 10A to Figure 10D and Table 15.
  • the CD40 agonistic activity of bispecific antibodies 1608, 1609, 1654, and 1655 was significantly enhanced by the PD-L1 cross-linking signal provided by CHO/PD-L1, representing that these bispecific antibodies Its activity is selective: it has lower activity in environments with low PD-L1 expression (such as blood); it has higher activity in environments with high PD-L1 expression (such as tumor environment), and has the ability to enhance drug efficacy and control side effects. Effect.
  • the bispecific antibodies 1605, 1606, 1607, 1652, and 1653 were less enhanced by CHO/PD-L1; the control CD40 monoclonal antibody CP-870893 was not enhanced by CHO/PD-L1.
  • HEK-Blue CD40L cells that highly express CD40 were used to test the blocking effect of bispecific antibodies on CD40/CD40L binding.
  • the specific method is as follows: collect HEK-Blue CD40L cells and rehydrate them with pre-cooled PBS containing 2% FBS. Suspension, the density is 1E7/mL, and then added to a 96-well plate, 50 ⁇ L per well, that is, 5E5 cells/well, and then add Biotin-CD40L (Mingji Biotechnology) and serially diluted bispecific antibodies, 50 ⁇ L per well, Biotin-CD40L The final concentration was 10 nM and incubated at 4°C for 1 hour. Wash 2 times with pre-chilled PBS.
  • Human PBMC cells were resuspended in complete culture medium (RPMI 1640 containing 10% FBS) and seeded in a 10cm cell culture dish, and incubated in a 37°C carbon dioxide incubator for 2 hours. Discard the culture supernatant and suspended cells. The adherent cells are monocytes. Mononuclear cells were cultured in complete medium containing 100ng/mL GM-CSF (PeproTech, Catalog No. 300-03) and 100ng/mL IL-4 (PeproTech, Catalog No. 200-04) for 6 days and replaced every 2 days. liquid to obtain imDC cells.
  • complete culture medium RPMI 1640 containing 10% FBS
  • Figure 11A compares the DC regulatory activities of different bispecific antibodies, using IL-12/IL-23 p40 as an indicator.
  • the heteroantibodies stimulate DCs to secrete IL-12 p40 in a concentration-dependent manner, and the activities of different bispecific antibodies vary greatly.
  • Figure 11B and Figure 11C compare the DC regulatory activities of the parental PD-L1 monoclonal antibody, the parental CD40 monoclonal antibody, the bispecific antibody (1609) and the control CD40 monoclonal antibody CP-870893.
  • the detection indicators are CD83 and IL-12/IL-23 p40, the data shows that the parental PD-L1 monoclonal antibody has no regulation on DC, and the activity of the parental CD40 monoclonal antibody is weak. After constructing a double antibody, its DC activity is significantly enhanced.
  • the MLR experimental system incubated with allogeneic DC cells and T cells was used to test the T cell regulatory activity of bispecific antibodies.
  • Allogeneic T cells are isolated from human PBMC cells.
  • Allogeneic T cells are isolated from human PBMC cells.
  • the results are shown in Figure 12A and Figure 12B.
  • the bispecific antibody enhanced T cell activation, and its T cell regulatory activity was significantly stronger than the parental PD-L1 monoclonal antibody, the parental CD40 monoclonal antibody, and the PD-L1 control monoclonal antibody Durvalumab.
  • Example 21 In vivo anti-tumor efficacy of anti-mouse PD-L1/mouse CD40 bispecific antibodies
  • This example detects the anti-tumor efficacy of bispecific antibodies in mice.
  • a surrogate anti-mouse PD-L1/mouse CD40 bispecific antibody 1058 expressing the bispecific antibody was constructed (the anti-PD-L1 sequence is from IMGT Database ID 9814, anti-CD40 sequences are No. 33_VH and No. 34_VL in WO2018185045A1).
  • the purpose of using this surrogate is to evaluate the in vivo effect of bispecific antibodies formed by antibodies targeting PD-L1 and antibodies targeting CD40 in wild-type mice, and is only used to verify the efficacy of such bispecific antibodies.
  • mice C57BL/6 mice, female, 6-8 weeks old, were purchased from Beijing Vitong Lever. Mice acclimate to environment for one week Afterwards, 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). When the tumor volume grows to about 100mm3 , they are divided into groups according to the tumor volume, with 8 mice in each group, respectively set as the vehicle control group, the anti-mouse PD-L1 monoclonal antibody administration group, and the anti-mouse CD40 monoclonal antibody administration group. group, anti-mouse PD-L1/mouse CD40 bispecific antibody group.
  • Example 22 In vivo anti-tumor efficacy of anti-PD-L1/CD40 bispecific antibodies
  • This example evaluates the anti-tumor efficacy of anti-PD-L1/CD40 bispecific antibodies in PD-L1/CD40 humanized mice.
  • the experiment was completed in cooperation with Beijing Agricultural College.
  • PD-L1/CD40 humanized mice female, 6-8 weeks old, purchased from Biocytogen.
  • each mouse was inoculated with 5E6 MC38/hPD-L1 mouse colon cancer cells.
  • the tumor volume grows to about 100 mm, they are divided into groups according to the tumor volume, with 6 mice in each group. They are given vehicle, anti-PD-L1 monoclonal antibody, anti-CD40 monoclonal antibody, and anti-PD-L1/anti-CD40 bispecific antibody 1654.
  • the experimental animals used were male cynomolgus monkeys, which were randomly divided into 5 groups (2 animals/group). They were given vehicle control, 12 mg/kg bispecific antibodies 1607, 1608, and 1609, and 10 mg/kg control CD40 monoclonal antibody CP- 870893.

Abstract

本发明公开了一种抗CD40抗体和抗PD-L1×CD40双特异抗体及其应用。所述抗CD40的抗体包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:64中的HCDR1、HCDR2和HCDR3，所述轻链可变区包含SEQ ID NO:65中的LCDR1、LCDR2和LCDR3。本发明的新的抗CD40激动性抗体能够有效地调控DC细胞的活化，具有更强的T细胞活化作用，但毒副作用低，适合用于肿瘤的免疫治疗。本发明的双特异性抗体可以最大化抗肿瘤药效，通过PD-L1依赖的CD40活化，提高CD40活化的选择性，降低CD40激动性抗体的毒副作用。

