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WO2020207470A1 - 抗cd40抗体及其用途 - Google Patents

抗cd40抗体及其用途 Download PDF

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WO2020207470A1
WO2020207470A1 PCT/CN2020/084186 CN2020084186W WO2020207470A1 WO 2020207470 A1 WO2020207470 A1 WO 2020207470A1 CN 2020084186 W CN2020084186 W CN 2020084186W WO 2020207470 A1 WO2020207470 A1 WO 2020207470A1
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amino acid
antibody
acid sequence
cells
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PCT/CN2020/084186
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English (en)
French (fr)
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张宏恺
王媛
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南开大学
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Priority to US17/594,255 priority Critical patent/US20220324988A1/en
Priority to CN202080027681.4A priority patent/CN113728008B/zh
Priority to EP20786934.8A priority patent/EP3954705A4/en
Priority to CA3136491A priority patent/CA3136491A1/en
Priority to AU2020256828A priority patent/AU2020256828A1/en
Priority to SG11202111188VA priority patent/SG11202111188VA/en
Priority to JP2021560225A priority patent/JP7535060B2/ja
Publication of WO2020207470A1 publication Critical patent/WO2020207470A1/zh

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    • 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
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to novel antibodies and antibody fragments that specifically bind to CD40, and compositions containing the antibodies or antibody fragments, as well as medicines, combination products or kits.
  • the present invention relates to nucleic acids encoding the antibodies or antibody fragments thereof, host cells containing them, and related uses.
  • the present invention relates to the therapeutic and diagnostic uses of these antibodies and antibody fragments.
  • T cells The complete activation of T cells requires two signals: antigen-presenting cells (APC) uptake and process tumor antigens to form MHC-antigen complexes, which are presented to T cells and combined with TCR on the surface of T cells.
  • APC antigen-presenting cells
  • the first signal; the second or costimulatory signal is transmitted through the interaction between CD28 and B7-1(CD80)/B7-2(CD86), as well as the costimulatory factors CD40, OX40, GITR, etc. and their ligands (Smith-Garvin , JE, GAKoretzky, and MSJordan, T cell activation. Annu Rev Immunol, 2009.27: p.591-619).
  • T cells may undergo non-responsiveness (anergy) or programmed cell death (apoptosis) after antigen stimulation.
  • CD40 is a member of the TNF receptor (TNFR) superfamily. It is mainly expressed on B cells, dendritic cells (DC cells), monocytes and macrophages and other antigen-presenting cells (Grewal, ISand RAFlavell, CD40 and CD154 in cell-mediated immunity. Annu Rev Immunol, 1998.16: p.111-35). CD40 forms a trimer on the cell surface, and the corresponding ligand CD40L (ie CD154) is mainly expressed on the surface of activated T cells. The interaction of CD40 and CD40L is a costimulatory signal for T cell activation. Depending on the specific cell type, CD40 participates in the role leading to specific gene expression patterns.
  • TNFR TNF receptor
  • CD40L and CD40 on T cells can activate a variety of pathways, including NF- ⁇ B (nuclear factor ⁇ B), MAPK (mitogen-activated protein kinase) and STAT3 (signal transducer and activator of transcription 3), etc.
  • NF- ⁇ B nuclear factor ⁇ B
  • MAPK mitogen-activated protein kinase
  • STAT3 signal transducer and activator of transcription 3
  • CD40 is not only expressed by normal immune cells, but also by many malignant cells. Specifically, CD40 is overexpressed in the following diseases: B-line NHL, chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), Hodgkin’s disease, multiple myeloma and bladder cancer, kidney cancer, ovarian cancer , Cervical cancer, breast cancer, lung cancer, nasopharyngeal cancer and malignant melanoma (Hassan, SB, et al., Anti-CD40-mediated cancer immunotherapy: an update of recent and longoing clinical trials.ImmunopharmacolImmunotoxicol, 2014.36(2) : P.96-104).
  • CLL chronic lymphocytic leukemia
  • HCL hairy cell leukemia
  • Hodgkin’s disease multiple myeloma and bladder cancer
  • kidney cancer ovarian cancer
  • Cervical cancer breast cancer
  • lung cancer nasopharyngeal cancer
  • CD40 agonist antibodies can fight tumor cells through a variety of mechanisms: First, CD40 agonist antibodies mediate stronger anti-tumor effects by activating the immune system. Specifically, CD40 agonist antibodies can activate DC cells to increase their antigen presentation ability, which is manifested in the increased expression of costimulatory molecules, such as the B7 family (CD80, CD86), and the promotion of cytokine secretion, such as interleukin 12. This will lead to a significant T cell response (Fong, L. and EG Engleman, Dendritic cells in cancer immunotherapy.
  • costimulatory molecules such as the B7 family (CD80, CD86)
  • cytokine secretion such as interleukin 12. This will lead to a significant T cell response (Fong, L. and EG Engleman, Dendritic cells in cancer immunotherapy.
  • CD40 agonist antibodies can promote the proliferation and immunity of resting B cells Globulin class changes, antibody secretion, and have an impact on the development of germinal centers and the survival of memory B cells, all of which are essential for humoral immune response (Beatty, GL, Y. Li, and KBLong, Cancer immunotherapy: activating innate and adaptive immunity through CD40 agonists. Expert Rev Anticancer Ther, 2017.17(2): p.175-186).
  • CD40 agonist antibody binds to CD40 expressed on the surface of tumor cells, it mediates antibody-dependent cytotoxicity (ADCC), and killer cells directly eliminate tumor cells with high CD40 expression (Vonderheide, RHand MJ Glennie, Agonistic CD40). antibodies and cancer therapy. Clin Cancer Res, 2013.19(5): p.1035-43).
  • CD40 agonist antibody binds to CD40 expressed on the surface of tumor cells, it directly inhibits tumor growth and promotes its apoptosis.
  • the CD40/CD40L signaling pathway can block the cell cycle of tumor cells and stop cells at G2-M
  • the interaction of CD40/CD40L can also promote the increase of Fas expression on the surface of tumor cells, and inhibit the growth of high-expressing CD40 tumor cells and promote their apoptosis through the Fas/FasL signaling pathway (Eliopoulos, AG, et al., CD40 induces apoptosis in carcinoma) Cells through activation of cytotoxic ligands of the tumor necrosis factor superfamily. Mol Cell Biol, 2000.20(15): p.5503-15).
  • CD40 agonist antibodies can be divided into two types according to their different agonistic methods.
  • the first type of CD40 agonist activity does not depend on cross-linking of Fc receptors (such as CP-870893 and CDX-1140), although the former shows encouraging anti-cancer activity Efficacy, but there is a dose-limiting toxicity, which can lead to systemic immune disorders, venous thromboembolism and cytokine release syndrome (Cytokine Release Syndrome, CRS); the other type requires Fc receptor cross-linking to have CD40 Agonistic activity.
  • Fc receptors such as CP-870893 and CDX-1140
  • CRS Cytokine Release Syndrome
  • CD40 antibodies in the field, such as Pfizer’s CP870893, there is still a need for some new CD40 antibodies with properties comparable to or better than existing antibodies, especially CD40 that relies on Fc receptor cross-linking.
  • Antibodies especially antibodies with better anti-cancer properties and higher safety.
  • the present invention therefore provides a new antibody that binds to CD40 (especially human CD40 or rhesus CD40), and antigen-binding fragments thereof.
  • the anti-CD40 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH), wherein the VH comprises
  • the anti-CD40 antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region (VL), wherein the VL comprises:
  • LCDR3 contains at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).
  • the anti-CD40 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH and a light chain variable region VL, wherein
  • the VH includes
  • HCDR3 contains at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions);
  • the VL includes:
  • LCDR3 contains at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).
  • the anti-CD40 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein
  • the VH includes
  • HCDR3 contains at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions);
  • the VL includes:
  • LCDR3 contains at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).
  • the anti-CD40 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH and a light chain variable region VL, wherein
  • the VH includes
  • HCDR3 contains at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions);
  • the VL includes:
  • LCDR3 contains at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).
  • the VH comprises an amino acid sequence selected from SEQ ID NO: 13, 58, 60, 62, or 14, or consists of the amino acid sequence.
  • VL comprises an amino acid sequence selected from SEQ ID NO: 15, 64, 66 or 16, or consists of said amino acid sequence.
  • the anti-CD40 antibody or antigen-binding fragment thereof of the present invention comprises
  • the three complementary regions determine the LCDR region.
  • the anti-CD40 antibody or antigen-binding fragment thereof of the present invention comprises
  • the anti-CD40 antibody or antigen-binding fragment thereof of the present invention comprises
  • the anti-CD40 antibody or antigen-binding fragment thereof of the present invention comprises:
  • the anti-CD40 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein
  • the VH includes complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence of SEQ ID NO: 1 or 2, or consists of the amino acid sequence, or HCDR1 includes the amino acid sequence of SEQ ID NO: 1.
  • CDRs complementarity determining regions
  • amino acid sequence of 2 has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) amino acid sequence
  • HCDR2 includes the amino acid sequence of SEQ ID NO: 3 or 4, or consists of the amino acid sequence
  • HCDR2 includes an amino acid sequence with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO: 3 or 4
  • HCDR3 includes SEQ ID NOs: 5, 51, The amino acid sequence of 52, 55 or 6 or consists of the amino acid sequence, or HCDR3 contains one, two or three changes (preferably amino acid sequence) compared with the amino acid sequence of SEQ ID NO: 5, 51, 52, 55 or 6. Substitution, preferably conservative substitution) amino acid sequence;
  • VL includes complementarity determining regions (CDR) LCDR1, LCDR2 and LCDR3, where LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 7 or 8, or LCDR1 includes the same as SEQ ID NO: 7.
  • CDR complementarity determining regions
  • the amino acid sequence of 8 has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) amino acid sequence;
  • LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9 or 10, or LCDR2 includes an amino acid sequence with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared with the amino acid sequence of SEQ ID NO: 9 or 10;
  • LCDR3 includes an amino acid sequence selected from SEQ ID NOs: 11, 53, 54
  • the amino acid sequence of, 56 or 12 or consists of the amino acid sequence, or LCDR3 contains one, two or three changes compared with the amino acid sequence of SEQ ID NO: 11, 53, 54, 56 or 12 (preferably amino acid substitution , Preferably conservative substitution) amino acid sequence.
  • the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein
  • the VH includes
  • HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5; or
  • HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 51; or
  • HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 52; or
  • HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; or
  • the VL includes
  • LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequence: SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11; or
  • LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequence: SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 53; or
  • LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 54; or
  • LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12; or
  • the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein
  • the VH includes
  • HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5; or
  • HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 51; or
  • HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 52; or
  • the VL includes
  • LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequence: SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11; or
  • LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequence: SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 53; or
  • LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 54; or
  • the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein
  • the VH includes
  • HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; or
  • the VL includes
  • LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12; or
  • the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof, which comprises
  • HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO:1, SEQ ID NO: 3 and SEQ ID NO: 5, and LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11; or
  • HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO:1, SEQ ID NO: 3 and SEQ ID NO: 51, and LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 53; or
  • HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO:1, SEQ ID NO: 3 and SEQ ID NO: 52, and/or LCDR1, LCDR2 and LCDR2 and LCDR3: SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 54; or
  • HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, and LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12;
  • the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein
  • the VH includes
  • HCDR1, HCDR2 and HCDR3 comprising or consisting of the following sequences: SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 55; or
  • the VL includes
  • LCDR1, LCDR2 and LCDR3 comprising or consisting of the following sequences: SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 56; or
  • amino acid sequence shown in SEQ ID NO: 55 is as follows:
  • ARERVGA-X1-PTYYY-X2-X3-DV wherein X1, X2 or X3 can be any amino acid, preferably, wherein X1 can be T, N, W, K, A or Y, more preferably T or N; wherein X2 Preferably it is W or Y; wherein X3 can be W, Y, M, F, T, more preferably M, W or Y.
  • amino acid sequence shown in SEQ ID NO: 56 is as follows:
  • X1, X2, X3 and X4 can be any amino acids, preferably, where X1 can be Q, N or P, more preferably Q or N; X2 can Is G, Q, F, S, Y or M; more preferably G or Q; X3 can be E, N, T, S or K; more preferably E or N; X4 can be T, Q, V , L, P or E; more preferably T, Q or V.
  • the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3 and said VL comprises (CDR) LCDR1, LCDR2 and LCDR3, wherein the combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contained in said antibody or antigen-binding fragment thereof is as follows (Table A) Shown:
  • Table A Exemplary combinations of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 in the antibody of the present invention or its antigen-binding fragment
  • the anti-CD40 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein,
  • (iii) Comprising one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, and an amino acid sequence selected from SEQ ID NO: 13, 58, 60, 62 or 14). 1) the amino acid sequence of the amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution), preferably, the amino acid change does not occur in the CDR region, preferably, the amino acid change occurs in the FR region;
  • amino acid sequence comprising SEQ ID NO: 15, 64, 66 or 16 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or consisting of it;
  • (iii) Comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) compared with an amino acid sequence selected from SEQ ID NO: 15, 64, 66 or 16.
  • the amino acid change does not occur in the CDR region, and preferably, the amino acid change occurs in the FR region.
  • the heavy chain variable region VH of the anti-CD40 antibody or antigen-binding fragment thereof of the present invention is provided.
  • amino acid sequence comprising SEQ ID NO: 13, 58, 60, or 62 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or consisting of it; or
  • (iii) Comprising one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) compared with an amino acid sequence selected from SEQ ID NO: 13, 58, 60 or 62
  • the amino acid change does not occur in the CDR region, and preferably, the amino acid change occurs in the FR region.
  • the light chain variable region VL of the anti-CD40 antibody or antigen-binding fragment thereof of the present invention is provided.
  • amino acid sequence comprising SEQ ID NO: 15, 64 or 66 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
  • the heavy chain variable region VH of the anti-CD40 antibody or antigen-binding fragment thereof of the present invention is provided.
  • amino acid changes preferably Amino acid substitutions, more preferably conservative substitutions of amino acids
  • the amino acid changes do not occur in the CDR region, preferably, the amino acid changes occur in the FR region.
  • the light chain variable region VL of the anti-CD40 antibody or antigen-binding fragment thereof of the present invention is provided.
  • amino acid changes (preferably not more than 5, 4, 3, 2, 1) compared with the amino acid sequence selected from SEQ ID NO: 16 Amino acid substitutions, more preferably conservative substitutions of amino acids), preferably, the amino acid changes do not occur in the CDR region, preferably, the amino acid changes occur in the FR region.
  • the anti-CD40 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein,
  • the heavy chain variable region VH includes or consists of the amino acid sequence of SEQ ID NO: 13, and the light chain variable region VL includes or consists of the amino acid sequence of SEQ ID NO: 15;
  • the heavy chain variable region VH comprises or consists of the amino acid sequence of SEQ ID NO: 58
  • the light chain variable region VL comprises or consists of the amino acid sequence of SEQ ID NO: 64;
  • the heavy chain variable region VH comprises or consists of the amino acid sequence of SEQ ID NO: 60
  • the light chain variable region VL comprises or consists of the amino acid sequence of SEQ ID NO: 66;
  • the heavy chain variable region VH includes or consists of the amino acid sequence of SEQ ID NO: 62, and the light chain variable region VL includes or consists of the amino acid sequence of SEQ ID NO: 66;
  • the heavy chain variable region VH includes or consists of the amino acid sequence of SEQ ID NO: 14, and the light chain variable region VL includes or consists of the amino acid sequence of SEQ ID NO: 16.
  • the amino acid sequence of the heavy chain variable region of the antibody of the present invention has at least 90%, 91%, 92% of the amino acid sequence of the heavy chain variable region in (i)-(v) described above. %, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or its amino acid sequence is compared with the above-mentioned (i)-(v) heavy chain variable region Amino acid sequence, comprising one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) amino acid sequence, preferably Preferably, the amino acid change does not occur in the CDR region, and preferably, the amino acid change occurs in the FR region.
  • the amino acid sequence of the light chain variable region of the antibody of the present invention and the amino acid sequence of the light chain variable region in (i)-(v) described above have at least 90%, 91%, 92%. %, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or its amino acid sequence is compared with the light chain variable region in (i)-(v) described above Amino acid sequence, comprising one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) amino acid sequence, preferably Preferably, the amino acid change does not occur in the CDR region, and preferably, the amino acid change occurs in the FR region.
  • the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the antibody or antigen-binding fragment thereof comprises
  • VH heavy chain variable region
  • VL light chain variable region
  • Table B Exemplary combinations of heavy chain variable region VH and light chain variable region VL in the antibody or antigen-binding fragment of the present invention
  • the anti-CD40 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and/or light chain, wherein
  • amino acid sequence comprising SEQ ID NO: 17, 18, 19, 67, 69, 70, or 20 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or consisting of;
  • amino acid sequence of amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR region of the heavy chain, more preferably, The amino acid change does not occur in the variable region of the heavy chain, and most preferably, the amino acid change occurs in the constant region of the heavy chain;
  • amino acid sequence comprising SEQ ID NO: 21, 68, 71, or 22 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or consisting of it;
  • (iii) Comprising one or more (preferably not more than 20 or 10, more preferably not more than 5, 4, 3, 2) compared with an amino acid sequence selected from SEQ ID NO: 21, 68, 71 or 22 , 1) amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) amino acid sequence, preferably, the amino acid change does not occur in the CDR region of the light chain, more preferably, the amino acid change does not occur in In the light chain variable region, most preferably, the heavy chain amino acid changes occur in the light chain constant region.
  • amino acid change preferably amino acid substitution, more preferably amino acid conservative substitution
  • the heavy chain contained in the anti-CD40 antibody or antigen-binding fragment thereof of the present invention is the heavy chain contained in the anti-CD40 antibody or antigen-binding fragment thereof of the present invention.
  • amino acid sequence comprising and being selected from SEQ ID NO: 17, 18, 19, 67, 69 or 70 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, or 99% identical amino acid sequence or consisting of it;
  • (iii) Comprising one or more (preferably not more than 20 or 10, more preferably not more than 5, 4) compared with an amino acid sequence selected from SEQ ID NO: 17, 18, 19, 67, 69 or 70 , 3, 2, 1) amino acid sequence of amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR region of the heavy chain, more preferably, the amino acid The change does not occur in the variable region of the heavy chain, and most preferably, the amino acid change of the heavy chain occurs in the constant region of the heavy chain.
  • amino acid sequence of amino acid changes preferably amino acid substitutions, more preferably amino acid conservative substitutions
  • the light chain contained in the anti-CD40 antibody or antigen-binding fragment thereof of the present invention is the light chain contained in the anti-CD40 antibody or antigen-binding fragment thereof of the present invention.
  • amino acid sequence comprising and selected from SEQ ID NO: 21, 68 or 71 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or 99% identical amino acid sequence or consisting of it;
  • (iii) Comprising one or more (preferably not more than 20 or 10, more preferably not more than 5, 4, 3, 2, 1) compared with an amino acid sequence selected from SEQ ID NO: 21, 68 or 71 (Preferably amino acid substitution, more preferably amino acid conservative substitution) amino acid sequence, preferably, the amino acid change does not occur in the CDR region of the light chain, more preferably, the amino acid change does not occur in the light chain In the variable region, most preferably, the heavy chain amino acid changes occur in the light chain constant region.
  • the heavy chain contained in the anti-CD40 antibody or antigen-binding fragment thereof of the present invention is the heavy chain contained in the anti-CD40 antibody or antigen-binding fragment thereof of the present invention.
  • (iii) Comprising one or more (preferably not more than 20 or 10, more preferably not more than 5, 4, 3, 2, 1) amino acids compared to the amino acid sequence selected from SEQ ID NO: 20 Change the amino acid sequence (preferably amino acid substitution, more preferably amino acid conservative substitution), preferably, the amino acid change does not occur in the CDR region of the heavy chain, more preferably, the amino acid change does not occur in the heavy chain variable region Most preferably, the heavy chain amino acid changes occur in the constant region of the heavy chain.
  • the light chain contained in the anti-CD40 antibody or antigen-binding fragment thereof of the present invention is the light chain contained in the anti-CD40 antibody or antigen-binding fragment thereof of the present invention.
  • (iii) Comprising one or more (preferably not more than 20 or 10, more preferably not more than 5, 4, 3, 2, 1) amino acids compared to the amino acid sequence selected from SEQ ID NO: 22 Change the amino acid sequence (preferably amino acid substitution, more preferably amino acid conservative substitution), preferably, the amino acid change does not occur in the CDR region of the light chain, more preferably, the amino acid change does not occur in the light chain variable region Most preferably, the amino acid change of the heavy chain occurs in the constant region of the light chain.
  • the anti-CD40 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and/or light chain, wherein,
  • the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 17, 18, 19, 67, 69 or 70
  • the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 21, 68 or 71
  • the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 17, 18 or 19, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 21;
  • the heavy chain includes or consists of the amino acid sequence of SEQ ID NO: 67, and the light chain includes or consists of the amino acid sequence of SEQ ID NO: 68;
  • the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 69 or 70, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 71;
  • the heavy chain includes or consists of the amino acid sequence of SEQ ID NO: 20, and the light chain includes or consists of the amino acid sequence of SEQ ID NO: 22.
  • the amino acid sequence of the heavy chain of the antibody of the present invention is at least 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence of the heavy chain in (i)-(v) above. , 95%, 96%, 97%, 98%, or 99% identity, or its amino acid sequence is compared with the heavy chain amino acid sequence in (i)-(v) above, and contains 1 or more (preferably not more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) amino acid sequence, preferably, the amino acid changes do not occur in the heavy In the CDR region of the chain, more preferably, the amino acid change does not occur in the variable region of the heavy chain, and most preferably, the amino acid change occurs in the constant region of the heavy chain.
  • the amino acid sequence of the light chain of the antibody of the present invention is at least 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence of the heavy chain in (i)-(v) above. , 95%, 96%, 97%, 98%, or 99% identity, or its amino acid sequence is compared with the light chain amino acid sequence in (i)-(v) above, and contains one or more (preferably not more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) amino acid sequence, preferably, the amino acid changes do not occur in light In the CDR region of the chain, more preferably, the amino acid change does not occur in the variable region of the light chain, and most preferably, the amino acid change occurs in the constant region of the light chain.
  • the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof, which comprises a heavy chain and a light chain, wherein the combination of the heavy chain and light chain contained in the antibody or the antigen-binding fragment thereof is as follows (Table C) shows:
  • Table C Exemplary combinations of heavy and light chains in the antibodies of the invention or antigen-binding fragments thereof
  • the heavy chain and/or light chain of the anti-CD40 antibody or fragment thereof of the present invention further comprises a signal peptide sequence, for example, the signal peptide sequence comprises or consists of the amino acid sequence shown in SEQ ID NO: 43.
  • the amino acid changes described herein include amino acid substitutions, insertions or deletions.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the changes occur in the CDR regions (especially CDR3 regions) of the heavy and/or light chains of the antibody. In some embodiments, there may be 1, 2, or 3 changes in the CDR regions, such as the CDR3 region.
  • the amino acid changes described in the present invention occur in regions outside the CDR (for example, in FR).
  • the change occurs in the FR region of the antibody, such as the FR region of the antibody heavy chain and/or light chain variable region, such as FR1, FR2, FR2, or F4.
  • the change occurs in the FR2 region.
  • amino acid changes described in the present invention occur in regions outside the variable region of the heavy chain and/or outside the variable region of the light chain, for example, in the constant region of the heavy chain and/or light chain.
  • substitutions are conservative substitutions.
  • Conservative substitution refers to the replacement of an amino acid by another amino acid in the same category, for example, an acidic amino acid is replaced by another acidic amino acid, a basic amino acid is replaced by another basic amino acid, or a neutral amino acid is replaced by another neutral amino acid. Replacement. Exemplary substitutions are shown in Table D below:
  • the substitution occurs in the CDR region of the antibody.
  • the obtained variant has a modification (e.g., improvement) in certain biological properties (e.g., increased affinity) relative to the parent antibody and/or will have certain biological properties substantially retained of the parent antibody.
  • An exemplary substitution variant is an affinity matured antibody.
  • the substitution occurs in the CDR3 region of the heavy chain and/or light chain of the antibody. In some embodiments, there may be 1, 2, or 3 substitutions in the CDR3 region.
  • the substitution occurs in the FR region of the antibody, such as the FR region of the antibody heavy chain and/or light chain variable region, such as FR1, FR2, FR2, or F4. In some embodiments, the substitution occurs in the FR2 region. In some embodiments, there may be 1, 2, or 3 substitutions in the FR region.
  • the invention provides antibodies comprising the variable region sequence disclosed herein or the variable region sequence having the CDR disclosed herein, and the constant domain of a modified Fc region, the modified Fc Compared to its affinity for other Fc receptors (ie, activated receptors), the region has an enhanced affinity for Fc ⁇ RIIb.
  • modified Fc region Compared to its affinity for other Fc receptors (ie, activated receptors), the region has an enhanced affinity for Fc ⁇ RIIb.
  • Such agonistic anti-CD40 antibodies with enhanced Fc ⁇ RIIb specificity are expected to exhibit excellent efficacy in cancer treatment and chronic infections (Li and Ravetch (2011) Science 333: 1030; White et al. (2011) J. Immunol. 187 : 1754).
  • Fc ⁇ RIIb-specific agonistic anti-CD40 antibodies can exhibit enhanced auxiliary effects by increasing the proliferation of cytotoxic CD8 + T cells and the maturation of activated dendritic cells, leading to enhanced anti-tumor responses .
  • the signal enhancement of the FcR-mediated agonist CD40 antibody due to the cross-linking of the present invention can be a major contributor to therapeutic efficacy.
  • Cross-linking of FcR-conjugated CD40 agonist antibodies through the Fc portion of the antibody can increase signal strength and thereby enhance cell activation.
  • Exemplary mutations in the Fc sequence include, for example, E233D, G237D, H268D, P271G, A330R, S267E, and/or L328F.
  • the antibody of the present invention comprises a human IgG1 constant domain with mutations that enhance the specificity of FcyRIIb, the mutations including E233D, G237D, H268D, P271G and A330R, or S267E and L328F. See, for example, SEQ ID NO: 18 or 19 for the sequence of an exemplary antibody heavy chain comprising a mutated human IgG1 constant domain that enhances Fc ⁇ RIIb specificity.
  • the antibodies provided herein can be further modified to contain other non-protein moieties known and readily available in the art.
  • the part suitable for antibody derivatization includes, but is not limited to, water-soluble polymers.
  • water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene Pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (such as glycerin), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated.
  • the addition or deletion of glycosylation sites of the antibody can be conveniently achieved by changing the amino acid sequence to create or remove one or more glycosylation sites.
  • the carbohydrate attached to it can be changed.
  • modifications to remove unwanted glycosylation sites may be useful, such as removing fucose moieties to improve antibody-dependent cellular cytotoxicity (ADCC) functions (see Shield et al. (2002) JBC277:26733 ).
  • ADCC antibody-dependent cellular cytotoxicity
  • galactosidation can be modified to modify complement dependent cytotoxicity (CDC).
  • cysteine engineered antibody such as a "thioMAb"
  • Cysteine engineered antibodies can be generated as described, for example, in U.S. Patent No. 7,521,541.
  • the anti-CD40 antibody or antigen-binding fragment thereof of the present invention shows the same or similar binding affinity and/or specificity as the antibody of the present invention for CD40; and/or inhibits (eg, competitively inhibits) the antibody of the present invention It binds to CD40 and/or binds to the same or overlapping epitope with the antibody of the present invention; and/or competes with the antibody of the present invention for binding to CD40; and/or has one or more biological characteristics of the antibody of the present invention.
  • the anti-CD40 antibody of the present invention is an antibody in the form of IgG1 or an antibody in the form of IgG2 or an antibody in the form of IgG3 or an antibody in the form of IgG4.
  • the anti-CD40 antibody is a monoclonal antibody.
  • the anti-CD40 antibody is humanized.
  • Different methods for humanizing antibodies are known to the skilled person, as reviewed by Almagro & Fransson, the contents of which are fully incorporated herein by reference (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13: 1619-1633).
  • the anti-CD40 antibody is a human antibody.
  • Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol 20: 450-459 (2008).
  • the anti-CD40 antibody is a chimeric antibody.
  • the anti-CD40 antibody of the present invention also encompasses its antibody fragments, preferably antibody fragments selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibodies (such as scFv) or (Fab' ) 2. Single domain antibody, diabody (dAb) or linear antibody.
  • the anti-CD40 antibody molecule is in the form of a bispecific or multispecific antibody molecule.
  • the bispecific antibody molecule has a first binding specificity for CD40 and a second binding specificity for PD-1 or PD-L1 or PD-L2 or OX40 or 4-1BB or GITR or the like.
  • the bispecific antibody molecule binds to CD40 and TNF or IL-17.