Description

抗CD40抗体和抗PD-L1×CD40双特异抗体及其应用
本申请要求申请日为2022/5/31的中国专利申请2022106137021、申请日为2022/5/31的中国专利申请2022106162803、申请日为2023/5/19的中国专利申请2023105733440的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物技术领域，具体涉及一种抗CD40抗体和抗PD-L1×CD40双特异抗体及其应用。
背景技术
近些年来，肿瘤治疗领域发展迅速，从经典的手术、放疗及化疗为主的治疗手段，发展出靶向治疗、免疫治疗等更先进的治疗方案。尤其是以PD-L1/PD-1抑制剂为代表的肿瘤免疫疗法，在多个肿瘤适应症中显著延长了肿瘤患者的生存期，也在多个肿瘤适应症中被定位为一线治疗(Nat Rev Immunol.2020 Nov；20(11):651-668.)。肿瘤免疫治疗作为最有可能治愈肿瘤的方案，成为抗肿瘤新药研发领域的中心焦点。
肿瘤免疫学的关键挑战之一是增加冷肿瘤中浸润性T细胞的数量和质量。在许多肿瘤患者中，T细胞启动不足是肿瘤微环境中缺乏T细胞的原因，而负载有肿瘤抗原的抗原递呈细胞，尤其是DC细胞，在启动T细胞应答上有着决定性的作用。CD40，I型跨膜蛋白，属于肿瘤坏死因子受体(TNFR)超家族成员，在抗原递呈细胞(DC细胞，细胞，B细胞)、血小板、部分非造血细胞和多种类型的肿瘤细胞中广泛表达，在固有免疫和适应性免疫中都有着重要作用，在DC细胞活化中更是起着关键作用(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.)。CD40被激活后，通过上调DC细胞上的共刺激分子和MHC以及诱导促炎症细胞因子，活化并授权DC细胞去促进抗肿瘤特异性T细胞的活化，该类T细胞具有彻底清除肿瘤细胞的潜力。
在小鼠的多个肿瘤模型中，利用CD40激动性抗体可以实现T细胞活化，并显示出稳健的抗肿瘤药效(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激动性抗体还可以与PD-L1/PD-1等免疫检查点抗体发挥协同抗肿瘤作用，其依赖于DC细胞和T细胞的交叉作用机制：DC通过上调共刺激分子和分 泌IL-12去刺激肿瘤特异性T细胞活化，T细胞活化后又通过分泌IFN-γ来活化DC细胞。CD40抗体，PD-L1/PD-1抗体作用于交叉作用的不同环节，增强正反馈，最大化抗肿瘤药效(Cancer Res.2016 Nov 1；76(21):6266-6277.Immunity.2018 Dec 18；49(6):1148–1161.e7.)。
目前，有多家生物制药公司在开发针对CD40的激动性单抗，其相关专利如WO2003040170、WO2014070934A1、US20180066053、US20140348836、WO2020108611、CN111763259等，并且有多个抗体已经进入临床测试阶段。辉瑞开发的CD40单抗Selicrelumab在首次人体单剂量研究中，在15名晚期黑色素瘤患者中有4名产生了客观部分反应(PR)，其中一名患者随后接受了一年的重复剂量的Selicrelumab，在15年后仍保持着完全缓解(CR)。然而在后续临床试验中Selicrelumab药效并不佳,其它的CD40单抗比如APX005M,SEA-CD40等也仅有很低的客观反应率(ORR)。CD40激动性单抗在临床上显示出了较多的不良反应，包括细胞因子释放综合征(CRS)、肝损伤和血小板减少等，Selicrelumab、APX005M及SEA-CD40的最大耐受剂量(MTD)分别为0.2,0.3和0.06mg/kg(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.)。因此，本领域亟需一种安全性高、药效佳的靶向CD40的抗体。
发明内容
为了解决上述技术问题，鉴于CD40抗体和/或PD-L1抗体的研究开发现状，本发明提供一种抗CD40抗体和抗PD-L1×CD40双特异抗体，本发明的新的抗CD40激动性抗体能够有效地调控DC细胞的活化，具有更强的T细胞活化作用，但毒副作用低，适合用于肿瘤的免疫治疗；本发明的抗PD-L1×CD40双特异抗体通过同时激动CD40和阻断PD-L1/PD-1，作用于DC细胞和T细胞的交叉作用的正反馈通路中，最大化抗肿瘤药效；也通过PD-L1依赖的CD40活化，提高CD40活化的选择性，降低CD40激动性抗体的毒副作用。
本发明第一方面提供：一种抗CD40的抗体，其包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:64中的HCDR1、HCDR2和HCDR3，所述轻链可变区包含SEQ ID NO:65中的LCDR1、LCDR2和LCDR3。
术语“CD40”包括由细胞天然表达的CD40的任何变体或同种型。本发明的抗体可特异性结合人CD40及猴CD40(如食蟹猴)。作为另一种选择，该抗体也可以是人CD40特异性的，可不表现出与其他物种的交叉反应性。CD40或其任何变体或同种型可从天然 表达它们的细胞或组织中分离而得，或使用本领域通用以及本文所述的那些技术通过重组技术产生。
在一些实施方案中，所述轻链可变区包含SEQ ID NO:38中的LCDR1、LCDR2和LCDR3，且所述重链可变区包含如SEQ ID NO:40中的HCDR1、HCDR2和HCDR3；
或所述轻链可变区包含SEQ ID NO:38中的LCDR1、LCDR2和LCDR3，且所述重链可变区包含如SEQ ID NO:39中的HCDR1、HCDR2和HCDR3；
或所述轻链可变区包含如SEQ ID NO:30中的LCDR1、LCDR2和LCDR3，且所述重链可变区包含如SEQ ID NO:31中的HCDR1、HCDR2和HCDR3；
或所述轻链可变区包含如SEQ ID NO:32中的LCDR1、LCDR2和LCDR3，且所述重链可变区包含如SEQ ID NO:33中的HCDR1、HCDR2和HCDR3；
或所述轻链可变区包含如SEQ ID NO:32中的LCDR1、LCDR2和LCDR3，且所述重链可变区包含如SEQ ID NO:34中的HCDR1、HCDR2和HCDR3；
或所述轻链可变区包含如SEQ ID NO:35中的LCDR1、LCDR2和LCDR3，且所述重链可变区包含如SEQ ID NO:36中的HCDR1、HCDR2和HCDR3；
或所述轻链可变区包含如SEQ ID NO:35中的LCDR1、LCDR2和LCDR3，且所述重链可变区包含如SEQ ID NO:37中的HCDR1、HCDR2和HCDR3；
或所述轻链可变区包含如SEQ ID NO:41中的LCDR1、LCDR2和LCDR3，且所述重链可变区包含如SEQ ID NO:42中的HCDR1、HCDR2和HCDR3；
或所述轻链可变区包含如SEQ ID NO:41中的LCDR1、LCDR2和LCDR3，且所述重链可变区包含如SEQ ID NO:43中的HCDR1、HCDR2和HCDR3。
在一些实施方案中，所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的，在一些具体实施方案中，CDR是根据Kabat编号规则确定的。
在一些实施方案中，本发明提供的第一方面所述的抗体，所述LCDR1包含如SEQ ID NO:1所示的氨基酸序列，所述LCDR2包含如X5X6SX7X8X9S所示的氨基酸序列，其中X5为Y或A；X6为T或A；X7为S、R或T；X8为L或R；X9为Q或D，所述LCDR3包含如SEQ ID NO:3所示的氨基酸序列；所述HCDR1包含如SEQ ID NO:4所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:5所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:6所示的氨基酸序列。
在本发明的优选实施方案中，所述LCDR1的氨基酸序列如SEQ ID NO:1所示，所述LCDR2的氨基酸序列如X5X6SX7X8X9S所示的氨基酸序列，其中X5为Y或A；X6为T或A；X7为S、R或T；X8为L或R；X9为Q或D，所述LCDR3的氨基酸序列如SEQ ID NO:3所示；所述HCDR1的氨基酸序列如SEQ ID NO:4所示，所述HCDR2的氨基 酸序列如SEQ ID NO:5所示，以及所述HCDR3的氨基酸序列如SEQ ID NO:6所示。
如本发明第一方面所述的抗体，所述LCDR1包含如SEQ ID NO:9、SEQ ID NO:8、SEQ ID NO:7或SEQ ID NO:10所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:14、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:11或SEQ ID NO:15所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:19、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:16或SEQ ID NO:20所示的氨基酸序列；所述HCDR1包含如SEQ ID NO:21或SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:23或SEQ ID NO:24所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:28、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:25或SEQ ID NO:29所示的氨基酸序列。
在本发明的优选实施方案中，所述抗体包含如SEQ ID NO:9、SEQ ID NO:8、SEQ ID NO:7或SEQ ID NO:10所示的LCDR1的氨基酸序列，如SEQ ID NO:14、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:11或SEQ ID NO:15所示的LCDR2的氨基酸序列，如SEQ ID NO:19、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:16或SEQ ID NO:20所示的LCDR3的氨基酸序列；如SEQ ID NO:21或SEQ ID NO:22所示的HCDR1的氨基酸序列，如SEQ ID NO:23或SEQ ID NO:24所示的HCDR2的氨基酸序列，以及如SEQ ID NO:28、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:25或SEQ ID NO:29所示的HCDR3的氨基酸序列。
在本发明的优选实施方案中，所述LCDR1的氨基酸序列如SEQ ID NO:9、SEQ ID NO:8、SEQ ID NO:7或SEQ ID NO:10所示，所述LCDR2的氨基酸序列如SEQ ID NO:14、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:11或SEQ ID NO:15所示，所述LCDR3的氨基酸序列如SEQ ID NO:19、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:16或SEQ ID NO:20所示；所述HCDR1的氨基酸序列如SEQ ID NO:21或SEQ ID NO:22所示，所述HCDR2的氨基酸序列如SEQ ID NO:23或SEQ ID NO:24所示，以及所述HCDR3的氨基酸序列如SEQ ID NO:28、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:25或SEQ ID NO:29所示。
如本发明第一方面所述的抗体，在本发明的较佳实施方案中：
A)所述LCDR1包含如SEQ ID NO:9所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:14所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:23所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:28所示的氨基酸序列；
B)所述LCDR1包含如SEQ ID NO:9所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:14所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列， 所述HCDR1包含如SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:24所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:28所示的氨基酸序列；
C)所述LCDR1包含如SEQ ID NO:7所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:16所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:23所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:25所示的氨基酸序列；
D)所述LCDR1包含如SEQ ID NO:7所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:17所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:24所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:26所示的氨基酸序列；
E)所述LCDR1包含如SEQ ID NO:7所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:17所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:23所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:26所示的氨基酸序列；
F)所述LCDR1包含如SEQ ID NO:8所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:13所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:18所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:24所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:27所示的氨基酸序列；
G)所述LCDR1包含如SEQ ID NO:8所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:13所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:18所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:23所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:27所示的氨基酸序列；
H)所述LCDR1包含如SEQ ID NO:10所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:15所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:20所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:24所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:29所示的氨基酸序列；或
I)所述LCDR1包含如SEQ ID NO:10所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:15所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:20所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:23所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:29所示的氨基酸序列。
在本发明的更佳实施方案中：
A)所述轻链可变区包含分别如SEQ ID NO:9、SEQ ID NO:14和SEQ ID NO:19所 示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:21、SEQ ID NO:23和SEQ ID NO:28所示的HCDR1、HCDR2和HCDR3；
B)所述轻链可变区包含分别如SEQ ID NO:9、SEQ ID NO:14和SEQ ID NO:19所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:21、SEQ ID NO:24和SEQ ID NO:28所示的HCDR1、HCDR2和HCDR3；
C)所述轻链可变区包含分别如SEQ ID NO:7、SEQ ID NO:11和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:21、SEQ ID NO:23和SEQ ID NO:25所示的HCDR1、HCDR2和HCDR3；
D)所述轻链可变区包含分别如SEQ ID NO:7、SEQ ID NO:12和SEQ ID NO:17所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:22、SEQ ID NO:24和SEQ ID NO:26所示的HCDR1、HCDR2和HCDR3；
E)所述轻链可变区包含分别如SEQ ID NO:7、SEQ ID NO:12和SEQ ID NO:17所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:26所示的HCDR1、HCDR2和HCDR3；
F)所述轻链可变区包含分别如SEQ ID NO:8、SEQ ID NO:13和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:22、SEQ ID NO:24和SEQ ID NO:27所示的HCDR1、HCDR2和HCDR3；
G)所述轻链可变区包含分别如SEQ ID NO:8、SEQ ID NO:13和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:27所示的HCDR1、HCDR2和HCDR3；
H)所述轻链可变区包含分别如SEQ ID NO:10、SEQ ID NO:15和SEQ ID NO:20所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:21、SEQ ID NO:24和SEQ ID NO:29所示的HCDR1、HCDR2和HCDR3；或，
I)所述轻链可变区包含分别如SEQ ID NO:10、SEQ ID NO:15和SEQ ID NO:20所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:21、SEQ ID NO:23和SEQ ID NO:29所示的HCDR1、HCDR2和HCDR3。
在本发明的优选实施方案中：
在一些实施方案中，所述抗体中，所述LCDR1的氨基酸序列如SEQ ID NO:9所示，所述LCDR2的氨基酸序列如SEQ ID NO:14所示，所述LCDR3的氨基酸序列如SEQ ID NO:19所示，所述HCDR1的氨基酸序列如SEQ ID NO:21所示，所述HCDR2的氨基酸序列如SEQ ID NO:23所示，以及所述HCDR3的氨基酸序列如SEQ ID NO:28所示。
在一些实施方案中，所述抗体中，所述LCDR1的氨基酸序列如SEQ ID NO:9所示， 所述LCDR2的氨基酸序列如SEQ ID NO:14所示，所述LCDR3的氨基酸序列如SEQ ID NO:19所示，所述HCDR1的氨基酸序列如SEQ ID NO:21所示，所述HCDR2的氨基酸序列如SEQ ID NO:24所示，以及所述HCDR3的氨基酸序列如SEQ ID NO:28所示。
在一些实施方案中，所述抗体中，所述LCDR1的氨基酸序列如SEQ ID NO:7所示，所述LCDR2的氨基酸序列如SEQ ID NO:11所示，所述LCDR3的氨基酸序列如SEQ ID NO:16所示，所述HCDR1的氨基酸序列如SEQ ID NO:21所示，所述HCDR2的氨基酸序列如SEQ ID NO:23所示，以及所述HCDR3的氨基酸序列如SEQ ID NO:25所示。
在一些实施方案中，所述抗体中，所述LCDR1的氨基酸序列如SEQ ID NO:7所示，所述LCDR2的氨基酸序列如SEQ ID NO:12所示，所述LCDR3的氨基酸序列如SEQ ID NO:17所示，所述HCDR1的氨基酸序列如SEQ ID NO:22所示，所述HCDR2的氨基酸序列如SEQ ID NO:24所示，以及所述HCDR3的氨基酸序列如SEQ ID NO:26所示。
在一些实施方案中，所述抗体中，所述LCDR1的氨基酸序列如SEQ ID NO:7所示，所述LCDR2的氨基酸序列如SEQ ID NO:12所示，所述LCDR3的氨基酸序列如SEQ ID NO:17所示，所述HCDR1的氨基酸序列如SEQ ID NO:22所示，所述HCDR2的氨基酸序列如SEQ ID NO:23所示，以及所述HCDR3的氨基酸序列如SEQ ID NO:26所示。
在一些实施方案中，所述抗体中，所述LCDR1的氨基酸序列如SEQ ID NO:8所示，所述LCDR2的氨基酸序列如SEQ ID NO:13所示，所述LCDR3的氨基酸序列如SEQ ID NO:18所示，所述HCDR1的氨基酸序列如SEQ ID NO:22所示，所述HCDR2的氨基酸序列如SEQ ID NO:24所示，以及所述HCDR3的氨基酸序列如SEQ ID NO:27所示。
在一些实施方案中，所述抗体中，所述LCDR1的氨基酸序列如SEQ ID NO:8所示，所述LCDR2的氨基酸序列如SEQ ID NO:13所示，所述LCDR3的氨基酸序列如SEQ ID NO:18所示，所述HCDR1的氨基酸序列如SEQ ID NO:22所示，所述HCDR2的氨基酸序列如SEQ ID NO:23所示，以及所述HCDR3的氨基酸序列如SEQ ID NO:27所示。
在一些实施方案中，所述抗体中，所述LCDR1的氨基酸序列如SEQ ID NO:10所示，所述LCDR2的氨基酸序列如SEQ ID NO:15所示，所述LCDR3的氨基酸序列如SEQ ID NO:20所示，所述HCDR1的氨基酸序列如SEQ ID NO:21所示，所述HCDR2的氨基酸序列如SEQ ID NO:24所示，以及所述HCDR3的氨基酸序列如SEQ ID NO:29所示。
在一些实施方案中，所述抗体中，所述LCDR1的氨基酸序列如SEQ ID NO:10所示，所述LCDR2的氨基酸序列如SEQ ID NO:15所示，所述LCDR3的氨基酸序列如SEQ ID NO:20所示，所述HCDR1的氨基酸序列如SEQ ID NO:21所示，所述HCDR2的氨基酸序列如SEQ ID NO:23所示，以及所述HCDR3的氨基酸序列如SEQ ID NO:29 所示。
在上述一些实施方案，所列CDR的氨基酸序列按照Kabat定义规则确定的。但是，本领域人员公知，在本领域中可以通过多种方法来定义抗体的CDR，例如基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath),Contact(University College London)，国际ImMunoGeneTics database(IMGT)(万维网imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应理解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。各种编号系统其对应CDR是本领域技术人员熟知的，如表1所示：
表1抗体CDR定义方法
备注：表1中，其中，Laa-Lbb指从抗体轻链的N端开始，按照其对应的编码规则从第aa位至第bb位的氨基酸序列；Haa-Hbb指从抗体重链的N端开始，按照其对应的编码规则从第aa位至第bb位的氨基酸序列。例如，表1第二行第二列的L24-L34指从抗体轻链可变区N端开始，按照Kabat编码规则从第24位至第34位残基所确定的氨基酸序列；其它依次类推。
如本发明第一方面所述的抗体，所述轻链可变区的框架区为人源框架区，且所述重链可变区的框架区为人源框架区。
如本发明第一方面所述的抗体，在本发明的较佳实施方案中：
a)所述轻链可变区包含与SEQ ID NO:38具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，所述重链可变区包含与SEQ ID NO:40或SEQ ID NO:39具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；
b)所述轻链可变区包含与SEQ ID NO:30具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，所述重链可变区包含与SEQ ID NO:31具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；
c)所述轻链可变区包含与SEQ ID NO:32具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，所述重链可变区包含与SEQ ID NO:33或SEQ ID NO:34具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；
d)所述轻链可变区包含与SEQ ID NO:35具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，所述重链可变区包含与SEQ ID NO:36或SEQ ID NO:37具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；或
e)所述轻链可变区包含与SEQ ID NO:41具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，所述重链可变区包含与SEQ ID NO:42或SEQ ID NO:43具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列。
在本发明的较佳实施方案中，所述具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列的可变区维持与原序列相同的抗原(例如人CD40)结合的功能。
如下进行序列之间序列同一性的计算。为确定两个氨基酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置处的氨基酸残基。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基占据时，则所述分子在这个位置处是相同的。可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。还可以使用PAM120加权余数表、空位长度罚分12、空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列之间的同一性百分数。额外地或备选地，可以进一步使用本发明所述的蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。
如本发明第一方面所述的抗体，在本发明的更佳实施方案中：
a)所述轻链可变区包含如SEQ ID NO:38所示的氨基酸序列，且所述重链可变区包含如SEQ ID NO:40或SEQ ID NO:39所示的氨基酸序列；
b)所述轻链可变区包含如SEQ ID NO:30所示的氨基酸序列，且所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列；
c)所述轻链可变区包含如SEQ ID NO:32所示的氨基酸序列，且所述重链可变区包含如SEQ ID NO:33或SEQ ID NO:34所示的氨基酸序列；
d)所述轻链可变区包含如SEQ ID NO:35所示的氨基酸序列，且所述重链可变区包含如SEQ ID NO:36或SEQ ID NO:37所示的氨基酸序列；或
e)所述轻链可变区包含如SEQ ID NO:41所示的氨基酸序列，且所述重链可变区包含如SEQ ID NO:42或SEQ ID NO:43所示的氨基酸序列。
如本发明第一方面所述的抗体，在本发明的进一步更佳实施方案中：
在一些实施方案中，所述抗体包含如SEQ ID NO:38所示的轻链可变区，且包含如SEQ ID NO:40所示的重链可变区。
在一些实施方案中，所述抗体包含如SEQ ID NO:38所示的轻链可变区，且包含如SEQ ID NO:39所示的重链可变区。
在一些实施方案中，所述抗体包含如SEQ ID NO:30所示的轻链可变区，且包含如SEQ ID NO:31所示的重链可变区。
在一些实施方案中，所述抗体包含如SEQ ID NO:32所示的轻链可变区，且包含如SEQ ID NO:33所示的重链可变区。
在一些实施方案中，所述抗体包含如SEQ ID NO:32所示的轻链可变区，且包含如SEQ ID NO:34所示的重链可变区。
在一些实施方案中，所述抗体包含如SEQ ID NO:35所示的轻链可变区，且包含如SEQ ID NO:36所示的重链可变区。
在一些实施方案中，所述抗体包含如SEQ ID NO:35所示的轻链可变区，且包含如SEQ ID NO:37所示的重链可变区。
在一些实施方案中，所述抗体包含如SEQ ID NO:41所示的轻链可变区，且包含如SEQ ID NO:42所示的重链可变区。
在一些实施方案中，所述抗体包含如SEQ ID NO:41所示的轻链可变区，且包含如SEQ ID NO:43所示的重链可变区。
在本发明的优选实施方案中：
在一些实施方案中，所述抗体中，所述轻链可变区的氨基酸序列如SEQ ID NO:38所示，且所述重链可变区的氨基酸序列如SEQ ID NO:40或SEQ ID NO:39所示。
在一些实施方案中，所述抗体中，所述轻链可变区的氨基酸序列如SEQ ID NO:30所示，且所述重链可变区的氨基酸序列如SEQ ID NO:31所示。
在一些实施方案中，所述抗体中，所述轻链可变区的氨基酸序列如SEQ ID NO:32所示，且所述重链可变区的氨基酸序列如SEQ ID NO:33或SEQ ID NO:34所示。
在一些实施方案中，所述抗体中，所述轻链可变区的氨基酸序列如SEQ ID NO:35所示，且所述重链可变区的氨基酸序列如SEQ ID NO:36或SEQ ID NO:37所示。
在一些实施方案中，所述抗体中，所述轻链可变区的氨基酸序列如SEQ ID NO:41所示，且所述重链可变区的氨基酸序列如SEQ ID NO:42或SEQ ID NO:43所示。
在本发明的更优选实施方案中：
在一些实施方案中，所述抗体中，所述轻链可变区的氨基酸序列如SEQ ID NO:38所示，且所述重链可变区的氨基酸序列如SEQ ID NO:40所示。
在一些实施方案中，所述抗体中，所述轻链可变区的氨基酸序列如SEQ ID NO:38所示，且所述重链可变区的氨基酸序列如SEQ ID NO:39所示。
在一些实施方案中，所述抗体中，所述轻链可变区的氨基酸序列如SEQ ID NO:30所示，且所述重链可变区的氨基酸序列如SEQ ID NO:31所示。
在一些实施方案中，所述抗体中，所述轻链可变区的氨基酸序列如SEQ ID NO:32所示，且所述重链可变区的氨基酸序列如SEQ ID NO:33所示。
在一些实施方案中，所述抗体中，所述轻链可变区的氨基酸序列如SEQ ID NO:32所示，且所述重链可变区的氨基酸序列如SEQ ID NO:34所示。
在一些实施方案中，所述抗体中，所述轻链可变区的氨基酸序列如SEQ ID NO:35所示，且所述重链可变区的氨基酸序列如SEQ ID NO:36所示。
在一些实施方案中，所述抗体中，所述轻链可变区的氨基酸序列如SEQ ID NO:35所示，且所述重链可变区的氨基酸序列如SEQ ID NO:37所示。
在一些实施方案中，所述抗体中，所述轻链可变区的氨基酸序列如SEQ ID NO:41所示，且所述重链可变区的氨基酸序列如SEQ ID NO:42所示。
在一些实施方案中，所述抗体中，所述轻链可变区的氨基酸序列如SEQ ID NO:41所示，且所述重链可变区的氨基酸序列如SEQ ID NO:43所示。
如本发明第一方面所述的抗体，所述抗体满足以下三项中的一项或多项：(1)所述抗体为全长抗体、Fab、Fab'、F(ab')2或Fv，所述Fv优选scFv；(2)所述抗体为单特异性抗体或多特异性抗体；(3)所述抗体为由上述抗体制得的单克隆抗体或多克隆抗体。
本发明的抗体包括单克隆抗体(缩写mAb或Ab)，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。
如本发明第一方面所述的抗体，所述抗体包含重链恒定区和/或轻链恒定区。
在一些实施方案中，所述抗体的重链恒定区源自人源抗体IgG1、IgG2、IgG3或IgG4的重链恒定区，和/或所述抗体的轻链恒定区源自人源抗体的κ链。
在一些实施方案中，所述恒定区包括不改变抗体可变区结构和功能的恒定区变体。现有技术中已公开多种此类恒定区变体，例如抗体的重链恒定区的Fc具有238、265、269、270、297、327和329(采用EU编号系统)中的一个或更多个氨基酸的替代(美国专利No.6,737,056)，或者抗体的重链恒定区的Fc具有234、235、265、329(采用EU编号系统)处的一个或更多个氨基酸的替代，或者抗体的重链恒定区的Fc具有238、252、254、256、265、272、286、303、305、307、311、312、317、340、356、360、362、376、378、380、382、413、424或434(采用EU编号系统)处的一个或更多个氨基酸的替代(参见美国专利No.7,371,826)等等。这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
在本发明的较佳实施方案中，所述重链恒定区包含如SEQ ID NO:45所示的氨基酸序列，且所述轻链恒定区包含如SEQ ID NO:44所示的氨基酸序列。
在本发明的更佳实施方案中，所述抗体的重链包含如SEQ ID NO:94所示的氨基酸序列，并所述抗体的轻链包含如SEQ ID NO:95所示的氨基酸序列。
在本发明的更佳实施方案中，所述抗体的重链包含如SEQ ID NO:96所示的氨基酸序列，并所述抗体的轻链包含如SEQ ID NO:81所示的氨基酸序列。
在本发明的更佳实施方案中，所述抗体的重链包含如SEQ ID NO:87所示的氨基酸序列，并所述抗体的轻链包含如SEQ ID NO:81所示的氨基酸序列。
在本发明的优选实施方案中，所述重链恒定区的氨基酸序列如SEQ ID NO:45所示，且所述轻链恒定区的氨基酸序列如SEQ ID NO:44所示。
在本发明的优选实施方案中，所述抗体的重链的氨基酸序列如SEQ ID NO:94所示，所述抗体的轻链的氨基酸序列如SEQ ID NO:95所示。
在本发明的优选实施方案中，所述抗体的重链的氨基酸序列如SEQ ID NO:96所示，所述抗体的轻链的氨基酸序列如SEQ ID NO:81所示。
在本发明的优选实施方案中，所述抗体的重链的氨基酸序列如SEQ ID NO:87所示，所述抗体的轻链的氨基酸序列如SEQ ID NO:81所示。
本发明第二方面提供：一种双特异性抗体，其包含特异性结合人CD40的第一抗原结合结构域和特异性结合人PD-L1的第二抗原结合结构域；其中，所述特异性结合人CD40的第一抗原结合结构域如本发明第一方面所述的抗CD40的抗体中所述定义。
本发明所述的术语“抗体”以最广义使用，其涵盖单克隆抗体、多克隆抗体，涵盖单特异性抗体、多特异性抗体(例如双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)，也涵盖传统抗体(由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构抗体)，以及具有抗原结合活性的Fab、Fab’、F(ab’)2、Fv、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体peptibody、结构域抗体(重链(VH)抗体、轻链(VL)抗体)。传统的抗体(也称“全长抗体”、“完全抗体”)通常是约150,000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)通过链间二硫键连接而成的四肽链结构。全长抗体每条重链由重链可变区(本发明中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本发明中缩写为VL)和轻链恒定区(本发明中缩写为CL)组成。轻链恒定区由一个结构域CL组成。哺乳动物重链分为α、δ、ε、γ和μ重链。哺乳动物轻链分为λ或κ轻链。包含α、δ、ε、γ和μ重链的免疫球蛋白为免疫球蛋白(Ig)A、IgD、IgE、IgG和IgM。完全抗体形成“Y”形状。Y的茎由两条重链的第二和第三恒定区(并且对于IgE和IgM，第四恒定区)结合在一起组成，二硫键(链间)在铰链中形成。重链γ、α和δ具有由三个串联(成一行)Ig结构域构成的恒定区，和用于增加柔性的铰链区；重链μ和ε具有由四个免疫球蛋白结构域构成的恒定区。第二和第三恒定区分别称为“CH2结构域”和“CH3结构域”。Y的每个臂包括结合到单个轻链的单个重链的可变区和第一恒定区(CH1)。“Fc”区是包含抗体的CH2和CH3结构域的两个重链片段,两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。现有技术中已公开多种Fc恒定区变体，例如抗体的重链恒定区的Fc具有238、265、269、270、297、327和329(采用EU编号系统)中的一个或更多个氨基酸的替代(美国专利No.6,737,056)，或者抗体的重链恒定区的Fc具有234、235、265、329(采用EU编号系统)处的一个或更多个氨基酸的替代，或者抗体的重链恒定区的Fc具有238、252、254、256、265、272、286、303、305、307、311、312、317、340、356、360、362、376、378、380、382、413、424或434(采用EU编号系统)处的一个或更多个氨基酸的替代(参见美国专利No.7,371,826)等等。这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
术语“可变区”或“可变域”指抗体重链或轻链中涉及抗体结合抗原的域。VHH、VH和VL各包含四个保守的框架区(FR)和三个互补决定区(CDR)。其中，术语“互补决定区”或“CDR”指可变结构域内主要促成与抗原结合的区域；“框架”或“FR”是指除CDR残基之外的可变结构域残基。VH或VHH包含3个CDR区：为便于区分，VH的3个CDR以HCDR1、HCDR2和HCDR3标识，VHH的3个CDR以VHH-CDR1、 VHH-CDR2和VHH-CDR3标识；VL包含3个CDR区：LCDR1、LCDR2和LCDR3。每个VH和VL由从氨基末端排到羧基末端按以下顺序排列的三个CDR和四个FR构成：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。单个VH或VL可能足以赋予抗原结合特异性。如本文所用，术语“VHH”和“纳米抗体”具有相同的含义并可互换使用，是指克隆抗体重链的可变区，仅由一个重链可变区组成的纳米抗体，它具有完整的抗原结合功能。VHH特异性结合表位无需其他抗原结合结构域一起识别(此与常规四肽链结构抗体不同，常规四肽链结构抗体的表位由VL与VH形成的结构对一起识别)。VHH是由单一重链可变结构域形成的小型稳定及高效的抗原识别单元。纳米抗体具有优良的生物学特性，分子量12-15kDa，是完整抗体的十分之一，具有很好的组织穿透性，特异性高，水溶性好。因其特殊的结构性质，兼具了传统抗体与小分子药物的优势，几乎完美克服了传统抗体的开发周期长，稳定性较低，保存条件苛刻等缺陷，逐渐成为新一代抗体治疗中的新兴力量，在免疫诊断和治疗中显示出广阔的应用前景。VHH包括但不限于经骆驼科动物产生的天然抗体，也可以是骆驼科动物产生的抗体后再经人源化的，也可以是经噬菌体体展示技术筛选获得的。获得结合特定抗原或表位的VHH的方法，先前已公开于例如以下文献中：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.2645-2652，17June，2009和WO94/04678。
术语“PD-L1”包括由细胞天然表达的PD-L1的任何变体或同种型。本发明的抗体可与非人物种(如食蟹猴)的PD-L1交叉反应。作为另一种选择，该抗体也可以是人PD-L1特异性的，可不表现出与其他物种的交叉反应性。PD-L1或其任何变体或同种型可从天然表达它们的细胞或组织中分离而得，或使用本领域通用以及本文所述的那些技术通过重组技术产生。
本发明所述的双特异性抗体，较佳地，所述第二抗原结合结构域至少包含一个VHH，所述VHH包括SEQ ID NO:66中的VHH-CDR1、VHH-CDR2和VHH-CDR3。在一些实施方案中，所述VHH-CDR1包含如X41YYX42X43C所示的氨基酸序列，其中为D或E，为S或T，为K或Q，所述VHH-CDR2包含如SEQ ID NO:76所示的氨基酸序列，并且所述VHH-CDR3包含如SEQ ID NO:77所示的氨基酸序列。
在一些实施方案中，所述VHH包含SEQ ID NO:49、SEQ ID NO:74、SEQ ID NO:72或SEQ ID NO:73中的VHH-CDR1、VHH-CDR2和VHH-CDR3。
在一些实施方案中，所述VHH-CDR1、VHH-CDR2和VHH-CDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的，在一些具体实施方案中，VHH-CDR1、VHH-CDR2和VHH-CDR3是根据Kabat编号规则确定的。在一些实施方案中，所述VHH- CDR1包含如SEQ ID NO:46、SEQ ID NO:67或SEQ ID NO:70所示的氨基酸序列，所述VHH-CDR2包含如SEQ ID NO:47、SEQ ID NO:68或SEQ ID NO:71所示的氨基酸序列，以及所述VHH-CDR3包含如SEQ ID NO:48或SEQ ID NO:69所示的氨基酸序列。
在一些实施方案中，所述VHH包含如SEQ ID NO:46、SEQ ID NO:67或SEQ ID NO:70所示的VHH-CDR1，如SEQ ID NO:47、SEQ ID NO:68或SEQ ID NO:71所示的VHH-CDR2，以及如SEQ ID NO:48或SEQ ID NO:69所示的VHH-CDR3。
在本发明更优选的实施方案中，所述VHH-CDR1包含如SEQ ID NO:46所示的氨基酸序列，VHH-CDR2包含如SEQ ID NO:47所示的氨基酸序列，VHH-CDR3包含如SEQ ID NO:48所示的氨基酸序列；
或，所述VHH-CDR1包含如SEQ ID NO:67所示的氨基酸序列，VHH-CDR2包含如SEQ ID NO:68所示的氨基酸序列，VHH-CDR3包含如SEQ ID NO:69所示的氨基酸序列；
或，所述VHH-CDR1包含如SEQ ID NO:70所示的氨基酸序列，VHH-CDR2包含如SEQ ID NO:71所示的氨基酸序列，VHH-CDR3包含如SEQ ID NO:48所示的氨基酸序列。
在一些实施方案中，所述VHH包括：
序列如SEQ ID NO:46所示的VHH-CDR1、序列如SEQ ID NO:47所示的VHH-CDR2和序列如SEQ ID NO:48所示的VHH-CDR3；
序列如SEQ ID NO:67所示的VHH-CDR1、序列如SEQ ID NO:68所示的VHH-CDR2和序列如SEQ ID NO:69所示的VHH-CDR3，
或，序列如SEQ ID NO:70所示的VHH-CDR1、序列如SEQ ID NO:71所示的VHH-CDR2和序列如SEQ ID NO:48所示的VHH-CDR3。
在一些实施方案中，所述VHH的氨基酸序列如SEQ ID NO:49、SEQ ID NO:72、SEQ ID NO:73或SEQ ID NO:74所示，或与如SEQ ID NO:49、SEQ ID NO:72、SEQ ID NO:73或SEQ ID NO:74所示的氨基酸序列具有至少90％、至少95％或者至少99％序列序列同一性。
在一些实施方案中，所述VHH为人源化的VHH。
在一些实施方案中，所述VHH包含框架区FR1、FR2、FR3和FR4，其中，所述FR1包含如SEQ ID NO:85所示的氨基酸序列，所述FR2包含如SEQ ID NO:79所示的氨基酸序列，所述FR3包含如SEQ ID NO:86所示的氨基酸序列，以及所述FR4包含如SEQ ID NO:84所示的氨基酸序列；
在一些实施方案中，所述FR1包含如SEQ ID NO:82或SEQ ID NO:78所示的氨基酸序列，所述FR2包含如SEQ ID NO:79所示的氨基酸序列，所述FR3包含如SEQ ID NO:83或SEQ ID NO:80所示的氨基酸序列，以及所述FR4包含如SEQ ID NO:84所示的氨基酸序列。
在一些实施方案中，所述FR1包含如SEQ ID NO:82所示的氨基酸序列，所述FR2包含如SEQ ID NO:79所示的氨基酸序列，所述FR3包含如SEQ ID NO:83所示的氨基酸序列，以及所述FR4包含如SEQ ID NO:84所示的氨基酸序列；
或，所述FR1包含如SEQ ID NO:78所示的氨基酸序列，所述FR2包含如SEQ ID NO:79所示的氨基酸序列，所述FR3包含如SEQ ID NO:80所示的氨基酸序列，以及所述FR4包含如SEQ ID NO:84所示的氨基酸序列；
或，所述FR1包含如SEQ ID NO:82所示的氨基酸序列，所述FR2包含如SEQ ID NO:79所示的氨基酸序列，所述FR3包含如SEQ ID NO:80所示的氨基酸序列，以及所述FR4包含如SEQ ID NO:84所示的氨基酸序列。
在一些实施方案中，所述VHH的氨基酸序列如SEQ ID NO:49、SEQ ID NO:72、SEQ ID NO:73或SEQ ID NO:74所示，或与SEQ ID NO:49、SEQ ID NO:72、SEQ ID NO:73或SEQ ID NO:74所示的氨基酸序列具有至少90％序列同一性。
在一些实施方案中，所述VHH的氨基酸序列如SEQ ID NO:49、SEQ ID NO:72、SEQ ID NO:73或SEQ ID NO:74所示。
在一些实施方案中，所述第二抗原结合结构域还包含重链恒定区。
优选地，所述重链恒定区选自IgG1、IgG2、IgG3或IgG4的重链恒定区；所述重链恒定区优选为人IgG1的Fc区；所述重链恒定区更优选为包含如SEQ ID NO:88所示的氨基酸序列。
更优选地，所述VHH与重链恒定区通过连接子连接；所述连接子优选为具有如(G4S)x所示的氨基酸序列的连接子，其中，x独立地选自1-20的整数，更优选为如SEQ ID NO:89所示的连接子。
进一步更优选地，所述第二抗原结合结构域的氨基酸序列如SEQ ID NO:93、SEQ ID NO:90、SEQ ID NO:91或SEQ ID NO:92所示，或与SEQ ID NO:93、SEQ ID NO:90、SEQ ID NO:91或SEQ ID NO:92所示的氨基酸序列具有至少90％序列同一性；
最优选地，所述第二抗原结合结构域具有2条氨基酸序列如SEQ ID NO:93所示的序列。
本发明中，“Fab”由一条轻链和一条重链的CH1及可变区组成。“Fab’”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的部分，两个Fab’ 片段的两条重链之间可形成链间二硫键以形成F(ab’)2分子。F(ab’)2片段由通过两条重链间的二硫键保持在一起的两个Fab’片段组成。术语“Fv”意指抗体的单臂的VL和VH结构域组成的抗体片段。
本发明中，所述的scFv(single chain antibody fragment，单链抗体)是指VH结构域和VL结构域通过连接子(也称接头)连接形成的多肽链。其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子[参见，例如，Bird等人，Science 242:423-426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)]。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的G4S氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(G4S)4或(G4S)3接头，但也可使用其变体。
术语“多特异性抗体”按其最广义使用，涵盖具有两个或多个表位特异性的抗体，例如双特异性抗体。这些多特异性抗体包括但不限于：包含重链可变区(VH)和轻链可变区(VL)的抗体，其中该VH-VL单元具有两个或多个表位特异性；具有两个或多个VL和VH区的抗体，每个VH-VL单元与不同的靶点或同一个靶点的不同表位结合；具有两个或更多个单可变区的抗体(例如VHH)，每个单可变区与不同的靶点或同一个靶点的不同的表位结合。
术语“表位”指能够与抗体特异性结合的抗原上的区域(area或region)。表位可以由连续氨基酸串(线性表位)形成或包含非连续氨基酸(构象表位)，例如因抗原的折叠(即通过蛋白质性质的抗原的三级折叠)而变成空间接近。构象表位和线性表位的差别在于：在变性溶剂的存在下，抗体对构象表位的结合丧失。表位包含处于独特空间构象的至少3，至少4，至少5，至少6，至少7，或8-10个氨基酸。筛选结合特定表位的抗体(即那些结合相同表位的)可以使用本领域例行方法来进行，例如但不限于丙氨酸扫描、肽印迹(见Meth.Mol.Biol.248(2004)443-463)、
术语“特异性结合”是指抗体以比针对其他抗原或表位更高的亲和力结合至某个抗原或该抗原内的表位。通常，抗体以约1×10-7M或更小(例如约1×10-8M或更小、约1×10-9M或更小、约1×10-10M或更小、约1×10-11M或更小，或者约1×10-12M或更小)的平衡解离常数(KD)结合抗原或抗原内的表位。在一些实施方式中，抗体与抗原结合的KD为该抗体结合至非特异性抗原(例如BSA、酪蛋白)的KD的10％，或1％。可使用标准程序来测量KD，例如通过表面等离子体共振测定法所测量的。然而，特异性结合至抗原或抗原内的表位的抗体可能对其它相关的抗原具有交叉反应性，例如，对来自其它物种(同源)(诸如人或猴，例如食蟹猕猴(Macaca fascicularis)(cynomolgus，cyno)、黑猩猩(Pan troglodytes)(chimpanzee，chimp))或狨猴(Callithrix  jacchus)(commonmarmoset，marmoset)的相同抗原具有交叉反应性。