  • Multispecific antibody molecules can have any combination of binding specificities for the aforementioned molecules.
  • the antibodies of the invention have one or more of the following characteristics:
  • CD40-expressing cells in a cross-linked or constitutive form, preferably in a cross-linked form, such as activating the NFkappa-B signaling pathway; in some embodiments, flow cytometry is used for detection; in some embodiments
  • the reporter cells used are cells expressing NF- ⁇ B-GFP and hCD40;
  • its equilibrium dissociation constant is less than or equal to about 5 ⁇ 10 -7 M, 4.5 ⁇ 10 -7 M, 4.4 ⁇ 10 -7 M, 4.3 ⁇ 10 -7 M, 4.2 ⁇ 10 -7 M, 4.1 ⁇ 10 -7 M, 4 ⁇ 10 -7 M, 3.9 ⁇ 10 -7 M, 3.8 ⁇ 10 -7 M, 3.7 ⁇ 10 -7 M, 3.6 ⁇ 10 -7 M, 3.5 ⁇ 10 -7 M, or 3.4 ⁇ 10 -7 M; in some embodiments, the measurement is a surface plasmon resonance technique.
  • binding to CD40 on cells expressing CD40 is less than or equal to about 50nM, 40nM, 30nM, 20nM, 15nM, 14nM, 13nM, 12nM, 10nM, 9nM, 8nM.
  • flow cytometry is used in combination; in some embodiments, the CD40-expressing cells are 293 cells, such as 293FT cells.
  • the EC50 is less than or equal to about 4nM, 3.5nM, 3nM, 2.9nM, 2.8nM, 2.7nM, or 2.6nM; in some embodiments, it is measured using flow cytometry.
  • the tumor cell is a Raji cell or Ramos cell.
  • Having agonist activity such as significantly activating (e.g., human) B cells or T cells or dendritic cells.
  • the CD40 antibody has one or more of the aforementioned properties when cross-linked.
  • the present invention also encompasses antibodies conjugated to other substances ("immunoconjugates").
  • Cytotoxic agents include any agents that are harmful to cells. Examples of cytotoxic agents (such as chemotherapeutics) or other substances suitable for forming immunoconjugates are known in the art, see, for example, WO2017/004006 or WO2017/059243.
  • the tag is for example a tag sequence, such as a peptide.
  • the tag amino acid sequence is a hexahistidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), etc. Many of them are commercially available of. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for example, hexahistidine provides convenient purification of the fusion protein.
  • peptide tags used for purification include, but are not limited to, hemagglutinin ("HA") tags, which correspond to epitopes derived from influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767) and "flag" label.
  • HA hemagglutinin
  • the indicated label may be a diagnostic agent or a detectable agent.
  • the obtained antibody conjugate can be used as part of a clinical test method (such as determining the efficacy of a specific therapy) for monitoring or predicting the onset, formation, progression, and/or severity of a disease or disorder.
  • Detectable or diagnostic agents include, but are not limited to, various enzymes, such as horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase or acetylcholinesterase; prosthetic groups, such as streptavidin/biotin And avidin/biotin; fluorescent substances, such as but not limited to umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinamide fluorescein, dansyl chloride or phycoerythrin; luminescence Substances, such as but not limited to luminol; bioluminescent substances, such as but not limited to luciferase, luciferin, and aequorin; radioactive substances, such as but not limited to iodine ( 131 I, 125 I, 123 I and 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115 In, 113 In
  • the therapeutic agent includes chemotherapeutics, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators.
  • the antibody molecule of the present invention can be conjugated to a therapeutic moiety (therapeutic agent) such as radioactive metal ions, such as ⁇ -emitters such as 213 Bi or can be used to make radioactive metal ions (including but not limited to 131 In, 131 LU, 131 Y, 131 Ho, 131 Sm) macrocyclic chelating agents conjugated to polypeptides.
  • a therapeutic moiety such as radioactive metal ions, such as ⁇ -emitters such as 213 Bi or can be used to make radioactive metal ions (including but not limited to 131 In, 131 LU, 131 Y, 131 Ho, 131 Sm) macrocyclic chelating agents conjugated to polypeptides.
  • the macrocyclic chelating agent is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), which can be passed through a linker The molecule attaches to the antibody.
  • the invention provides a nucleic acid encoding any antibody or fragment or any chain thereof described herein.
  • a vector comprising the nucleic acid is provided.
  • the vector is an expression vector, such as a pFuse vector.
  • a host cell comprising the nucleic acid or the vector is provided.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (such as CHO cells or 293 cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof.
  • the host cell is prokaryotic.
  • the nucleic acid of the present invention includes:
  • the nucleic acid selected from SEQ ID NO: 39-42, or the nucleic acid selected from SEQ ID NO: 39-42 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95% %, 96%, 97%, 98%, or 99% identical nucleic acids.
  • the present invention also covers nucleic acids that hybridize with the following nucleic acids under stringent conditions or have one or more substitutions (such as conservative substitutions), deletions or insertions with the following nucleic acids: comprising a code selected from SEQ ID NO: 1- A nucleic acid comprising a nucleic acid sequence of the amino acid sequence shown in any one of 9; or a nucleic acid comprising a coding and an amino acid sequence selected from any one of SEQ ID NO: 13-22 with at least 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of the amino acid sequence; or the nucleic acid comprising the nucleic acid sequence selected from the group consisting of SEQ ID NO: 39-42 Nucleic acid, or comprising a nucleic acid sequence selected from SEQ ID NO: 39-42 with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9
  • one or more vectors comprising the nucleic acid are provided.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YAC).
  • YAC yeast artificial chromosomes
  • pFuse vector Once the expression vector or DNA sequence for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this goal, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques.
  • the cells are grown in culture and screened for appropriate activity.
  • the methods and conditions for culturing the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on the methods known in this specification and the prior art, depending on the specific expression vector and Changes or optimization of mammalian host cells.
  • the marker may, for example, provide prototrophy, biocidal resistance (e.g., antibiotics), or heavy metal (e.g. copper) resistance, etc., to the auxotrophic host.
  • 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 elements may also be required for optimal mRNA synthesis. These elements can include splicing signals, as well as transcription promoters, enhancers, and termination signals.
  • a host cell comprising one or more polynucleotides of the invention.
  • a host cell comprising the expression vector of the invention is provided.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof.
  • the present invention provides a method for preparing the antibody molecule of the present invention or a fragment thereof (preferred antigen-binding fragment), wherein the method includes a method suitable for expressing the antibody molecule of the present invention or a fragment thereof (preferred antigen-binding fragment).
  • the host cell is cultured under the condition of nucleic acid, and the antibody or fragment thereof (preferably antigen-binding fragment) is optionally isolated.
  • the method further comprises recovering the antibody molecule of the invention or a fragment thereof (preferably an antigen-binding fragment) from the host cell.
  • a method for preparing an antibody molecule of the present invention comprises, under conditions suitable for expression of the antibody, culturing the antibody encoding the antibody (for example, any one polypeptide chain and/or multiple polypeptide chains)
  • the nucleic acid or the host cell containing the expression vector of the nucleic acid, as provided above, and the antibody is optionally recovered from the host cell (or host cell culture medium).
  • the nucleic acid encoding the antibody (such as the antibody described above, such as any one polypeptide chain and/or multiple polypeptide chains) is isolated, and inserted into one or more vectors for use in the host Further cloning and/or expression in the cell.
  • Such nucleic acids are easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding to genes encoding antibody heavy and light chains).
  • the antibody molecules prepared as described herein can be purified by known existing techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography and the like.
  • the actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these will be obvious to those skilled in the art.
  • the purity of the antibody molecule of the present invention can be determined by any one of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
  • the present invention also provides methods for identifying, screening or characterizing the physical/chemical properties and/or biological activities of the antibody molecules of the present invention.
  • the antibody of the present invention is tested for its antigen binding activity, for example, by a known method such as ELISA, Western blot, and the like.
  • the binding to CD40 can be determined using methods known in the art, and exemplary methods are disclosed herein.
  • surface plasmon resonance assays e.g., affinity measurement
  • ELISA assays are used.
  • the invention also provides an assay method for identifying anti-CD40 antibodies with biological activity.
  • Biological activities may include, for example, binding to CD40 (such as binding to human and/or rhesus CD40), improving CD40-mediated signal transduction (such as improving NFkappa-B signaling pathway), and reducing expression of CD40 by directly inducing tumor cell apoptosis.
  • Cells such as Raji or Ramos cells
  • activating dendritic cells or B cells or T cells such as by increasing T cell cytokine production
  • promoting T cells such as CD8 + T cells, such as activated CD8 + T cells
  • B cells proliferate or inhibit tumor growth.
  • the antibodies of the invention are tested for such biological activity.
  • T cells eg CD8+ T cells
  • dendritic cells or B cells can be measured using methods known in the art. For example, by the level (eg, expression) of the cell activation marker CD8 (T cell) or CD86 (dendritic cell or B cell). Methods known in the art can also be used to measure CD40 signaling (for example, NF- ⁇ B signaling pathway) to measure T cell activation.
  • a transgenic cell expressing human CD40 and a reporter gene including the NF-kappa B promoter fused to a reporter gene (e.g., ⁇ -luciferase, GFP) is generated.
  • the addition of anti-CD40 antibodies to cells leads to an increase in NF-kappa B transcription, which is detected using assays for reporter genes (such as luciferase reporter assays).
  • the ADCC effect of antibodies can be determined using methods known in the art. For example, by detecting its induction of tumor cell apoptosis (for example, by apoptosis marker molecules, such as CD95), the inhibition of tumor cell growth, or the inhibition of tumor growth in vivo.
  • apoptosis marker molecules such as CD95
  • T cells eg, CD8+ T cells, such as activated CD8+ T cells
  • B cells eg, CD8+ T cells, such as activated CD8+ T cells
  • the proliferation of B cells can be measured by a luminescence method, such as the CellTiter-Glo luminescence method.
  • the proliferation of CD8+ T cells can be detected by the OVA-specific OT-I cell method.
  • Cells for use in any of the above in vitro assays include cell lines that naturally express CD40 or are engineered to express CD40. Such cells include T cells that naturally express CD40L (for example, CD8+ T cells, such as activated CD8+ T cells), B cells or dendritic cells that naturally express CD40. Such cells also include cell lines that express CD40 and are transfected with CD40-encoding DNA that does not normally express CD40.
  • T cells that naturally express CD40L for example, CD8+ T cells, such as activated CD8+ T cells
  • B cells or dendritic cells that naturally express CD40.
  • Such cells also include cell lines that express CD40 and are transfected with CD40-encoding DNA that does not normally express CD40.
  • immunoconjugates of the present invention can be used to replace or supplement the anti-CD40 antibody to perform any of the aforementioned assays.
  • any of the aforementioned assays can be performed using anti-CD40 antibodies and other active agents.
  • the invention provides pharmaceutical compositions and combination products comprising the antibodies of the invention.
  • the present invention provides a composition comprising any antibody molecule described herein or a fragment thereof (preferably an antigen-binding fragment thereof) or an immunoconjugate thereof, preferably the composition is a pharmaceutical composition.
  • the composition further comprises pharmaceutical excipients.
  • the present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising the antibodies of the present invention or immunoconjugates thereof and/or compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising polynucleotides encoding the antibodies of the present invention .
  • the composition comprises one or more antibodies of the invention or fragments thereof or one or more polynucleotides encoding one or more antibodies or fragments thereof of the invention.
  • compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.
  • pharmaceutical carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, etc. that are physiologically compatible.
  • Pharmaceutical carriers suitable for the present invention can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. It is also possible to use saline solutions and aqueous dextrose and glycerol solutions as liquid carriers, especially for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dry skim milk, glycerin , Propylene, glycol, water, ethanol, etc.
  • excipients see also "Handbook of Pharmaceutical Excipients", fifth edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.
  • composition may also contain small amounts of wetting or emulsifying agents, or pH buffering agents.
  • composition of the invention can be in a variety of forms. These forms include, for example, liquid, semi-solid and solid dosage forms such as liquid solutions (for example, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories.
  • liquid solutions for example, injectable solutions and infusible solutions
  • dispersions or suspensions for example, liposomes, and suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic use. Commonly preferred compositions are in the form of injectable solutions or infusible solutions.
  • the preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection.
  • the antibody molecule is administered by intravenous infusion or injection.
  • the antibody molecule is administered by intramuscular, intraperitoneal or subcutaneous injection.
  • the agonistic antibody that specifically binds to human CD40 of the present invention can be lyophilized for storage, and can be restored in a suitable carrier before use. This technique has been proven to be effective for conventional protein preparations, and well-known freeze-drying and reconstitution techniques can be used.
  • the pharmaceutical composition or formulation of the present invention may also contain other therapeutic agents that are required for the specific indication being treated, preferably those that have complementary activities that do not adversely affect each other.
  • therapeutic agents preferably those that have complementary activities that do not adversely affect each other.
  • chemotherapeutics cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs or immunomodulators, etc.
  • the present invention also provides a combination product, which comprises the antibody of the present invention or an antigen-binding fragment thereof, or an immunoconjugate thereof, and one or more other therapeutic agents (such as chemotherapeutics, other antibodies, Cytotoxic agents, vaccines, anti-infective agents, small molecule drugs or immunomodulators, etc.).
  • a combination product which comprises the antibody of the present invention or an antigen-binding fragment thereof, or an immunoconjugate thereof, and one or more other therapeutic agents (such as chemotherapeutics, other antibodies, Cytotoxic agents, vaccines, anti-infective agents, small molecule drugs or immunomodulators, etc.).
  • two or more components of the combination product may be administered to the subject in combination sequentially, separately, or simultaneously.
  • the present invention also provides a kit comprising the antibody, pharmaceutical composition, immunoconjugate or combination product of the present invention, and optionally a package insert to guide administration.
  • the present invention also provides a pharmaceutical product comprising the antibody, pharmaceutical composition, immunoconjugate, and combination product of the present invention.
  • the pharmaceutical product further includes a package insert to guide administration.
  • the other therapeutic agent includes, for example, one or more of the following: anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-TIGIT antibody, anti-OX40 (also known as CD134, TNFRSF4, ACT35 and/or TXGP1L) antibody, anti-LAG-3 antibody, anti-CD73 antibody, anti-CD137 antibody, anti-CD27 antibody, anti-CSF-1R antibody, TLR agonist, or small molecule antagonist of IDO or TGF ⁇ . See also WO2017/059243 or WO2017/004006 for examples of therapeutic agents that can be combined with the antibody of the present invention.
  • the present invention also provides a method for enhancing an immune response (e.g., antigen-specific T cell response) in a subject, which comprises administering to the subject an effective amount of the anti-CD40 antibody or antigen thereof of the present invention By binding the fragments, the immune response in the individual is enhanced.
  • an immune response e.g., antigen-specific T cell response
  • the individual has a tumor.
  • the subject has a viral infection, such as a chronic viral infection.
  • the present invention provides a method for activating T cells (such as CD8+ T cells) and/or dendritic cells and/or B cells in a subject using the antibodies of the present invention, which comprises giving the subject An effective amount of the anti-CD40 antibody or antigen-binding fragment thereof of the present invention is administered.
  • the present invention also provides a method for promoting the proliferation of T cells (such as activated CD8+ T cells) or B cells using the method of the present invention, which comprises administering to a subject an effective amount of the anti-CD40 antibody or antigen-binding fragment thereof of the present invention .
  • T cell responses such as antigen-specific T cell responses
  • the T cells may be T eff cells, such as CD4+T eff cells, CD8+T eff cells, T helper (T h ) cells, and T toxic (T c ) cells.
  • the present invention provides the use of the antibody molecules of the present invention to treat or prevent diseases that require modulation (e.g., enhancement) of an immune response in a subject, such as tumors or infections, such as chronic infections. Therefore, the present invention also provides a method for inhibiting tumor growth in a subject, which comprises administering the anti-CD40 antibody or antigen-binding fragment thereof of the present invention to the subject so that tumor growth is inhibited.
  • diseases that require modulation e.g., enhancement
  • an immune response in a subject such as tumors or infections, such as chronic infections. Therefore, the present invention also provides a method for inhibiting tumor growth in a subject, which comprises administering the anti-CD40 antibody or antigen-binding fragment thereof of the present invention to the subject so that tumor growth is inhibited.
  • the disease is a CD40-related disease, for example, the disease is a disease in which CD40 expression or activity is reduced (e.g., compared to healthy controls), or the disease is a disease in which CD40 gene and/or protein levels are reduced (E.g. compared to healthy controls); or the disease benefits from activating CD40 activity, such as activating the CD40 signaling pathway, and/or activating T cells or B cells or dendritic cells.
  • the present invention relates to a method of activating antigen activity or activating an antigen-mediated signaling pathway in an individual, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof disclosed herein.
  • activating CD40 activity or activating CD40-mediated signaling pathway refers to activating the CD40 signaling pathway.
  • the antibody or antigen-binding fragment thereof of the present invention can cause antibody-dependent cell-mediated cytotoxicity (ADCC), thereby killing tumor cells. Therefore, the present invention also relates to a method of treating tumors, which comprises administering an effective amount of the antibody or antigen-binding fragment thereof of the present invention to an individual.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the present invention relates to a method of preventing or treating a tumor (e.g., cancer) in a subject, the method comprising administering to the subject an effective amount of the antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein.
  • a tumor e.g., cancer
  • the tumor is cancer.
  • the tumor may be a solid tumor or a liquid tumor, such as a malignant hematological tumor.
  • the tumor is an immunogenic tumor.
  • the tumor is non-immunogenic.
  • the tumor is PD-L1 positive.
  • the tumor is PD-L1 negative.
  • tumors treated and/or prevented with antibody molecules include, but are not limited to, solid tumors, hematological cancers (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), and metastatic lesions.
  • the cancer is a solid tumor.
  • solid tumors include malignant tumors, for example, sarcomas and cancers of multiple organ systems, such as those that invade the lung, breast, ovary, lymphoid, gastrointestinal (for example, colon), anus, genitals, and genitourinary tract (for example, Kidney, bladder epithelium, bladder cells, prostate), pharynx, CNS (for example, brain, neural or glial cells), head and neck, skin (for example, melanoma), nasopharyngeal (for example, differentiated or undifferentiated Metastatic or locally recurrent nasopharyngeal carcinoma) and those of the pancreas, as well as adenocarcinomas, including malignant tumors such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small bowel cancer and esophageal cancer.
  • the cancer can be early, middle or late or metastatic cancer.
  • the cancer is selected from colorectal cancer (e.g., CRC), melanoma, e.g., advanced melanoma (e.g., stage II-IV melanoma) or HLA-A2-positive-melanoma; pancreas Cancer, for example, advanced pancreatic cancer; breast cancer, for example, metastatic breast cancer or triple negative breast cancer; head and neck cancer (e.g., HNSCC); esophageal cancer; renal cell carcinoma (RCC), for example, renal clear cell carcinoma (ccRCC) Or metastatic renal cell carcinoma (MRCC); lung cancer (e.g., NSCLC); cervical cancer; bladder cancer; or hematological malignancies, e.g., leukemia (e.g., lymphocytic leukemia) or lymphoma (e.g., Hodgkin’s lymphoma) (HL), non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma
  • CRC
  • examples of cancer further include, but are not limited to, B-cell proliferative disorders, which further include, but are not limited to, lymphoma (eg, B-cell non-Hodgkin's lymphoma (NHL)) and lymphocytic leukemia.
  • lymphoma eg, B-cell non-Hodgkin's lymphoma (NHL)
  • NHL lymphocytic leukemia
  • the tumor is a tumor that requires activation of T cells or B cells or dendritic cells, such as cancer, such as a tumor or cancer with T cell dysfunction.
  • the tumor is a tumor in which the expression or activity of OX40 is reduced.
  • the tumor is a tumor that benefits from activation of the OX40 signaling pathway, such as cancer.
  • the cancer described herein is lymphoma, colon cancer, colorectal cancer, rectal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, and metastatic cancers thereof.
  • the antibodies or antigen-binding fragments thereof of the present invention are not suitable for the treatment of blood cancers with CD40 expression, which may be exacerbated by treatment with CD40 agonists.
  • Certain cancers may be known to express CD40 and therefore experience such exacerbations and therefore can be excluded in category.
  • specific tumor samples are tested for CD40 expression and are excluded from therapies with CD40 antibodies of the invention based on the test results.
  • compositions disclosed herein can be used to treat metastatic lesions associated with the aforementioned cancers.
  • the antibodies or antigen-binding fragments thereof that specifically bind to human CD40 of the present invention can also be administered prophylactically to reduce the risk of cancer, delay the onset of events in cancer progression, and/or reduce the risk of recurrence after cancer remission. This may be especially useful for patients whose tumors are difficult to locate and are known to have tumors due to other biological factors.
  • the present invention relates to a method of preventing or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of the antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein Or combination products or kits.
  • the infectious disease is a chronic infection.
  • the infection is a viral infection.
  • the infection is acute or chronic.
  • the chronic infection is a persistent infection, a latent infection, or a slow infection.
  • the chronic infection is caused by a pathogen selected from bacteria, viruses, fungi, and protozoa.
  • viruses include HIV, hepatitis (hepatitis A, hepatitis B, and hepatitis C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein-Barr virus), adenovirus, influenza Virus, flavivirus, Echo virus, rhinovirus, Coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue fever virus , Papilloma virus, molluscum virus, polio virus, rabies virus, JC virus and arbo encephalitis virus; some examples of bacteria include Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus , Meningococcus and Neisseria
  • herpes viruses e
  • the infectious diseases treated and/or prevented with antibody molecules include pathogens for which no effective vaccine currently exists or pathogens for which conventional vaccines are not fully effective. These include but are not limited to HIV, (A, B and C) hepatitis, influenza, herpes, Giardia, malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa (Pseudomonas aeruginosa).
  • the antibody or pharmaceutical composition or immunoconjugate or combination product or kit of the present invention can also be administered in combination with one or more other therapies, such as treatment modalities and/or other therapeutic agents, for use herein Said prevention and/or treatment.
  • therapies such as treatment modalities and/or other therapeutic agents, for use herein Said prevention and/or treatment.
  • the treatment modality includes surgery (e.g., tumor resection); radiation therapy (e.g., external particle beam therapy, which involves three-dimensional conformal radiotherapy in which the irradiation area is designed), localized irradiation (e.g., directed to a preselected target Or organ irradiation) or focused irradiation) and so on.
  • radiation therapy e.g., external particle beam therapy, which involves three-dimensional conformal radiotherapy in which the irradiation area is designed
  • localized irradiation e.g., directed to a preselected target Or organ irradiation
  • focused irradiation can be selected from stereotactic radiosurgery, segmented stereotactic radiosurgery, and intensity-modulated radiotherapy.
  • the focused irradiation may have a radiation source selected from particle beams (protons), cobalt-60 (photons), and linear accelerators (X-rays).
  • Radiation therapy can be administered by one of several methods or a combination of methods, including but not limited to external particle beam therapy, internal radiation therapy, implant irradiation, stereotactic radiosurgery, whole body radiation therapy, radiotherapy, and permanent or transient Interstitial brachytherapy.
  • the therapeutic agent is selected from chemotherapeutics, cytotoxic agents, vaccines, other antibodies, anti-infective agents, or immunomodulators (e.g., activators of costimulatory molecules or inhibitors of immune checkpoint molecules).
  • Exemplary other antibodies include, but are not limited to, immune checkpoint inhibitors (e.g., anti-CTLA-4, anti-TIM-3, anti-CEACAM); antibodies that stimulate immune cells (e.g., agonistic GITR antibody or CD137 antibody); anti-cancer Antibodies (e.g., rituximab Trastuzumab Tositumomab Ibritomab Alemtuzumab Iparizumab Bevacizumab Erlotinib Cetuximab and many more.
  • the other antibodies may be anti-PD-L1 antibodies, anti-LAG-3 antibodies, anti-PD-1 antibodies, or anti-CLA-4 antibodies.
  • Exemplary vaccines include but are not limited to cancer vaccines.
  • the vaccine can be a DNA-based vaccine, an RNA-based vaccine or a virus-transduction-based vaccine.
  • Cancer vaccines can be preventive or therapeutic, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immunostimulatory cytokines (He et al. (2004) J. Immunol. 173: 4919-28).
  • Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigen, Trp-2, MART1 and/or tyrosinase, or transfected to express the cytokine GM-CSF Tumor cells.
  • the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine.
  • the peptide cancer vaccine is a multivalent long peptide, a multipeptide, a peptide mixture, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, for example, Yamada et al., Cancer Sci, 104: 14-21 , 2013).
  • the administration of the antibody or fragment thereof of the invention is combined with the administration of a tumor antigen.
  • the antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen.
  • the tumor antigen comprises a protein.
  • the tumor antigen comprises nucleic acid.
  • the tumor antigen is a tumor cell.
  • anti-infective agents include, but are not limited to, antiviral agents, antifungal agents, antiprotozoal agents, antibacterial agents, such as the nucleoside analogue zidovudine (AST), ganciclovir, foscarnet or cidovir etc.
  • antiviral agents include, but are not limited to, antiviral agents, antifungal agents, antiprotozoal agents, antibacterial agents, such as the nucleoside analogue zidovudine (AST), ganciclovir, foscarnet or cidovir etc.
  • Immunomodulators include immune checkpoint molecular inhibitors and costimulatory molecular activators.
  • the inhibitor of immune checkpoint molecules is CTLA-4, TIM-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (e.g., CEACAM-1 and/or CEACAM-5) and/ Or TGFR inhibitor.
  • the inhibition of molecules can be done at the DNA, RNA or protein level.
  • inhibitory nucleic acids eg, dsRNA, siRNA, or shRNA
  • the inhibitor of the immune checkpoint molecule is a polypeptide that binds to the immune checkpoint molecule, for example, a soluble ligand or antibody or antibody fragment.
  • the immunomodulator is an activator or agonist of a costimulatory molecule.
  • the agonist of the costimulatory molecule is selected from agonists of the following molecules (for example, agonistic antibodies or antigen-binding fragments thereof, or soluble fusions): OX40, CD2, CD27, CDS, ICAM-1, LFA -1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand.
  • the antibodies or fragments thereof of the present invention can be administered in combination with treatments comprising adoptive transfer of T cells expressing chimeric antigen receptors (CAR) (eg, cytotoxic T cells or CTL).
  • CAR chimeric antigen receptors
  • the antibodies or fragments thereof of the present invention can be administered in combination with anti-tumor agents or oncolytic viruses.
  • CD40 agonism can be combined with other forms of immunotherapy such as cytokine therapy (eg interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy, which provides enhanced tumor Antigen presentation.
  • cytokine therapy eg interferon, GM-CSF, G-CSF, IL-2
  • bispecific antibody therapy which provides enhanced tumor Antigen presentation. See, for example, Holliger (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak (1994) Structure 2: 1121-1123.
  • the antibodies or fragments thereof of the present invention can be combined with conventional methods for enhancing host immune function, including but not limited to: (i) APC enhancement, such as (a) injection of encoding heterologous MHC into tumors DNA of allogeneic antigen, or (b) transfected biopsy tumor cells with genes that increase the possibility of immune antigen recognition (such as immunostimulatory cytokines, GM-CSF, costimulatory molecules B7.1, B7.2), ( iii) Adoptive cellular immunotherapy, or treatment with activated tumor-specific T cells.
  • APC enhancement such as (a) injection of encoding heterologous MHC into tumors DNA of allogeneic antigen, or (b) transfected biopsy tumor cells with genes that increase the possibility of immune antigen recognition (such as immunostimulatory cytokines, GM-CSF, costimulatory molecules B7.1, B7.2),
  • Adoptive cellular immunotherapy or treatment with activated tumor-specific T cells.
  • Adoptive cellular immunotherapy includes the isolation of tumor-infiltrating host T lymphocytes, such as stimulation by IL-2 or tumor or both, to expand the population in vitro; in addition, dysfunctional isolated T cells can also be used in vitro by applying the antibody of the present invention To activate, the T cells thus activated can then be re-administered to the host.
  • T lymphocytes such as stimulation by IL-2 or tumor or both
  • Such combination therapies encompass combined administration (in which two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case, other therapies, such as treatment modality and
  • other therapies such as treatment modality and
  • the administration of the antibody of the invention occurs before, at the same time, and/or after the therapeutic agent.
  • Antibody molecules and/or other therapies, such as therapeutic agents or treatment modalities can be administered during active disease or during remission or less active disease.
  • the antibody molecule can be administered before other treatments, concurrently with other treatments, after treatment, or during disease remission.
  • immunoconjugates or compositions or combination products or kits of the present invention can be used to replace or supplement the antibodies of the present invention for any treatment.