术语“亲和力”是指分子(例如，抗体)的单个结合部位与其结合配体(例如，抗原)之间非共价相互作用的总体的强度。除非另外指明，如本文所用，“亲和力”是指内部结合亲和力，其反映出结合对(例如，抗体与抗原)的成员之间1:1相互作用。分子X对其配体Y的亲和力通常可以由解离常数(KD)表示。亲和力可以通过本领域已知的常规方法(包括本文所述的那些)测量。术语“kassoc”或“ka”指特定抗体-抗原相互作用的缔合速率，而如本文所使用的术语“kdis”或“kd”指特定抗体-抗原相互作用的解离速率。如本文所使用的，术语“KD”指解离常数，其获得自kd与ka的比率(即kd/ka)并且表示为摩尔浓度(M)。可以使用本领域良好建立的方法测定抗体的KD值。用于测定抗体KD的方法包括使用生物传感系统例如系统测量表面等离子体共振，或通过溶液平衡滴定法(SET)测量溶液中的亲和力。
术语“抗CD40抗体”、“特异结合CD40的抗体”是指能够以足够的亲和力结合CD40的抗体，使得该抗体可用作靶向CD40的诊断剂和/或治疗剂。在某些实施例中，与CD40结合的抗体具有以下解离常数(KD)<约1μM、<约100nM、<约10nM、<约1nM、<约0.1nM、<约0.01nM或<约0.001nM(例如10-8M或更小、例如10-8M至10-12M、例如10-9M至10-10M)。在某些实施例中，抗CD40抗体结合来自不同物种的CD40中保守的抗原表位。
术语“抗PD-L1抗体”、“特异结合PD-L1的抗体”是指能够以足够的亲和力结合PD-L1的抗体，使得该抗体可用作靶向PD-L1的诊断剂和/或治疗剂。在某些实施例中，与PD-L1结合的抗体具有以下解离常数(KD)<约1μM、<约100nM、<约10nM、<约1nM、<约0.1nM、<约0.01nM或<约0.001nM(例如10-8M或更小、例如10-8M至10-12M、例如10-9M至10-10M)。在某些实施例中，抗PD-L1抗体结合来自不同物种的PD-L1中保守的抗原表位。
在某些较佳实施例中，所述第一抗原结合结构域与所述第二抗原结合结构域直接或通过连接子可操作地连接。
优选地，所述第二抗原结合结构域连接于所述第一抗原结合结构域的轻链可变区或重链可变区的N端、或轻链恒定区的C端、或所述IgG的C端。
所述连接子优选为肽序列，更优选包含或组成为(G4S)nG，其中n＝1-10且为整数，例如n为3。
本发明所述的双特异性抗体中，含有两条第一多肽链和两条第二多肽链。
在某些较佳实施例中，所述第一多肽链的氨基酸序列如SEQ ID NO:50所示或与SEQ ID NO:50具有至少99％、至少95％、或者至少90％序列同一性，和/或，所述第二多肽 链的氨基酸序列如SEQ ID NO:51所示或与SEQ ID NO:51具有至少99％、至少95％、或者至少90％序列同一性。
在某些较佳实施例中，所述第一多肽链的氨基酸序列如SEQ ID NO:52所示或与SEQ ID NO:52具有至少99％、至少95％、或者至少90％序列同一性，和/或，所述第二多肽链的氨基酸序列如SEQ ID NO:53或SEQ ID NO:60所示或与SEQ ID NO:53或SEQ ID NO:60具有至少99％、至少95％、或者至少90％序列同一性。
在某些较佳实施例中，所述第一多肽链的氨基酸序列如SEQ ID NO:54所示或与SEQ ID NO:54具有至少99％、至少95％、或者至少90％序列同一性，和/或，所述第二多肽链的氨基酸序列如SEQ ID NO:55或SEQ ID NO:61所示或与SEQ ID NO:55或SEQ ID NO:61具有至少99％、至少95％、或者至少90％序列同一性。
在某些较佳实施例中，所述第一多肽链的氨基酸序列如SEQ ID NO:56所示或与SEQ ID NO:56具有至少99％、至少95％、或者至少90％序列同一性，和/或，所述第二多肽链的氨基酸序列如SEQ ID NO:57或SEQ ID NO:62所示或与SEQ ID NO:62具有至少99％、至少95％、或者至少90％序列同一性。
在某些较佳实施例中，所述第一多肽链的氨基酸序列如SEQ ID NO:58所示或与SEQ ID NO:58具有至少99％、至少95％、或者至少90％序列同一性，和/或，所述第二多肽链的氨基酸序列如SEQ ID NO:59或SEQ ID NO:63所示或与SEQ ID NO:59或SEQ ID NO:63具有至少99％、至少95％、或者至少90％序列同一性。
本发明第三方面提供：一种双特异性抗体，其包含特异性结合人CD40的第一抗原结合结构域和特异性结合人PD-L1的第二抗原结合结构域，其中：所述第二抗原结合结构域至少包含一个VHH，所述VHH的序列如本发明第二方面所述的双特异性抗体中所定义。
在某些较佳实施例中，所述第一抗原结合结构域如本发明第一方面所述的抗CD40抗体中所定义。
在某些较佳实施例中，所述第一抗原结合结构域与所述第二抗原结合结构域直接或通过连接子可操作地连接。
优选地，所述第二抗原结合结构域连接于所述第一抗原结合结构域的轻链可变区或重链可变区的N端、或轻链恒定区的C端、或所述IgG的C端。
所述连接子优选为肽序列，更优选包含或组成为(G4S)nG，其中n＝1-10且为整数，例如n为3。
本发明所述的双特异性抗体中，含有两条第一多肽链和两条第二多肽链。
在某些较佳实施例中，所述第一多肽链的氨基酸序列如SEQ ID NO:50所示，和/或， 所述第二多肽链的氨基酸序列如SEQ ID NO:51所示。
在某些较佳实施例中，所述第一多肽链的氨基酸序列如SEQ ID NO:52所示，和/或，所述第二多肽链的氨基酸序列如SEQ ID NO:53或SEQ ID NO:60所示。
在某些较佳实施例中，所述第一多肽链的氨基酸序列如SEQ ID NO:54所示，和/或，所述第二多肽链的氨基酸序列如SEQ ID NO:55或SEQ ID NO:61所示。
在某些较佳实施例中，所述第一多肽链的氨基酸序列如SEQ ID NO:56所示，和/或，所述第二多肽链的氨基酸序列如SEQ ID NO:57或SEQ ID NO:62所示。
在某些较佳实施例中，所述第一多肽链的氨基酸序列如SEQ ID NO:58所示，和/或，所述第二多肽链的氨基酸序列如SEQ ID NO:59或SEQ ID NO:63所示。
本发明第四方面提供：一种分离的核酸，其编码本发明第一方面所述的抗CD40抗体、第二方面或第三方面所述的双特异性抗体。
如本领域已知，在本发明中“核酸”是指任何长度的核苷酸链，并且包括DNA和RNA。核苷酸可以是脱氧核糖核苷酸、核糖核苷酸、修饰的核苷酸或碱基、和/或它们的类似物、或者能够通过DNA或RNA聚合酶掺入链的任何底物。
本发明第五方面提供：一种重组表达载体，其包含本发明第四方面所述的分离的核酸。
较佳地，所述重组表达载体为质粒、粘粒、噬菌体或病毒载体。
例如，所述质粒的骨架为pcDNA3.4。
术语“重组表达载体”意指基因修饰的寡核苷酸或聚核苷酸构筑体，当构筑体包含编码mRNA、蛋白质、多肽或肽的核苷酸序列，并且载体在足以使mRNA、蛋白质、多肽、或肽在细胞内表达的条件下与细胞接触时，所述构筑体准许由宿主细胞表达mRNA、蛋白质、多肽或肽。本公开的载体总体上不是天然存在的。然而，载体的部分可以是天然存在的。本发明的重组表达载体可以包含任何类型的核苷酸，包括但不限于如下DNA和RNA：其可以是单链或双链的，合成的或部分地从天然来源中获得，并且其可以含有天然、非天然或改变的核苷酸。重组表达载体可以包含天然存在或非天然存在的核苷酸间连接，或这两种类型的连接。在示范性方面中，改变的核苷酸或非天然存在的核苷酸间连接不阻碍载体的转录或复制。
本发明的重组表达载体可以是任何合适的重组表达载体，其能够用于转化或转染将一种或多种所关注的基因或序列递送入任何合适的宿主细胞并且优选在宿主细胞中表达所述基因或序列。合适的载体包括经过设计用于扩展和扩增或用于表达或以上两项的那些载体，载体的实例包括但不限于病毒载体、裸DNA或RNA表达载体、质粒、粘粒或噬菌体载体、与阳离子凝聚剂相关的DNA或RNA表达载体、包囊化于脂质体中的DNA 或RNA表达载体以及某些真核细胞，例如生产细胞。
本发明第六方面提供：一种转化体，其包含本发明第五方面所述的重组表达载体。
优选地，所述转化体的宿主细胞为原核细胞或真核细胞。
更优选地，所述真核细胞为酵母细胞或哺乳动物细胞。
所述哺乳动物细胞例如为EXPI-293细胞或CHO细胞。
如本文所使用，术语“宿主细胞”是指可以含有本文所描述的核酸或载体的任何类型的细胞。宿主细胞可以是真核细胞，例如植物、动物、真菌或海藻；或宿主细胞可以是原核细胞，例如细菌或原生动物。如本文所描述，宿主细胞可以是起源于或获自个体的细胞。宿主细胞可以来源于或获自哺乳动物。如本文所使用，术语“哺乳动物”是指任何哺乳动物，包括但不限于啮齿目(order Rodentia)哺乳动物，如小鼠和仓鼠；和兔形目(order Lagomorpha)哺乳动物，如兔。优选地，哺乳动物来自食肉目(order Carnivora)，包括猫科动物(猫)和犬科动物(犬)。更优选地，哺乳动物来自偶蹄目(order Artiodactyla)，包括牛科动物(牛)和猪科动物(猪)，或属于奇蹄目(order Perssodactyla)，包括马科动物(马)。最优选地，哺乳动物属于灵长目(order Primate)、新世界猴类(Ceboids)或狐猴类(Simoids)(猴)或属于类人猿亚目(order Anthropoids)(人类和猿)。特别优选的哺乳动物是人类。
可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因编辑(CRISPR-Cas系统、ZFN系统或TALEN系统)、转座子(Sleeping Beauty或PiggyBAC)、基因枪、基于脂质的转染或其他常规技术。在原生质体融合的情况下，将细胞在培养基中培育并且筛选适宜的活性。用于培养所产生的转染细胞和用于回收产生的抗体分子的方法和条件是本领域技术人员已知的并且可以基于本说明书和现有技术已知的方法，根据使用的特定表达载体和哺乳动物宿主细胞变动或优化。另外，可以通过引入允许选择已转染的宿主细胞的一个或多个标记物，选出已经稳定将DNA掺入至其染色体中的细胞。标记物可以例如向营养缺陷型宿主提供原养型、杀生物抗性(例如，抗生素)或重金属(如铜)抗性等。可选择标记基因可以与待表达的DNA序列直接连接或通过共转化引入相同的细胞中。也可能需要额外元件以便最佳合成mRNA。这些元件可以包括剪接信号，以及转录启动子、增强子和终止信号。
本发明第七方面提供：一种抗CD40的抗体或双特异性抗体的制备方法，其包含以下步骤：培养如本发明第六方面所述的转化体，从培养物中获得抗CD40的抗体或双特异性抗体。
本发明第八方面提供：一种药物组合物，其包含本发明第一方面所述的抗CD40抗 体、第二方面或第三方面所述的双特异性抗体，以及药学上可接受的载体。
较佳地，所述药物组合物还含其它药剂，在一些实施方案中，所述其它药剂选自由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
“药学上可接受的载体”是常规使用的那些载体中的任一种，并且仅受到物理-化学考虑因素(如溶解性和与靶向CD40的抗体的反应性的缺乏)限制，并且受给药途径限制。本文所描述的药学上可接受的载体，例如媒剂、佐剂、赋形剂和稀释剂为所属领域的技术人员所熟知并且公众可容易获得。在一个方面中，药学上可接受的载体是对药物组合物的活性成分具有化学惰性的载体，并且是在使用条件下不具有不利的副作用或毒性的载体。在一些实施例中，当向动物或人类给予时，载体不产生不良、过敏或其它不适当的反应。在一些方面中，药物组合物不含热原质以及会对人类或动物有害的其它杂质。药学上可接受的载体包括任何和所有溶剂、分散介质、涂料、抗细菌剂和抗真菌剂、等张剂和吸收延迟剂等等；其用途在所属领域中是众所周知。
适用于实施本文所公开的方法的组合物的治疗配制物，如多肽、聚核苷酸或抗体，可以通过以冻干饼或水溶液的形式将具有所期望纯度的所选择组合物与任选的生理学上药学上可接受的载体、赋形剂或稳定剂混合(《雷明顿的药物科学(Remington′sPharmaceutical Sciences)》，第18版，A.R.Gennaro编，马克出版公司(Mack PublishingCompany)(1990))来制备用于储存。药物组合物可以通过与一种或多种合适的载体或佐剂掺合来制造，所述载体或佐剂如水、矿物油、聚乙二醇、淀粉、滑石、乳糖、增稠剂、稳定剂、悬浮剂等等。这类组合物可以呈溶液、悬浮液、片剂、胶囊、乳膏、油膏、软膏的形式或呈其它常规形式。
体内给药所用的组合物应为无菌的。这通过在冻干和复原之前或之后经由无菌过滤膜过滤容易地实现。治疗性组合物一般置于具有无菌接入端口的容器中，例如具有可被皮下注射针刺穿的塞子的静脉内溶液袋或小瓶。适用于可注射用途的医药形式包括无菌水溶液或分散液和用于临时制备无菌可注射溶液或分散液的无菌粉末。在一些情况下，所述形式应为无菌的，并且应是流体，以达到能够易于注射的程度。其应在制造和储存的条件下稳定并且应被保存以免遭微生物如细菌和真菌的污染作用。用于肠胃外给药的组合物通常将以冻干形式或以溶液形式储存。
载体可以是含有例如水或其合适的混合物和植物油的溶剂或分散介质。适当的流动性可以例如通过使用涂层如卵磷脂、在分散液情况下通过维持所需粒径和通过使用表面活性剂来维持载体的选择将部分地通过药物组合物的特定类型以及药物组合物的施用途 径来确定。相应地，可配制各种合适的药物组合物的配制物。
本发明的药物组合物可以包含任何药学上可接受的成份，包括例如酸化剂、添加剂、吸附剂、气雾剂推进剂(aerosol propellant)、空气置换剂、碱化剂、防结块剂、抗凝剂、抗微生物防腐剂、抗氧化剂、抗菌剂(antiseptic)、基质、粘合剂、缓冲剂、螯合剂、涂布剂、着色剂、干燥剂、清洁剂、稀释剂、消毒剂(disinfectant)、崩解剂、分散剂、溶解增强剂、染料、润肤剂、乳化剂、乳液稳定剂、填充剂、成膜剂、香味增强剂、调味剂、流动增强剂、胶凝剂、粒化剂、保温剂、润滑剂、粘膜粘着剂、软膏基质、软膏、油性媒剂、有机碱、锭剂基质、颜料、增塑剂、抛光剂、防腐剂、多价螯合剂、皮肤渗透剂、增溶剂、溶剂、稳定剂、栓剂基质、表面活性剂(surface active agent)、表面活性剂(surfactant)、悬浮剂、甜味剂、治疗剂、增稠剂、张力剂、毒性剂、增粘剂、吸水剂、水可混溶性共溶剂、水软化剂或湿润剂。
在一些实施例中，包含本文所描述的双特异性抗体的药物组合物经过配制用于肠胃外给药、皮下给药、静脉内给药、肌内给药、动脉内给药、鞘内给药或腹膜内给药。在其它实施例中，药物组合物通过经鼻、喷雾、口服、气雾剂、直肠或阴道给药来给予。组合物可以通过输注、快速注射或通过植入装置来给予。
局部配制物为所属领域的技术人员所熟知。这类配制物在本发明的背景下尤其适于施用到皮肤。
在一些实施例中，本文所描述的药物组合物经过配制用于肠胃外给药。出于本文的目的，肠胃外给药包括但不限于静脉内、动脉内、肌内、脑内、脑室内、心内、皮下、骨内、皮内、鞘内、腹膜内、眼球后、肺内、膀胱内和阴茎海绵体内注射或输注。也涵盖通过在特定部位处手术植入的给药。
可注射配制物是根据本发明。所属领域的普通技术人员熟知用于可注射组合物的有效医药载体的要求(参见例如《制药学和药学实践(Pharmaceutics and PharmacyPractice)》，J.B.利平科特公司(J.B.Lippincott Company)，宾夕法尼亚州费城(Philadelphia)，Banker和Chalmers编，第238-250页(1982)；和《可注射药物的ASHP手册(ASHP Handbook on Injectable Drugs)》，Toissel，第4版，第622-630页(1986))。
所属领域的技术人员应了解，除上述药物组合物以外，本发明的组合物可以被配制成包合物，如环糊精包合物，或脂质体。
本发明第九方面提供：如本发明第一方面所述的抗CD40抗体、第二方面或第三方面所述的双特异性抗体和/或如本发明第八方面所述的药物组合物在制备预防和/或治疗肿瘤的药物中的应用。
所述肿瘤优选PD-L1阳性和/或CD40阳性的肿瘤。
在本发明的优选实施方案中，所述肿瘤为淋巴癌、乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠直肠癌、膀胱癌、横纹肌肉瘤、食道癌、宫颈癌、多发性骨髓瘤、白血病、胆囊癌、胶质母细胞瘤或黑色素瘤，但不限制于此。
本发明第十方面提供：一种试剂盒，其包括本发明第一方面所述的抗CD40抗体、第二方面或第三方面所述的双特异性抗体或如本发明第八方面所述的药物组合物。
较佳地，所述试剂盒还包括(i)施用抗体或药物组合物的装置；和/或(ii)使用说明。
本发明第十一方面提供：一种套装药盒，其包含药盒A和药盒B，其中：
所述药盒A含有如本发明第一方面所述的抗CD40抗体、第二方面或第三方面所述的双特异性抗体和/或如本发明第八方面所述的药物组合物；
所述药盒B含有其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
本发明第十二方面提供：一种免疫检测，或者测定CD40和/或PD-L1的方法，其包括使用如本发明第一方面所述的抗CD40抗体、第二方面或第三方面所述的双特异性抗体和/或如本发明第八方面所述的药物组合物。
在本发明的优选实施方案中，所述检测为非诊断目的的检测，仅适用于科研目的。
本发明第十三方面提供：一种预防和/或治疗肿瘤的方法，其包括向有需要的患者施用治疗有效量的如本发明第一方面所述的抗CD40抗体、第二方面或第三方面所述的双特异性抗体和/或如本发明第八方面所述的药物组合物，或者如本发明第十一方面所述的套装药盒。
例如，所述肿瘤为淋巴癌、乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠直肠癌、膀胱癌、横纹肌肉瘤、食道癌、宫颈癌、多发性骨髓瘤、白血病、胆囊癌、胶质母细胞瘤或黑色素瘤，但不限制于此。
如本发明所用，术语“有效量”表示引发例如研究者或临床医师所追求的组织、系统、动物或人的生物学或药学响应的药物或药剂的量。此外，术语“治疗有效量”表示，与没有接受该量的相应受试者相比，引起疾病、病症或副作用的改进治疗、治愈、预防或减轻的量，或者使疾病或病况的进展速率降低的量。该术语在其范围内还包括有效增强正常生理功能的量。
本发明第十四方面提供：一种联合疗法，其包括分别向有需要的患者施用如本发明第一方面所述的抗CD40抗体、第二方面或第三方面所述的双特异性抗体和/或如本发明第八方面所述的药物组合物，和第二治疗剂；
所述第二治疗剂较佳地包含其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
本发明第十五方面提供：用作药物的如本发明第一方面所述的抗CD40抗体、第二方面或第三方面所述的双特异性抗体和/或如本发明第八方面所述的药物组合物；在一些技术方案中，所述药物用于预防和/或治疗肿瘤。
优选地，所述肿瘤为PD-L1阳性和/或CD40阳性的肿瘤。
例如，所述肿瘤为淋巴癌、乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠直肠癌、膀胱癌、横纹肌肉瘤、食道癌、宫颈癌、多发性骨髓瘤、白血病、胆囊癌、胶质母细胞瘤或黑色素瘤。
优选地，所述肿瘤为结肠癌。
本发明还听提供一种抗体药物偶联物，其包含与一种或更多种治疗剂或放射性同位素缀合的如本发明第一方面所述的抗CD40抗体、第二方面或第三方面所述的双特异性抗体。
优选地，所述治疗剂为细胞毒性剂、化学治疗剂、药物、生长抑制剂和/或毒素；和/或，所述缀合为使用接头将所述抗体与所述治疗剂或放射性同位素连接。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明的新的抗CD40激动性抗体能够有效地调控DC细胞的活化，具有更强的T细胞活化作用，但毒副作用低，适合用于肿瘤的免疫治疗。本发明的双特异抗体，其通过同时激动CD40和阻断PD-L1/PD-1，作用于DC细胞和T细胞的交叉作用的正反馈通路中，最大化抗肿瘤药效；也通过PD-L1依赖的CD40活化，提高CD40活化的选择性，降低CD40激动性抗体的毒副作用。
附图说明
图1为本发明实施例7中报告基因法测定单抗的CD40的激动活性。
图2为本发明实施例8中单抗的DC细胞调控活性，以CD83为DC活化指标。
图3为本发明实施例9中单抗的T细胞调控活性，以IFN-γ为T细胞活化指标。
图4A～图4C为本发明实施例10中单抗在人CD40基因敲入小鼠体内的安全性。其中图4A为小鼠的体重改变；图4B为小鼠的肝功和血液学改变；图4C为小鼠的脏器系数的改变。
图5为本发明实施例12中报告基因法测定的单抗的PD-L1/PD-1阻断活性。
图6为本发明实施例13中单抗的T细胞调控活性。
图7为本发明实施例4中双特异抗体的结构。
图8为本发明实施例15中双特异抗体同时结合PD-L1和CD40。
图9为本发明实施例16中报告基因法测定的双特异抗体的PD-L1/PD-1阻断活性。
图10为本发明实施例17中报告基因法测定的双特异抗体的CD40的激动活性：图10A：1605、1606、1607的CD40激动活性；图10B：1608、1609的CD40激动活性；图10C：1652、1653的CD40激动活性；图10D：1654、1655、CP-870893的CD40激动活性。
图11为本发明实施例19中双特异抗体的DC调控活性：图11A比较不同双特异抗体的活性，以IL-12p40为指标；图11B和图11C比较亲本单抗和双特异抗体的活性，图11B展示的是CD83，图11C展示的是IL-12p40。
图12A和图12B为本发明实施例20中双特异抗体的T细胞调控活性。
图13为本发明实施例21中双特异抗体在小鼠体内抗MC38肿瘤的药效。
图14为抗PD-L1/CD40双特异抗体的体内抗MC38/hPD-L1结肠癌肿瘤药效。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1 CD40抗体和PD-L1抗体噬菌体文库建立
根据human CD40蛋白晶体结构，设计human CD40抗体的variable region序列，并通过基因合成技术获得single chain Fv序列(VL-G4S linker-VH)，使用限制性内切酶装入噬菌体载体(pComb3XSS，购自北京庄盟国际生物基因科技有限公司)中，获得重组质粒库。使用携带scFv基因的噬菌体质粒电击穿孔转化到SS320大肠杆菌感受态细胞。待SS320大肠杆菌(购自Lucigen)增殖到对数期后，加入辅助噬菌体(M13K07，购自NEB)感染，过夜培养，从培养物上清中提取噬菌体，获得CD40抗体噬菌体文库。
根据human PD-L1蛋白晶体结构，设计human PD-L1纳米抗体的variable region序列，并通过基因合成技术获得VHH序列，使用限制性内切酶装入噬菌体载体中，获得重组质粒库。使用携带VHH基因的噬菌体质粒电击穿孔转化到SS320大肠杆菌感受态细胞。待SS320增殖到对数期后，加入辅助噬菌体感染，过夜培养，从培养物上清中提取噬菌体，获得PD-L1抗体噬菌体文库。
实施例2从抗体噬菌体文库中筛选CD40抗体和PD-L1抗体
从CD40抗体噬菌体文库中筛选CD40抗体，从PD-L1抗体噬菌体文库中筛选PD-L1抗体。
采用基于蛋白的噬菌体抗体淘选技术，进行了三轮噬菌体展示文库的淘选。第一轮筛选方法如下：在酶标板中包被human CD40-mFc抗原或者human PD-L1-mFc抗原(购自百普赛斯)，取对应的噬菌体文库加入等体积的2％脱脂奶预混孵育，将预混液加入包被孔中反应后，无菌PBST洗涤去除预混液。使用75mM的柠檬酸钠缓冲液对孔板中吸附的噬菌体进行洗脱；中和噬菌体文库后，使用M13K07辅助噬菌体扩增100倍筛选后的噬菌体文库。然后以类似第一轮筛选的方法进行第二轮和第三轮筛选。经过三轮筛选后得到富集的噬菌体文库。噬菌体的富集情况通过每一轮筛选的起始噬菌体用量和筛选后收集到的噬菌体滴度监测。
使用富集后的CD40噬菌体文库和PD-L1噬菌体文库分别侵染SS320大肠杆菌，然后涂于琼脂糖平板培养。挑取单克隆菌落，放入96孔深孔板中用含有氨苄青霉素和卡那霉素的2YT培养基于37℃震荡培养，得到含有单克隆噬菌体的上清。将单克隆噬菌体上清与包被有human CD40-mFc抗原或者human PD-L1-mFc抗原的酶标板进行孵育1小时，然后用无菌PBST进行清洗，再加入Anti-M13-HRP(购自义翘神州)，并于4℃孵育30分钟。随后使用酶标仪检测噬菌体和抗原的结合，筛选出与抗原结合高的CD40单克隆噬菌体和PD-L1单克隆噬菌体。
实施例3重组CD40单克隆抗体的制备和重组PD-L1单克隆抗体的制备
将筛选出的CD40单克隆噬菌体的重链和轻链可变区的cDNA序列分别克隆到已含有抗体恒定区的pcDNA3.4载体(Invitrogen)中，即获得多个抗CD40单克隆抗体，分别命名为1605CD、1606CD、1607CD、1608CD、1609CD、1652CD、1653CD、1654CD、1655CD。使用PEI法将重链和轻链重组质粒共转染到EXPI-293细胞(Invitrogen)中，瞬时转染7-10天，离心并收取上清液。将上清液经过protein A纯化，获得纯化的抗CD40单克隆抗体。CD40抗体的CDR序列参考表7(由Kabat CDR系统确定)；重链可变区和轻链可变区的氨基酸序列参考表8。
示例性地，1654CD的全长氨基酸序列如下：
1654CD的重链的氨基酸序列(SEQ ID NO:94)：

1654CD的轻链的氨基酸序列(SEQ ID NO:95)：
1606CD的全长氨基酸序列如下：
1606CD的重链的氨基酸序列(SEQ ID NO:96)：
1606CD的轻链的氨基酸序列(SEQ ID NO:81)：
1652CD的全长氨基酸序列如下：
1652CD的重链的氨基酸序列(SEQ ID NO:87)：

1652CD的轻链的氨基酸序列(SEQ ID NO:81)：
将筛选出的PD-L1单克隆噬菌体的可变区的cDNA序列(VHH)克隆到已含有抗体恒定区的pcDNA3.4载体(Invitrogen)中，即获得多个抗PD-L1单克隆抗体，分别命名为，1029，1031，1102，1541。PD-L1单克隆抗体的互补决定区(CDR)序列如表2所示(由Kabat CDR系统确定)、单克隆抗体的框架区(FR)序列如表3所示、可变区(VHH)的氨基酸序列如表4所示、单克隆抗体的连接子和恒定区的氨基酸序列如表5所示以及单克隆抗体的全长氨基酸序列如表6所示。使用PEI法将质粒转染到EXPI-293细胞(Invitrogen)中，瞬时转染7-10天，离心并收取上清液。将上清液经过protein A纯化，获得纯化的抗PD-L1单克隆抗体。
表2 PD-L1单克隆抗体的CDR序列
注：表内加粗并带下划线的为与1029相比，区别的氨基酸。其中为D或E；为S或T；为K或Q；为G或S；为S或T；为D或E；为K或N。
表3 PD-L1单克隆抗体的FR序列
注：表内加粗并带下划线的为与1029相比，区别的氨基酸。其中X48为L或F；X49为A或S；X50为K或R；X51为A或T。
表4 PD-L1单克隆抗体的VHH的氨基酸序列
注：表内斜体下划线部分的为上述的CDR序列；加粗并带下划线的为与1029相比，区别的氨基酸。
VHH的通式的氨基酸序列(SEQ ID NO:66)：

其中为D或E；为S或T；为K或Q；为G或S；为S或T；为D或E；为K或N，X48为L或F；X49为A或S；X50为K或R；X51为A或T。
表5 PD-L1单克隆抗体的连接子和恒定区的氨基酸序列
表6 PD-L1单克隆抗体的全长氨基酸序列

实施例4重组抗PD-L1/CD40双特异抗体的制备
重组抗PD-L1/CD40双特异抗体使用PD-L1单克隆抗体1541与CD40单克隆抗体1605CD、1606CD、1607CD、1608CD、1609CD、1652CD、1653CD、1654CD、1655CD。
将筛选出的CD40单克隆噬菌体的重链和轻链可变区的cDNA序列分别克隆到已含有抗体恒定区的pcDNA3.4载体(Invitrogen)中；将筛选出的PD-L1单克隆噬菌体的可变区的cDNA序列(VHH)克隆到已含有CD40-VH和抗体恒定区的pcDNA3.4载体(Invitrogen)中，VHH序列装入抗体恒定区的C端，并在恒定区与VHH之间引入(G4S)linker；即获得多个抗PD-L1/CD40双特异抗体(结构如图7所示)，分别命名为1605、1606、1607、1608、1609、1652、1653、1654、1655。双特异抗体的CDR序列如表7所示(由Kabat CDR系统确定)；重链可变区和轻链可变区的氨基酸序列如表8所示；双特异抗体的重链和轻链的氨基酸序列如表9所示。使用PEI法将重链和轻链重组质粒共转染到EXPI-293细胞(Invitrogen)中，瞬时转染7-10天，离心并收取上清液。将上清液经过protein A纯化，获得纯化的抗PD-L1/CD40双特异抗体。
表7双特异抗体的CDR序列

注：RASQX1IX2X3YLX4(SEQ ID NO:1)中，X1为D、G、S或T；X2为R或S，X3为N或S；X4为N或A。X5X6SX7X8X9S中，X5为Y或A；X6为T或A；X7为S、R或T；X8为L或R；X9为Q或D。QQGX10X11X12PW(SEQ ID NO:3)中，X10为K、I、N、Q或S；X11为S、A、N或T；X12为L、Y或F。SX13YYMS(SEQ ID NO:4)中，X13为N或D。FIRNKANX14YT(SEQ ID NO:5)中，X14为A或G。YGGX15X16X17GWYFDX18(SEQ ID NO:6)中，X15为L或I；X16为K或R；X17为V、K、I或Q；X18为L或V。
表8双特异抗体的重链可变区和轻链可变区的氨基酸序列

其中，Anti-CD40的重链可变区的共用序列为：
EVQLVESGGGLVQPGGSLRLSCAASGFTFSX19YYMSWVRQAPGKGLEWVX20FIRNKANX21YTTEYAASVKGRFTISRDNSKX22TLYLQMNX23LRAEDTAVYYCARYGGX24X25X26GWYFDX27WGQGTLVTVSS(SEQ ID NO:64)：X19为N或D；X20为G或A；X21为A或G；X22为N或S；X23为R或S；X24为L或I；X25为K或R；X26为V、K、I或Q；X27为L或V。
Anti-CD40的轻链可变区的共用序列为(SEQ ID NO:65)：
DIQMTQSPSSLSASVGDRVTITCRASQX28IX29X30YLX31WYQQKPGKAPKLLIYX32X33SX34X35X36SGVPSRFSGSGSGTDYTLTISSLQPEDFATYX37CQQGX38X39X40PWTFGGGTKVEIK：X28为D、G、S或T；X29为R或S；X30为N或S；X31为N或A；X32为Y或A；X33为T或A；X34为S、R或T；X35为L或R；X36为Q或D；X37为Y或F；X38为K、I、N、Q或S；X39为S、A、N或T；X40为L、Y或F。
表9双特异抗体的重链和轻链的氨基酸序列