  • the administration mode of the antibodies of the present invention can be any suitable route, such as parenteral administration, such as intradermal, intramuscular, and intraperitoneal , Intravenous or subcutaneous, mucosal (oral, intranasal, intravaginal, rectal) or other methods as understood by those skilled in the art.
  • parenteral administration such as intradermal, intramuscular, and intraperitoneal
  • Intravenous or subcutaneous such as intravenous or subcutaneous, mucosal (oral, intranasal, intravaginal, rectal) or other methods as understood by those skilled in the art.
  • the agonistic antibody that specifically binds to CD40 can be administered intratumorally to the drainage site of the lymph node for local delivery to the tumor using known methods.
  • the agonistic antibody that specifically binds to human CD40 of the present invention can be administered to a patient by any suitable route, for example, parenterally by intravenous (i.v.) infusion or bolus injection, intramuscular or subcutaneous or intraperitoneal injection.
  • i.v. intravenous
  • bolus injection intramuscular or subcutaneous or intraperitoneal injection.
  • medication schedules are covered herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration and pulse infusion.
  • the appropriate dose of the antibody of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and progress of the disease , Whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician.
  • the antibody is suitably administered to the patient in one treatment or over a series of treatments. Generally, the clinician administers the composition until the dose to achieve the desired effect is achieved.
  • the antibody of the present invention can therefore be administered in a single dose, or in two or more doses (which may contain the same or different amounts of the desired molecule) within a certain period of time, or by continuous infusion via an implanted device or catheter.
  • the appropriate dose can be determined by using appropriate dose response data.
  • the antibody can be administered to the patient for an extended period of time. In certain embodiments, the antibody is administered every week, every two weeks, every month, every two months, every three months, every four months, every five months, or every six months.
  • the anti-CD40 antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of CD40 in biological samples.
  • detection includes quantitative or qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (for example, FACS), antibody molecule complexed magnetic beads, ELISA assays Method, PCR-technology (for example, RT-PCR).
  • the biological sample is blood, serum, or other liquid samples of biological origin.
  • the biological sample comprises cells or tissues.
  • the biological sample is derived from the relevant lesions of the diseases described herein (e.g., tumors or infections)
  • CD40 is human CD40 or cyno CD40.
  • the method includes contacting a biological sample with an anti-CD40 antibody or antigen-binding fragment thereof as described herein under conditions that allow the anti-CD40 antibody to bind to CD40, and detecting whether the anti-CD40 antibody is between the anti-CD40 antibody and CD40 Whether to form a complex. The formation of the complex indicates the presence of CD40.
  • the method can be an in vitro or in vivo method.
  • anti-CD40 antibodies are used to select subjects suitable for treatment with anti-CD40 antibodies, for example where CD40 is the biomarker used to select the subject.
  • the antibodies of the invention can be used to diagnose the diseases described herein, for example to evaluate (eg, monitor) the treatment or progression, diagnosis, and/or staging of the diseases described herein in a subject.
  • labeled anti-CD40 antibodies are provided.
  • Labels include, but are not limited to, labels or parts that are directly detected (such as fluorescent labels, chromophore labels, electron dense labels, chemiluminescence labels, and radioactive labels), and parts that are indirectly detected, such as enzymes or ligands, for example, Through enzymatic reactions or molecular interactions.
  • Exemplary labels include, but are not limited to, the radioisotopes 32 P, 14 C, 125 I, 3 H and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl ( dansyl), umbelliferone, luceriferase, for example, firefly luciferase and bacterial luciferase (US Patent No.
  • the sample is obtained before treatment with the anti-CD40 antibody. In some embodiments, the sample is obtained prior to treatment with the disease drugs described herein. In some embodiments, the sample is formalin fixed, paraffin coated (FFPE). In some embodiments, the sample is a biopsy (e.g., a core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.
  • FFPE formalin fixed, paraffin coated
  • the sample is a biopsy (e.g., a core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.
  • CD40 is detected before treatment, for example, before the initial treatment or before some treatment after the treatment interval.
  • a detection kit comprising the antibody of the present invention or an antigen-binding fragment thereof is provided for the diagnosis of diseases described herein, such as tumors or infections.
  • a method of treating diseases described herein, such as tumors or infections comprising: testing a subject (e.g., a sample) (e.g., a sample of the subject) for the presence of CD40, thereby Determine the CD40 value, compare the CD40 value to the control value, and if the CD40 value is less than the control value, then administer to the subject a therapeutically effective amount of an anti-CD40 antibody optionally in combination with one or more other therapies (for example, as described herein The aforementioned anti-CD40 antibodies), thereby treating the diseases described herein, such as tumors or infections.
  • the present invention therefore also relates to the use of the antibody or antigen-binding fragment thereof of the present invention in the above-mentioned method, and the use of the antibody or antigen-binding fragment thereof of the present invention in the preparation of a medicine or composition or combination product or kit used in the above-mentioned method Use, and/or use of the antibody or antigen-binding fragment thereof of the present invention in preparing a kit for diagnosing the diseases described herein.
  • the methods and uses applicable to the antibody or antigen-binding fragment thereof of the present invention are also applicable to immunoconjugates, compositions, combination products or kits comprising the antibody or antigen-binding fragment thereof of the present invention.
  • Figure 1 shows the monoclonal activation of Jurkat/NF- ⁇ B-GFP+hCD40 reporter cells by HEL and CD40L produced by 293F cells measured by flow cytometry.
  • the reporter cells have GFP expression after activation.
  • the GFP positive rate is detected by FITC channel .
  • Figure 2 shows the positive rate of CD40-binding antibodies in the antibody library obtained from the second round ( Figure 2A) and the third round ( Figure 2B) of phage display enrichment by phage ELISA.
  • the binding signal of antibody to CD40 is greater than that of BSA binding signal. More than three times is defined as a positive antibody.
  • Figure 3 shows that the supernatant containing negative control N27 and positive CD40 agonist antibody produced by the Jurkat/NF- ⁇ B-GFP+hCD40 reporter cell monoclonal was determined by flow cytometry to Jurkat/NF- ⁇ B-GFP+hCD40 reporter cell Activation.
  • the fluorescence intensity of GFP detected by the FITC channel represents the activation degree of the monoclonal.
  • Figure 4 shows the determination of the properties of the NK003 polymer of the antibody of the present invention produced by 293F cells by size exclusion chromatography.
  • Figure 5A shows that the antibody NK003 of the present invention produced by 293F cells specifically binds to CD40, but does not bind or lowly binds to other TNFR family members OX40, 4-1BB, and GITR.
  • Figure 5B shows the specific binding of the antibody NK004 of the present invention produced by 293F cells to CD40 measured by ELISA.
  • Figure 6 shows that the antibody NK003 of the present invention produced by 293F cells competitively binds to CD40 with CD40L as determined by ELISA.
  • Figure 7A shows that the antibody NK003 of the present invention produced by 293F cells was determined by flow cytometry to activate Jurkat/NF- ⁇ B-GFP+hCD40 reporter cells in a cross-linked form
  • Figure 7B shows the flow cytometry measurement of 293F cells produced by The invented antibody NK004 does not rely on cross-linking and activates Jurkat/NF- ⁇ B-GFP+hCD40 reporter cells in a constitutive manner.
  • the FITC channel detects GFP
  • MFI is defined as the product of the Geometrical Mean of GFP-positive cells and the percentage of GFP-positive cells.
  • Figure 8 shows the binding of the antibody NK003 of the present invention produced by 293F cells to 293FT cells overexpressing human CD40 ( Figure 8A) and 293FT cells overexpressing rhesus CD40 ( Figure 8B) measured by flow cytometry.
  • Figure 9 shows that the antibody NK003 of the present invention produced by 293F cells induced apoptosis of Raji cells (Figure 9A) and Ramos cells ( Figure 9B) by flow cytometry, and the apoptosis index is the expression of CD95.
  • MFI is defined as the product of the geometric mean number of CD95 positive cells and the percentage of CD95 positive cells.
  • Figure 10 shows the determination of the antibody NK003 of the present invention produced by 293F cells by flow cytometry on dendritic cells in PBMC when no crosslinker (Figure 10A) and crosslinker anti-Fc ( Figure 10B) are added. activation.
  • the index of dendritic cell activation is the expression of CD86.
  • MFI is defined as the product of the geometric mean number of CD86 positive cells and the percentage of CD86 positive cells.
  • Figure 11 shows the proliferation effect of the antibody NK003 of the present invention produced by 293F cells on B cells in PBMC measured by the CellTiter-Glo method.
  • the cell proliferation index is fluorescence intensity (RLU). The larger the RLU value, the more cells.
  • Fig. 12 shows the activation of B cells in PBMC by flow cytometry measurement of the antibody NK003 of the present invention produced by 293F cells without the addition of a cross-linking agent (Fig. 12A) and the addition of a cross-linking agent anti-Fc (Fig. 12B).
  • the B cell activation index is the expression of CD86.
  • MFI is defined as the product of the geometric mean number of CD86 positive cells and the percentage of CD86 positive cells.
  • Figure 13 shows the antibody NK003 and negative control HEL of the present invention produced in 293F cells while being inoculated with Raji cells in SCID mice, individual mouse tumor growth curves (Figure 13A) and mouse survival rate statistics (Figure 13B) .
  • Figure 14 shows the immune system activation of the antibody NK003 produced in 293F cells and negative control HEL in MC38 tumor-bearing CD40 humanized mice, the number of OT1 cells, the ratio of OT1 cells to CD8 cells (OT1/CD8 + Ratio) and the increase in the ratio of CD8 cells to CD4 cells (CD8/CD4 ratio) can indicate that the immune system is activated.
  • Figure 15 shows the tumor growth curve of individual mice (Figure 15A) and the weight change of the antibody NK003, negative control HEL and positive control CP870893 produced in 293F cells administered to MC38 tumor-bearing CD40 humanized mice Curve ( Figure 15B).
  • Figure 16 shows that the antibodies of the present invention NK003, Fc ⁇ RIIB enhanced mutant NK003-V12, Fc ⁇ RIIA/Fc ⁇ RIIB enhanced mutant NK003-S267E/L328F and negative control HEL produced by 293F cells determined by flow cytometry were measured in 293FT-Fc ⁇ RIIA cells, respectively ( Figure 16A) and 293FT-Fc ⁇ RIIB cells ( Figure 16B) cross-linked to activate Jurkat/NF- ⁇ B-GFP+hCD40 reporter cells.
  • FITC channel detects GFP
  • MFI is defined as the product of the geometric mean number of GFP-positive cells and the percentage of GFP-positive cells.
  • Figure 17 shows the plasmid map of NK003 inserted into the pcomb3 vector in Fab format.
  • Figure 18 shows the positive rate of CD40-binding antibodies in the antibody library obtained from the third round of phage display measured by phage ELISA.
  • the binding signal of antibodies to CD40 is more than three times the signal to BSA is defined as positive antibodies.
  • Figure 19 shows the third-generation sequencing results of NK003VH (Figure 19A) and VL ( Figure 19B) complementarity determining region CDR3 affinity maturation.
  • Figure 20 shows that the antibodies NK003-AM-9 and NK003-AM-18 of the present invention produced by 293F cells were determined by flow cytometry to activate Jurkat/NF- ⁇ B-GFP+hCD40 reporter cells in a cross-linked form, and the FITC channel detects GFP, MFI is defined as the product of the Geometrical Mean of GFP-positive cells and the percentage of GFP-positive cells.
  • Figure 21 shows the third-generation sequencing results of NK003VH (Figure 21A) and VL (Figure 21B) framework region affinity maturation.
  • Figure 22 shows that the antibody NK003-AM-18-EP1 of the present invention produced by 293F cells is determined by flow cytometry to activate Jurkat/NF- ⁇ B-GFP+hCD40 reporter cells in a cross-linked form.
  • the FITC channel detects GFP, and MFI is defined as GFP The product of the Geometrical Mean of positive cells and the percentage of GFP positive cells.
  • the term “comprising” or “including” means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps.
  • the term “comprising” or “including” when used, unless otherwise specified, it also covers the combination of the stated elements, integers or steps.
  • an antibody variable region that "comprises” a specific sequence it is also intended to encompass the antibody variable region composed of the specific sequence.
  • CD40 is known in the art, such as human CD40 or rhesus CD40.
  • CD40 is also known as tumor necrosis factor receptor superfamily member 5 (TNFRSF5), CD4OL receptor, or CD154 receptor.
  • TNFRSF5 tumor necrosis factor receptor superfamily member 5
  • CD4OL receptor CD4OL receptor
  • CD154 receptor CD154 receptor.
  • the human full-length CD40 protein is a type I membrane protein with 277 amino acids, see, for example, NCBI, NM_001250.5.
  • NCBI NM_001265862.1.
  • anti-CD40 antibody refers to antibodies that are capable of having sufficient affinity Binding (human or rhesus monkey) CD40 subunit or fragments thereof so that the antibody can be used as a diagnostic and/or therapeutic agent in targeting (human or rhesus monkey) CD40.
  • the degree of binding of an anti-CD40 antibody to a non-(human or rhesus monkey) CD40 protein is about 10%, about 20%, or about 30% of the binding of the antibody to (human or rhesus monkey) CD40 protein. , About 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or more, as measured, for example, by radioimmunoassay (RIA) or biooptical interferometry or MSD assay.
  • RIA radioimmunoassay
  • Antibodies that specifically bind to human CD40 may have cross-reactivity with other related antigens, for example, with the same antigen from other species (homologous) (such as rhesus monkeys). Although monospecific antibodies specifically bind to one antigen or one epitope, bispecific antibodies specifically bind to two different antigens or two different epitopes.
  • a “complementarity determining region” or “CDR region” or “CDR” is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen contact residues ( "Antigen contact point”) area.
  • CDR is mainly responsible for binding to antigen epitopes.
  • the CDRs of the heavy and light chains are usually called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • the CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3.
  • each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, which include For example: Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et al.
  • the residues of each CDR are as follows.
  • the CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (for example, any of the exemplary CDRs of the present invention).
  • variable region residues in an antibody refers to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues).
  • the CDR of the antibody of the present invention determines the boundary by IMGT rules, for example, the IMGT database is used to determine the boundary.
  • the CDR boundaries of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Therefore, when it comes to defining antibodies with specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences include the specific CDR sequences, but due to the application of different schemes (for example Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.
  • Fc region or “Fc domain” or “Fc” refers to the C-terminal region of the heavy chain of an antibody, which mediates the binding of immunoglobulin to host tissues or factors, including binding to various cells located in the immune system (e.g., effector Fc receptors on cells) or bind to the first component of the classical complement system (C1q). Therefore, the Fc region contains the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL).
  • first constant region immunoglobulin domain e.g., CH1 or CL
  • the Fc region contains the CH2 and CH3 constant regions in each of the two heavy chains of the antibody; the IgM and IgE Fc regions contain three heavy chain constant domains (CH structure) in each polypeptide chain. Domain 2-4).
  • the Fc region contains immunoglobulin domains C ⁇ 2 and C ⁇ 3 and the hinge between C ⁇ 1 and C ⁇ 2.
  • the Fc region of a human IgG heavy chain is usually defined as the amino acid residue at position C226 or P230 (or the amino acid between these two amino acids) to the carboxyl group of the heavy chain
  • the fragments at the end where numbering is based on the EU index in Kabat. Kabat et al. (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD; see also Figures 3c-3f of US Patent Application Publication No. 2008/0248028.
  • the CH2 domain of the Fc region of human IgG extends from about amino acid 231 to about amino acid 340, and the CH3 domain is located at the C-terminal side of the CH2 domain in the Fc region, that is, it extends from about amino acid 341 to about amino acid 447 (including C-terminal lysine).
  • the Fc region can be a native sequence Fc, including any allotype variant, or a variant Fc (such as a non-naturally occurring Fc).
  • Fc can also refer to this region in isolation or in the context of Fc-containing protein polypeptides such as "Fc region-containing binding proteins" also known as "Fc fusion proteins” (eg, antibodies or immunoadhesins).
  • epitopes within protein antigens can be formed from contiguous amino acids (usually linear epitopes) or discontinuous amino acids (usually conformational epitopes) juxtaposed by the tertiary folding of the protein. Epitopes formed by consecutive amino acids usually (but not always) remain exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation.
  • An antibody that binds to the same or overlapping epitope refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% of the reference antibody in a competition assay. Antigen binding, on the contrary, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% of the binding of the antibody to its antigen in a competition assay.
  • An antibody that competes with a reference antibody for binding to its antigen refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% of the binding of the antibody to its antigen in a competition assay.
  • Many types of competitive binding assays can be used to determine whether one antibody competes with another, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition Determination (see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253).
  • An antibody that inhibits (for example, competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen .
  • the reference antibody inhibits 50%, 60%, 70%, 80%, 90% or 95% of the binding of the antibody to its antigen.
  • the binding of an antibody to its antigen can be measured by affinity (e.g. equilibrium dissociation constant). Methods for determining affinity are known in the art.
  • An antibody that shows the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody that can have at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody Affinity and/or specificity. This can be determined by any method known in the art for determining binding affinity and/or specificity.
  • Antibody in the form of IgG refers to the form of IgG to which the constant region of the heavy chain of the antibody belongs.
  • the heavy chain constant regions of all antibodies of the same type are the same, and the heavy chain constant regions of antibodies of different types are different.
  • an antibody in the form of IgG1 means that the Ig domain of its heavy chain constant region is the Ig domain of IgG1.
  • Human antibody refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • a “humanized” antibody refers to an antibody in which some, most or all of the amino acids outside the CDR domain of a non-human antibody (such as a mouse antibody) have been replaced by corresponding amino acids derived from human immunoglobulin. In one embodiment of the humanized form of the antibody, some, most, or all of the amino acids outside the CDR domain have been replaced by amino acids from human immunoglobulins, while some, most of the amino acids in one or more CDR regions Or all amino acids have not changed. Small additions, deletions, insertions, substitutions, or modifications of amino acids are allowed, as long as they do not eliminate the ability of the antibody to bind to a specific antigen. "Humanized” antibodies retain antigen specificity similar to the original antibody.
  • chimeric antibody refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody .
  • antibody fragment refers to a molecule that is different from an intact antibody, which contains a part of an intact antibody and binds to the antigen to which the intact antibody binds.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, human CD40).
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabody; linear antibody; single chain antibody (such as scFv); single domain antibody; bivalent or bispecific Antibodies or fragments thereof; camelid antibodies; and bispecific antibodies or multispecific antibodies formed from antibody fragments.
  • multispecific refers to an antibody that specifically binds to at least two different antigens or two different epitopes within an antigen, such as three, four, or five different antigens or epitopes.
  • bispecific refers to antibodies that specifically bind to two different antigens or two different epitopes within the same antigen. Bispecific antibodies can be cross-reactive to other related antigens, or can bind epitopes shared between two or more different antigens.
  • cross-linking refers to the higher-order multimerization of CD40 on cells induced by the binding of an antibody that specifically binds human CD40 to cis or trans Fc ⁇ RIIb, resulting in the induction of CD40 agonistic activity.
  • the anti-human F(ab')2 as described herein can be used as a cross-linking agent to evaluate cross-linking in vitro.
  • cross-reactivity refers to the ability of the antibodies described herein to bind CD40 from different species.
  • the antibodies described herein that bind human CD40 also bind CD40 from another species (e.g., cynomolgus CD40).
  • cross-reactivity can be measured by detecting specific reactivity or binding with purified antigens in binding assays (e.g. SPR, ELISA) or otherwise functionally interacting with cells that physiologically express CD40.
  • binding assays e.g. SPR, ELISA
  • Methods for determining cross-reactivity include standard binding assays as described herein, for example by Surface plasmon resonance (SPR) analysis use 2000SPR instrument (Biacore AB, Uppsala, Sweden) or flow cytometry technology.
  • SPR Surface plasmon resonance
  • an “immunoconjugate” is an antibody conjugated to one or more other substances, including but not limited to cytotoxic agents or labels.
  • label refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the reagent to which it is conjugated or fused.
  • the label itself may be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of enzymatic labeling, may catalyze a chemical change of a detectable substrate compound or composition.
  • the term is intended to cover the direct labeling of a probe or antibody by coupling (ie, physically linking) a detectable substance to the probe or antibody and the indirect labeling of the probe or antibody by reaction with another reagent that is directly labeled.
  • Examples of indirect labels include detection of primary antibodies using fluorescently labeled secondary antibodies and end labeling of DNA probes with biotin so that they can be detected with fluorescently labeled streptavidin.
  • Vector refers to a polynucleotide that can replicate within a biological system or can move between such systems.
  • Vector polynucleotides usually contain elements such as origin of replication, polyadenylation signals or selection markers, whose function is to promote the replication or maintenance of these polynucleotides in biological systems. Examples of such biological systems may include cells, viruses, animals, plants, and biological systems reconstructed with biological components capable of replicating vectors.
  • the polynucleotide containing the vector may be a DNA or RNA molecule or a hybrid of these molecules.
  • Expression vector refers to a vector that can be used in a biological system or a reconstructed biological system to direct the translation of the polypeptide encoded by the polynucleotide sequence present in the expression vector.
  • an “isolated” antibody is an antibody that has been separated from a component of its natural environment.
  • the antibody is purified to more than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC) confirmed.
  • electrophoresis e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reverse phase HPLC
  • nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule exists outside the chromosome or at a chromosomal location different from its natural chromosomal location.
  • the sequences are aligned for optimal comparison purposes (for example, the first and second amino acid sequences or nucleic acid sequences can be used for optimal alignment. Gaps can be introduced in one or both or non-homologous sequences can be discarded for comparison purposes).
  • the length of the compared reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.
  • Mathematical algorithms can be used to achieve sequence comparison and percent identity calculation between two sequences.
  • the Needlema and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm (at http://www.gcg.com) that has been integrated into the GAP program of the GCG software package is used. (Available), use Blossum 62 matrix or PAM250 matrix and gap weight 16, 14, 12, 10, 8, 6 or 4 and length weight 1, 2, 3, 4, 5 or 6, to determine the difference between two amino acid sequences Percent identity.
  • the GAP program in the GCG software package (available at http://www.gcg.com) is used, the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 are used. Length weights 1, 2, 3, 4, 5, or 6, determine the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and a parameter set that should be used unless otherwise specified) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, for example, to identify other family member sequences or related sequences.
  • hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions describes hybridization and washing conditions. Instructions for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and non-aqueous methods are described in the references and either method can be used.
  • the specific hybridization conditions mentioned in this article are as follows: 1) Low stringency hybridization conditions are in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by at least 50°C (for low stringency conditions, you can Increase the washing temperature to 55°C) Wash twice in 0.2X SSC, 0.1% SDS; 2) Medium stringency hybridization conditions are about 45°C in 6X SSC, and then at 60°C in 0.2X SSC, 0.1% SDS Wash one or more times in medium; 3) High-stringency hybridization conditions are in 6X SSC at about 45°C, and then wash one or more times in 0.2X SSC, 0.1% SDS at 65°C; and preferably 4) Very high The stringent hybridization conditions are washing one or more times in 0.5M sodium phosphate, 7% SDS at 65°C, and then in 0.2X SSC, 0.1% SDS at 65°C.
  • the very high stringency condition (4) is the preferred condition and one that should be used unless otherwise specified.
  • host cell refers to cells into which exogenous nucleic acid is introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages.
  • the offspring may not be exactly the same as the parent cell in nucleic acid content, but may contain mutations. Included herein is the mutant progeny with the same function or biological activity screened or selected in the initially transformed cell.
  • Suitable host cells suitable for use in the present invention include prokaryotic microorganisms such as E. coli.
  • the host cell can also be eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells, such as insect cells. Vertebrate cells can also be used as hosts. For example, a mammalian cell line that has been adapted to grow in suspension can be used.
  • Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK 293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells ( CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver Cells (Hep G2), Chinese Hamster Ovary cells (CHO cells), CHOK1SV cells, CHOK1SV GS-KO cells, CHOS cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63 and Sp2/0.
  • the host cell is a CHO cell, such as CHOS cell CHOK1SV cell or CHOK1SV GS-KO, or the host cell is 293 cell, such as HEK293 cell.
  • agonist refers to an antibody that specifically binds to human CD40, and induces the proliferation or activation of B cells and/or dendritic cells (DC) or T cells when binding to CD40.
  • the proliferation or activation of B cells and DC and T cells can be measured by measuring increased B cell proliferation, or measuring any surface markers CD23, CD80, CD83, CD86 on B cells, or CD80, CD83, CD86 and CD80 on DC. Up-regulation of HLA-DR. When compared with a control sample that does not contain antibodies, the agonist can induce B cell and/or DC and/or T cell activation in a statistically significant manner.
  • “Inhibition of tumor cell/tumor growth” refers to the growth of tumor cells or tumors in vitro or in vivo when contacted with a therapeutic agent or a combination of therapeutic agents, compared with the growth of the same tumor cell or tumor in the absence of the therapeutic agent. The measured drop.
  • the inhibition of tumor cells or tumor growth in vitro or in vivo can be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100%.
  • therapeutic agent encompasses any substance that is effective in preventing or treating diseases, such as tumors (such as cancer) and infections (such as chronic infections), including chemotherapeutics, cytotoxic agents, vaccines, other antibodies, and anti-infections. Active agents, small molecule drugs or immunomodulators.
  • “Chemotherapeutic agents” include chemical compounds useful in the treatment of immune system diseases, including but not limited to alkylating agents; antimetabolites; anti-microtubule inhibitors, natural products; antibiotics; enzymes; miscellaneous agents; hormones and antagonists; Anti-estrogens; anti-androgens; non-steroidal anti-androgens, topoisomerase inhibitors, receptor tyrosine kinase inhibitors, angiogenesis inhibitors, etc.
  • chemotherapeutics of the invention such as Anastrozole Bicalutamide Bleomycin Sulfate Busulfan Busulfan injection Capecitabine N4-Pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin Carmustine Chlorambucil Cisplatin Claribine Cyclophosphamide Cytarabine, Cytosine Arabinoside Cytarabine liposome injection dacarbazine Dactinomycin (actinomycin D, Cosmegan), daunorubicin hydrochloride Daunorubicin Citrate Liposome Injection Dexamethasone, Docetaxel Doxorubicin hydrochloride Etoposide Fludarabine phosphate 5-fluorouracil Flutamide tezacitibine, gemcitabine (difedeoxycytidine), hydroxyurea Idabycin Ifosfamide Irinotecan L-Asparaginase Leucovorin, Melphalan
  • This definition also includes anti-hormonal drugs such as anti-estrogens used to modulate or inhibit the effects of hormones on tumors, including, for example, tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxyl Tamoxifen, trovoxifene, keoxifene, LY117018, onlastone and toremifene and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide acetate and ge Serrelin; and a pharmaceutically acceptable salt, acid or derivative of any of the above.
  • anti-hormonal drugs such as anti-estrogens used to modulate or inhibit the effects of hormones on tumors, including, for example, tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxyl Tamoxifen, trovoxifene, keoxifene, LY117018, onlastone and
  • cytotoxic agent refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction.
  • cytotoxic agents include, but are not limited to: radioisotopes, such as iodine (131 I, 125 I, 123 I and 121 I), carbon (14 C), sulfur (35 S), tritium (3 H), indium ( 115 In, 113 In, 112 In and 111 In), technetium ( 99 Tc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe) ), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 S
  • exemplary cytotoxic agents of the present invention include anti-microtubule drugs, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, anthracyclines, vinblastine alkaloids , Intercalators, agents capable of interfering with signal transduction pathways, pro-apoptotic agents, proteasome inhibitors, and irradiation (e.g., local or whole body irradiation (e.g., gamma radiation).
  • irradiation e.g., local or whole body irradiation (e.g., gamma radiation).
  • small molecule drugs refers to low molecular weight organic compounds capable of regulating biological processes.
  • Small molecules are defined as molecules with a molecular weight of less than 10 kD, usually less than 2 kD and preferably less than 1 kD.
  • Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As a therapeutic agent, small molecules can penetrate cells better than large molecules, are less susceptible to degradation, and are less likely to trigger an immune response.
  • anti-infective active agent includes any molecule that specifically inhibits or eliminates the growth of microorganisms, such as viruses, bacteria, fungi, or protozoa, such as parasites, at the applied concentration and dosing interval, but is not lethal to the host.
  • anti-infective agent includes antibiotics, antibacterial agents, antiviral agents, antifungal agents, and antiprotozoal agents.
  • the anti-infective active agent is non-toxic to the host at the applied concentration and dosing interval.
  • Antibacterial anti-infective agents or antibacterial agents can be broadly classified as bactericidal (ie, direct killing) or bacteriostatic (ie, preventing division). Antibacterial anti-infective active agents can be further classified as narrow-spectrum antibacterial agents (i.e., affect only small bacterial subtypes, for example, Gram-negative, etc.) or broad-spectrum antibacterial agents (i.e., affect a wide range of species). Examples include amikacin, gentamicin, geldanamycin, herbimycin, mupirocin, nitrofurantoin, pyrazinamide, quinupristin/dalfopristin, rifampicin/isophos Amide or tinidazole, etc.