实施例5重组CD40单克隆抗体与CD40的结合检测
利用酶联免疫吸附实验(ELISA)检测单克隆抗体对human CD40的结合能力，以及种属特异性。具体方法如下：用pH＝9.6的碳酸盐缓冲溶液在酶标板中分别包被抗原human CD40,cynomolgus CD40,rat CD40,mouse CD40(均购自百普赛斯)，1μg/mL，每孔100μL，4℃过夜孵育。PBST洗涤5次。用300μL/孔含1％BSA的PBST封闭，室温孵育1小时。PBST洗涤5次。加入用含1％BSA的PBST梯度稀释的单克隆抗体，并加入对照CD40单抗CP-870893(IMGT数据库ID 10523)，每孔100μL，室温孵育1小时。PBST洗涤5次。加入用含1％BSA的PBST稀释的HRP标记的抗人IgG抗体(Jackson ImmunoResearch，货号109-035-088)，每孔100μL，室温孵育1小时。PBST洗涤5次。加入比色底物TMB，每孔100μL，室温下显色10分钟，随后加入1M硫酸终止反应。在酶标仪上读取OD450nm，分析结果并用4参数拟合结合曲线计算EC50。
结果如表10所示，单克隆抗体1605CD、1606CD、1607CD、1608CD、1609CD、1652CD、1653CD、1654CD、1655CD均可与human CD40，cynomolgus CD40结合，且与人和食蟹猴抗原的结合力相当。与rat CD40，mouse CD40不能结合。
表10单克隆抗体与抗原的结合
实施例6抗CD40单克隆抗体对CD40/CD40L结合的影响
利用高表达CD40的HEK-Blue CD40L细胞(购自InvivoGen)测试单克隆抗体对CD40/CD40L结合的阻断作用。具体方法如下：收集HEK-Blue CD40L细胞，用预冷的含2％FBS的PBS重悬，密度为1×107/mL，然后加入96孔板中，每孔50μL，即5×105细胞/孔，然后加入Biotin-CD40L(明济生物)和梯度稀释的CD40单克隆抗体，每孔50μL，Biotin-CD40L的终浓度为10nM，在4℃孵育1个小时。预冷的PBS洗涤2次。加入稀释于预冷的含2％FBS的PBS的Streptavidin-PE(BioLegend，货号405203)，在4℃孵育30分钟。预冷的PBS洗涤2次。然后再用预冷的含2％FBS的PBS重悬细胞，上流式分析仪检测。
结果如表11所示，单克隆抗体1605CD、1606CD、1607CD、1608CD、1609CD、1652CD、1653CD、1654CD、1655CD阻碍CD40/CD40L的相互作用，而对照CD40单抗CP-870893对CD40/CD40L相互作用无影响。
表11单克隆抗体对CD40/CD40L结合的影响
实施例7报告基因法检测抗CD40单克隆抗体的CD40激动活性
利用HEK-Blue CD40L测试单克隆抗体的CD40激动活性。HEK-Blue CD40L细胞购自InvivoGen，高表达有CD40，以及在NF-κB反应元件控制下的SEAP报告基因。当HEK-Blue CD40L细胞上的CD40被激活后，诱导下游信号NF-κB的活化，继而诱导SEAP的产生。通过QUANTI-Blue试剂(InvivoGen)检测分泌的SEAP的量，即可监测CD40的活化情况。具体方法如下：收集HEK-Blue CD40L细胞，3×105/mL重悬于完全培养基(RPMI 1640含10％FBS)，均匀铺至96孔板中，100μL/孔，即每孔3×104个细胞；100μL/孔加入用完全培养基序列稀释的抗CD40单克隆抗体样品以及对照抗体(CP-870893)，在37℃、5％CO2孵箱中孵育20-24小时。孵育结束后，取出96孔板用离心机300g离心5分钟，吸取40μL/孔上清转移至新的96孔板中，向上清中加入160μL/孔QUANTI-Blue 试剂，在37℃、5％CO2孵箱中孵育20-30分钟，在酶标仪上读取OD655nm，分析结果。
结果如图1所示，单克隆抗体的CD40激动活性大致可以分为两类，一类抗体1608CD、1609CD、1654CD、1655CD的CD40激动活性较弱，弱于对照CD40单抗CP-870893；另一类抗体1605CD、1606CD、1607CD、1652CD、1653CD的活性较强，强于对照CD40单抗CP-870893。
实施例8抗CD40单克隆抗体的DC调控活性
测试CD40单克隆抗体的树突状细胞(DC)调控活性。
将捐献的人PBMC细胞重悬于完全培养基(RPMI 1640含10％FBS)并接种在10cm细胞培养皿中，在37℃二氧化碳培养箱中孵育2小时。弃除培养上清及悬浮细胞，贴壁的即为单核细胞。将单核细胞在含100ng/mL GM-CSF(PeproTech，货号300-03)和100ng/mL IL-4(PeproTech，货号200-04)的完全培养基中培养，孵育6天，每2天换液，即得imDC细胞。收集imDC细胞，用完全培养基重悬，接种于24孔板，并加入100nM的抗体样品以及对照抗体(CP-870893)。将培养板置于37℃二氧化碳培养箱中孵育2天。孵育结束之后，取出孔内上清，按照试剂盒使用手册检测细胞因子IL-12/IL-23 p40(R&D,货号DY1240)；同时收集孔板中细胞，与检测抗体(APC anti-human CD83 Antibody,Biolegend,305312)孵育后，上流式细胞分析仪检测DC细胞上的CD83表达。
图2比较了本发明抗CD40单克隆抗体的DC调控活性，不同单抗均能显著上调DC细胞上的CD83。
实施例9抗CD40单克隆抗体的T细胞调控活性
CD40激动性抗体活化授权DC细胞后，继而活化T细胞是其最重要的抗肿瘤生物学效应。用异体DC细胞和T细胞孵育的MLR实验体系，测试CD40单克隆抗体的T细胞调控活性。
用与实施例8一致的方法，获取DC细胞。异体的T细胞，从人PBMC细胞中分离获得，具体的分离方法参照Pan T细胞分离试剂盒(Miltenyi Biotech，货号130-096-535)使用说明书。简要的说，先用PBS洗涤PBMC一次，再将PBMC按1E7细胞每40μL分离缓冲液(PBS含2mM EDTA,0.5％BSA,pH＝7.2)重悬(以下使用量均按1E7细胞计)，加入10μL Pan T cell Biotin Antibody Cocktail，在4度孵育5分钟。再加入30μL分离缓冲液和20μL Pan T cell MicroBead Cocktail，在4度孵育10分钟。过MACS分离柱，即得T细胞。
收集获得的人DC细胞和人T细胞，重悬于完全培养基(RPMI 1640含10％FBS) 中，接种于96孔板，接种的DC细胞和T细胞分别为1E4/孔，1E5/孔，混合培养。并加入用完全培养基系列稀释的单克隆抗体样品以及对照抗体。将培养板置于37℃二氧化碳培养箱中孵育5天。孵育结束之后，取出孔内上清，按照试剂盒使用手册检测细胞因子IFN-γ(Biolegend，货号430101)。
结果如图3所示，本发明的强CD40激动活性单抗1606CD和弱CD40激动活性单抗1654CD，在低比例DC的条件下均能增强T细胞活化，而对照单抗CP-870893基本没有作用。
实施例10抗CD40单克隆抗体的初步安全性评价
抗CD40单克隆抗体的初步安全性评价在人CD40基因敲入小鼠体内进行。实验动物购自百奥赛图，随机分为4组(5只/组)，分别给予溶媒对照，21mg/kg单抗1606CD，21mg/kg单抗1654CD，以及21mg/kg的对照CD40单抗CP-870893。腹腔注射，每周给药2次，连续给药2周，共给药4次。试验期间，监测动物的临床症状、体重、肝功能生化和血细胞计数，给药期结束后，所有动物于末次药后次日按计划安乐死，进行解剖，观察是否有异常并进行脏器称重。
如图4A～图4C所示，反复注射强CD40激动活性单抗1606CD、弱CD40激动活性单抗1654CD的小鼠未见明显异常，仅在血液学中可见淋巴细胞和粒细胞的下降。而反复注射单抗CP-870893的小鼠，可见体重下降，ALT升高，RBC，HGB，PLT等的显著下降，并于解剖时，可见明显的脏器坏死及脏器增大。结果提示，单抗1606CD、1654CD较单抗CP-870893具有更好的安全性。
实施例11：重组PD-L1单克隆抗体与PD-L1的结合检测
利用酶联免疫吸附实验(ELISA)检测单克隆抗体对人PD-L1的结合能力，以及种属特异性。具体方法如下：用pH＝9.6的碳酸盐缓冲溶液在酶标板中分别包被抗原人(human)PD-L1，食蟹猴(cynomolgus)PD-L1，大鼠(rat)PD-L1，小鼠(mouse)PD-L1(均购自百普赛斯)，1μg/mL，每孔100μL，4℃过夜孵育。PBST洗涤5次。用300μL/孔含1％BSA的PBST封闭，室温孵育1小时。PBST洗涤5次。加入用含1％BSA的PBST梯度稀释的单克隆抗体，并加入对照PD-L1单抗Durvalumab(IMGT数据库ID 10010)，每孔100μL，室温孵育1小时。PBST洗涤5次。加入用含1％BSA的PBST稀释的HRP标记的抗人IgG抗体(Jackson ImmunoResearch，货号109-035-088)，每孔100μL，室温孵育1小时。PBST洗涤5次。加入比色底物TMB，每孔100μL，室温下显色10分钟，随后加入1M硫酸终止反应。在酶标仪上读取OD450nm，分析结果并用4参数 拟合结合曲线计算EC50。
结果如表12所示，单克隆抗体1029，1031，1102，1541均可与人PD-L1和食蟹猴PD-L1结合，且与人和食蟹猴抗原的结合力与对照抗体相当。与大鼠PD-L1，小鼠PD-L1不能结合。
表12单克隆抗体与抗原的结合

NB：不结合
利用基于生物层干涉(BLI)原理的非标记生物分子相互作用分析仪Gator检测单克隆抗体与抗原之间的相互作用。具体方法如下：使用PA探针，将单抗稀释到50nM加入探针板，从而单抗被PA探针捕获。然后加入从200nM起始做梯度稀释的抗原人PD-L1，抗原与被PA探针捕获的双特异抗体相互作用，通过检测探针表面反射干涉光谱的信号变化，对相互作用进行分析，最终计算出抗体的结合动力学常数。
结果如表13所示，单克隆抗体1029，1031，1102，1541与人PD-L1的结合动力学常数在4.37至8.30nM之间。
表13单克隆抗体与抗原的结合动力学
实施例12：报告基因法检测PD-L1单克隆抗体的PD-L1/PD-1阻断活性
利用明济生物构建的Jurkat/PD-1-NFAT-luciferase细胞(高表达有PD-1，以及在NFAT反应元件控制下的luciferase报告基因)和WIL2S/PD-L1(高表达有PD-L1)细胞，以及 抗CD20/CD3双特异抗体，建立PD-L1/PD-1阻断活性的报告基因检测方法。具体方法如下：收集WIL2S/PD-L1细胞并按细胞密度为4×106/mL重悬于完全培养基(RPMI 1640含10％FBS)，50μL/孔，即每孔2×105个细胞，均匀铺至96孔板中；收集Jurrkat-PD-1-NFAT-luciferase细胞并按细胞密度为4×106/mL重悬于完全培养基(RPMI 1640含10％FBS)，50μL/孔，即每孔2×105个细胞，均匀加至上述96孔板中；完全培养基稀释抗CD20/CD3双特异抗体，25μL/孔加至上述96孔板中；完全培养基序列稀释抗PD-L1单克隆抗体样品以及对照抗体，25μL/孔加至上述96孔板中；然后在37℃、5％CO2孵箱中孵育6小时。孵育结束后，向上述96孔板中加入50μL/孔的one-glo试剂(Promega，货号E6120)，放置于震板器上震荡5分钟，静置10分钟。然后在酶标仪(MD，SpectraMax iD3)上使用化学发光模块读取相对化学发光单位值(RLU)，分析结果。
结果如图5所示，单克隆抗体1029，1031，1102，1541均可以阻断PD-L1传递给PD-1的负信号，抗体的活性相似，但较PD-L1对照单抗Durvalumab的活性略强。
实施例13：PD-L1单克隆抗体的T细胞调控活性
用异体DC细胞和T细胞孵育的MLR实验体系，测试单克隆抗体的T细胞调控活性。
将捐献的人PBMC细胞重悬于完全培养基(RPMI 1640含10％FBS)并接种在10cm细胞培养皿中，在37℃二氧化碳培养箱中孵育2小时。弃除培养上清及悬浮细胞，贴壁的即为单核细胞。将单核细胞在含100ng/mL GM-CSF(PeproTech，货号300-03)和100ng/mL IL-4(PeproTech，货号200-04)的完全培养基中培养，孵育6天，每2天换液，再加入TNFα和IL-1β(均购自PeproTech)孵育2天，即得到DC细胞。
异体的T细胞，从捐献的人PBMC细胞中分离获得，具体的分离方法参照Pan T细胞分离试剂盒(Miltenyi Biotech，货号130-096-535)使用说明书。简要的说，先用PBS洗涤PBMC一次，再将PBMC按1×107细胞每40μL分离缓冲液(PBS含2mM EDTA,0.5％BSA,pH＝7.2)重悬(以下使用量均按1×107细胞计)，加入10μL Pan T cell Biotin Antibody Cocktail，在4℃孵育5分钟。再加入30μL分离缓冲液和20μL Pan T cell MicroBead Cocktail，在4℃孵育10分钟。过MACS分离柱，即得T细胞。
收集获得的人DC细胞和人T细胞，重悬于完全培养基(RPMI 1640含10％FBS)中，接种于96孔板，接种的DC细胞和T细胞分别为1×104/孔和1×105/孔，混合培养。并加入用完全培养基序列稀释的抗体样品。将培养板置于37℃二氧化碳培养箱中孵育5天。孵育结束之后，取出孔内上清，按照试剂盒使用手册检测细胞因子IFN-γ(Biolegend，货号430101)。
结果如图6所示，单克隆抗体明显增强T细胞活化。
实施例14重组抗PD-L1/CD40双特异抗体与PD-L1、CD40的结合检测
利用酶联免疫吸附实验(ELISA)检测双特异抗体对human PD-L1，human CD40的结合能力，以及种属特异性。具体方法如下：用pH＝9.6的碳酸盐缓冲溶液在酶标板中分别包被抗原human PD-L1、cynomolgus PD-L1、rat PD-L1、mouse PD-L1、human CD40,cynomolgus CD40、rat CD40、mouse CD40(均购自百普赛斯)，1μg/mL，每孔100μL，4℃过夜孵育。PBST洗涤5次。用300μL/孔含1％BSA的PBST封闭，室温孵育1小时。PBST洗涤5次。加入用含1％BSA的PBST梯度稀释的双特异抗体，并加入对照PD-L1单抗Durvalumab(IMGT数据库ID 10010)和CD40单抗CP-870893(IMGT数据库ID 10523)，每孔100μL，室温孵育1小时。PBST洗涤5次。加入用含1％BSA的PBST稀释的HRP标记的抗人IgG抗体(Jackson ImmunoResearch，货号109-035-088)，每孔100μL，室温孵育1小时。PBST洗涤5次。加入比色底物TMB，每孔100μL，室温下显色10分钟，随后加入1M硫酸终止反应。在酶标仪上读取OD450nm，分析结果并用4参数拟合结合曲线计算EC50。
结果如表14所示，双特异抗体1605、1606、1607、1608、1609、1652、1653、1654、1655均可与human PD-L1和CD40，cynomolgus PD-L1和CD40结合，且与人和食蟹猴抗原的结合力相当。与rat PD-L1和CD40，mouse PD-L1和CD40不能结合。
表14双特异抗体与抗原的结合

NB：不结合
实施例15重组抗PD-L1/CD40双特异抗体与PD-L1、CD40的同时结合检测
用pH＝9.6的碳酸盐缓冲溶液在酶标板中包被抗原human CD40(购自百普赛斯)，1μg/mL，每孔100μL，4℃过夜孵育。PBST洗涤5次。用300μL/孔含1％BSA的PBST封闭，室温孵育1小时。PBST洗涤5次。加入用含1％BSA的PBST梯度稀释的双特异 抗体，并加入对照PD-L1单抗Durvalumab和CD40单抗CP-870893，每孔100μL，室温孵育1小时。PBST洗涤5次。加入用含1％BSA的PBST稀释的Biotin标记的human PD-L1(明济生物制药)，每孔100μL，室温孵育1小时。PBST洗涤5次。加入用含1％BSA的PBST稀释的Streptavidin-HRP(BioLegend，货号405210)，每孔100μL，室温孵育30分钟。加入比色底物TMB，每孔100μL，室温下显色10分钟，随后加入1M硫酸终止反应。在酶标仪上读取OD450nm，分析结果并用4参数拟合结合曲线计算EC50。
结果如图8所示，双特异抗体1605、1606、1607、1608、1609、1652、1653、1654、1655均可同时结合human PD-L1和human CD40，而对照单抗没有信号，不能同时结合两种抗原。
实施例16报告基因法检测抗PD-L1/CD40双特异抗体的PD-L1/PD-1阻断活性
Jurkat/PD-1-NFAT-luciferase细胞(高表达有PD-1，以及在NFAT反应元件控制下的luciferase报告基因)和WIL2S/PD-L1细胞(高表达有PD-L1)参照文献方法(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.)，通过慢病毒转染构建获得。利用此Jurkat和WIL2S细胞，抗CD20/CD3双特异抗体，建立PD-L1/PD-1阻断活性的报告基因检测方法。具体方法如下：收集WIL2S/PD-L1细胞并按细胞密度为4E6/mL重悬于完全培养基(RPMI 1640含10％FBS)，50μL/孔，即每孔2E5个细胞，均匀铺至96孔板中；收集Jurrkat-PD-1-NFAT-luciferase细胞并按细胞密度为4E6/mL重悬于完全培养基(RPMI 1640含10％FBS)，50μL/孔，即每孔2E5个细胞，均匀加至上述96孔板中；完全培养基稀释抗CD20/CD3双特异抗体,25μL/孔加至上述96孔板中；完全培养基序列稀释抗PD-L1/CD40双特异抗体样品以及对照抗体，25μL/孔加至上述96孔板中；然后在37℃、5％CO2孵箱中孵育6小时。孵育结束后，向上述96孔板中加入50μl/孔的one-glo试剂(Promega，货号E6120)，放置于振板器上振荡5分钟，静置10分钟。然后在酶标仪(MD，SpectraMax iD3)上使用化学发光模块读取相对化学发光单位值(RLU)，分析结果。
结果如图9所示，双特异抗体1605、1606、1607、1608、1609、1652、1653、1654、1655可以阻断PD-L1传递给PD-1的负信号，不同双特异抗体的活性相似，与PD-L1对照单抗Durvalumab的活性也相似。
实施例17报告基因法检测抗PD-L1/CD40双特异抗体的CD40激动活性
利用HEK-Blue CD40L测试双特异抗体的CD40激动活性。HEK-Blue CD40L细胞 购自InvivoGen，高表达有CD40，以及在NF-κB反应元件控制下的SEAP报告基因。当HEK-Blue CD40L细胞上的CD40被激活后，诱导下游信号NF-κB的活化，继而诱导SEAP的产生。通过QUANTI-Blue试剂(InvivoGen)检测分泌的SEAP的量，即可监测CD40的活化情况。利用CHO/PD-L1细胞与HEK-Blue CD40L细胞共同孵育测试在PD-L1存在时，双特异抗体的CD40激动活性。具体方法如下：收集HEK-Blue CD40L细胞，3E5/mL重悬于完全培养基(RPMI 1640含10％FBS)，均匀铺至96孔板中，100μL/孔，即每孔3E4个细胞；收集CHO/PD-L1细胞，6E5/mL重悬于完全培养基(RPMI 1640含10％FBS)，50μL/孔，即每孔3E4个细加入96孔板中，不需要CHO/PD-L1细胞的则加入50μL/孔完全培养基；50μL/孔加入用完全培养基序列稀释的抗PD-L1/CD40双特异抗体样品以及对照抗体，在37℃、5％CO2孵箱中孵育20-24小时。孵育结束后，取出96孔板用离心机300g离心5分钟，吸取40μL/孔上清转移至新的96孔板中，向上清中加入160μL/孔QUANTI-Blue试剂，在37℃、5％CO2孵箱中孵育20-30分钟，在酶标仪上读取OD655nm，分析结果。
结果如图10A至图10D和表15所示，双特异抗体1608、1609、1654、1655的CD40激动活性，明显被CHO/PD-L1提供的PD-L1 cross-linking信号增强，代表这些双抗的活性具有选择性：在PD-L1低表达环境(比如在血液)中活性较低；在PD-L1高表达的环境(比如肿瘤环境)中活性较高，具有增强药效、控制副作用的治疗效果。而双特异抗体1605、1606、1607、1652、1653被CHO/PD-L1增强程度较低；对照CD40单抗CP-870893没有被CHO/PD-L1增强。
表15双特异抗体的CD40激动活性
实施例18抗PD-L1/CD40双特异抗体对CD40/CD40L结合的影响
利用高表达CD40的HEK-Blue CD40L细胞测试双特异抗体对CD40/CD40L结合的阻断作用。具体方法如下：收集HEK-Blue CD40L细胞，用预冷的含2％FBS的PBS重 悬，密度为1E7/mL，然后加入96孔板中，每孔50μL，即5E5细胞/孔，然后加入Biotin-CD40L(明济生物)和梯度稀释的双特异抗体，每孔50μL，Biotin-CD40L的终浓度为10nM，在4℃孵育1个小时。预冷的PBS洗涤2次。加入稀释于预冷的含2％FBS的PBS的Streptavidin-PE(BioLegend，货号405203)，在4℃孵育30分钟。预冷的PBS洗涤2次。然后再用预冷的含2％FBS的PBS重悬细胞，上流式分析仪检测。
结果如表16所示，双特异抗体1605、1606、1607、1608、1609、1652、1653、1654、1655阻碍CD40/CD40L的相互作用，而对照CD40单抗CP-870893对CD40/CD40L相互作用无影响。
表16双特异抗体对CD40/CD40L结合的影响
实施例19抗PD-L1/CD40双特异抗体的DC调控活性
测试双特异抗体的树突状细胞(DC)调控活性。
将人PBMC细胞重悬于完全培养基(RPMI 1640含10％FBS)并接种在10cm细胞培养皿中，在37℃二氧化碳培养箱中孵育2小时。弃除培养上清及悬浮细胞，贴壁的即为单核细胞。将单核细胞在含100ng/mL GM-CSF(PeproTech,货号300-03)和100ng/mL IL-4(PeproTech,货号200-04)的完全培养基中培养，孵育6天，每2天换液，即得imDC细胞。收集imDC细胞，用完全培养基重悬，接种于24孔板，并加入梯度稀释的双特异抗体样品以及对照抗体。将培养板置于37℃二氧化碳培养箱中孵育2天。孵育结束之后，取出孔内上清，按照试剂盒使用手册检测细胞因子IL-12/IL-23 p40(R&D,货号DY1240)；在一些实验中，同时收集孔板中细胞，与检测抗体(APC anti-human CD83 Antibody、Biolegend、305312；PE/Cyanine7 anti-human CD86 Antibody、Biolegend、374210)孵育后，上流式细胞分析仪检测DC细胞上的CD83、CD86表达。
图11A比较了不同双特异抗体的DC调控活性，以IL-12/IL-23 p40作为指标，双特 异抗体浓度依赖的刺激DC分泌IL-12 p40，不同双特异抗体的活性差异较大。图11B和图11C比较了亲本PD-L1单抗，亲本CD40单抗、双特异抗体(1609)以及对照CD40单抗CP-870893的DC调控活性，检测指标为CD83和IL-12/IL-23 p40，数据显示亲本PD-L1单抗对DC无调控，亲本CD40单抗活性较弱，构建成双抗后其DC活性明显增强。
实施例20抗PD-L1/CD40双特异抗体的T细胞调控活性
用异体DC细胞和T细胞孵育的MLR实验体系，测试双特异抗体的T细胞调控活性。
用与实施例19一致的方法，获取DC细胞。异体的T细胞，从人PBMC细胞中分离获得，具体的分离方法参照Pan T细胞分离试剂盒(Miltenyi Biotech，货号130-096-535)使用说明书。简要的说，先用PBS洗涤PBMC一次，再将PBMC按1E7细胞每40μL分离缓冲液(PBS含2mM EDTA,0.5％BSA,pH＝7.2)重悬(以下使用量均按1E7细胞计)，加入10μL Pan T cell Biotin Antibody Cocktail，在4度孵育5分钟。再加入30μL分离缓冲液和20μL Pan T cell MicroBead Cocktail，在4度孵育10分钟。过MACS分离柱，即得T细胞。
收集获得的人DC细胞和人T细胞，重悬于完全培养基(RPMI 1640含10％FBS)中，接种于96孔板，接种的DC细胞和T细胞分别为1E4/孔，1E5/孔，混合培养。并加入用完全培养基序列稀释的双特异抗体样品以及对照抗体。将培养板置于37℃二氧化碳培养箱中孵育5天。孵育结束之后，取出孔内上清，按照试剂盒使用手册检测细胞因子IFN-γ(Biolegend，货号430101)。
结果如图12A和图12B所示，双特异抗体增强T细胞活化，其T细胞调控活性显著强于亲本PD-L1单抗，亲本CD40单抗以及PD-L1对照单抗Durvalumab。
实施例21抗小鼠PD-L1/小鼠CD40双特异抗体的体内抗肿瘤药效
本实施例检测双特异抗体在小鼠体内的抗肿瘤药效。为了更方便的评价抗PD-L1/CD40双特异抗体的体内药效，构建表达了双特异抗体的替代品抗小鼠PD-L1/小鼠CD40双特异抗体1058(抗PD-L1序列来自IMGT数据库ID 9814，抗CD40序列为WO2018185045A1中的No.33_VH和No.34_VL)。使用此替代品的目的是在野生型小鼠中评价靶向PD-L1的抗体与靶向CD40的抗体形成的双特异抗体的体内效果，仅用于验证此类双特异抗体的药效。
小鼠选用C57BL/6小鼠，雌性，6-8周龄，购自北京维通利华。小鼠适应环境一周 后，每只小鼠接种3E5个MC38小鼠结肠癌细胞(购自中国医学科学院基础医学研究所基础医学细胞中心)。待肿瘤体积长至约100mm3时，按肿瘤体积分组，每组8只小鼠，分别设定为溶媒对照组，抗小鼠PD-L1单抗给药组，抗小鼠CD40单抗给药组，抗小鼠PD-L1/小鼠CD40双特异抗体组。给药剂量为35nmol/kg、腹腔注射，每周给药1次，连续给药2周。自给药之日起，每周测量3次肿瘤体积，测量其长径a，短径b，计算肿瘤体积(mm3)＝(a x b2)/2。
结果如图13所示，双特异抗体的替代品抗小鼠PD-L1/小鼠CD40双特异抗体1058显著抑制了小鼠体内的MC38结肠癌移植瘤的生长，显示了良好的抗肿瘤药效，其药效强于PD-L1单抗，也强于CD40单抗。
实施例22抗PD-L1/CD40双特异抗体的体内抗肿瘤药效
本实施例评价抗PD-L1/CD40双特异抗体在PD-L1/CD40人源化小鼠体内的抗肿瘤药效。实验与北京农学院合作完成。PD-L1/CD40人源化小鼠，雌性，6-8周龄，采购自百奥赛图。小鼠适应环境一周后，每只小鼠接种5E6个MC38/hPD-L1小鼠结肠癌细胞。待肿瘤体积长至约100mm3时，按肿瘤体积分组，每组6只小鼠，分别给予溶媒，抗PD-L1单抗，抗CD40单抗，抗PD-L1/抗CD40双特异性抗体1654，腹腔注射，每周给药2次，连续给药2周。自给药之日起，监测小鼠临床表现，小鼠体重、以及肿瘤体积。肿瘤测量其长径a，短径b，计算肿瘤体积(mm3)＝(a x b2)/2。
结果如图14所示，1654显著抑制PD-L1/CD40人源化小鼠体内的MC38/hPD-L1移植瘤的生长，在低至7nmol/kg剂量下，肿瘤生长抑制率(TGI)即达到72％；强于20nmol/kg剂量的PD-L1单抗或者CD40单抗，两者的TGI均为28％。实验过程中，溶媒对照组有一只小鼠死亡，其它小鼠无异常。小鼠体重无异常。
实施例23抗PD-L1/CD40双特异抗体的初步安全性评价
抗PD-L1/CD40双特异抗体的初步安全性评价委托昭衍(苏州)新药研究中心有限公司进行。
实验动物使用的是雄性食蟹猴，随机分为5组(2只/组)，分别给予溶媒对照，12mg/kg双特异抗体1607、1608、1609，以及10mg/kg的对照CD40单抗CP-870893。使用注射泵于动物前肢或后肢皮下静脉输注给药，给药容量为10mL/kg，给药速度为0.5mL/kg/min。每周给药1次，连续给药2周，共给药3次。试验期间，定期监测动物的临床症状、体重、食量、体温、血细胞计数、凝血功能、血液生化和尿液分析，免疫细胞表型，细胞因子，并检测血药浓度和抗药抗体。给药期结束后，第2-5组所有存活动物于末次药后次日 按计划实施安乐死，进行大体解剖，观察是否有异常并进行脏器称重。
双特异抗体1607，1608、1609 12mg/kg剂量组动物的临床观察、体重、体重增长、食量、体温、血细胞计数、凝血功能、血液生化、尿液分析、脏器重量和病理大体解剖均未见供试品相关的异常改变。仅可见免疫细胞表型和部分细胞因子升高，与药理作用机制相关。而CD40单抗CP-870893 10m/kg剂量组动物分别于D7-D8、D10-D11和D7-D13可见少量软便/稀便；D14的体重增长较药前可见轻微降低；从D5开始食量明显减少；RBC、HGB、HCT和PLT可见降低趋势；免疫细胞表型改变和部分细胞因子升高；并于食蟹猴解剖时发现了胸腺的脏器重量、脏体比和脏脑比降低，脾脏的脏器重量、脏体比和脏脑比升高，变化幅度明显。
结果提示，抗PD-L1/CD40双特异抗体较CD40单抗具有更好的安全性。

Claims (23)