  • antiviral agent includes any substance that inhibits or eliminates the growth, pathogenicity, and/or survival of a virus. This includes, for example, acyclovir, cidofovir, zidovudine, didanosine (ddI, VIDEX), zacitabine (ddC, HIVID), stavudine (d4T, ZERIT), lamie Fudine (3TC, EPIVIR)), abacavir (ZIAGEN), emtricitabine (EMTRIVA) and so on.
  • antifungal agent includes any substance that inhibits or eliminates the growth, pathogenicity, and/or survival of fungi. This includes, for example, natamycin, schizotonin, filipin, nystatin, amphotericin B, candesine, patchouli, neem seed oil, coconut Oil (Coconut Oil) etc.
  • antiprotozoal agent includes any substance that inhibits or eliminates the growth, morbidity, and/or survival of protozoan organisms, such as parasites.
  • antiprotozoal agents include antimalarial agents such as quinine, quinidine and the like.
  • immunomodulator refers to natural or synthetic active agents or drugs that inhibit or modulate the immune response.
  • the immune response can be a humoral response or a cellular response.
  • Immunomodulators include immunosuppressive agents.
  • immune checkpoint molecule refers to a type of inhibitory signal molecule present in the immune system, which avoids tissue damage by regulating the persistence and intensity of the immune response in peripheral tissues, and participates in maintaining tolerance to self-antigens (Pardoll DM. , The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12(4): 252-264).
  • Immune checkpoint molecules include but are not limited to programmed death 1 (PD-1), PD-L1, PD-L2, cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG-3 and TIM-3.
  • costimulatory molecule refers to a corresponding binding partner on T cells that specifically binds to a costimulatory ligand to mediate a costimulatory response (such as but not limited to proliferation) of the T cell.
  • Co-stimulatory molecules are cell surface molecules that contribute to an effective immune response in addition to antigen receptors or their ligands.
  • Co-stimulatory molecules include but are not limited to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, OX40 , CD40, GITR, 4-1BB (ie CD137), CD27 and CD28.
  • the "costimulatory molecule” is OX40, GITR, 4-1BB (ie CD137), CD27, and/or CD28.
  • cytokine is a general term for proteins that are released by a cell population and act as intercellular mediators on another cell.
  • cytokines are lymphokines, monocytes, interleukins (IL), such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factor, such as TNF- ⁇ or TNF- ⁇ ; and other polypeptide factors, including LIF and kit ligand (KL) and Gamma interferon.
  • IL interleukins
  • cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of natural sequence cytokines, including small molecular entities produced by artificial synthesis, and their pharmaceutically acceptable Derivatives and salts.
  • inhibitor or "antagonist” includes substances that reduce certain parameters (e.g., activity) of a given molecule (e.g., immune checkpoint molecule).
  • this term includes substances that cause the given molecule to be inhibited by at least 5%, 10%, 20%, 30%, 40% or more of the activity (eg, PD-L1 activity). Therefore, the inhibitory effect does not have to be 100%.
  • activator includes substances that increase certain parameters (e.g., activity) of a given molecule (e.g., co-stimulatory molecule). For example, this term includes substances that increase the activity of a given molecule by at least 5%, 10%, 20%, 30%, 40%, or more (eg, OX40 activity). Therefore, the activation does not have to be 100%.
  • pharmaceutical excipients refers to diluents, adjuvants (for example Freund's adjuvant (complete and incomplete)), excipients, carriers or stabilizers, etc. administered together with the active substance.
  • composition refers to a composition that is present in a form that allows the biological activity of the active ingredients contained therein to be effective, and does not contain additional substances that have unacceptable toxicity to the subject to which the composition is administered. Ingredients.
  • combination product refers to a fixed or non-fixed combination in the form of a dosage unit or a kit of parts for combined administration, in which two or more therapeutic agents can be independently at the same time or within a time interval Separate administration, especially when these time intervals allow the combination partner to exhibit cooperation, for example, a synergistic effect.
  • fixed combination means that the antibody of the present invention and the combination partner (such as other therapeutic agents, such as immunomodulators, such as immunosuppressive agents or anti-inflammatory agents) are simultaneously administered to a patient in the form of a single entity or dosage.
  • non-fixed combination means that the antibody of the present invention and the combination partner (such as other therapeutic agents, such as immunomodulators, such as immunosuppressive agents or anti-inflammatory agents) are administered to a patient simultaneously, concurrently or sequentially as separate entities, without specific Time constraints, where such administration provides therapeutically effective levels of both compounds in the patient.
  • combination partner such as other therapeutic agents, such as immunomodulators, such as immunosuppressive agents or anti-inflammatory agents
  • cocktail therapy such as the administration of three or more therapeutic agents.
  • the drug combination is a non-fixed combination.
  • Immunogenicity refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic, and enhancing tumor immunogenicity helps clear tumor cells through an immune response.
  • Immuno response refers to a biological response in vertebrates against foreign substances that protects the organism from these substances and diseases caused by them.
  • the immune response is determined by cells of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and by these Mediated by the action of soluble macromolecules (including antibodies, cytokines, and complement) produced by cells or liver, which lead to the selective targeting, binding, damage, destruction and/or elimination of invading pathogens, cells or infected pathogens from vertebrate organisms Tissues, cancerous or other abnormal cells, or (in the case of autoimmune or pathological inflammation) normal human cells or tissues.
  • T lymphocytes for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils
  • soluble macromolecules including antibodies
  • T effector T cells for example, effector T cells or Th cells, such as CD4+ or CD8+ T cells, or suppression or depletion of Treg cells.
  • T effector ("T eff ") cells refer to T cells with cytolytic activity (such as CD4+ and CD8+ T cells) and T helper (Th) cells (which secrete cytokines and activate and guide other immune cells), but Does not include regulatory T cells (Treg cells).
  • the term "effective amount” refers to the amount or dose of the antibody or fragment or conjugate or composition of the present invention that, after administration to the patient in single or multiple doses, produces the desired effect in the patient in need of treatment or prevention.
  • the effective amount can be easily determined by the attending physician as a person skilled in the art by considering various factors such as the species of mammal; its size, age and general health; the specific disease involved; the degree or severity of the disease; The response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.
  • Therapeutically effective amount refers to the amount that is effective to achieve the desired therapeutic result at the required dose and for the required period of time.
  • the therapeutically effective amount of the antibody or antibody fragment or its conjugate or composition may vary according to various factors such as disease state, the age, sex and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual.
  • a therapeutically effective amount is also an amount in which any toxic or harmful effects of the antibody or antibody fragment or its conjugate or composition are less than the therapeutically beneficial effects.
  • a "therapeutically effective amount” preferably inhibits a measurable parameter (such as swelling rate, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%. % And still more preferably at least about 80% or 90%.
  • a measurable parameter such as swelling rate, etc.
  • the ability of a compound to inhibit a measurable parameter can be evaluated in an animal model system predicting efficacy in human autoimmune diseases or inflammation.
  • prophylactically effective amount refers to an amount that effectively achieves the desired preventive result at the required dose and for the required period of time. Generally, since the prophylactic dose is used in the subject before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • the "individual” or “subject” described herein can be used interchangeably, including mammals. Mammals include, but are not limited to, domestic animals (for example, cattle, sheep, cats, dogs, and horses), primates (for example, human and non-human primates such as monkeys), rabbits, and rodents (for example, , Mice and rats).
  • the individual or subject may be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., suffering from a disease described herein or having a disease described herein Risk of patients).
  • the subject has or is at risk of suffering from a disease described herein (eg, a tumor or infection as described herein).
  • the subject has received or has received other treatments, such as chemotherapy treatment and/or radiation therapy.
  • the subject is immunocompromised due to infection or is at risk of being immunocompromised due to infection.
  • combination therapy refers to the administration of two or more therapeutic agents or treatment modalities (e.g. radiation therapy or surgery) to treat IL-23 related diseases as described in this disclosure.
  • administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, for example, in a single capsule having a fixed ratio of active ingredients.
  • administration includes co-administration of the respective active ingredients in multiple or in separate containers (e.g., tablets, capsules, powders and liquids).
  • the powder and/or liquid can be reconstituted or diluted to the desired dose before administration.
  • such administration also includes the use of each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effects of the drug combination in the treatment of the conditions or conditions described herein.
  • treatment refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • prevention includes the inhibition of the occurrence or development of a disease or condition or the symptoms of a specific disease or condition.
  • subjects with a family history of immune system disease are candidates for preventive regimens.
  • prevention refers to the occurrence of symptoms or symptoms of immune system diseases (autoimmune diseases or inflammation), especially in the presence of immune system diseases (autoimmune diseases or inflammation).
  • Pre-drug administration in subjects at risk of immune disease or inflammation.
  • Subject/patient sample refers to a collection of cells or fluids obtained from a patient or subject.
  • the source of the tissue or cell sample can be solid tissue, such as fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid) ), peritoneal fluid (ascites), or interstitial fluid; cells from the subject's pregnancy or development at any time.
  • Tissue samples may contain compounds that are not naturally mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and so on.
  • NF- ⁇ B transcription factor binding elements in the promoter of the NF- ⁇ B-GFP reporter gene lentiviral system (QIAGEN, CCS-013G).
  • the activation of this promoter can drive the expression of GFP.
  • Jurkat cells ATCC, TIB-152
  • Jurkat cells infected with lentivirus were screened with puromycin (InvivoGen, ant-pr-1).
  • TNF ⁇ protein (Sino Biological, 10602-HNAE) to stimulate the cells selected with puromycin, and 24 hours later, flow cytometry (BD, FACSAria III) sorts the GFP fluorescent cell subsets to obtain Jurkat/NF- ⁇ B-GFP cells.
  • the positive control CD40L sequence used herein comes from patent WO 2016/177771 (SEQ ID NO: 15), and the negative control HEL sequence is shown in Table 10.
  • the DNA sequences of CD40L and HEL are fully synthesized in Jinweizhi Company.
  • the DNA sequence was inserted into the eukaryotic expression vector pFUSE (InvivoGen, pfuse-hg1fc1) and transfected with PEI into 293F cells (Thermo Fisher Scientific, R79007) using standard procedures; Freestyle medium (Lifetechnologies, 12338026) ) Culture the cells on a shaker at 37°C. After 7 days of culture, the supernatant was collected and purified on the AKTA system (GE) using a Superdex TM 200 Increase prepacked column (GE, 28-9909-44).
  • CD40 signal transduction can activate the NF- ⁇ B pathway. If CD40 is successfully expressed on the Jurkat/NF- ⁇ B-GFP cell membrane surface, CD40L stimulation will induce GFP expression.
  • the constructed Jurkat/NF- ⁇ B-GFP cells sorted as described above were infected with the human CD40 lentiviral supernatant obtained above, and after 16 hours, 100nM of the above-obtained CD40L protein (as a positive control) and HEL (as a negative control) were added ) Stimulate, 24h later, flow cytometer single cell sorting the cells with the strongest GFP fluorescence into 96-well plates.
  • the cells in the above 96-well plate were cultured in RPMI1640 medium (Lifetechnologies, C11875500CP) containing 10% fetal bovine serum (Biological Industries, 04-001-1A) for 2 weeks, and after the single cells grew up, 100 nM of the above was added separately Trimerize CD40L protein (as a positive control) and HEL (as a negative control), select a CD40L-positive and HEL-negative single clone as Jurkat/NF- ⁇ B-GFP+hCD40 (referred to as NF- ⁇ B-GFP+hCD40) cells For follow-up experiments. The results are shown in Figure 1. The positive rate of Jurkat/NF- ⁇ B-GFP+hCD40 monoclonal GFP after CD40L stimulation was 94.7%, and HEL stimulation was not activated.
  • the peripheral blood of 30 healthy adults was purchased from Tianjin Blood Center and Shanghai Miaotong Biological Company, and PBMC was obtained by centrifugation with ficoll separation solution (Haoyang, LDS1075). Centrifugation conditions: 20°C, 2000rpm, up 5 down 0 mode for 30 minutes.
  • the human natural antibody library was constructed using the PBMC obtained as described above, and the library was constructed by a conventional method (phage display, Tim Clackson and Henry B. Lowman). The obtained antibody heavy chain and light chain variable regions are randomly combined and displayed on the N-terminus of phage capsid protein pIII in the form of single-chain antibody scFv to obtain a phage display antibody library with a storage capacity of 10 10 .
  • Phage screening is a conventional technology (Phage Display: A Laboratory Manual, Carlos F Barbas III).
  • the biotinylated CD40 protein (acrobiosystems, TN5-H82F9) was incubated with the phage display antibody library obtained as described above for 2 hours at room temperature. After incubation, add 150ul streptavidin magnetic beads Dynabeads M280 (Lifetechnologies, 11205D) directly, and incubate on a mixer at room temperature for 30 minutes.
  • PBS-tween PBS: Lifetechnologies, 70011044; Tween: Sigma-Aldrich, 9005-64-5
  • PBS Lifetechnologies, 70011044; Tween: Sigma-Aldrich, 9005-64-5
  • PBS Lifetechnologies, 70011044; Tween: Sigma-Aldrich, 9005-64-5
  • PH2.2 glycine-hydrochloric acid
  • Bacteriophage conjugated The eluted phage was used to infect E. coli XL1-blue (Agilent, 200236), and the helper phage VCSM13 (Agilent, 200251) was added for amplification and used in the next round of screening. There are three rounds of screening. After three rounds, the antibody phages that bind to CD40 were enriched, and the screening results are shown in Table 1.
  • An ELISA plate (Corning, 3690) was coated with 1 ⁇ g/ml of human CD40 (Acrobiosystems, CD0-H5228) and incubated overnight at 4°C. Add the overnight induced phage supernatant to the ELISA plate after overnight incubation, incubate at room temperature for 1 hour, and wash 8 times with 0.05% PBST.
  • BSA Solarbio, A8020-100
  • HRP-conjugated anti-M13 GE, 27-9421-01, M13 is phage capsid protein
  • a positive clone was defined as a CD40 binding signal that was more than 3 times that of the BSA control. The results are shown in Figure 2. The positive rates of the second and third rounds of screening were 6.9% and 27.6%, respectively.
  • N27 ScFv into secretory lentiviral vector pCDH as a negative control.
  • the sequence of N27 ScFv is shown in Table 11.
  • the lentiviral core plasmid and helper plasmids pPACKH1-GAG, pPACKH1-REV, pVSVG were co-transfected into 293FT cells at a ratio of 1:1:1:1, and the medium was changed 6 hours after transfection.
  • the cells were cultured at 37°C with 10% fetal bovine serum ( In the DMEM medium of Biological Industries, 04-001-1A), continue to culture for 48 hours to collect the supernatant containing the CD40-binding antibody lentiviral library; infect HEK293 cells with 50,000 pg of the CD40-binding antibody lentiviral library, and after infection for 16 hours, Centrifuge to remove the medium containing the lentivirus, add fresh medium, and sort the infected HEK293 cells into a 96-well plate using flow sorting technology so that each well contains only one infected HEK293 cell. Culture the cells for 3 weeks. After the cells reach a certain number, take the supernatant.
  • 10% fetal bovine serum In the DMEM medium of Biological Industries, 04-001-1A
  • the supernatant was added with 3 ⁇ 10 5 Jurkat/NF- ⁇ B-GFP+hCD40 reporter cells obtained as described above, and 2.5 ⁇ g/ml goat anti-human Fc (SouthernBiotech, SBA-2048-01) secondary antibody was added for cross-linking
  • the antibody secreted by the cells enhances the activation strength of the agonist antibody; the activity of the cell supernatant is tested after 24 hours of culture, and the detection result of the positive antibody is shown in Figure 3.
  • the antibody gene is extracted from the HEK293 cells with positive cell supernatant activity and constructed into pFUSE
  • the vector was sequenced to obtain the positive antibody VH and VL sequences, and the two positive antibodies obtained were numbered NK003 and NK004.
  • Table 4 to Table 8 for their sequences (including CDR, VH/VL, heavy chain, and light chain), please refer to Table 4 to Table 8 in the sequence list the sequence of.
  • the selected agonist antibodies NK003, NK004 heavy chain and light chain DNA (Jin Weizhi) were synthesized and cloned into the vector pFUSE.
  • PEI was used to convert the plasmids containing heavy and light chains. They were transiently co-transfected into 293F suspension cells at 1:1 to express full-length antibody.
  • the AKTA system was purified using Superdex TM 200Increase prepacked column.
  • NK003 The binding selectivity of NK003 was evaluated by direct ELISA with TNFR family members OX40, 4-1BB and GITR. Coated on the ELISA plate containing 1 ⁇ g/ml human OX40 (Acrobiosystems, OX0-H5224), 4-1BB (Acrobiosystems, 41B-H5227), GITR (Acrobiosystems, GIR-H5228) and CD40 (Acrobiosystems, CD0-H5228) Incubate in PBS solution overnight at 4°C.
  • human OX40 Acrobiosystems, OX0-H5224
  • 4-1BB Acrobiosystems, 41B-H5227
  • GITR Acrobiosystems, GIR-H5228
  • CD40 Acrobiosystems, CD0-H5228
  • NK003 diluted to different concentrations with PBS as obtained in Example 4: 0.625 ⁇ g/ml, 1.25 ⁇ g/ml, 2.5 ⁇ g/ml and 5 ⁇ g/ml, after incubating for 30min at room temperature, wash with 0.05% PBS-Tween 8 times. Added goat anti-human HRP-conjugated Fc antibody (SouthernBiotech, 2048-05) to detect bound NK003. As shown in Figure 5A, NK003 selectively binds to human CD40, with low or no binding to other tested TNFR family proteins.
  • NK004 In order to evaluate the binding of NK004 to CD40, a 1 ⁇ g/ml human CD40 (Acrobiosystems, CD0-H5228) PB solution was coated on an ELISA plate and incubated overnight at 4°C. Add the PBS obtained in Example 4 and dilute to different concentrations NK004: 0.002nM, 0.016nM, 0.13nM, 1nM, 8nM, 64nM and 500nM. HEL is a negative control antibody. After incubating at room temperature for 30 minutes, wash with 0.05% PBS-Tween 8 times . Add goat anti-human HRP-conjugated Fc antibody to detect bound NK004. As shown in Figure 5B, NK004 can bind to human CD40.
  • ELISA was used to evaluate the effect of NK003 on the binding of CD40L and CD40.
  • NK003 and NK004 activate NF- ⁇ B-GFP+hCD40 reporter cell line in cross-linked form
  • flow cytometry was used to detect the activation of NF- ⁇ B-GFP+hCD40 reporter cell lines by NK003 and NK004 antibodies, and to determine the EC50 of the two antibodies, the specific steps are as follows:
  • Example 4 Take 3 ⁇ 10 5 cells/tube to obtain NF- ⁇ B-GFP+hCD40 reporter cells as in Example 1, and add NK003 or NK004 diluted to different concentrations as obtained in Example 4: 0.001 ⁇ g/ml, 0.005 ⁇ g /ml, 0.01 ⁇ g/ml, 0.05 ⁇ g/ml, 0.1 ⁇ g/ml, 0.5 ⁇ g/ml, 1 ⁇ g/ml, 5 ⁇ g/ml and 10 ⁇ g/ml, 10 ⁇ g/ml, using HEL antibody as a negative control; for cross-linking, Each group was added with a 2.5 ⁇ g/ml secondary antibody, namely goat anti-human Fc.
  • a 2.5 ⁇ g/ml secondary antibody namely goat anti-human Fc.
  • NK004 on CD40 does not depend on the cross-linking of the secondary antibody, and activates the NF- ⁇ B-GFP+hCD40 reporter cell line in a constitutive mode.
  • the EC50 when the secondary antibody is added is 4Nm ( Figure 7B).
  • Biacore T200 (GE Healthcare) is used to detect the affinity of antibodies NK003 and NK004. Flow NK003 and NK004 through the Protein A chip at 10 ⁇ L/min and capture them on the chip.
  • Use Running buffer HBS-EP+, GE
  • the concentration is set to 2 ⁇ M, 1 ⁇ M, 500nM, 250nM, 125nM, 62.5nM, 31.25nM.
  • the above-mentioned CD40 of different concentrations was flowed through the antibody-captured chip at a flow rate of 30 ⁇ L/min, combined for 120s, and then the chip was flowed through the Running buffer at a flow rate of 30 ⁇ L/min for 240s, and the antigen gradually dissociated from the antibody-captured chip.
  • Data processing was performed using BIAevaluation software S200, which is the supporting software of Biacore T200 instrument, to calculate the binding constant (Ka), dissociation constant (Kd) and equilibrium dissociation constant (KD). The results are shown in Table 2.
  • the binding of 293FT cells expressing rhesus and human CD40 to NK003 was detected by flow cytometry, and the cross-reactivity of NK003 with rhesus and human CD40 was determined.
  • the CDS regions of rhesus monkey CD40 (NCBI, NM_001265862.1) and human CD40 (NCBI, NM_001250.5) were cloned into pCDH vector, and were transiently transfected into 293FT cells with PEI using standard procedures. After 6 hours of staining, the medium was changed and cultured in DMEM medium containing 10% fetal bovine serum (Biological Industries, 04-001-1A) at 37°C. After 48 hours of expression, cells expressing rhesus monkey CD40 and cells expressing human CD40 were obtained. .
  • NK003 diluted to different concentrations with PBS prepared as described in Example 4: 0.001 ⁇ g/ml, 0.005 ⁇ g /ml, 0.01 ⁇ g/ml, 0.05 ⁇ g/ml, 0.1 ⁇ g/ml, 0.5 ⁇ g/ml, 1 ⁇ g/ml, 5 ⁇ g/ml and 10 ⁇ g/ml, HEL 10 ⁇ g/ml as a negative control, the cells and different concentrations of NK003 4 Incubate at °C for 30 min, wash with PBS three times, add Alexa Fluro 488 goat anti-human Fc fluorescent secondary antibody (Lifetechnologies, A11013) at 1:100, incubate at 4°C for 30 min in the dark, and analyze by flow cytometer.
  • GraphPad Prism 6.0 was used to fit the curve and calculate the EC50. The results are shown in Figure 8. It can be seen that NK003 cross-reacts with rhesus CD40 (Figure 8B) and human CD40 ( Figure 8A). The EC50 combined with rhesus CD40 is 8nM , And human CD40 binding EC50 is 10nM.
  • Lymphoma cells Raji and Ramos have CD40 expression on the membrane surface.
  • Raji cells ATCC, CRL-7936
  • Ramos cells ATCC, CRL-1596
  • Raji and Ramos cells were seeded on a 24-well plate at a density of 3 ⁇ 10 5 /mL, cultured in RPMI 1640 medium containing 10% fetal bovine serum (Lifetechnologies, 10091148), and the cells were seeded at the same time as in Example 4 above
  • the prepared PBS is diluted to different concentrations of NK003: 0.03 ⁇ g/ml, 0.06 ⁇ g/ml, 0.09 ⁇ g/ml, 0.3 ⁇ g/ml, 0.6 ⁇ g/ml, 0.9 ⁇ g/ml, 3 ⁇ g/ml, 6 ⁇ g/ml and 9 ⁇ g/ml, HEL9 ⁇ g/ml were used as negative controls, and goat anti-human Fc was added as a crosslinking agent at a concentration of 2.5 ⁇ g/ml to each well.
  • NK003 promoted the apoptosis of Raji with an EC50 of 2.6 nM ( Figure 9A); NK003 promoted the apoptosis of Ramos cells with an EC50 of 3 nM ( Figure 9B).
  • PBMC Human peripheral blood mononuclear cells
  • NK003 to activate dendritic cells
  • the isolation of DC cells is a conventional method, specifically as follows: PBMC (Hemacare, PB009C-3) is cultured in RPMI 1640 medium containing 10% fetal bovine serum (Lifetechnologies, 10091148) at 37°C, and adherent mononuclei are harvested 6 hours later Cells; remove the supernatant, add serum-free RPMI1640 medium containing 100ng/mL GM-CSF (R&D Systems, 204-IL) and 10ng/mL IL4 (R&D Systems, 215-GM), and culture at 37°C; three days later Replace half of the medium, supplement GM-CSF and IL4 to 100ng/mL and 10ng/mL; on the sixth day of culture, change the suspended cells to a new culture flask, and use the same 100ng/mL GM-CSF and 10ng /mL
  • the DC cells obtained by the above induction were seeded in a 96-well plate at 1 ⁇ 10 5 cells/well, and different concentrations of NK003 were added: 0.01 ⁇ g/ml, 0.05 ⁇ g/ml, 0.1 ⁇ g/ml, 0.5 ⁇ g/ml, 1 ⁇ g/ml, 10 ⁇ g/ml and 100 ⁇ g/ml, 100 ⁇ g/ml HEL were used as negative controls; for cross-linking, each group was added 2.5 ⁇ g/ml goat anti-human Fc at the same time.
  • NK003 can significantly activate human DC cells, with an EC50 of 2nM ( Figure 10A), and an EC50 of 8nM after adding crosslinker (goat anti-human Fc, anti-Fc) ( Figure 10B) And the cross-linked form of NK003 activates DC with a significantly stronger strength than uncross-linked (MFI is significantly higher).
  • PBMC Human peripheral blood mononuclear cells
  • CD19 immunomagnetic bead kit (Miltenyi Biotec, 130-050-301) was used to sort CD19 + B cells from human PBMC (Hemacare, PB009C-3) according to the kit instructions.
  • the B cells obtained by the above sorting were cultured in RPMI1640 medium containing 10% fetal bovine serum (Lifetechnologies, 10091148), and 10ng/mL IL4 was added. Inoculate B cells in a 96-well plate at a rate of 1 ⁇ 10 5 cells/well, and add different concentrations of NK003: 0.01 ⁇ g/ml, 0.05 ⁇ g/ml, 0.1 ⁇ g/ml, 0.5 ⁇ g/ml, 1 ⁇ g/ml, 10 ⁇ g /ml and 100 ⁇ g/ml, 100 ⁇ g/ml HEL as a negative control.
  • NK003 can promote the proliferation of human B cells with an EC50 of 4 nM.
  • PBMC peripheral blood mononuclear cells
  • CD19 + B cells were sorted from human PBMC using CD19 immunomagnetic bead kit.
  • the B cells obtained by the above sorting were cultured in RPMI 1640 medium containing 10% fetal bovine serum (Lifetechnologies, 10091148). Inoculate B cells in a 96-well plate at a rate of 1 ⁇ 10 5 cells/well, and add different concentrations of NK003: 0.01 ⁇ g/ml, 0.05 ⁇ g/ml, 0.1 ⁇ g/ml, 0.5 ⁇ g/ml, 1 ⁇ g/ml, 10 ⁇ g /ml and 100 ⁇ g/ml, 100 ⁇ g/ml HEL as a negative control.
  • EC50 is 70Nm without crosslinking agent (goat anti-human Fc, anti-Fc) ( Figure 12A), and EC50 is 4nM after crosslinking agent ( Figure 12A). 12B), and the cross-linked form of NK003 activates B cells with significantly stronger strength than uncross-linked (significantly higher CD86 expression).
  • Antibody-dependent cell-mediated cytotoxicity refers to that the Fab fragment of an antibody binds to the epitope of tumor cells, and its Fc fragment binds to the FcR on the surface of killer cells (NK cells, macrophages, etc.) to mediate killing The cell directly kills the target cell.
  • NK cells killer cells
  • SCID mice to inoculate tumor cells expressing CD40.
  • SCID mice Eight five-week-old male SCID mice were purchased from Weitong Lihua and raised in an SPF animal room. After one week of feeding, they were randomly divided into two groups. The back was inoculated with Raji cells subcutaneously in the number of 2.5 ⁇ 10 6 cells per mouse. Four cells in each group were inoculated. The day of inoculation was recorded as Day 0. The first group was administered NK003 antibody, and the second group was administered HEL control antibody.
  • NK003 activates the immune system in CD40 humanized mice
  • the CD40 humanized mouse model was constructed and provided by Li Fubin of Shanghai Jiaotong University, and was kept in an SPF animal room.
  • OT-1 mice were provided by Li Fubin of Shanghai Jiaotong University and were kept in an SPF animal room. The OT-1 mice were killed by cervical dislocation and their spleens were removed, and the spleen was crushed to collect spleen cells.
  • Use the immunomagnetic bead kit (R&D Systems, MAGM203) to isolate CD8 + T cells according to the kit instructions, that is, OT-1CD8 + T cells.
  • mice Eight-week-old male CD40 humanized mice were selected to be injected with OT-1 CD8 + T cells in the tail vein at the number of 2 ⁇ 10 6 /mouse, and 0.1 mg/kg DEC-OVA (Sigma-Aldrich, SAB4700735) and 5mg/kg NK003 (the same dose of HEL is the control antibody). Seven days after cell inoculation, the mice were sacrificed by cervical spine method to separate their spleens. Grind the spleen to obtain a single cell suspension.
  • CD45.1 + CD8 + TCR-V ⁇ 2 + subgroup is OT-1CD8 + T cells (OT1 cells).
  • the CD45.1 + CD8 + subpopulation is CD8 cells, and the CD45.1 + CD4 + subpopulation is CD4 cells.
  • the results are shown in Figure 14. The proportion of OT-1CD8 + T cells in the NK003 group increased significantly, the number of cells increased significantly, and the ratio of CD8 to CD4 increased correspondingly. NK003 can activate CD40 humanized mouse immune system.
  • NK003 inhibits tumor growth in CD40 humanized mice
  • CD40 humanized mice constructed and provided by Li Fubin of Shanghai Jiaotong University as described in 7.2 to carry the tumor cell MC38 (Institute of Basic Medicine, Chinese Academy of Medical Sciences, Basic Medical Cell Center, 3111C0001CCC000523) inoculation.
  • the positive control anti-CD40 antibody CP870893 sequence used herein comes from patent US 7338660 (see SEQ ID NO: 46 and SEQ ID NO: 48 for light chain and heavy chain sequences). Transfect the light chain and heavy chain of CP870893 synthesized by Jin Weizhi into pFUSE vector, and transiently co-transfect 293F suspension cells at 1:1 to express the full-length antibody. After 1 week of expression, use Superdex TM 200 Increase pre-installed on the AKTA system Column purification (the specific steps are the same as those described in Example 4).
  • mice Eight-week-old male CD40 humanized mice were subcutaneously inoculated with MC38 cells at the back of 2 ⁇ 10 6 /mouse.
  • Tumor inhibition rate (average volume of control group-average volume of experimental group)/average volume of control group ⁇ 100%.
  • NK003-V12, NK003-S267E/L328F Fc region mutants increase binding to Fc ⁇ RIIB and CD40 agonist activity
  • glutamic acid (E) at residue 233 in the Fc region of NK003 was mutated to aspartic acid (D)
  • residue at 237 Glycine (G) was mutated to aspartic acid (D)
  • histidine (H) at residue 268 was mutated to aspartic acid (D)
  • proline (P) at residue 271 was mutated to glycine (G)
  • the 330th residue alanine (A) was mutated to arginine (R), resulting in the NK003-V12 mutant.
  • NK003Fc region IgG1, EU numbering
  • E glutamic acid
  • L 328th residue Leucine
  • F phenylalanine
  • the light and heavy chains of the NK003-V12 and NK003-S267E/L328F mutants synthesized by Jin Weizhi were transfected into the pFUSE vector according to 1: 1 Transient co-transfection of 293F suspension cells to express the full-length antibody, and after one week of expression, it was purified on the AKTA system using a Superdex TM 200Increase prepacked column (the specific steps are the same as those described in Example 4).
  • the Fc ⁇ RIIA (NCBI, NM_001136219.1) and Fc ⁇ RIIB (NCBI, NM_004001.4) CDS regions synthesized by Jin Weizhi were cloned into pCDH vector, and transiently transfected into 293FT cells with PEI using standard procedures, and the medium was changed after 6 hours of transfection. It was cultured in DMEM medium containing 10% fetal bovine serum (Biological Industries, 04-001-1A) at 37°C. After 48 hours of expression, cells expressing Fc ⁇ RIIA and 293FT cells expressing Fc ⁇ RIIB were obtained.
  • NK003, NK003-V12 and NK003-S267E/L328F were used as negative controls; after co-cultivation at 37°C for 24 hours, washed with PBS three times and analyzed by flow cytometry to detect the MFI of GFP. The results are shown in Figure 16. Compared with wild-type NK003, NK003-V12, NK003-S267E/L328F have significantly enhanced agonist activity.
  • a mutation library was constructed for the complementarity determining regions and framework regions of NK003 VH and VL, and inserted into the pcomb3 phage vector (Biovector Inc. 108925) in the form of Fab ( Figure 17).
  • the phage affinity maturation method was used to complete the optimization of NK003.
  • the light chain VL and CL fragments were amplified using the primer pair VL1/VL2, CL1/CL2, respectively. Reaction conditions: 95°C for 2min, 1cycle; 95°C for 30s, 65°C for 30s, 72°C for 10s, 35cycle; 72°C for 5min, 1cycle. Use Tiangen Recovery Kit to recover PCR target fragments.
  • the primers are as follows:
  • VL1 5’-GCGGCCGAGCTCGATGTTGTGATGACTCAG-3’
  • VL2 5'-GGTCCCCTGGCCAAA AGT GTA CGG AGT TCA TAG ACC TTG CAT
  • NK003 light chain mutation library The above two parts of PCR products were subjected to overlap PCR amplification to obtain the NK003 light chain mutation library. Reaction conditions: 95°C 2min, 1cycle; 95°C 30s, 65°C 30s, 72°C 20s, 8cycle; adding VL1/CL2 primer, 95°C 30s, 65°C 30s, 72°C 20s, 27cycle; 72°C 5min, 1cycle. SacI (NEB, R3156L) and PacI (NEB, R0547L) were used to digest the NK003 light chain mutation library and pcomb3 vector with double restriction enzymes, and the Tiangen recovery kit was used to recover the PCR target fragments.
  • the mutation library gene and the carrier were ligated at a molar ratio of 3:1 with T4 ligase (NEB, M0202L) at 25°C for 3 hours.
  • the ligation product was transferred to the XL1-Blue electroporation competence by electroporation method, and the library capacity was 5 ⁇ 10 5 light chain mutation library, named pcomb3-NK003-LCDR3.
  • VH1/VH2 and CH1-1/CH1-2 heavy chain fragments were amplified using the primer pairs VH1/VH2, CH1-1/CH1-2, respectively. Reaction conditions: 95°C for 2min, 1cycle; 95°C for 30s, 65°C for 30s, 72°C for 10s, 35cycle; 72°C for 5min, 1cycle.
  • the primers are as follows:
  • VH1 5’-TGCAGCTGCTCGAGCAGGTACAGCTGGTGCAGTC-3’
  • VH2 5'-CTTTGCCCCAGACGTC CAT GTA GTA GTA GTA GTA GTA GGT TCA AGT AGC TCC CAC TCT TTC TCTCGCACAG-3' (two random mutations at the horizontal line to MNN, high-throughput synthesis by Jinweizhi)
  • CH1-1 5’-GACGTCTGGGGCAAAGGGACCACGGTC-3’
  • the above two parts of PCR products were amplified by overlap PCR to obtain the NK003 heavy chain mutation library.
  • the reaction conditions 95°C 2min, 1cycle; 95°C 30s, 65°C 30s, 72°C 20s, 8cycle; add VH1/CH1-2 primer, 95 °C30s, 65°C30s, 72°C20s, 27cycle; 72°C5min, 1cycle.
  • SpeI NEB, R3133L
  • XhoI NEB, R0146L
  • the mutant library gene and the carrier were ligated at a molar ratio of 3:1 with T4 ligase at 25°C for 3h.
  • the ligation product was transferred to XL1-Blue electrocompetence by electroporation, and the library capacity was 2.4 ⁇ 10 7 light and heavy chain double mutation library, named pcomb3-NK003-AM.
  • the phage eluted in the third round was infected with XL1-blue, and then coated with a plate. After mixing with a scraper, the NK003-AM plasmid was extracted using the Tiangen plasmid small extraction kit. After double digestion with SacI and SpeI, the target fragment of about 1500 bp was recovered and sent to Jinweizhi for third-generation sequencing.
  • the sequencing results are shown in Figure 19.
  • the heavy chain and light chain DNA of antibodies NK003-AM-9 and NK003-AM-18 (Jin Weizhi) selected were synthesized and cloned into the vector pFUSE.
  • the specific method of purification and expression is shown in Example 4.
  • flow cytometry was used to detect the activation of the NF- ⁇ B-GFP+hCD40 reporter cell line by NK003-AM-9 and NK003-AM-18 antibodies.
  • the specific steps are as follows:
  • NK003-AM-9, NK003-AM-18 and NK003 0.01 ⁇ g/ml, 0.05 ⁇ g/ml, 0.1 ⁇ g/ml, 0.5 ⁇ g/ml and 1 ⁇ g/ml; for cross-linking, each group was added with a 2.5 ⁇ g/ml secondary antibody at the same time, namely goat anti-human Fc (SouthernBiotech, SBA-2048-01).
  • NK003-AM-9 and NK003-AM-18 activate the NF- ⁇ B-GFP+hCD40 reporter cell line in a cross-linked form.
  • the EC50 is 0.25nM and 0.3nM, respectively. The activities of the two are similar, and both are more similar than the wild type. NK003 improved by nearly 10 times.
  • NK003 light chain DNA (SEQ ID NO: 74) as template, using random mutation PCR kit (Agilent Technologies, 200550) and primer pairs VL1/VL2, VL3/VL4 to carry out Nested PCR amplification of light chain VL region, reaction conditions :95°C2min, 1cycle; 95°C30s, 65°C30s, 72°C30s, 28cycle; 72°C10min, 1cycle.
  • primer pair CL1/CL2 was used to amplify light chain CL region, reaction conditions: 95°C 2min, 1cycle; 95°C 30s, 65°C 30s, 72°C 30s, 30cycle ; 72°C5min, 1cycle.
  • the primers are as follows:
  • VL1 5’-CTATCGCGATTGCAGTGGCACTGGCTG-3’
  • VL2 5’-CCAGATTTCAACTGCTCATCAGATGGC-3’
  • VL3 5’-CTACCGTGGCCCAGGCGGCCGAGCTC-3’
  • VL4 5’-GAAGACAGATGGTGCAGCCACAGTTCG-3’
  • the VL and CL PCR products were subjected to overlap PCR amplification to obtain the NK003 light chain mutation library. Reaction conditions: 95°C for 2min, 1cycle; 95°C for 30s, 65°C for 30s, 72°C for 40s, 8cycle; adding VL3/CL2 primer, 95°C for 30s, 65°C for 30s, 72°C for 40s, 27cycle; 72°C for 10min, 1cycle.
  • the NK003 light chain mutation library and pcomb3 vector were double digested with SacI and PacI, and the target fragments of PCR were recovered using the Tiangen recovery kit.
  • the mutant library gene and the carrier were ligated at a molar ratio of 3:1 with T4 ligase at 25°C for 3h.
  • the ligation product was transferred to XL1-Blue electrocompetent state by electroporation, and the library capacity was 1 ⁇ 10 5 light chain mutation library, named pcomb3-NK003-VL.
  • NK003 heavy chain DNA (SEQ ID NO: 75) as a template, using primer pairs VH1/VH2, VH3/VH4 to perform Nested PCR amplification of the heavy chain VH region, reaction conditions 3: 95°C 2min, 1 cycle; 95°C 30s, 65°C30s, 72°C30s, 28cycle; 72°C10min, 1cycle.
  • the primer pair CH1-1/CH1-2 was used to amplify the heavy chain CH1 region, reaction conditions: 95°C 2min, 1 cycle; 95°C 30s, 65°C 30s, 72 °C30s, 32cycle; 72°C10min, 1cycle.
  • Use Tiangen Recovery Kit to recover PCR target fragments.
  • the primers are as follows:
  • VH1 5’-GCCGCTGGATTGTTATTACTCGCTGC-3’
  • VH2 5’-CAGAGGTGCTCTTGGAGGAGGGTGCC-3’
  • VH3 5’-GCCATGGCCGAGGTGCAGCTGCTCGAG-3’
  • VH4 5’-GAAGACCGATGGGCCCTTGGTGGAGGC-3’
  • CH1-1 5’-GCCTCCACCAAGGGCCCATCGGTCTTC-3’
  • the above-mentioned VH and CH1 PCR products were subjected to overlap PCR amplification to obtain the NK003 heavy chain mutation library.
  • Reaction conditions 95°C2min, 1cycle; 95°C30s, 65°C30s, 72°C40s, 8cycle; adding VH3/CH1-2 primer, 95°C30s, 65°C30s, 72°C40s, 27cycle; 72°C10min, 1cycle .
  • the NK003 heavy chain mutation library and pcomb3-NK003-VL vector were double-enzyme digested with SpeI and XhoI, and the target fragments of PCR were recovered using Tiangen Recovery Kit.
  • the mutant library gene and the carrier were ligated at a molar ratio of 3:1 with T4 ligase at 25°C for 3h.
  • the ligated product was transferred to XL1-Blue electrocompetent state by electroporation method, and the library capacity was 2.4 ⁇ 10 7 light and heavy chain double mutation library, named pcomb3-NK003-EP.
  • the phage eluted in the third round was infected with XL1-blue, and then coated with a plate. After mixing with a scraper, the NK003-EP plasmid was extracted using the Tiangen plasmid small extraction kit. After double digestion with SacI and SpeI, the target fragment of about 1500 bp was recovered and sent to Jinweizhi for third-generation sequencing. The sequencing results are shown in Figure 21. According to the results of the third-generation sequencing, the antibody NK003-AM-18-EP1 heavy chain DNA (Jin Weizhi) was synthesized and cloned into the vector pFUSE.
  • NK003-AM-18-EP1 The plasmids containing the light chain of NK003-AM-18 and the heavy chain of NK003-AM-18-EP1 were transiently co-transfected into 293F suspension cells with PEI using standard procedures to express the full-length NK003-AM- 18-EP1 antibody. See Example 4 for specific methods of purification and expression. See sequence list 6 for NK003-AM-18-EP1 VH sequence.
  • flow cytometry was used to detect the activation of the NF- ⁇ B-GFP+hCD40 reporter cell line by the NK003-AM-18-EP1 antibody.
  • the specific steps are as follows:
  • NK003-AM-18, NK003-AM-18-EP1 and NK003 0.0075nM, 0.025 nM, 0.075nM, 0.25nM, 0.75nM, 2.5nM and 7.5nM; for cross-linking, each group was added with a 2.5 ⁇ g/ml secondary antibody at the same time, namely goat anti-human Fc.
  • NK003-AM-18-EP1 activates the NF- ⁇ B-GFP+hCD40 reporter cell line in a cross-linked form with an EC50 of 0.35 nM, which is similar to the activity of NK003-AM-18.

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Abstract

提供了特异性结合CD40的新型抗体和抗体片段以及含有所述抗体或抗体片段的组合物、药物、组合产品和试剂盒。此外，还提供了编码所述抗体或其抗体片段的核酸及包含其的宿主细胞，以及相关用途。此外，还提供了这些抗体和抗体片段的治疗和诊断用途。

Description

抗CD40抗体及其用途
本发明涉及特异性结合CD40的新型抗体和抗体片段以及含有所述抗体或抗体片段的组合物，以及药物、组合产品或试剂盒。此外，本发明涉及编码所述抗体或其抗体片段的核酸及包含其的宿主细胞，以及相关用途。此外，本发明涉及这些抗体和抗体片段的治疗和诊断用途。
背景技术
T细胞的完全激活需要两个信号：抗原呈递细胞(APC)对肿瘤抗原进行摄取、加工，形成MHC-抗原复合物，呈递给T细胞并与T细胞表面的TCR结合，这是T细胞活化的第一信号；第二或共刺激信号则是通过CD28与B7-1(CD80)/B7-2(CD86)，以及共刺激因子CD40、OX40、GITR等与其配体相互作用来传递(Smith-Garvin，J.E.，G.A.Koretzky，and M.S.Jordan，T cell activation.Annu Rev Immunol，2009.27：p.591-619)。在缺乏共刺激信号时，T细胞可能发生无应答性(无反应力)或抗原刺激后的细胞程序性死亡(细胞凋亡)。
CD40是TNF受体(TNFR)超家族的成员，主要表达在B细胞、树突状细胞(DC细胞)、单核细胞和巨噬细胞等多种抗原呈递细胞上(Grewal，I.S.and R.A.Flavell，CD40 and CD154 in cell-mediated immunity.Annu Rev Immunol，1998.16：p.111-35)。CD40在细胞表面形成三聚体，相应配体CD40L(即CD154)主要表达于激活的T细胞表面。CD40和CD40L的相互作用是T细胞活化的共刺激信号。根据特定的细胞类型，CD40参与作用导致特定的基因表达模式。T细胞上CD40L与CD40的结合可以激活多种通路，包括NF-κB(核因子κB)，MAPK(裂原活化蛋白激酶)和STAT3(信号转导子和转录激活子3)等(Rothe，M.，et al.，TRAF2-mediated activation ofNF-kappa B by TNF receptor 2and CD40.Science，1995.269(5229)：p.1424-7)。
CD40不仅由正常的免疫细胞表达，同时也由许多恶性细胞表达。具体而言，CD40在以下疾病中过表达：B系NHL、慢性淋巴细胞性白血病(CLL)、多毛细胞白血病(HCL)、霍奇金病、多发性骨髓瘤以及膀胱癌、肾癌、卵巢癌、宫颈癌、乳腺癌、肺癌、鼻咽癌和恶性黑色素瘤等(Hassan，S.B.，et al.，Anti-CD40-mediated cancer immunotherapy：an update of recent and ongoing clinical trials.Immunopharmacol Immunotoxicol，2014.36(2)：p.96-104)。
CD40激动剂抗体可以通过多种机制对抗肿瘤细胞：第一，CD40激动剂抗体通过激活免疫系统，介导更强的抗肿瘤效应。具体而言，CD40激动剂抗体可以激活DC细胞增加其抗原递呈能力，这表现在共刺激分子，如B7家族(CD80，CD86)的表达增加，以及促进细胞因子分泌，如白细胞介素12，这将导致显著的T细胞应答(Fong，L.and E.G.Engleman，Dendritic cells in cancer immunotherapy.Annu Rev Immunol，2000.18：p.245-73)；CD40激动剂抗体可以促进静息B细胞的增殖、免疫球蛋白类别转变、抗体分泌，并且对生发中心的发展和记忆性B细胞的存活都有影响，所有这一切都是体液免疫应答必不可少的(Beatty，G.L.，Y.Li，and K.B.Long，Cancer immunotherapy：activating innate and adaptive immunity through CD40 agonists.Expert Rev Anticancer Ther，2017.17(2)：p.175-186)。第二，CD40激动剂抗体与肿瘤细胞表面表达的CD40结合后，介导抗体依赖性细胞毒性(ADCC)作用，由杀伤细胞直接清除高表达CD40的肿瘤细胞(Vonderheide，R.H.and M.J.Glennie，Agonistic CD40 antibodies and cancer therapy.Clin Cancer Res，2013.19(5)：p.1035-43)。第三，CD40激动剂抗体与肿瘤细胞表面表达的CD40结合后，直接抑制肿瘤的生长并促进其凋亡，例如CD40/CD40L信号通路可阻断 肿瘤细胞的细胞周期，使细胞停止于G2-M期，CD40/CD40L的相互反应还可促进肿瘤细胞表面Fas表达上升，通过Fas/FasL信号通路抑制高表达CD40肿瘤细胞生长并促进其凋亡(Eliopoulos，A.G.，et al.，CD40 induces apoptosis in carcinoma cells through activation of cytotoxic ligands of the tumor necrosis factor superfamily.Mol Cell Biol，2000.20(15)：p.5503-15)。
CD40的激动剂抗体按照其激动方式不同可分两类，第一类CD40激动活性不依赖Fc受体的交联(如CP-870893和CDX-1140)，尽管前者显示出令人鼓舞的抗癌疗效，但是存在剂量限制性毒性，会导致全身免疫功能的紊乱、静脉血栓性栓塞症和细胞因子释放综合征(Cytokine Release Syndrome，CRS)的发生；另一类需要Fc受体交联才具有CD40激动活性。肿瘤组织及周围引流淋巴结有更多肿瘤相关炎性细胞的存在，FcγR2b受体在肿瘤细胞周围有更多聚集。因此，这样的“交联抗体”激动剂就具有了更高的组织选择性，在肿瘤微环境中抗体才能产生明显的激动作用，而在身体正常组织部位，作用能力会保持在低水平，这样就能提高治疗的安全窗。
因此，尽管在本领域中已存在一些CD40抗体，例如辉瑞制药的CP870893，但是仍然需要一些新的具有与现有抗体相当或更佳性质的CD40抗体，特别是依赖于Fc受体交联的CD40抗体，尤其是具有更佳抗癌性质且安全性更高的抗体。
发明内容
本发明因此提供了一种新的结合CD40(特别是人CD40或恒河猴CD40)的抗体，以及其抗原结合片段。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段包含重链可变区(VH)，其中所述VH包含
(i)如SEQ ID NO：13、58、60、62或14所示的VH中所含的三个互补决定区域(CDR)HCDR1、HCDR2和HCDR3，或
(ii)相对于(i)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列，或
(iii)相对于(i)的序列，在HCDR3上包含至少一个且不超过3、2或1个氨基酸改变(优选地氨基酸置换，优选地保守置换)。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段包含轻链可变区(VL)，其中所述VL包含：
(i)如SEQ ID NO：15、64、66或16所示的VL中所含的三个互补决定区域(CDR)LCDR1、LCDR2和LCDR3；或
(ii)相对于(i)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列，或
(iii)相对于(i)的序列，在LCDR3上包含至少一个且不超过3、2或1个氨基酸改变(优选地氨基酸置换，优选地保守置换)。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段包含重链可变区VH和轻链可变区VL，其中