  1. 一种抗CD40的抗体，其包含重链可变区和轻链可变区，其特征在于，所述重链可变区包含SEQ ID NO:64中的HCDR1、HCDR2和HCDR3，所述轻链可变区包含SEQ ID NO:65中的LCDR1、LCDR2和LCDR3；
    优选地，所述LCDR1包含如SEQ ID NO:1所示的氨基酸序列，所述LCDR2包含如X5X6SX7X8X9S所示的氨基酸序列，其中X5为Y或A，X6为T或A，X7为S、R或T，X8为L或R，X9为Q或D，所述LCDR3包含如SEQ ID NO:3所示的氨基酸序列；所述HCDR1包含如SEQ ID NO:4所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:5所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:6所示的氨基酸序列。
  2. 如权利要求1所述的抗体，其特征在于，所述LCDR1包含如SEQ ID NO:9、SEQ ID NO:8、SEQ ID NO:7或SEQ ID NO:10所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:14、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:11或SEQ ID NO:15所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:19、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:16或SEQ ID NO:20所示的氨基酸序列；所述HCDR1包含如SEQ ID NO:21或SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:23或SEQ ID NO:24所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:28、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:25或SEQ ID NO:29所示的氨基酸序列；
    较佳地，所述LCDR1包含如SEQ ID NO:9所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:14所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:23所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:28所示的氨基酸序列；
    所述LCDR1包含如SEQ ID NO:9所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:14所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:24所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:28所示的氨基酸序列；
    所述LCDR1包含如SEQ ID NO:7所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:16所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:23所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:25所示的氨基酸序列；
    所述LCDR1包含如SEQ ID NO:7所示的氨基酸序列，所述LCDR2包含如SEQ ID  NO:12所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:17所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:24所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:26所示的氨基酸序列；
    所述LCDR1包含如SEQ ID NO:7所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:17所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:23所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:26所示的氨基酸序列；
    所述LCDR1包含如SEQ ID NO:8所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:13所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:18所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:24所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:27所示的氨基酸序列；
    所述LCDR1包含如SEQ ID NO:8所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:13所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:18所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:23所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:27所示的氨基酸序列；
    所述LCDR1包含如SEQ ID NO:10所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:15所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:20所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:24所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:29所示的氨基酸序列；或
    所述LCDR1包含如SEQ ID NO:10所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:15所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:20所示的氨基酸序列，所述HCDR1包含如SEQ ID NO:21所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:23所示的氨基酸序列，以及所述HCDR3包含如SEQ ID NO:29所示的氨基酸序列；
    更佳地，所述轻链可变区包含分别如SEQ ID NO:9、SEQ ID NO:14和SEQ ID NO:19所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:21、SEQ ID NO:23和SEQ ID NO:28所示的HCDR1、HCDR2和HCDR3；
    所述轻链可变区包含分别如SEQ ID NO:9、SEQ ID NO:14和SEQ ID NO:19所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:21、SEQ ID NO:24和SEQ ID NO:28所示的HCDR1、HCDR2和HCDR3；
    所述轻链可变区包含分别如SEQ ID NO:7、SEQ ID NO:11和SEQ ID NO:16所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:21、SEQ ID NO:23和SEQ ID NO:25所示的HCDR1、HCDR2和HCDR3；
    所述轻链可变区包含分别如SEQ ID NO:7、SEQ ID NO:12和SEQ ID NO:17所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:22、SEQ ID NO:24和SEQ ID NO:26所示的HCDR1、HCDR2和HCDR3；
    所述轻链可变区包含分别如SEQ ID NO:7、SEQ ID NO:12和SEQ ID NO:17所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:26所示的HCDR1、HCDR2和HCDR3；
    所述轻链可变区包含分别如SEQ ID NO:8、SEQ ID NO:13和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:22、SEQ ID NO:24和SEQ ID NO:27所示的HCDR1、HCDR2和HCDR3；
    所述轻链可变区包含分别如SEQ ID NO:8、SEQ ID NO:13和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:27所示的HCDR1、HCDR2和HCDR3；
    所述轻链可变区包含分别如SEQ ID NO:10、SEQ ID NO:15和SEQ ID NO:20所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:21、SEQ ID NO:24和SEQ ID NO:29所示的HCDR1、HCDR2和HCDR3；或，
    所述轻链可变区包含分别如SEQ ID NO:10、SEQ ID NO:15和SEQ ID NO:20所示的LCDR1、LCDR2和LCDR3，并且所述重链可变区包含分别如SEQ ID NO:21、SEQ ID NO:23和SEQ ID NO:29所示的HCDR1、HCDR2和HCDR3。
  3. 如权利要求2所述的抗体，其特征在于，所述轻链可变区的框架区为人源框架区，且所述重链可变区的框架区为人源框架区；
    较佳地，所述轻链可变区包含与SEQ ID NO:38具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，所述重链可变区包含与SEQ ID NO:40或SEQ ID NO:39具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；
    所述轻链可变区包含与SEQ ID NO:30具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，所述重链可变区包含与SEQ ID NO:31具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；
    所述轻链可变区包含与SEQ ID NO:32具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，所述重链可变区包含与SEQ ID NO:33或SEQ ID NO:34具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；
    所述轻链可变区包含与SEQ ID NO:35具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，所述重链可变区包含与SEQ ID NO:36或SEQ ID NO:37具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；
    或者，
    所述轻链可变区包含与SEQ ID NO:41具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，所述重链可变区包含与SEQ ID NO:42或SEQ ID NO:43具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；
    更佳地，所述轻链可变区包含如SEQ ID NO:38所示的氨基酸序列，且所述重链可变区包含如SEQ ID NO:40或SEQ ID NO:39所示的氨基酸序列；
    所述轻链可变区包含如SEQ ID NO:30所示的氨基酸序列，且所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列；
    所述轻链可变区包含如SEQ ID NO:32所示的氨基酸序列，且所述重链可变区包含如SEQ ID NO:33或SEQ ID NO:34所示的氨基酸序列；
    所述轻链可变区包含如SEQ ID NO:35所示的氨基酸序列，且所述重链可变区包含如SEQ ID NO:36或SEQ ID NO:37所示的氨基酸序列；或，
    所述轻链可变区包含如SEQ ID NO:41所示的氨基酸序列，且所述重链可变区包含如SEQ ID NO:42或SEQ ID NO:43所示的氨基酸序列；
    进一步更佳地，所述抗体包含如SEQ ID NO:38所示的轻链可变区，且包含如SEQ ID NO:40所示的重链可变区；
    优选地，所述具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列的可变区维持与原序列相同的抗原结合功能。
  4. 如权利要求3所述的抗体，其特征在于，所述抗体满足以下三项中的一项或多项：
    (1)所述抗体为全长抗体、Fab、Fab'、F(ab')2或Fv，所述Fv优选scFv；
    (2)所述抗体为单特异性抗体或多特异性抗体；
    (3)所述抗体为由上述抗体制得的单克隆抗体或多克隆抗体。
  5. 如权利要求4所述的抗体，其特征在于，所述抗体包含重链恒定区和/或轻链恒定区；
    优选地，所述抗体的重链恒定区源自人源抗体IgG1、IgG2、IgG3或IgG4的重链恒定区，和/或所述抗体的轻链恒定区源自人源抗体的κ链；
    更优选地，所述重链恒定区包含如SEQ ID NO:45所示的氨基酸序列，且所述轻链恒定区包含如SEQ ID NO:44所示的氨基酸序列；
    最优选地，所述抗体的重链包含如SEQ ID NO:94所示的氨基酸序列，并所述抗体的轻链包含如SEQ ID NO:95所示的氨基酸序列；或，所述抗体的重链包含如SEQ ID NO:96所示的氨基酸序列，并所述抗体的轻链包含如SEQ ID NO:81所示的氨基酸序列；或，所述抗体的重链包含如SEQ ID NO:87所示的氨基酸序列，并所述抗体的轻链包含如SEQ  ID NO:81所示的氨基酸序列。
  6. 一种双特异性抗体，其包含特异性结合人CD40的第一抗原结合结构域和特异性结合人PD-L1的第二抗原结合结构域，其中，所述第一抗原结合结构域如权利要求1～5任一项所述抗CD40的抗体中所定义。
  7. 如权利要求6所述的双特异性抗体，其特征在于，所述第二抗原结合结构域至少包含一个VHH，所述VHH包括SEQ ID NO:66中的VHH-CDR1、VHH-CDR2和VHH-CDR3；
    较佳地，所述VHH-CDR1包含如X41YYX42X43C所示的氨基酸序列，其中为D或E，为S或T，为K或Q，所述VHH-CDR2包含如SEQ ID NO:76所示的氨基酸序列，并且所述VHH-CDR3包含如SEQ ID NO:77所示的氨基酸序列；
    更佳地，所述VHH包含SEQ ID NO:49、SEQ ID NO:74、SEQ ID NO:72或SEQ ID NO:73中的VHH-CDR1、VHH-CDR2和VHH-CDR3；
    进一步更佳地，所述VHH-CDR1包含如SEQ ID NO:46、SEQ ID NO:67或SEQ ID NO:70所示的氨基酸序列，所述VHH-CDR2包含如SEQ ID NO:47、SEQ ID NO:68或SEQ ID NO:71所示的氨基酸序列，以及所述VHH-CDR3包含如SEQ ID NO:48或SEQ ID NO:69所示的氨基酸序列。
  8. 如权利要求7所述的双特异性抗体，其特征在于，所述VHH-CDR1包含如SEQ ID NO:46所示的氨基酸序列，VHH-CDR2包含如SEQ ID NO:47所示的氨基酸序列，VHH-CDR3包含如SEQ ID NO:48所示的氨基酸序列；
    或，所述VHH-CDR1包含如SEQ ID NO:67所示的氨基酸序列，VHH-CDR2包含如SEQ ID NO:68所示的氨基酸序列，VHH-CDR3包含如SEQ ID NO:69所示的氨基酸序列；
    或，所述VHH-CDR1包含如SEQ ID NO:70所示的氨基酸序列，VHH-CDR2包含如SEQ ID NO:71所示的氨基酸序列，VHH-CDR3包含如SEQ ID NO:48所示的氨基酸序列；
    较佳地，所述VHH包括：
    序列如SEQ ID NO:46所示的VHH-CDR1、序列如SEQ ID NO:47所示的VHH-CDR2和序列如SEQ ID NO:48所示的VHH-CDR3，
    序列如SEQ ID NO:67所示的VHH-CDR1、序列如SEQ ID NO:68所示的VHH-CDR2和序列如SEQ ID NO:69所示的VHH-CDR3，
    或，序列如SEQ ID NO:70所示的VHH-CDR1、序列如SEQ ID NO:71所示的VHH-CDR2和序列如SEQ ID NO:48所示的VHH-CDR3；
    进一步更佳地，所述VHH的氨基酸序列如SEQ ID NO:49、SEQ ID NO:72、SEQ ID NO:73或SEQ ID NO:74所示；或与SEQ ID NO:49、SEQ ID NO:72、SEQ ID NO:73或SEQ ID NO:74具有至少90％序列同一性。
  9. 一种双特异性抗体，其包含特异性结合人CD40的第一抗原结合结构域和特异性结合人PD-L1的第二抗原结合结构域，其中：所述第二抗原结合结构域至少包含一个VHH，所述VHH的序列如权利要求7或8所述的双特异性抗体中所定义；较佳地，所述第一抗原结合结构域如权利要求1～5任一项所述抗CD40的抗体中所定义。
  10. 如权利要求6～9任一项所述的双特异性抗体，其特征在于，所述第一抗原结合结构域与所述第二抗原结合结构域直接或通过连接子可操作地连接；
    较佳地，所述第二抗原结合结构域连接于所述第一抗原结合结构域的轻链可变区或重链可变区的N端、或轻链恒定区的C端、或所述IgG的C端；所述连接子优选为肽序列，更优选包含或组成为(G4S)nG，其中n＝1-10且为整数，例如n＝3。
  11. 如权利要求6～10任一项所述的双特异性抗体，其含有两条第一多肽链和两条第二多肽链，其中：
    所述第一多肽链的氨基酸序列如SEQ ID NO:56所示或与SEQ ID NO:56具有至少90％序列同一性，和/或，所述第二多肽链的氨基酸序列如SEQ ID NO:62或SEQ ID NO:57所示，或与SEQ ID NO:62或SEQ ID NO:57具有至少90％序列同一性；
    所述第一多肽链的氨基酸序列如SEQ ID NO:50所示或与SEQ ID NO:50具有至少90％序列同一性，和/或，所述第二多肽链的氨基酸序列如SEQ ID NO:51所示或与SEQ ID NO:51具有至少90％序列同一性；
    所述第一多肽链的氨基酸序列如SEQ ID NO:52所示或与SEQ ID NO:52具有至少90％序列同一性，和/或，所述第二多肽链的氨基酸序列如SEQ ID NO:53或SEQ ID NO:60所示，或与SEQ ID NO:53或SEQ ID NO:60具有至少90％序列同一性；
    所述第一多肽链的氨基酸序列如SEQ ID NO:54所示或与SEQ ID NO:54具有至少90％序列同一性，和/或，所述第二多肽链的氨基酸序列如SEQ ID NO:55或SEQ ID NO:61所示，或与SEQ ID NO:55或SEQ ID NO:61具有至少90％序列同一性；或，
    所述第一多肽链的氨基酸序列如SEQ ID NO:58所示或与SEQ ID NO:58具有至少90％序列同一性，和/或，所述第二多肽链的氨基酸序列如SEQ ID NO:59或SEQ ID NO:63所示，或与SfEQ ID NO:59或SEQ ID NO:63具有至少90％序列同一性。
  12. 一种分离的核酸，其特征在于，其编码如权利要求1～5任一项所述的抗CD40的抗体或如权利要求6～11任一项所述的双特异性抗体。
  13. 一种重组表达载体，其包含如权利要求12所述的分离的核酸；
    较佳地，所述重组表达载体为质粒、粘粒、噬菌体或病毒载体；
    更佳地，所述质粒的骨架为pcDNA3.4。
  14. 一种转化体，其包含如权利要求13所述的重组表达载体；优选地，所述转化体的宿主细胞为原核细胞或真核细胞；更优选地，所述真核细胞为酵母细胞或哺乳动物细胞；其中，所述哺乳动物细胞例如为EXPI-293细胞或CHO细胞。
  15. 一种抗CD40的抗体或双特异性抗体的制备方法，其包含以下步骤：
    培养如权利要求14所述的转化体，从培养物中获得抗CD40的抗体或双特异性抗体。
  16. 一种药物组合物，其包含如权利要求1～5任一项所述的抗CD40的抗体或如权利要求6～11任一项所述的双特异性抗体，以及药学上可接受的载体；
    较佳地，所述药物组合物还含其它药剂；其它药剂优选自有由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
  17. 如权利要求1～5任一项所述的抗CD40的抗体、如权利要求6～11任一项所述的双特异性抗体，和/或如权利要求16所述的药物组合物在制备预防和/或治疗肿瘤的药物中的应用；优选地，所述肿瘤为为淋巴癌、乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠直肠癌、膀胱癌、横纹肌肉瘤、食道癌、宫颈癌、多发性骨髓瘤、白血病、胆囊癌、胶质母细胞瘤或黑色素瘤。
  18. 一种试剂盒，其包括如权利要求1～5任一项所述的抗CD40的抗体、如权利要求6～11任一项所述的双特异性抗体或如权利要求16所述的药物组合物；
    较佳地，所述试剂盒还包括(i)施用抗体或药物组合物的装置；和/或(ii)使用说明。
  19. 一种套装药盒，其包含药盒A和药盒B，其中：
    所述药盒A含有如权利要求1～5任一项所述的抗CD40的抗体、如权利要求6～11任一项所述的双特异性抗体和/或如权利要求16所述的药物组合物；
    所述药盒B含有其他抗肿瘤抗体或者包含其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
  20. 一种免疫检测，或者测定CD40和/或PD-L1的方法，其特征在于，其包括使用如权利要求1～5任一项所述的抗CD40的抗体、如权利要求6～11任一项所述的双特异性抗体和/或如权利要求16所述的药物组合物；优选地，所述检测为非诊断目的的检测。
  21. 一种预防和/或治疗肿瘤的方法，其特征在于，包括向有需要的患者施用治疗有效量的如权利要求1～5任一项所述的抗CD40的抗体、如权利要求6～11任一项所述的双 特异性抗体和/或如权利要求16所述的药物组合物，或者如权利要求19所述的套装药盒；优选地，所述肿瘤为PD-L1阳性和/或CD40阳性的肿瘤。
  22. 一种联合疗法，其特征在于，其包括分别向有需要的患者施用如权利要求1～5任一项所述的抗CD40的抗体、如权利要求6～11任一项所述的双特异性抗体和/或如权利要求16所述的药物组合物，和第二治疗剂；
    所述第二治疗剂较佳地包含其他抗肿瘤抗体或者包含其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
  23. 用作药物的如权利要求1～5任一项所述的抗CD40的抗体、如权利要求6～11任一项所述的双特异性抗体和/或如权利要求16所述的药物组合物；优选地，所述药物用于预防和/或治疗肿瘤；更优选地，所述肿瘤为淋巴癌、乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠直肠癌、膀胱癌、横纹肌肉瘤、食道癌、宫颈癌、多发性骨髓瘤、白血病、胆囊癌、胶质母细胞瘤或黑色素瘤。
PCT/CN2023/097156 2022-05-31 2023-05-30 抗cd40抗体和抗pd-l1×cd40双特异抗体及其应用 WO2023232036A1 (zh)

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