(a)所述VH包含
(i)如SEQ ID NO：13、58、60、62或14所示的VH中所含的三个互补决定区域(CDR)HCDR1、HCDR2和HCDR3，或
(ii)相对于(i)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列；
(iii)相对于(i)的序列，在HCDR3上包含至少一个且不超过3、2或1个氨基酸改变(优选地氨基酸置换，优选地保守置换)；和/或
(b)所述VL包含：
(i)如SEQ ID NO：15、64、66或16所示的VL中所含的三个互补决定区域(CDR)LCDR1、LCDR2和LCDR3；或
(ii)相对于(i)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列；或
(iii)相对于(i)的序列，在LCDR3上包含至少一个且不超过3、2或1个氨基酸改变(优选地氨基酸置换，优选地保守置换)。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段包含重链可变区VH和/或轻链可变区VL，其中
(a)所述VH包含
(i)如SEQ ID NO：13、58、60或62所示的VH中所含的三个互补决定区域(CDR)HCDR1、HCDR2和HCDR3，或
(ii)相对于(i)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列；
(iii)相对于(i)的序列，在HCDR3上包含至少一个且不超过3、2或1个氨基酸改变(优选地氨基酸置换，优选地保守置换)；和/或
(b)所述VL包含：
(i)如SEQ ID NO：15、64或66所示的VL中所含的三个互补决定区域(CDR)LCDR1、LCDR2和LCDR3；或
(ii)相对于(i)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列；或
(iii)相对于(i)的序列，在LCDR3上包含至少一个且不超过3、2或1个氨基酸改变(优选地氨基酸置换，优选地保守置换)。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段包含重链可变区VH和轻链可变区VL，其中
(a)所述VH包含
(i)如SEQ ID NO：14所示的VH中所含的三个互补决定区域(CDR)HCDR1、HCDR2和HCDR3，或
(ii)相对于(i)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列；
(iii)相对于(i)的序列，在HCDR3上包含至少一个且不超过3、2或1个氨基酸改变(优选地氨基酸置换，优选地保守置换)；和/或
(b)所述VL包含：
(i)如SEQ ID NO：16所示的VL中所含的三个互补决定区域(CDR)LCDR1、LCDR2和LCDR3；或
(ii)相对于(i)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列；或
(iii)相对于(i)的序列，在LCDR3上包含至少一个且不超过3、2或1个氨基酸改变(优选地氨基酸置换，优选地保守置换)。
在优选的实施方案中，VH包含选自SEQ ID NO：13、58、60、62或14所示的氨基酸序列，或由所述氨基酸序列组成。
在优选的实施方案中，VL包含选自SEQ ID NO：15、64、66或16所示的氨基酸序列，或由所述氨基酸序列组成。
在优选的实施方案中，本发明抗CD40抗体或其抗原结合片段包含
如SEQ ID NO：13、58、60、62或14所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：15、64、66或16所示的轻链可变区的3个互补决定区LCDR。
在优选的实施方案中，本发明抗CD40抗体或其抗原结合片段包含
如SEQ ID NO：13、58、60或62所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：15、64或66所示的轻链可变区的3个互补决定区LCDR。
在优选的实施方案中，本发明抗CD40抗体或其抗原结合片段包含
如SEQ ID NO：14所示的重链可变区的3个互补决定区HCDR，以及如SEQ ID NO：16所示的轻链可变区的3个互补决定区LCDR。
在优选的实施方案中，本发明抗CD40抗体或其抗原结合片段包含：
(i)如SEQ ID NO：13所示的重链可变区的3个互补决定区HCDR1、HCDR2和HCDR3，以及如SEQ ID NO：15所示的轻链可变区的3个互补决定区LCDR1、LCDR2和LCDR3；
(ii)如SEQ ID NO：58所示的重链可变区的3个互补决定区HCDR1、HCDR2和HCDR3，以及如SEQ ID NO：64所示的轻链可变区的3个互补决定区LCDR1、LCDR2和LCDR3；
(iii)如SEQ ID NO：60或62所示的重链可变区的3个互补决定区HCDR1、HCDR2和HCDR3，以及如SEQ ID NO：66所示的轻链可变区的3个互补决定区LCDR1、LCDR2和LCDR3；
(iv)如SEQ ID NO：14所示的重链可变区的3个互补决定区HCDR1、HCDR2和HCDR3，以及如SEQ ID NO：16所示的轻链可变区的3个互补决定区LCDR1、LCDR2和LCDR3；
(v)(i)-(iv)中任一项的CDR组合，其中相比HCDR3和/或LCDR3，在所述序列上包含至少一个且不超过3、2或1个氨基酸改变(优选地氨基酸置换，优选地保守置换)。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段包含重链可变区(VH)和/或轻链可变区(VL)，其中
(i)所述VH包含互补决定区域(CDR)HCDR1、HCDR2和HCDR3，其中HCDR1包含SEQ ID NO：1或2的氨基酸序列，或由所述氨基酸序列组成，或者HCDR1包含与SEQ ID NO：1或2的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列；HCDR2包含SEQ ID NO：3或4的氨基酸序列，或由所述氨基酸序列组成，或者HCDR2包含与选自SEQ ID NO：3或4的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列；HCDR3包含SEQ ID NO：5、51、52、55或6的氨基酸序列或由所述氨基酸序列组成，或者HCDR3包含与SEQ ID NO：5、51、52、55或6的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列；
和/或
(ii)其中所述VL包含互补决定区域(CDR)LCDR1、LCDR2和LCDR3，其中LCDR1包含SEQ ID NO：7或8的氨基酸序列或由所述氨基酸序列组成，或者LCDR1包含与SEQ ID NO：7或8的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列；LCDR2包含SEQ ID NO：9或10的氨基酸序列或由所述氨基酸序列组成，或者LCDR2包含与SEQ ID NO：9或10的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列；LCDR3包含选自SEQ ID NO：11、53、54、56或12的氨基酸序列或由所述氨基酸序列组成，或者LCDR3包含与SEQ ID NO：11、53、54、56或12的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在优选的实施方案中，本发明提供抗CD40抗体或其抗原结合片段，其包含重链可变区(VH)和轻链可变区(VL)，其中
(a)所述VH包含
(i)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：5；或者
(ii)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：51；或者
(iii)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：52；或者
(iv)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：2、SEQ ID NO：4和SEQ ID NO：6；或者
(v)(i)-(iv)中任一项的HCDR组合，其中在所述三个CDR区上共包含至少一个且不超过 5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)；或者
(vi)(i)-(iv)中任一项的HCDR组合，其中在所述HCDR3包含至少一个且不超过3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)；
和/或
(b)所述VL包含
(i)包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：11；或者
(ii)包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：53；或者
(iii)包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：54；或者
(iv)包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：8、SEQ ID NO：10和SEQ ID NO：12；或者
(v)(i)-(iv)中任一项的LCDR组合，其中在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)；或者
(vi)(i)-(iv)中任一项的LCDR组合，其中在所述HCDR3包含至少一个且不超过3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)。
在优选的实施方案中，本发明提供抗CD40抗体或其抗原结合片段，其包含重链可变区(VH)和轻链可变区(VL)，其中
(a)所述VH包含
(i)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：5；或者
(ii)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：51；或者
(iii)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：52；或者
(iv)(i)-(iii)中任一项的HCDR组合，其中在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)；或者
(v)(i)-(iii)中任一项的HCDR组合，其中在所述HCDR3包含至少一个且不超过3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)；
和/或
(b)所述VL包含
(i)包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：11；或者
(ii)包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：53；或者
(iii)包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：54；或者
(iv)(i)-(iii)中任一项的LCDR组合，其中在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)；或者
(v)(i)-(iii)中任一项的LCDR组合，其中在所述LCDR3包含至少一个且不超过3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)。
在优选的实施方案中，本发明提供抗CD40抗体或其抗原结合片段，其包含重链可变区(VH)和轻链可变区(VL)，其中
(a)所述VH包含
(i)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：2、SEQ ID NO：4和SEQ ID NO：6；或者
(ii)(i)的HCDR组合，其中在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)；或者
(iii)(i)中的HCDR组合，其中在所述HCDR3包含至少一个且不超过3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)；
和/或
(b)所述VL包含
(i)包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：8、SEQ ID NO：10和SEQ ID NO：12；或者
(ii)(i)中LCDR组合，其中在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)；或者
(iii)(i)中的LCDR组合，其中在所述LCDR3包含至少一个且不超过3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)。
在优选的实施方案中，本发明提供抗CD40抗体或其抗原结合片段，其包含
(i)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：5，以及包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：11；或者
(ii)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：51，以及包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：53；或者
(iii)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：52，以及/或者包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：54；或者
(iv)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：2、SEQ ID NO：4和SEQ ID NO：6，以及包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：8、SEQ ID NO：10和SEQ ID NO：12；
(v)(i)-(iv)中任一项的HCDR和LCDR组合，其中在所述三个HCDR区上和/或在所述三个LCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)；或者
(vi)(i)-(iv)中任一项的HCDR和LCDR组合，其中在所述HCDR3和/或LCDR3中包含至少一个且不超过3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)。
在优选的实施方案中，本发明提供抗CD40抗体或其抗原结合片段，其包含重链可变区(VH)和轻链可变区(VL)，其中
(a)所述VH包含
(i)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：55；或
(ii)(i)中的HCDR的组合，其中在所述三个HCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)；
和/或
(b)所述VL包含
(iii)包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：56；或
(iv)(i)中的LCDR的组合，其中在所述三个LCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)。
在一些实施方案中，SEQ ID NO：55所示的氨基酸序列如下：
A-R-E-R-V-G-A-X1-P-T-Y-Y-Y-X2-X3-DV，其中X1、X2或X3可以任何氨基酸，优选地，其中X1可以为T、N、W、K、A或Y，更优选的T或N；其中X2优选的为W或Y；其中X3可以为W、Y、M、F、T，更优选地为M、W或Y。
在一些实施方案中，SEQ ID NO：56所示的氨基酸序列如下：
M-X1-X2-L-X3-X4-P-Y-T，其中X1、X2、X3和X4可以为任何氨基酸，优选地，其中X1可以为Q、N或P，更优选地为Q或N；X2可以为G、Q、F、S、Y或M；更优选的为G或Q；X3可以为E、N、T、S或K；更优选地为E或N；X4可以为T、Q、V、L、P或E；更优选地为T、Q或V。
在优选的实施方案中，本发明提供抗CD40抗体或其抗原结合片段，其包含重链可变区(VH)和轻链可变区(VL)，其中所述VH包含互补决定区域(CDR)HCDR1、HCDR2和HCDR3并且所述VL包含(CDR)LCDR1、LCDR2和LCDR3，其中所述抗体或其抗原结合片段所包含的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3的组合如下表(表A)所示：
表A：本发明抗体或其抗原结合片段中HCDR1、HCDR2、HCDR3、LCDR1、LCDR2 和LCDR3的示例性组合
Figure PCTCN2020084186-appb-000001
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段包含重链可变区VH和/或轻链可变区VL，其中，
(a)重链可变区VH
(i)包含与选自SEQ ID NO：13、58、60、62或14的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者
(ii)包含选自SEQ ID NO：13、58、60、62或14的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：13、58、60、62或14的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中，优选地，所述氨基酸改变发生在FR区中；
和/或
(b)轻链可变区VL
(i)包含与选自SEQ ID NO：15、64、66或16的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO：15、64、66或16的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：15、64、66或16的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中，优选地，所述 氨基酸改变发生在FR区中。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段的重链可变区VH
(i)包含与选自SEQ ID NO：13、58、60或62的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者
(ii)包含选自SEQ ID NO：13、58、60或62的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：13、58、60或62的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中，优选地，所述氨基酸改变发生在FR区中。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段的轻链可变区VL
(i)包含与选自SEQ ID NO：15、64或66的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO：15、64或66的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：15、64或66的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中，优选地，所述氨基酸改变发生在FR区中。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段的重链可变区VH
(i)包含与选自SEQ ID NO：14的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者
(ii)包含选自SEQ ID NO：14的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：14的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中，优选地，所述氨基酸改变发生在FR区中。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段的轻链可变区VL
(i)包含与选自SEQ ID NO：16的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO：16的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：16的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中，优选地，所述氨基酸改变发生在FR区中。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段包含重链可变区VH和/或轻链可变区VL，其中，
(i)重链可变区VH包含SEQ ID NO：13的氨基酸序列或由其组成，和轻链可变区VL包含SEQ ID NO：15的氨基酸序列或由其组成；
(ii)重链可变区VH包含SEQ ID NO：58的氨基酸序列或由其组成，和轻链可变区VL包含SEQ ID NO：64的氨基酸序列或由其组成；
(iii)重链可变区VH包含SEQ ID NO：60的氨基酸序列或由其组成，和轻链可变区VL包含SEQ ID NO：66的氨基酸序列或由其组成；
(iv)重链可变区VH包含SEQ ID NO：62的氨基酸序列或由其组成，和轻链可变区VL包含SEQ ID NO：66的氨基酸序列或由其组成；
(v)重链可变区VH包含SEQ ID NO：14的氨基酸序列或由其组成，和轻链可变区VL包含SEQ ID NO：16的氨基酸序列或由其组成。
在一些实施方案中，本发明的抗体的重链可变区的氨基酸序列与上文所述的(i)-(v)中重链可变区的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，或者其氨基酸序列相比上文所述的(i)-(v)中重链可变区的氨基酸序列，包含1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中，优选地，所述氨基酸改变发生在FR区中。
在一些实施方案中，本发明的抗体的轻链可变区的氨基酸序列与上文所述的(i)-(v)中轻链可变区的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，或者其氨基酸序列相比上文所述的(i)-(v)中轻链可变区的氨基酸序列，包含1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中，优选地，所述氨基酸改变发生在FR区中。
在优选的实施方案中，本发明提供抗CD40抗体或其抗原结合片段，其包含重链可变区(VH)和轻链可变区(VL)，其中所述抗体或其抗原结合片段所包含的重链可变区VH和轻链可变区VL的组合如下表(表B)所示：
表B：本发明抗体或其抗原结合片段中重链可变区VH和轻链可变区VL的示例性组合
Figure PCTCN2020084186-appb-000002
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段包含重链和/或轻链，其中
(a)重链
(i)包含与选自SEQ ID NO：17、18、19、67、69、70或20的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO：17、18、19、67、69、70或20的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：17、18、19、67、69、70或20的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在重链的CDR区中，更优选地，所述氨基酸改变不发生在重链可变区中，最优选地，所述氨基酸改变发生在重链恒定区中；
和/或
(b)轻链
(i)包含与选自SEQ ID NO：21、68、71或22的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO：21、68、71或22的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：21、68、71或22的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在轻链的CDR区中，更优选地，所述氨基酸改变不发生在轻链可变区中，最优选的，所述重链氨基酸改变发生在轻链恒定区中。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段包含的重链
(i)包含与选自SEQ ID NO：17、18、19、67、69或70的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO：17、18、19、67、69或70的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：17、18、19、67、69或70的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在重链的CDR区中，更优选地，所述氨基酸改变不发生在重链可变区中，最优选的，所述重链氨基酸改变发生在重链恒定区中。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段包含的轻链
(i)包含与选自SEQ ID NO：21、68或71的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO：21、68或71的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：21、68或71的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在轻链的CDR区中，更优选地，所述氨基酸改变不发生在轻链可变区中，最优选的，所述重链氨基酸改变发生在轻 链恒定区中。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段包含的重链
(i)包含与选自SEQ ID NO：20的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO：20的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：20的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在重链的CDR区中，更优选地，所述氨基酸改变不发生在重链可变区中，最优选的，所述重链氨基酸改变发生在重链恒定区中。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段包含的轻链
(i)包含与选自SEQ ID NO：22的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
(ii)包含选自SEQ ID NO：22的氨基酸序列或由其组成；或者
(iii)包含与选自SEQ ID NO：22的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在轻链的CDR区中，更优选地，所述氨基酸改变不发生在轻链可变区中，最优选的，所述重链氨基酸改变发生在轻链恒定区中。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段包含重链和/或轻链，其中，
(i)重链包含SEQ ID NO：17、18、19、67、69或70的氨基酸序列或由其组成，和轻链包含SEQ ID NO：21、68或71的氨基酸序列或由其组成，
(ii)重链包含SEQ ID NO：17、18或19的氨基酸序列或由其组成，和轻链包含SEQ ID NO：21的氨基酸序列或由其组成；
(iii)重链包含SEQ ID NO：67的氨基酸序列或由其组成，和轻链包含SEQ ID NO：68的氨基酸序列或由其组成；
(iv)重链包含SEQ ID NO：69或70的氨基酸序列或由其组成，和轻链包含SEQ ID NO：71的氨基酸序列或由其组成；
(v)重链包含SEQ ID NO：20的氨基酸序列或由其组成，和轻链包含SEQ ID NO：22的氨基酸序列或由其组成。
在一些实施方案中，本发明抗体的重链的氨基酸序列与上述(i)-(v)中的重链的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，或其氨基酸序列与上述(i)-(v)中的重链氨基酸序列相比，包含1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在重链的CDR区中，更优选地，所述氨基酸改变不发生在重链可变区中，最优选的，所述氨基酸改变发生在重链的恒定区中。
在一些实施方案中，本发明抗体的轻链的氨基酸序列与上述(i)-(v)中的重链的氨基 酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，或其氨基酸序列与上述(i)-(v)中的轻链氨基酸序列相比，包含1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在轻链的CDR区中，更优选地，所述氨基酸改变不发生在轻链可变区中，最优选的，所述氨基酸改变发生在轻链的恒定区中。
在优选的实施方案中，本发明提供抗CD40抗体或其抗原结合片段，其包含重链和轻链，其中所述抗体或其抗原结合片段所包含的重链和轻链的组合如下表(表C)所示：
表C：本发明抗体或其抗原结合片段中重链和轻链的示例性组合
Figure PCTCN2020084186-appb-000003
在一些实施方案中，本发明抗CD40抗体或其片段的重链和/或轻链还包含信号肽序列，例如所述信号肽序列包含SEQ ID NO：43所示的氨基酸序列或由其组成。
在本发明的一个实施方案中，本文所述的氨基酸改变包括氨基酸的置换、插入或缺失。优选的，本文所述的氨基酸改变为氨基酸置换，优选地保守置换。
在一些实施方案中，改变发生在抗体的重链和/或轻链的CDR区(尤其是CDR3区)中。在一些实施方案中，在CDR区，例如CDR3区中可以存在1、2或3个改变。
在优选的实施方案中，本发明所述的氨基酸改变发生在CDR外的区域(例如在FR中)。在某些实施方案中，改变发生在抗体的FR区，例如抗体重链和/或轻链可变区的FR区，例如FR1、FR2、FR2或F4区。在一些实施方案中，改变发生在FR2区。在一些实施方案中，FR区可以存在1、2或3个改变。
更优选地，本发明所述的氨基酸改变发生在重链可变区外和/或轻链可变区外的区域，例如在重链和/或轻链的恒定区中。
在一些实施方案中，置换为保守性置换。保守置换是指一个氨基酸经相同类别内的另一氨基酸置换，例如一个酸性氨基酸经另一酸性氨基酸置换，一个碱性氨基酸经另一碱性氨基酸置换，或一个中性氨基酸经另一中性氨基酸置换。示例性的置换如下表D所示：
表D
原始残基 示例性置换 优选的保守氨基酸置换
Ala(A) Val、Leu、Ile Val
Arg(R) Lys、Gln、Asn Lys
Asn(N) Gln、His、Asp、Lys、Arg Gln
Asp(D) Glu、Asn Glu
Cys(C) Ser、Ala Ser
Gln(Q) Asn、Glu Asn
Glu(E) Asp、Gln Asp
Gly(G) Ala Ala
His(H) Asn、Gln、Lys、Arg Arg
Ile(I) Leu、Val、Met、Ala、Phe、正亮氨酸 Leu
Leu(L) 正亮氨酸、Ile、Val、Met、Ala、Phe Ile
Lys(K) Arg、Gln、Asn Arg
Met(M) Leu、Phe、Ile Leu
Phe(F) Trp、Leu、Val、Ile、Ala、Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val、Ser Ser
Trp(W) Tyr、Phe Tyr
Tyr(Y) Trp、Phe、Thr、Ser Phe
Val(V) Ile、Leu、Met、Phe、Ala、正亮氨酸 Leu
在某些实施方案中，置换发生在抗体的CDR区。通常，获得的变体相对于亲本抗体在某些生物学特性方面(例如，增加的亲和力)具有修饰(例如，改善)和/或将具有亲本抗体的基本上保留的某些生物学特性。示例性置换变体是亲和力成熟抗体。在一些实施方案中，置换发生在抗体的重链和/或轻链的CDR3区中。在一些实施方案中，在CDR3区中可以存在1、2或3个置换。
在某些实施方案中，置换发生在抗体的FR区，例如抗体重链和/或轻链可变区的FR区，例如FR1、FR2、FR2或F4区。在一些实施方案中，置换发生在FR2区。在一些实施方案中，FR区可以存在1、2或3个置换。
在某些实施方案中，本发明提供包含本文公开的可变区序列或具有本文公开的CDR的可变区序列，以及具有经修饰的Fc区的恒定结构域的抗体，所述经修饰的Fc区相比于其对其他Fc受体(即活化受体)的亲和力，具有对FcγRIIb增强的亲和力。预期具有增强的FcγRIIb特异性的此类激动性抗CD40抗体在癌症治疗和慢性感染中展示出优秀的效力(Li和Ravetch(2011)Science 333：1030；White等人(2011)J.Immunol.187：1754)。旨在不受理论束缚， 此类FcγRIIb特异性激动性抗CD40抗体可以通过增加促进细胞毒性CD8 +T细胞的增殖和活化的树突细胞的成熟、导致抗肿瘤应答增强来展示出增强的辅助作用。旨在不受理论束缚，由于本发明的交联导致的FcR介导的激动剂CD40抗体的信号增强可以是治疗效力的主要促进因素。通过抗体的Fc部分的经FcR接合的CD40激动剂抗体的交联可以增加信号强度并由此增强细胞活化。
能够产生对FcγRIIb增强的亲和力的Fc序列中的突变是本领域已知的，例如描述于Yu等人(2013)J.Am.Chem.Soc.135：9723和WO 2014/184545，Chu等人(2008)Mol.Immunol.45：3926，和Mimoto等人(2013)Protein Engineering Design & Selection 26：589。关于Fc区中突变的位置(编号)的命名法根据EU索引，如在Kabat等人(1981)Sequences of Proteins of Immunological Interest，第5版Public Health Service，National Institutes of Health，Bethesda，Md.)，其便于比较具有不同可变结构域长度的抗体中等同位置处的Fc序列。
示例性的Fc序列中的突变包括例如E233D，G237D，H268D，P271G、A330R、S267E和/或L328F。在一个优选的实施方案中，本发明的抗体包含增强FcγRIIb特异性的突变的人IgG1恒定结构域，所述突变包括E233D，G237D，H268D，P271G和A330R，或者S267E和L328F。示例性的包含增强FcγRIIb特异性的突变的人IgG1恒定结构域的抗体重链的序列参见例如SEQ ID NO：18或19。
在某些实施方案中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1，3-二烷、聚-1，3，6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。
在某些实施方案中，本文中所提供的抗体经改变以增加或降低抗体经糖基化的程度。对抗体的糖基化位点的添加或缺失可通过改变氨基酸序列以便产生或移除一或多个糖基化位点而方便地实现。当抗体包含Fc区时，可以改变附着于其的糖类。在一些应用中，除去不想要的糖基化位点的修饰可以是有用的，例如除去岩藻糖模块以提高抗体依赖性细胞性细胞毒性(ADCC)功能(参见Shield等(2002)JBC277：26733)。在其它应用中，可以进行半乳糖苷化修饰以修饰补体依赖性细胞毒性(CDC)。
在某些实施方案中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基置换。可以如，例如美国专利号7,521,541中所述地生成半胱氨酸改造的抗体。
在一些实施方案中，本发明的抗CD40抗体或其抗原结合片段显示与本发明抗体对CD40相同或相似的结合亲和力和/或特异性；和/或抑制(例如，竞争性抑制)本发明抗体与CD40的结合和/或与本发明抗体结合相同或重叠的表位；和/或与本发明抗体竞争结合CD40；和/或有本发明抗体的一个或多个生物学特性。
在一些实施方案中，本发明的抗CD40抗体是IgG1形式的抗体或IgG2形式的抗体或IgG3形式的抗体或IgG4形式的抗体。
在一些实施方案中，抗CD40抗体是单克隆抗体。
在一些实施方案中，抗CD40抗体是人源化的。用于使抗体人源化的不同方法是技术人员已知的，如由Almagro&Fransson综述的，其内容通过提述完整并入本文(Almagro JC和Fransson J(2008)Frontiers inBioscience13：1619-1633)。
在一些实施方案中，抗CD40抗体是人抗体。可使用本领域中已知的各种技术来制备人抗体。人抗体一般描述于van Dijk和van de Winkel，Curr.Opin.Pharmacol 5：368-74(2001)以及Lonberg，Curr.Opin.Immunol 20：450-459(2008)。
在一些实施方案中，抗CD40抗体是嵌合抗体。
在一个实施方案中，本发明的抗CD40抗体还涵盖其抗体片段，优选地选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体(例如scFv)或(Fab’)2、单结构域抗体、diabody(dAb)或线性抗体。
在某些实施方案中，抗CD40抗体分子处于双特异性或多特异性抗体分子形式。在一个实施方案中，双特异性抗体分子具有针对CD40的第一结合特异性和针对PD-1或PD-L1或PD-L2或OX40或4-1BB或GITR等的第二结合特异性。在一个实施方案中，双特异性抗体分子与CD40和TNF或IL-17结合。多特异性抗体分子可以具有任何针对前述分子的结合特异性的组合。
在一些实施方案中，本发明的抗体具有一个或多个如下的特性：
(1)选择性结合CD40，其结合能力高于与其他TNFR家族蛋白的结合，或其不与其他TNFR家族的蛋白相结合；在一些实施方案中，该结合利用ELISA检测。
(2)阻断CD40与CD40配体(CD40L)的结合。
(3)以交联形式或组成型形式，优选地以交联形式激活表达CD40的细胞，例如激活NFkappa-B信号通路；在一些实施方案中，用流式细胞术进行检测；在一些实施方案中，使用的报告细胞为表达NF-κB-GFP和hCD40的细胞；
(4)与CD40，例如人CD40结合，其平衡解离常数小于或等于大约5×10 -7M、4.5×10 -7M、4.4×10 -7M、4.3×10 -7M、4.2×10 -7M、4.1×10 -7M、4×10 -7M、3.9×10 -7M、3.8×10 -7M、3.7×10 -7M、3.6×10 -7M、3.5×10 -7M、或3.4×10 -7M；在一些实施方案中，所述测量为表面等离子共振技术。
(5)特异性地的与表达CD40(例如人CD40或恒河猴CD40)的细胞上的CD40结合，；在一些实施方案中，与CD40(例如人CD40和/或恒河猴CD40)结合的EC50小于或等于大约50nM、40nM、30nM、20nM、15nM、14nM、13nM、12nM、10nM、9nM、8nM。在一些实施方案中，结合采用流式细胞术检测；在一些实施方案中，表达CD40的细胞是293细胞，例如293FT细胞。
(6)诱导肿瘤细胞凋亡。在一些实施方案中，EC50小于或等于大约4nM、3.5nM、3nM、2.9nM、2.8nM、2.7nM、或2.6nM；在一些实施方案中，使用流式细胞术进行测量。在一些实施方案中，肿瘤细胞是Raji细胞或Ramos细胞。
(7)具有激动剂活性，例如显著激活(例如人)B细胞或T细胞或树突细胞。
(8)促进(例如人)B细胞或T细胞的增殖。
(9)增强免疫应答。
(10)抑制肿瘤生长，优选地同时维持个体体重。
在一些实施方案中，CD40抗体在交联的情况下具有上述一个或多个特性。
在一些实施方案中，本发明还涵盖与其他物质缀合的抗体(“免疫缀合物”)。
在一些实施方案中，其它物质例如治疗剂或标记，如细胞毒性剂或免疫抑制剂或化疗剂。细胞毒性剂包括任何对细胞有害的药剂。适合于形成免疫缀合物的细胞毒性剂(例如化疗剂)或其他物质的例子是本领域中已知的，参见例如WO2017/004006或WO2017/059243等。
在一些实施方案中，标记例如标记序列，例如肽。在优选的实施方案中，标记氨基酸序列是六组氨酸肽，如pQE载体(QIAGEN，Inc.，9259 Eton Avenue，Chatsworth，CA，91311)等中提供的标签，它们中的许多是可商业获得的。如Gentz等人，1989，Proc.Natl.Acad.Sci.USA 86：821-824中所述，例如，六组氨酸提供融合蛋白的便利纯化。用于纯化的其他肽标签包括但不限于血凝素(“HA”)标签，其对应于源自流感血凝素蛋白的表位(Wilson等人，1984，Cell 37：767)和“flag”标签。
在其他实施方案中，所示标记可以是诊断剂或可检测剂。所获得的抗体缀合物可以作为临床检验方法的部分(如确定特定疗法的效力)，用于监测或预测疾病或病症的发作、形成、进展和/或严重性。可检测剂或诊断剂包括但不限于多种酶，例如辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶；辅基，例如链霉亲和素/生物素和抗生物素蛋白/生物素；荧光物质，如但不限于伞形酮、荧光素、异硫氰酸荧光素、罗丹明、二氯三嗪胺荧光素、丹磺酰氯或藻红蛋白；发光物质，如但不限于鲁米诺；生物发光物质，如但不限于萤光素酶、萤光素和水母发光蛋白；放射性物质，如但不限于碘( 131I、 125I、 123I和 121I)、碳( 14C)、硫( 35S)、氚( 3H)、铟( 115In、 113In、 112In和 111In)、锝( 99Tc)、铊( 201Ti)、镓( 68Ga、 67Ga)、钯( 103Pd)、钼( 99Mo)、氙( 133Xe)、氟( 18F)、 153Sm、 177Lu、 159Gd、 149Pm、 140La、 175Yb、 166Ho、 90Y、47Sc、 186Re、 188Re、 142Pr、 105Rh、 97Ru、 68Ge、 57Co、 65Zn、 85Sr、 32P、 153Gd、 169Yb、 51Cr、 54Mn、 75Se、 113Sn和 117Tin；和用于各种正电子发射成像术中的正电子发射金属和非放射性顺磁金属离子。
在一些实施方案中，所述治疗性剂包括化疗剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂。
另外，本发明的抗体分子可以缀合至治疗性部分(治疗剂)如放射性金属离子，如α-发射体如 213Bi或可用于使放射金属离子(包括但不限于 131In、 131LU、 131Y、 131Ho、 131Sm)缀合至多肽的大环螯合剂。在某些实施方案中，大环螯合剂是1，4，7，10-四氮杂环十二烷-N，N’，N”，N”’-四乙酸(DOTA)，其可通过接头分子附着到抗体上。这类接头分子是本领域公知的并且在Denardo等人，1998，Clin Cancer Res.4(10)：2483-90中描述，所述文献每篇通过引用的方式完整并入。
用于治疗性部分与抗体缀合的技术是熟知的，参见，例如Arnon等人，“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”，引自Monoclonal Antibodies And Cancer Therapy，Reisfeld等人(编著)，第243-256页(Alan R.Liss，Inc.1985)。
在一些实施方案中，本发明提供了编码本文所述任何抗体或其片段或其任一条链的核酸。在一个实施方案中，提供包含所述核酸的载体。在一个实施方案中，载体是表达载体，例如pFuse载体。在一个实施方案中，提供包含所述核酸或所述载体的宿主细胞。在一个实施方案 中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或293细胞)或适用于制备抗体或其抗原结合片段的其它细胞。在另一个实施方案中，宿主细胞是原核的。
例如，本发明的核酸包含：
编码选自SEQ ID NO：13-22中任一项所示氨基酸序列的核酸，或编码与选自SEQ ID NO：13-22中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸；或者
选自SEQ ID NO：39-42所示的核酸，或与选自SEQ ID NO：39-42所示的核酸具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的核酸。
本发明还涵盖与下述核酸在严格性条件下杂交的核酸或与下述核酸具有一个或多个置换(例如保守性置换)、缺失或插入的核酸：包含编码选自SEQ ID NO：1-9中任一项所示氨基酸序列的核酸序列的核酸；或包含编码与选自SEQ ID NO：13-22中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸序列的核酸；或包含选自SEQ ID NO：39-42所示的核酸序列的核酸，或包含与选自SEQ ID NO：39-42所示的核酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的核酸序列的核酸。
在一个实施方案中，提供包含所述核酸的一个或多个载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。例如pFuse载体。一旦已经制备了用于表达的表达载体或DNA序列，则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。
在一个实施方案中，提供了包含一种或多种本发明多核苷酸的宿主细胞。在一些实施方案中，提供了包含本发明表达载体的宿主细胞。在一些实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞。
在一个实施方案中，本发明提供制备本发明抗体分子或其片段(优选的抗原结合片段)的方法，其中所述方法包括在适于表达编码本发明抗体分子或其片段(优选的抗原结合片段)的核酸的条件下培养所述宿主细胞，以及任选地分离所述抗体或其片段(优选地抗原结合片段)。在某个实施方案中，所述方法还包括从宿主细胞回收本发明抗体分子或其片段(优选地抗原结合片段)。
在一个实施方案中，提供了制备本发明抗体分子的方法，其中所述方法包括，在适合抗体表达的条件下，培养包含编码所述抗体(例如任意一条多肽链和/或多条多肽链)的核酸 或包含所述核酸的表达载体的宿主细胞，如上文所提供的，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述抗体。
为了重组产生本发明抗体分子，分离编码抗体(例如上文所描述的抗体，例如任意一条多肽链和/或多条多肽链)的核酸，并将其插入一个或多个载体，用于在宿主细胞中进一步克隆和/或表达。此类核酸易于使用常规规程分离和测序(例如通过使用能够与编码抗体重链和轻链的基因特异性结合的寡核苷酸探针进行)。
如本文所述制备的抗体分子可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。可以通过多种熟知分析方法中的任一种方法确定本发明的抗体分子的纯度，所述熟知分析方法包括大小排阻层析、凝胶电泳、高效液相色谱等。
在一些实施方案中，本发明还提供了对本发明的抗体分子进行鉴定、筛选或表征其物理/化学性质和/或生物学活性的方法。
一方面，对本发明的抗体测试其抗原结合活性，例如通过已知的方法诸如ELISA，Western印迹，等来进行。可使用本领域已知方法来测定对CD40的结合，本文中公开了例示性方法。在一些实施方案中，使用表面等离子共振测定法(例如亲和测量)或ELISA测定法。
本发明还提供了用于鉴定具有生物学活性的抗CD40抗体的测定法。生物学活性可以包括例如结合CD40(例如结合人和/或恒河猴CD40)，提高CD40介导的信号转导(例如提高NFkappa-B信号通路)，通过直接诱导肿瘤细胞凋亡消减表达CD40的细胞(例如Raji或Ramos细胞)，激活树突细胞或B细胞或T细胞(例如通过提高T细胞的细胞因子生成)、促进T细胞(例如CD8+T细胞，例如激活的CD8+T细胞)或B细胞增殖或抑制肿瘤生长。
在某些实施方案中，对本发明的抗体测试此类生物学活性。
可以使用本领域已知的方法来测定T细胞((例如CD8+T细胞))或树突细胞或B细胞的激活。例如，通过细胞活化标志物CD8(T细胞)或CD86(树突细胞或B细胞)的水平(例如表达)。还可以使用本领域公知的方法来测定CD40信号传导(例如NF-κB信号通路)，从而测定T细胞的激活。在一个实施方案中，生成表达人CD40和报告基因(包含融合至报告基因(例如β荧光素酶，GFP)的NF-kappa B启动子)的转基因细胞。对细胞添加抗CD40抗体导致NF-kappa B转录升高，这使用针对报告基因的测定法(例如荧光素酶报告基因测定法)来检测。
可以使用本领域已知的方法来测定抗体的ADCC效应。例如通过检测其对肿瘤细胞凋亡的诱导(例如通过细胞凋亡标志分子，例如CD95)、对肿瘤细胞生长的抑制或对体内肿瘤生长的抑制。
可以使用本领域已知的方法来测定T细胞(例如CD8+T细胞，例如活化的CD8+T细胞)或B细胞的增殖。例如通过发光法，例如CellTiter-Glo发光法，来测定B细胞的增殖。还例如，通过OVA特异性的OT-I细胞法，检测CD8+T细胞的增殖。
供任何上述体外测定法使用的细胞包括天然表达CD40或经改造而表达CD40细胞系。此类细胞包括天然表达CD40L的T细胞((例如CD8+T细胞，例如活化的CD8+T细胞))，天然表达CD40的B细胞或树突细胞。此类细胞还包括表达CD40和并非正常情况下表达CD40的编码CD40 DNA转染的细胞系。
可以理解的是，能够使用本发明的免疫缀合物替换或补充抗CD40抗体来进行任何上述测定法。
可以理解的是，能够使用抗CD40抗体和别的活性剂来进行任何上述测定法。
在一些实施方案中，本发明提供了包含本发明抗体的药物组合物和组合产品。
在一些实施方案中，本发明提供包含本文所述的任何抗体分子或其片段(优选地其抗原结合片段)或其免疫缀合物的组合物，优选地组合物为药物组合物。在一个实施方案中，所述组合物还包含药用辅料。
本发明还包括包含本发明抗体或其免疫缀合物的组合物(包括药物组合物或药物制剂)和/或包含编码本发明抗体的多核苷酸的组合物(包括药物组合物或药物制剂)。在某些实施方案中，组合物包含一种或多种本发明抗体或其片段或一种或多种编码一种或多种本发明抗体或其片段的多核苷酸。
这些组合物还可以包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。
如本文所用，“药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。适用于本发明的药用载体可以是无菌液体，如水和油，包括那些石油、动物、植物或合成来源的，如花生油、大豆油、矿物油、芝麻油等。当静脉内施用药物组合物时，水是优选的载体。还可以将盐水溶液和水性右旋糖以及甘油溶液用作液体载体，特别是用于可注射溶液。
合适的赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、甘油单硬脂酸酯、滑石、氯化钠、干燥的脱脂乳、甘油、丙烯、二醇、水、乙醇等。对于赋形剂的使用及其用途，亦参见“Handbook of PharmaceuticalExcipients”，第五版，R.C.Rowe，P.J.Seskey和S.C.Owen，PharmaceuticalPress，London，Chicago。
若期望的话，所述组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂。
本发明的组合物可以处于多种形式。这些形式例如包括液体、半固体和固体剂型，如液态溶液剂(例如，可注射用溶液剂和可输注溶液剂)、分散体剂或混悬剂、脂质体剂和栓剂。优选的形式取决于预期的施用模式和治疗用途。常见的优选组合物处于可注射用溶液剂或可输注溶液剂形式。优选的施用模式是肠胃外(例如，静脉内、皮下、腹腔(i.p.)、肌内)注射。在一个优选实施方案中，通过静脉内输注或注射施用抗体分子。在另一个优选实施方案中，通过肌内、腹腔或皮下注射施用抗体分子。
本发明的特异性结合人CD40的激动性抗体可冻干以便于储存，使用之前可在合适的载体中复原。此项技术已被证实对常规的蛋白制剂有效，并且可采用熟知的冻干和复原技术。
本发明的药物组合物或制剂还可以包含其他治疗剂，所述治疗剂是被治疗的特定适应证所需的，优选具有不会不利地彼此影响的互补活性的那些治疗剂。例如，化疗剂、细胞毒性剂、疫苗、其它抗体、抗感染活性剂、小分子药物或免疫调节剂等。
在一些实施方案中，本发明还提供了组合产品，其包含本发明的抗体或其抗原结合片段，或其免疫缀合物，以及一种或多种其它治疗剂(例如化疗剂、其他抗体、细胞毒性剂、疫苗、抗感染活性剂、小分子药物或免疫调节剂等)。
在一些方案中，所述组合产品中的两种或多种成分可以依次、分开或同时联合施用给受试者。
在一些实施方案中，本发明还提供了包含本发明的抗体、药物组合物、免疫缀合物或组合产品的试剂盒，以及任选的指导施用的包装插页。
在一些实施方案中，本发明还提供了包含本发明的抗体、药物组合物、免疫缀合物、组合产品的药物制品，任选地，所述药物制品还包括指导施用的包装插页。
在一些实施方案中，所述其他治疗剂包括例如以下中的一种或多种：抗CTLA-4抗体、抗PD-1抗体、抗PD-L1抗体、抗TIGIT抗体、抗OX40(也称为CD134、TNFRSF4、ACT35和/或TXGP1L)抗体、抗LAG-3抗体、抗CD73抗体、抗CD137抗体、抗CD27抗体、抗CSF-1R抗体、TLR激动剂、或IDO或TGFβ的小分子拮抗剂。可以与本发明的抗体组合的治疗剂的例子还参见WO2017/059243或WO2017/004006。
在一些实施方案中，本发明还提供了增强受试者中的免疫应答(例如抗原特异性T细胞应答)的方法，其包括向受试者施用有效量的本发明的抗CD40抗体或其抗原结合片段，使得所述个体中的免疫应答得到增强。在一些实施方案中，个体具有肿瘤。在另一实施方案中，主体具有病毒感染，例如慢性病毒感染。
在一些实施方案中，本发明提供了利用本发明的抗体在受试者中激活T细胞(例如CD8+T细胞)和/或树突细胞和/或B细胞的方法，其包括向受试者施用有效量的本发明的抗CD40抗体或其抗原结合片段。本发明还提供了利用本发明的方法促进T细胞(例如激活的CD8+T细胞)或B细胞增殖的方法，其包括向受试者施用有效量的本发明的抗CD40抗体或其抗原结合片段。因此，在一些实施方案中，考虑到本文所述的抗CD40抗体增强T细胞应答例如抗原特异性T细胞应答的共刺激的能力，本文提供使用本文所述的抗体刺激、增强或上调抗原特异性T细胞应答例如抗肿瘤T细胞应答的体外和体内方法。CD4+和CD8+T细胞应答可以使用抗CD40抗体增强。T细胞可以是T eff细胞，例如CD4+T eff细胞、CD8+T eff细胞、T辅助(T h)细胞和T毒性(T c)细胞。
在一些实施方案中，本发明提供了使用本发明的抗体分子用来治疗或预防需要在受试者中调节(例如增强)免疫应答的疾病，例如肿瘤或感染，例如慢性感染。因此，本发明还提供抑制受试者中的肿瘤生长的方法，其包括向主体施用本发明的抗CD40抗体或其抗原结合片段，使得肿瘤生长得到抑制。
在一些实施方案中，所述疾病是CD40相关疾病，例如所述疾病是CD40表达或活性降低的疾病(例如与健康对照相比)，或者所述疾病是CD40基因和/或蛋白质水平降低的疾病(例如与健康对照相比)；或所述疾病受益于激活CD40活性，例如激活CD40信号通路，和/或激活T细胞或B细胞或树突细胞。在一些实施方案中，本发明涉及在个体中激活抗原活性或激活抗原介导的信号通路的方法，该方法包括向对象施用有效量的本文公开的抗体或其抗原结合片段。在一些实施方案中，激活CD40活性或激活CD40介导的信号通路是指激活CD40信号通路。
在一些实施方案中，本发明的所述抗体或其抗原结合片段能够引起抗体依赖性细胞介导的细胞毒性(ADCC)，进而杀伤肿瘤细胞。因此，本发明还涉及治疗肿瘤的方法，其包括向个体中施用有效量的本发明的抗体或其抗原结合片段。
在另一方面中，本发明涉及预防或治疗受试者的肿瘤(例如癌症)的方法，所述方法包 括向所述受试者施用有效量的本文公开的抗体分子或药物组合物或免疫缀合物或组合产品或试剂盒。在一些实施方案中，所述肿瘤是癌症。肿瘤可以是实体瘤或液体瘤，例如恶性血液肿瘤。在某些实施方案中，肿瘤是免疫原性肿瘤。在某些实施方案中，肿瘤是非免疫原性的。在某些实施方案中，肿瘤是PD-L1阳性的。在某些实施方案中，肿瘤是PD-L1阴性的。
在一些实施方案中，用抗体分子治疗和/或预防的肿瘤包括但不限于实体瘤、血液学癌(例如，白血病、淋巴瘤、骨髓瘤，例如，多发性骨髓瘤)及转移性病灶。在一个实施方案中，癌是实体瘤。实体瘤的例子包括恶性肿瘤，例如，多个器官系统的肉瘤和癌，如侵袭肺、乳房、卵巢、淋巴样、胃肠道的(例如，结肠)、肛门、生殖器和生殖泌尿道(例如，肾、膀胱上皮、膀胱细胞、前列腺)、咽、CNS(例如，脑、神经的或神经胶质细胞)、头和颈、皮肤(例如，黑素瘤)、鼻咽(例如，分化或未分化的转移性或局部复发性鼻咽癌)和胰的那些癌、以及腺癌，包括恶性肿瘤，如结肠癌、直肠癌、肾细胞癌、肝癌、非小细胞肺癌、小肠癌和食道癌。癌症可以处于早期、中期或晚期或是转移性癌。
在一些实施方案中，癌症选自结直肠癌(例如，CRC)、黑素瘤，例如，晚期黑素瘤(例如，II-IV期黑素瘤)或HLA-A2阳性-黑素瘤；胰腺癌，例如，晚期胰腺癌；乳腺癌，例如，转移性乳腺癌或三阴性乳腺癌；头颈癌(例如，HNSCC)；食管癌；肾细胞癌(RCC)，例如，肾透明细胞癌(ccRCC)或转移性肾细胞癌(MRCC)；肺癌(例如，NSCLC)；宫颈癌；膀胱癌；或血液学恶性病，例如，白血病(例如，淋巴细胞白血病)或淋巴瘤(例如，霍奇金淋巴瘤(HL)、非霍奇金淋巴瘤(NHL)、弥漫性大B细胞淋巴瘤(DLBCL)、套细胞淋巴瘤(MCL)或CLL，例如，复发性或顽固性慢性淋巴细胞白血病)。
在一些实施方案中，癌症的例子进一步包括但不限于B细胞增殖性病症，其进一步包括但不限于淋巴瘤(例如B细胞非霍奇金氏淋巴瘤(NHL))和淋巴细胞性白血病。
在一些实施方案中，所述肿瘤是需要激活T细胞或B细胞或树突细胞的肿瘤，例如癌症，例如具有T细胞功能障碍的肿瘤或癌症。在一些实施方案中，所述肿瘤是OX40的表达或活性被降低的肿瘤。在一些实施方案中，所述肿瘤是受益于OX40信号通路激活的肿瘤，例如癌症。
在一些实施方案中，本文所述的癌症是淋巴瘤、结肠癌、结直肠癌、直肠癌、肺癌(例如非小细胞肺癌)、肝癌、胃癌及其转移性癌症。
在一些实施方案中，本发明的抗体或其抗原结合片段不适用于治疗具有CD40表达的血液癌症，其可能被用CD40激动剂治疗而恶化。某些癌症可能已知表达CD40并且因此经历此类恶化，因此可以在类别上被排除。在具体的实施方案中，测试特异性肿瘤样品的CD40表达并且基于测试结果从用本发明的CD40抗体的疗法中排除。
本文公开的方法和组合物可用于治疗与前述癌症相关的转移性病灶。
本发明的特异性结合人CD40的抗体或其抗原结合片段也可预防性地施用，以降低罹患癌症的风险、延迟癌症进展中事件的发作和/或在癌症缓解后降低复发的风险。这对于肿瘤难以定位而由于其它生物因素已知患有肿瘤的患者可能尤其有用。
在另一方面中，本发明涉及预防或治疗受试者的感染性疾病的方法，所述方法包括向所述受试者施用有效量的本文公开的抗体分子或药物组合物或免疫缀合物或组合产品或试剂盒。在一个实施方案中，感染性疾病是慢性感染。在一些实施方案中，所述感染是病毒感染。
在一些实施方案中，所述感染是急性的或慢性的。在一些实施方案中，所述慢性感染是 持续性的感染、潜伏的感染或缓慢感染。在一些实施方案中，所述慢性感染是由选自细菌、病毒、真菌和原生动物的病原体导致的。
在一些实施方案中，病毒的一些实例包括HIV、肝炎(甲肝、乙肝和丙肝)、疱疹病毒(例如VZV、HSV-1、HAV-6、HSV-II和CMV、EB病毒)、腺病毒、流感病毒、黄病毒、埃可病毒、鼻病毒、柯萨奇病毒、冠状病毒、呼吸道合胞病毒、腮腺炎病毒、轮状病毒、麻疹病毒、风疹病毒、细小病毒、牛痘病毒、HTLV病毒、登革热病毒、乳头瘤病毒、软疣病毒、脊髓灰质炎病毒、狂犬病病毒、JC病毒和虫媒脑炎病毒；细菌的一些实例包括衣原体、立克次体、分枝杆菌、葡萄球菌、链球菌、肺炎球菌、脑膜炎球菌和淋球菌、克雷伯氏菌、变形杆菌、沙雷菌、假单胞菌、军团菌、白喉、沙门氏菌、芽孢杆菌、霍乱、破伤风、肉毒中毒、炭疽、鼠疫、钩端螺旋体病和莱姆病病菌；真菌的一些实例包括假丝酵母(白色念珠菌、克柔假丝酵母、光滑假丝酵母、热带假丝酵母等)、新型隐球菌(Cryptococcus neoformans)、曲霉(烟曲霉、黑曲霉等)、属毛霉目(毛霉属、犁头霉属、根霉属)、申克氏胞丝菌(Sporothrix schenkii)、皮炎芽生菌(Blastomyces dermatitidis)、巴西副球孢子菌(Paracoccidioides brasiliensis)、粗球孢子菌(Coccidioides immitis)和荚膜组织胞浆菌(Histoplasma capsulatum)；寄生虫的一些实例包括痢疾变形虫(Entamoeba histolytica)、结肠小袋纤毛虫(Balantidium coli)、福氏耐格里变形虫(Naegleriafowleri)、棘阿米巴属种(Acanthamoeba sp.)、蓝氏贾第虫(Giardia lambia)、隐孢子虫属种(Cryptosporidium sp.)、卡氏肺孢子虫(Pneumocystis carinii)、间日疟原虫(Plasmod ium viva x)、微小巴贝虫(Ba besia microti)、布氏锥虫(Try pa nosomabrucei)、克氏锥虫(Trypanosoma cruzi)、杜氏利什曼原虫(Leishmania donovani)、刚地弓形虫(Toxoplasma gondii)、巴西日圆线虫(Nippostrongylus brasiliensis)。
在一些实施方案中，用抗体分子治疗和/或预防的感染性疾病包括目前不存在有效疫苗的病原体或常规疫苗对其未及完全有效的病原体。这些包括但不限于HIV、(甲型、乙型和丙型)肝炎、流感、疱疹、贾弟鞭毛虫(Giardia)、疟疾、利什曼原虫(Leishmania)、金黄色葡萄球菌(Staphylococcus aureus)、铜绿假单胞菌(Pseudomonas aeruginosa)。
可以用本发明公开的方法和组合物治疗的疾病还参见WO2017/059243或WO2017/004006。
在一些实施方案中，本发明的抗体或药物组合物或免疫缀合物或组合产品或试剂盒还能与一种或多种其它疗法例如治疗方式和/或其它治疗剂组合施用，用于本文所述的预防和/或治疗。
在一些实施方案中，治疗方式包括外科手术(例如肿瘤切除术)；放射疗法(例如，外粒子束疗法，它涉及其中设计照射区域的三维适形放射疗法)、局部照射(例如，指向预选靶或器官的照射)或聚焦照射)等。聚焦照射可以选自立体定位放射手术、分割立体定位放射手术和强度调节型放射疗法。聚焦照射可以具有选自粒子束(质子)、钴-60(光子)和直线加速器(X射线)的辐射源。放射疗法可以通过几种方法之一或方法组合施用，所述方法包括而不限于外粒子束疗法、内部放射疗法，植入物照射、立体定位放射手术、全身放射疗法、放疗法和永久或短暂间质近距放射疗法。
在一些实施方案中，治疗剂选自化疗剂、细胞毒性剂、疫苗、其它抗体、抗感染活性剂或免疫调节剂(例如共刺激分子的激活剂或免疫检查点分子的抑制剂)。
示例性的其它抗体包括但不限于免疫检查点抑制剂(例如，抗CTLA-4、抗TIM-3、抗CEACAM)；刺激免疫细胞的抗体(例如，激动性GITR抗体或CD137抗体)；抗癌抗体(例如，利妥昔单抗
Figure PCTCN2020084186-appb-000004
曲妥珠单抗
Figure PCTCN2020084186-appb-000005
托西莫单抗
Figure PCTCN2020084186-appb-000006
替伊 莫单抗
Figure PCTCN2020084186-appb-000007
阿来组单抗
Figure PCTCN2020084186-appb-000008
依帕珠单抗
Figure PCTCN2020084186-appb-000009
贝伐珠单抗
Figure PCTCN2020084186-appb-000010
厄洛替尼
Figure PCTCN2020084186-appb-000011
西妥昔单抗
Figure PCTCN2020084186-appb-000012
等等。例如，其他抗体可以是抗PD-L1抗体、抗LAG-3抗体、抗PD-1抗体或抗CLA-4抗体。
示例性的疫苗包括但不限于癌症疫苗。疫苗可以是基于DNA的疫苗、基于RNA的疫苗或基于病毒转导的疫苗。癌症疫苗可以是预防性的或治疗性的，诸如癌细胞、纯化的肿瘤抗原(包括重组蛋白、肽和碳水化物分子)、细胞和转染有编码免疫刺激细胞因子的基因的细胞(He等人(2004)J.Immunol.173：4919-28)。可以使用的肿瘤疫苗的非限制性实例包括黑素瘤抗原的肽，诸如gp100的肽、MAGE抗原、Trp-2、MART1和/或酪氨酸酶、或经转染以表达细胞因子GM-CSF的肿瘤细胞。在一些实施方案中，癌症疫苗是肽癌症疫苗，其在一些实施方案中是个性化肽疫苗。在一些实施方案中，该肽癌症疫苗是多价长肽、多重肽、肽混合物、杂合肽，或经肽脉冲的树突细胞疫苗(参见例如Yamada等人，Cancer Sci，104：14-21，2013)。在本发明的任何方法的一些实施方案中，本发明的抗体或其片段的施用与肿瘤抗原的施用组合。抗原可以例如是肿瘤抗原、病毒抗原、细菌性抗原或来自病原体的抗原。在一些实施方案中，肿瘤抗原包含蛋白质。在一些实施方案中，肿瘤抗原包含核酸。在一些实施方案中，肿瘤抗原是肿瘤细胞。
示例性的抗感染活性剂包括但不限于，抗病毒剂、抗真菌剂、抗原生动物剂、抗细菌剂，例如核苷类似物齐多夫定(AST)、更昔洛韦、膦甲酸或cidovir等。
免疫调节剂包括免疫检查点分子抑制剂和共刺激性分子激活剂。
在一些实施方案中，免疫检查点分子的抑制剂是CTLA-4、TIM-3、VISTA、BTLA、TIGIT、LAIR1、CD160、2B4、CEACAM(例如，CEACAM-1和/或CEACAM-5)和/或TGFR的抑制剂。对分子的抑制可以在DNA、RNA或蛋白质水平进行。在一些实施方案中，抑制性核酸(例如，dsRNA、siRNA或shRNA)可以用来抑制免疫检查点分子的表达。在其他实施方案中，免疫检查点分子的抑制剂是与免疫检查点分子结合的多肽，例如，可溶性配体或抗体或抗体片段。
在一些实施方案中，免疫调节剂是共刺激分子的激活剂或激动剂。在一个实施方案中，共刺激分子的激动剂选自以下分子的激动剂(例如，激动性抗体或其抗原结合片段、或可溶性融合物)：OX40、CD2、CD27、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、4-1BB(CD137)、GITR、CD30、CD40、BAFFR、HVEM、CD7、LIGHT、NKG2C、SLAMF7、NKp80、CD160、B7-H3或CD83配体。
在一些实施方案中，本发明的抗体或其片段可以与包含过继转移表达嵌合抗原受体(CAR)的T细胞(例如细胞毒性T细胞或CTL)的治疗联合施用。
在一些实施方案中，本发明的抗体或其片段可以与抗肿瘤剂或溶瘤病毒联合施用。
在所有上述方法中，CD40激动作用可以与其他形式的免疫疗法诸如细胞因子治疗(例如干扰素、GM-CSF、G-CSF、IL-2)或双特异性抗体疗法组合，其提供增强的肿瘤抗原呈递。参见例如Holliger(1993)Proc.Natl.Acad.Sci.USA 90：6444-6448；Poljak(1994)Structure 2：1121-1123。
在一些实施方案中，本发明的抗体或其片段可以与增强宿主免疫功能的常规方法组合，所述常规方法包括但不限于：(i)APC增强，诸如(a)向肿瘤注射编码异源MHC同种异体抗原的DNA，或(b)用增加免疫抗原识别可能性的基因(例如免疫刺激细胞因子、GM-CSF、共刺激分子B7.1、B7.2)转染活检的肿瘤细胞，(iii)过继性细胞免疫疗法，或用活化的肿瘤特异 性T细胞治疗。过继性细胞免疫疗法包括分离肿瘤浸润的宿主T淋巴细胞，诸如通过IL-2或肿瘤或两者刺激体外扩增该群体；此外，功能障碍的分离的T细胞也可通过体外应用本发明的抗体来活化，如此活化的T细胞可然后重新施用给宿主。
上文所述的各种组合疗法可以进一步组合以用于治疗。
更多的本发明抗体与其他治疗方式或治疗剂的组合的例子可以参见WO2017/059243或WO2017/004006等。
此类组合疗法涵盖组合施用(其中两种或更多种治疗剂包含在同一配制剂或分开的配制剂中)，和分开施用，在该情况中，可以在施用别的疗法，例如治疗方式和/或治疗剂之前，同时，和/或之后发生本发明的抗体的施用。抗体分子和/或其他疗法，例如治疗剂或治疗方式可以在活动性疾病期间或在缓解或活动度更小的疾病期间施用。抗体分子可以在其他治疗前、与其他治疗同时、治疗后或在疾病缓解期间施用。
可以理解的是，能够使用本发明的免疫缀合物或组合物或组合产品或试剂盒替换或补充本发明的抗体来进行任何治疗。
本发明的抗体(以及包含其的药物组合物或免疫缀合物，以及任何另外的治疗剂)的施用模式可为任何合适的途径，诸如胃肠外施用，例如真皮内、肌肉内、腹膜内、静脉内或皮下、粘膜(口腔、鼻内、阴道内、直肠)或如本领域所熟知的技术人员了解的其它方式。特异性结合CD40的激动性抗体可使用已知的方法瘤内施用到淋巴结引流部位以局部递送到肿瘤中。本发明的特异性结合人CD40的激动性抗体可通过任何合适的途径，例如经肠胃外通过静脉(i.v.)输注或弹丸式注射、肌内或皮下或腹膜内注射施用于患者。
本文中涵盖各种用药时程，包括，但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。
为了预防或治疗疾病，本发明的抗体的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、抗体的类型、疾病的严重性和进程、所述抗体是以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，和主治医师的判断力。所述抗体以一次治疗或经过一系列治疗合适地施用于患者。通常，临床医师施用组合物直到达到获得预期效果的剂量。本发明的抗体因此可以单一剂量施用，或者在一定时间内以两次或更多次剂量(其可包含相同或不同量的所需分子)施用，或者通过植入装置或导管连续输注施用。适当的剂量可通过使用适当的剂量反应数据确定。在某些实施方案中，可在延长的时间期限内向患者施用抗体。在某些实施方案中，抗体是每周、每两周、每月、每两个月、每三个月、每四个月、每五个月或每六个月给药。
在某些实施方案中，本文中提供的抗CD40抗体或其抗原结合片段可以用于检测CD40在生物样品中的存在。术语“检测”用于本文中时，包括定量或定性检测，示例性的检测方法可以涉及免疫组织化学、免疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的磁珠、ELISA测定法、PCR-技术(例如，RT-PCR)。在某些实施方案中，生物样品是血、血清或生物来源的其他液体样品。在某些实施方案中，生物样品包含细胞或组织。在一些实施方案中，生物样品来自本文所述疾病(例如肿瘤或感染)的相关病灶
在一些实施方案中，CD40是人CD40或食蟹猴CD40。在一些实施方案中，所述方法包括将生物样品与如本文所述的抗CD40抗体或其抗原结合片段在允许抗CD40抗体与CD40结合的条件下接触，并检测在抗CD40抗体和CD40之间是否形成复合物。复合物的形成表示存在CD40。该方法可以是体外或体内方法。在一个实施方案中，抗CD40抗体被用于选择 适合利用抗CD40抗体的治疗的受试者，例如其中CD40是用于选择所述受试者的生物标记物。
在一个实施方案中，可以使用本发明抗体诊断本文所述疾病，例如评价(例如，监测)对象中本文所述疾病的治疗或进展、其诊断和/或分期。在某些实施方案中，提供标记的抗CD40抗体。标记包括但不限于，被直接检测的标记或部分(如荧光标记、发色团标记、电子致密标记、化学发光标记和放射性标记)，以及被间接检测的部分，如酶或配体，例如，通过酶促反应或分子相互作用。示例性标记包括但不限于，放射性同位素 32P、 14C、 125I、 3H和 131I，荧光团如稀土螯合物或荧光素及其衍生物，罗丹明及其衍生物，丹酰(dansyl)，伞形酮(umbelliferone)，荧光素酶(luceriferase)，例如，萤火虫荧光素酶和细菌荧光素酶(美国专利号4,737,456)，荧光素，2，3-二氢酞嗪二酮，辣根过氧化物酶(HR)，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶解酶，糖类氧化酶，例如，葡萄糖氧化酶，半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，杂环氧化酶如尿酸酶和黄嘌呤氧化酶，以及利用过氧化氢氧化染料前体的酶如HR，乳过氧化物酶，或微过氧化物酶(microperoxidase)，生物素/亲和素，自旋标记，噬菌体标记，稳定的自由基，等等。
在本文中提供的任何发明的一些实施方案中，样品是在用抗CD40抗体治疗之前获得的。在一些实施方案中，样品是在用本文所述疾病药物治疗之前获得的。在一些实施方案中，样品是福尔马林固定、石蜡包膜(FFPE)的。在一些实施方案中，样品是活检(例如芯活检)，手术标本(例如来自手术切除的标本)，或细针吸出物。
在一些实施方案中，在治疗之前，例如，在起始治疗之前或在治疗间隔后的某次治疗之前检测CD40。
在一些实施方案中，提供了包含本发明抗体或其抗原结合片段的检测试剂盒，用于诊断本文所述疾病，例如肿瘤或感染。
在一些实施方案中，提供了一种治疗本文所述疾病例如肿瘤或感染的方法，所述方法包括：对受试者(例如，样品)(例如，受试者样品)检验CD40的存在，因而确定CD40值，将CD40值与对照值比较，并且如果CD40值小于对照值，则向受试者施用治疗有效量的任选与一种或多种其他疗法组合的抗CD40抗体(例如，本文所述的抗CD40抗体)，因而治疗本文所述疾病，例如肿瘤或感染。
本发明因此还涉及本发明的抗体或其抗原结合片段用于上述方法的用途，以及本发明的抗体或其抗原结合片段在制备用于上述方法的药物或组合物或组合产品或试剂盒中的用途，和/或本发明的抗体或其抗原结合片段在制备用于诊断本文所述疾病的试剂盒中的用途。
适用于本发明的抗体或其抗原结合片段的方法和用途，同样适用于包含本发明抗体或其抗原结合片段的免疫缀合物、组合物、组合产品或试剂盒。
在下面的附图进一步说明本发明。然而，这些附图和本发明的具体实施方案不应被认为限制本发明的范围，并且本领域技术人员容易想到的改变将包括在本发明的精神和所附权利要求的保护范围内。
附图说明
图1显示了通过流式细胞术测定293F细胞产生的HEL及CD40L对Jurkat/NF-κB-GFP+hCD40报告细胞单克隆的激活，报告细胞激活后有GFP的表达，GFP阳性率由FITC通道检测。
图2显示了通过噬菌体ELISA测定第二轮(图2A)、第三轮(图2B)噬菌体展示富集得到的抗体库中CD40结合抗体的阳性率，抗体与CD40结合信号大于与BSA结合信号的三倍以上定义为阳性抗体。
图3显示了通过流式细胞术测定Jurkat/NF-κB-GFP+hCD40报告细胞单克隆产生的含有阴性对照N27、阳性CD40激动剂抗体的上清对Jurkat/NF-κB-GFP+hCD40报告细胞的激活。FITC通道检测的GFP荧光强度代表单克隆的激活程度。
图4显示了通过分子排阻色谱法测定293F细胞产生的本发明抗体NK003聚体性质。
图5A显示了通过ELISA测定293F细胞产生的本发明抗体NK003与CD40特异性结合，与其他TNFR家族成员OX40、4-1BB、GITR不结合或低结合。图5B显示了通过ELISA测定293F细胞产生的本发明抗体NK004与CD40的特异性结合。
图6显示了通过ELISA测定293F细胞产生的本发明抗体NK003与CD40L竞争性结合CD40。
图7A显示了通过流式细胞术测定293F细胞产生的本发明抗体NK003以交联形式激活Jurkat/NF-κB-GFP+hCD40报告细胞，图7B显示了通过流式细胞术测定293F细胞产生的本发明抗体NK004不依赖于交联，以组成型形式激活Jurkat/NF-κB-GFP+hCD40报告细胞。FITC通道检测GFP，MFI定义为GFP阳性细胞几何平均数(Geometrical Mean)与GFP阳性细胞百分比的乘积。
图8显示了通过流式细胞术测定293F细胞产生的本发明抗体NK003分布与过表达人CD40的293FT细胞(图8A)、过表达恒河猴CD40的293FT细胞(图8B)的结合。
图9显示了通过流式细胞术测定293F细胞产生的本发明抗体NK003诱导Raji细胞(图9A)和Ramos细胞(图9B)的凋亡，细胞凋亡指标为CD95的表达。MFI定义为CD95阳性细胞几何平均数与CD95阳性细胞百分比的乘积。
图10显示了通过流式细胞术测定293F细胞产生的本发明抗体NK003分别在不加交联剂(图10A)及加入交联剂anti-Fc(图10B)时对PBMC中树突状细胞的激活。树突状细胞激活指标为CD86的表达。MFI定义为CD86阳性细胞几何平均数与CD86阳性细胞百分比的乘积。
图11显示了通过CellTiter-Glo法测定293F细胞产生的本发明抗体NK003对PBMC中B细胞的增殖作用，细胞增殖指标为荧光强度(RLU)，RLU值越大表示细胞越多。
图12显示了通过流式细胞术测定293F细胞产生的本发明抗体NK003分别在不加交联剂(图12A)及加入交联剂anti-Fc(图12B)时对PBMC中B细胞的激活。B细胞激活指标为CD86的表达。MFI定义为CD86阳性细胞几何平均数与CD86阳性细胞百分比的乘积。
图13显示了SCID小鼠中接种Raji细胞的同时给药在293F细胞中产生的本发明抗体NK003及阴性对照HEL，个体小鼠肿瘤生长曲线(图13A)及小鼠存活率统计(图13B)。
图14显示了MC38荷瘤CD40人源化小鼠中给药在293F细胞中产生的本发明抗体NK003及阴性对照HEL对免疫系统的激活，OT1细胞数、OT1细胞与CD8细胞比值(OT1/CD8 +比值)及CD8细胞与CD4细胞比值(CD8/CD4比值)的增加均可说明免疫系统有激活。
图15显示了MC38荷瘤CD40人源化小鼠中给药在293F细胞中产生的本发明抗体NK003、阴性对照HEL及阳性对照CP870893，个体小鼠肿瘤生长曲线(图15A)及小鼠体重变化曲线(图15B)。
图16显示了通过流式细胞术测定293F细胞产生的本发明抗体NK003、FcγRIIB增强型突变体NK003-V12、FcγRIIA/FcγRIIB增强型突变体NK003-S267E/L328F及阴性对照HEL分别在293FT-FcγRIIA细胞(图16A)和293FT-FcγRIIB细胞(图16B)交联下对Jurkat/NF-κB-GFP+hCD40报告细胞的激活。FITC通道检测GFP，MFI定义为GFP阳性细胞几何平均数与GFP阳性细胞百分比的乘积。
图17显示了NK003以Fab形式插入pcomb3载体的质粒图谱。
图18显示了通过噬菌体ELISA测定第三轮噬菌体展示得到的抗体库中CD40结合抗体的阳性率，抗体与CD40结合信号大于与BSA结合信号的三倍以上定义为阳性抗体。
图19显示了NK003VH(图19A)、VL(图19B)互补决定区CDR3亲和力成熟三代测序结果。
图20显示了通过流式细胞术测定293F细胞产生的本发明抗体NK003-AM-9、NK003-AM-18以交联形式激活Jurkat/NF-κB-GFP+hCD40报告细胞，FITC通道检测GFP，MFI定义为GFP阳性细胞几何平均数(Geometrical Mean)与GFP阳性细胞百分比的乘积。
图21显示了NK003VH(图21A)、VL(图21B)骨架区亲和力成熟三代测序结果。
图22显示了通过流式细胞术测定293F细胞产生的本发明抗体NK003-AM-18-EP1以交联形式激活Jurkat/NF-κB-GFP+hCD40报告细胞，FITC通道检测GFP，MFI定义为GFP阳性细胞几何平均数(Geometrical Mean)与GFP阳性细胞百分比的乘积。
定义
应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项。
如本文所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也 涵盖由所述及的要素、整数或步骤组合的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
如本文所述，术语“CD40”是本领域已知的，例如人CD40或恒河猴CD40。CD40也被称为肿瘤坏死因子受体超家族成员5(TNFRSF5)、CD4OL受体或CD154受体。人全长CD40蛋白质是具有277个氨基酸的I型膜蛋白，参见例如NCBI，NM_001250.5。恒河猴CD40参见例如NCBI，NM_001265862.1。
本文所用的术语“抗CD40抗体”、“抗CD40”、“CD40抗体”或“结合CD40的抗体”或“特异性结合CD40的抗体”是指这样的抗体，所述抗体能够以足够的亲合力结合(人或恒河猴)CD40亚基或其片段以致所述抗体可以用作靶向(人或恒河猴)CD40中的诊断剂和/或治疗剂。在一个实施方案中，抗CD40抗体与非(人或恒河猴)CD40蛋白结合的程度低于所述抗体与(人或恒河猴)CD40结合的约10％、约20％、约30％、约40％、约50％、约60％、约70％、约80％或约90％或以上，如例如通过放射性免疫测定(RIA)或生物光干涉测定法或MSD测定法测量的。
特异性地结合人CD40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)(诸如恒河猴)的相同抗原具有交叉反应性。虽然单特异性抗体特异性地结合一个抗原或一个表位，而双特异性抗体特异性地结合两个不同的抗原或两个不同的表位。
“互补决定区”或“CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：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”，Joumal 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)(在万维网上imgt.cines.fr/上)，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。
例如，根据不同的CDR确定方案，每一个CDR的残基如下所述。
Figure PCTCN2020084186-appb-000013
Figure PCTCN2020084186-appb-000014
CDR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，Md.(1991))的编号位置。
在一个实施方案中，本发明抗体的CDR通过IMGT规则确定边界，例如利用IMGT数据库确定边界。
应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
“Fc区”或“Fc结构域”或“Fc”指抗体的重链的C末端区域，其介导免疫球蛋白与宿主组织或因子的结合，包括结合位于免疫系统的各种细胞(例如效应细胞)上的Fc受体或结合经典补体系统的第一组分(C1q)。因此，Fc区包含排除第一恒定区免疫球蛋白结构域(例如CH1或CL)的抗体的恒定区。在IgG、IgA和IgD同种型中，Fc区包含抗体的两条重链各自中的CH2和CH3恒定区；IgM和IgE Fc区包含各多肽链中的三个重链恒定结构域(CH结构域2-4)。对于IgG，Fc区包含免疫球蛋白结构域Cγ2和Cγ3以及Cγ1和Cγ2之间的铰链。尽管免疫球蛋白重链的Fc区的边界可以改变，但人IgG重链Fc区通常定义为从位置C226或P230处的氨基酸残基(或这两个氨基酸之间的氨基酸)至重链的羧基末端的片段，其中编号根据Kabat中的EU索引。Kabat等人(1991)Sequences of Proteinsof Immunological Interest，National Institutes of Health，Bethesda，MD；还参见美国专利申请公开号2008/0248028的图3c-3f。人IgG Fc区的CH2结构域从约氨基酸231延伸至约氨基酸340，而CH3结构域位于Fc区中CH2结构域的C末端侧，即，其从IgG的约氨基酸341延伸至约氨基酸447(包括C末端赖氨酸)。如本文所用，Fc区可以为天然序列Fc，包括任何同种异型变体，或变体Fc(例如非天然存在的Fc)。Fc也可以孤立地或在含Fc蛋白多肽诸如“包含Fc区的结合蛋白”也称为“Fc融合蛋白”(例如抗体或免疫黏附素)的背景下指这一区域。
如本文所用，术语“表位”指抗原(例如，CD40)中与抗体分子特异性相互作用的部分。蛋白抗原内的表位可以从连续的氨基酸(通常为线性表位)或通过蛋白的三级折叠而并列的不连续氨基酸(通常为构象表位)形成。由连续的氨基酸形成的表位通常(但并非总是如此)对变性溶剂保持暴露，而由三级折叠形成的表位通常在用变性溶剂处理时丧失。表位通常包括独特空间构象中的至少3、4、5、6、7、8、9、10、11、12、13、14或15个氨基酸。
与参照抗体“结合相同或重叠表位的抗体”是指这样的抗体，其在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的所述参照抗体与其抗原的结合，反言之，参照抗体在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的该抗体与其抗原的结合。
与参照抗体竞争结合其抗原的抗体是指这样的抗体，其在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的所述参照抗体与其抗原的结合。反言之，参照抗体在竞争测定中阻断50％、60％、70％、80％、90％或95％以上的该抗体与其抗原的结合。众多类型的竞争性结合测定可用于确定一种抗体是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methods in Enzymology 9：242-253)。
抑制(例如竞争性抑制)参照抗体与其抗原的结合的抗体是指这样的抗体，其抑制50％、60％、70％、80％、90％或95％以上的所述参照抗体与其抗原的结合。反言之，参照抗体抑制50％、60％、70％、80％、90％或95％以上的该抗体与其抗原的结合。抗体与其抗原的结合可以亲和力(例如平衡解离常数)衡量。测定亲和力的方法是本领域已知的。
与参照抗体显示相同或相似的结合亲和力和/或特异性的抗体是指这样的抗体，其能够具有参照抗体的至少50％、60％、70％、80％、90％或95％以上的结合亲和力和/或特异性。这可以通过本领域已知的任何测定结合亲和力和/或特异性的方法进行测定。
“IgG形式的抗体”是指抗体的重链恒定区所属于的IgG形式。所有同一型的抗体的重链恒定区都是相同的，不同型的抗体之间的重链恒定区不同。例如，IgG1形式的抗体是指其重链恒定区Ig结构域为IgG1的Ig结构域。
“人”抗体(HuMAb)指具有其中框架区和CDR区域均来源于人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体含有恒定区，则恒定区也来源于人种系免疫球蛋白序列。
“人源化”抗体指这样的抗体，所述抗体中一些、大部分或所有非人抗体(例如小鼠抗体)的CDR结构域外面的氨基酸被来源于人免疫球蛋白的相应氨基酸所替代。在抗体的人源化形式的一个实施方案中，CDR结构域外部的一些、大部分或所有氨基酸已被来自人免疫球蛋白的氨基酸所替代，而一个或多个CDR区内的一些、大部分或所有氨基酸没有改变。氨基酸的小的添加、缺失、插入、取代或修改是容许的，只要它们没有消除抗体结合特定抗原的能力。“人源化”抗体保留与原始抗体类似的抗原特异性。
如本文所用，“嵌合抗体”指这样的抗体，其中可变区来源于一个物种且恒定区来源于另一物种，诸如其中可变区来源于小鼠抗体且恒定区来源于人抗体的抗体。
如本文所用，“抗体片段”指与完整抗体不同的分子，其包含完整抗体的一部分且结合完整抗体所结合的抗原。如本文所用，术语“抗原结合片段”如本文所用指保留特异性结合抗原(例如，人CD40)的能力的抗体的一种或多种片段。抗体片段的例子包括但不限于Fv，Fab，Fab’，Fab’-SH，F(ab’) 2；diabody；线性抗体；单链抗体(例如scFv)；单结构域抗体；双价或双特异性抗体或其片段；骆驼科抗体；和由抗体片段形成的双特异性抗体或多特异性抗体。
如本文所用，“多特异性”是指特异性结合至少两个不同抗原或抗原内两个不同表位，例如三个、四个或五个不同的抗原或表位的抗体。
如本文所用，“双特异性”是指特异性结合两个不同抗原或同一抗原内的两个不同表位的抗体。双特异性抗体可对其它相关抗原具有交叉反应性，或者可结合两个或更多个不同抗原之间所共享的表位。
如本文所用，术语“交联”是指在由特异性结合人CD40的抗体结合顺式或反式FcγRIIb所诱导的细胞上CD40的更高级多聚化，导致诱导CD40的激动活性。可在体外将如本文所述的抗人F(ab’)2用作交联剂对交联进行评估。
如本文所用，术语“交叉反应”指本文所述抗体结合来自不同物种的CD40的能力。例如，结合人CD40的本文所述的抗体也结合来自另一物种的CD40(例如食蟹猴CD40)。如本文所用，交叉反应性可以通过在结合测定(例如SPR、ELISA)中用纯化的抗原检测特异性反应性或结合或以其他方式功能性地与生理表达CD40的细胞相互作用来测量。用于测定交叉反应性的方法包括如本文所述的标准结合测定，例如通过
Figure PCTCN2020084186-appb-000015
表面等离子共振(SPR)分析使用
Figure PCTCN2020084186-appb-000016
2000SPR仪器(Biacore AB，Uppsala，Sweden)或流式细胞术技术。
“免疫缀合物”是与一个或多个其它物质(包括但不限于细胞毒性剂或标记)缀合的抗体。
本文所使用的术语“标记”是指被直接或间接缀合或融合至试剂(诸如多核苷酸探针或抗体)并且促进其所缀合或融合的试剂的检测的化合物或组合物。标记本身可以是可检测的(例如，放射性同位素标记或荧光标记)或在酶促标记的情况下可以催化可检测的底物化合物或组合物的化学改变。术语旨在涵盖通过将可检测物质偶联(即，物理连接)至探针或抗体来直接标记探针或抗体以及通过与直接标记的另一种试剂反应来间接标记探针或抗体。间接标记的实例包括使用荧光标记的二级抗体进行的一级抗体的检测和具有生物素的DNA探针的末端标记，使得其可以用荧光标记的链霉抗生素蛋白来检测。
“载体”是指能够在生物系统内复制或可在这类系统之间移动的多核苷酸。载体多核苷酸通常含有诸如复制起点、聚腺苷酸化信号或选择标记的元件，其功能是促进这些多核苷酸在生物系统中的复制或保持。此类生物系统的示例可包括细胞、病毒、动物、植物和用能够复制载体的生物组分再造的生物系统。包含载体的多核苷酸可为DNA或RNA分子或这些分子的杂合分子。“表达载体”是指可用于在生物系统或再造生物系统中以指导由存在于表达载体中的多核苷酸序列所编码的多肽进行翻译的载体。
“分离的”抗体是这样的抗体，其已经与其天然环境的组分分离。在一些实施方案中，将抗体纯化至超过95％或99％纯度，如通过例如电泳(例如，SDS-PAGE，等电聚焦(IEF)，毛细管电泳)或层析(例如，离子交换或反相HPLC)确定的。对于用于评估抗体纯度的方法的综述，参见，例如，Flatman等，J.Chromatogr.B848：79-87(2007)。
“分离的”核酸是指这样的核酸分子，其已经与其天然环境的组分分离。分离的核酸包括包含在通常包含该核酸分子的细胞中的核酸分子，但是该核酸分子存在于染色体外或在不同于其天然染色体位置的染色体位置处。
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或两个核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或核酸序列之一或二者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或核苷酸位置处的氨基酸残 基或核苷酸。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或核苷酸占据时，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。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http：//www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷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或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“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。
如本文所用，术语“在低严格性、中等严格性、高严格性或极高严格性条件下杂交”描述了杂交和洗涤条件。进行杂交反应的指导可以在通过引用方式并入的Current Protocols in Molecular Biology，John Wiley & Sons，N.Y.(1989)，6.3.1-6.3.6中找到。参考文献中描述了含水方法和非含水方法并且可以使用任一方法。本文中提及的特异性杂交条件如下：1)低严格性杂交条件是在约45℃于6X氯化钠/柠檬酸钠(SSC)中，随后至少在50℃(对于低严格性条件，可以增加洗涤的温度至55℃)于0.2X SSC，0.1％SDS中洗涤两次；2)中等严格性杂交条件是在约45℃于6X SSC中、随后在60℃在0.2X SSC、0.1％SDS中洗涤一次或多次；3)高严格性杂交条件是在约45℃在6X SSC中、随后在65℃于0.2X SSC、0.1％SDS中洗涤一次或多次；并且优选地4)极高严格性杂交条件是在65℃于0.5M磷酸钠、7％SDS中、随后在65℃于0.2X SSC、0.1％SDS中洗涤一次或多次。极高严格性条件(4)是优选的条件和除非另外说明，否则应当使用的一个条件。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换地使用且是指其中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括初级转化的细胞和来源于其的后代，而不考虑传代的数目。后代在核酸内容上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。适用于本发明的合适的宿主细胞包括原核微生物，如大肠杆菌。宿主细胞还可以是真核微生物如丝状真菌或酵母，或各种真核细胞，例如昆虫细胞等。也可以将脊椎动物细胞用作宿主。例如，可以使用被改造以适合于悬浮生长的哺乳动物细胞系。有用的哺乳动物宿主细胞系的例子包括SV40转化的猴肾CV1系(COS-7)；人胚肾系(HEK 293或293F细胞)、293细胞、幼仓鼠肾细胞(BHK)、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、人宫颈癌细胞(HELA)、犬肾细胞(MDCK)、布法罗大鼠肝脏细胞(BRL 3A)、人肺细胞(W138)、人肝脏细胞(Hep G2)、中国仓鼠卵巢细胞(CHO细胞)、CHOK1SV细胞、CHOK1SV GS-KO细胞、CHOS细胞、NSO细胞、骨髓瘤细胞系如Y0、NS0、P3X63和Sp2/0等。适于产生蛋白质的哺乳动物宿主细胞系的综述参见例如Yazaki和Wu，Methods in Molecular Biology，第248卷(B.K.C.Lo编著，Humana Press，Totowa，NJ)，第255-268页(2003)。在一个优选的实 施方案中，所述宿主细胞是CHO细胞，例如CHOS细胞CHOK1SV细胞或CHOK1SV GS-KO，或所述宿主细胞是293细胞，例如HEK293细胞。
术语“激动剂”或“激动性”是指特异性结合人CD40的抗体，在结合CD40时诱导B细胞和/或树突细胞(DC)或T细胞的增殖或激活。B细胞和DC和T细胞的增殖或激活可通过如下方式测定：测定增加的B细胞增殖，或者测定B细胞上任何表面标记物CD23、CD80、CD83、CD86，或DC上CD80、CD83、CD86和HLA-DR的上调。在与不含抗体的对照样本进行比较时，激动剂能够以统计意义上显著的方式诱导B细胞和/或DC和/或T细胞活化。
“抑制肿瘤细胞/肿瘤生长”是指与在不存在治疗剂的情况下相同肿瘤细胞或肿瘤的生长相比，在与治疗剂或治疗剂的组合接触时体外或体内肿瘤细胞生长或肿瘤的可测量的下降。体外或体内肿瘤细胞或肿瘤生长的抑制可为至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、99％或100％。
本文所述的术语“治疗剂”涵盖在预防或治疗疾病，例如肿瘤(例如癌症)和感染(例如慢性感染)中有效的任何物质，包括化疗剂、细胞毒性剂、疫苗、其它抗体、抗感染活性剂、小分子药物或免疫调节剂。
“化疗剂”包括在治疗免疫系统疾病中有用的化学化合物，包括但不限于烷化剂；抗代谢物；抗微管抑制剂、天然产物；抗生素；酶；杂类试剂；激素和拮抗剂；抗雌激素；抗雄激素；非类固醇抗雄激素、拓扑异构酶抑制剂、受体酪氨酸激酶抑制剂、血管生成抑制剂等等。本发明的示例性的化疗剂例如阿那曲唑
Figure PCTCN2020084186-appb-000017
比卡鲁胺
Figure PCTCN2020084186-appb-000018
硫酸博来霉素
Figure PCTCN2020084186-appb-000019
白消安
Figure PCTCN2020084186-appb-000020
白消安注射剂
Figure PCTCN2020084186-appb-000021
卡培他滨
Figure PCTCN2020084186-appb-000022
N4-戊氧羰基-5-脱氧-5-氟胞苷、卡铂
Figure PCTCN2020084186-appb-000023
卡莫司汀
Figure PCTCN2020084186-appb-000024
苯丁酸氮芥
Figure PCTCN2020084186-appb-000025
顺铂
Figure PCTCN2020084186-appb-000026
克拉立滨
Figure PCTCN2020084186-appb-000027
环磷酰胺
Figure PCTCN2020084186-appb-000028
阿糖胞苷、胞嘧啶阿拉伯糖苷
Figure PCTCN2020084186-appb-000029
阿糖胞苷脂质体注射剂
Figure PCTCN2020084186-appb-000030
达卡巴嗪
Figure PCTCN2020084186-appb-000031
更生霉素(dactinomycin)(放线菌素D、Cosmegan)、盐酸道诺霉素
Figure PCTCN2020084186-appb-000032
柠檬酸道诺霉素脂质体注射剂
Figure PCTCN2020084186-appb-000033
地塞米松、多西紫杉醇
Figure PCTCN2020084186-appb-000034
盐酸多柔比星
Figure PCTCN2020084186-appb-000035
Figure PCTCN2020084186-appb-000036
依托泊苷
Figure PCTCN2020084186-appb-000037
磷酸氟达拉滨
Figure PCTCN2020084186-appb-000038
5-氟尿嘧啶
Figure PCTCN2020084186-appb-000039
氟他胺
Figure PCTCN2020084186-appb-000040
tezacitibine、吉西他滨(双氟脱氧胞苷)、羟基脲
Figure PCTCN2020084186-appb-000041
伊达比星
Figure PCTCN2020084186-appb-000042
异环磷酰胺
Figure PCTCN2020084186-appb-000043
伊立替康
Figure PCTCN2020084186-appb-000044
L-天冬酰胺酶
Figure PCTCN2020084186-appb-000045
亚叶酸钙、美法仑
Figure PCTCN2020084186-appb-000046
6-巯基嘌呤
Figure PCTCN2020084186-appb-000047
甲氨蝶呤
Figure PCTCN2020084186-appb-000048
米托蒽醌(米托蒽醌)、米罗他(mylotarg)、紫杉醇
Figure PCTCN2020084186-appb-000049
phoenix(钇90/MX-DTPA)、喷司他丁、聚苯丙生20联用卡莫司汀植入物
Figure PCTCN2020084186-appb-000050
柠檬酸他莫昔芬
Figure PCTCN2020084186-appb-000051
替尼泊苷
Figure PCTCN2020084186-appb-000052
6-硫鸟嘌呤、塞替派、替拉扎明
Figure PCTCN2020084186-appb-000053
注射用盐酸拓扑替康
Figure PCTCN2020084186-appb-000054
长春碱
Figure PCTCN2020084186-appb-000055
长春新碱
Figure PCTCN2020084186-appb-000056
长春瑞滨(长春瑞滨)、依鲁替尼、吉利德(idelalisib)和贝伦妥单抗-维多汀(brentuximab vedotin)，以及任何上述物质的药学上可接受的盐、酸或衍生物。。该定义还包括用于调节或抑制激素对肿瘤的作用的抗激素药物诸如抗雌激素药物，包括例如他莫昔芬、雷洛昔芬、芳香化酶抑制4(5)-咪唑、4-羟基他莫昔芬、曲沃昔芬、keoxifene、LY117018、奥那司酮和托瑞米芬和抗雄激素，诸如氟他胺、尼鲁米特、比卡鲁胺、醋酸亮丙瑞林和戈舍瑞林；以及上述任一者的药学上可接受的盐、酸或衍生物。
术语“细胞毒性剂”用在本发明中指抑制或防止细胞功能和/或引起细胞死亡或破坏的物质。示例性的细胞毒性剂包括但不限于：放射性同位素，例如碘( 131I、 125I、 123I和 121I)、碳( 14C)、硫( 35s)、氚( 3H)、铟( 115In、 113In、 112In和 111In)、锝( 99Tc)、铊( 201Ti)、镓( 68Ga、 67Ga)、钯( 103Pd)、钼( 99Mo)、氙( 133Xe)、氟( 18F)、 153Sm、 177Lu、 159Gd、 149Pm、 140La、 175Yb、 166Ho、 90Y、47Sc、 186Re、 188Re、 142Pr、 105Rh、 97Ru、 68Ge、 57Co、 65Zn、 85Sr、 32P、 153Gd、 169Yb、 51Cr、 54Mn、 75Se、 113Sn和 117Tin；生长抑制剂；烷化剂(包括但不限于氮芥、乙烯亚胺衍生物、烷基磺酸盐、亚硝基脲和三氮烯)，例如尿嘧啶氮芥、氮芥(Chlormethine)、环磷酰胺(CYTOXAN TM)、异环磷酰胺(fosfamide)、美法仑、苯丁酸氮芥、胍血生、三乙撑蜜胺、三亚乙基硫代磷胺(Triethylenethiophosphoramine)、白消安、卡莫司汀、洛莫司汀、链脲霉素、达卡巴嗪和替莫唑胺；或抗代谢物(包括但不限于叶酸拮抗剂、嘧啶类似物、嘌呤类似物和腺苷脱氨酶抑制剂)：甲氨蝶呤、5-氟尿嘧啶、氟尿苷、阿糖胞苷、6-巯嘌呤、6-硫鸟嘌呤、磷酸氟达拉滨、喷司他丁(Pentostatine)和吉西他滨。在一些实施方案中，本发明的示例性的细胞毒性剂包括抗微管药物、拓扑异构酶抑制剂、抗代谢药、有丝分裂抑制剂、烷基化剂、蒽环类、长春碱类生物碱、嵌入剂、能够干扰信号转导途径的活性剂、促凋亡活性剂、蛋白酶体抑制剂和照射(例如，局部或全身照射(例如、γ辐射)。
术语“小分子药物”是指低分子量的能够调节生物过程的有机化合物。“小分子”被定义为分子量小于10kD、通常小于2kD和优选小于1kD的分子。小分子包括但不限于无机分子、有机分子、含无机组分的有机分子、含放射性原子的分子、合成分子、肽模拟物和抗体模拟物。作为治疗剂，小分子可以比大分子更能透过细胞、对降解更不易感和更不易于引发免疫应答。有关小分子，例如抗体和细胞因子的肽模拟物，以及小分子毒素的描述参见例如Casset等，(2003)Biochem.Biophys.Res.Commun.307：198-205；Muyldermans(2001)J.Biotechnol.74：277-302；Li(2000)Nat.Biotechnol.18：1251-1256；Apostolopoulos等，(2002)Curr.Med.Chem.9：411-420；Monfardini等，(2002)Curr.Pharm.Des.8：2185-2199；Domingues等，(1999)Nat.Struct.Biol.6：652-656；Sato和Sone(2003)Biochem.J.371：603-608；美国专利号6,326,482。
术语“抗感染活性剂”包括在施用浓度和给药间隔下特异性抑制或消除微生物生长但对宿主不致命的任何分子，所述微生物诸如病毒、细菌、真菌或原生动物，例如寄生虫。用于本文时，术语抗感染活性剂包括抗生素、抗细菌剂、抗病毒剂、抗真菌剂和抗原生动物剂。在一个具体方面中，抗感染活性剂在施用浓度和给药间隔对宿主是无毒的。
抗细菌的抗感染活性剂或抗细菌剂可广泛的分类为杀菌的(即，直接杀死)或抑菌的(即，阻止分裂)。抗菌的抗感染活性剂可进一步再分类为窄谱抗菌剂(即，仅影响小类细菌亚型，例如，革兰氏阴性等)或广谱抗菌剂(即，影响广泛种类)。实例包括阿米卡星、庆大霉素、格尔德霉素、除莠霉素、莫匹罗星、呋喃妥因、吡嗪酰胺、奎奴普丁/达福普汀、利福平/异福酰胺或替硝唑等。
术语“抗病毒剂”包括抑制或消除病毒生长、致病和/或存活的任何物质。这包括例如阿昔洛韦、西多福韦、齐多夫定、去羟肌苷(ddI，VIDEX)、扎西他滨(ddC，HIVID)、司他夫定(d4T，ZERIT)、拉米夫定(3TC，EPIVIR))、阿巴卡韦(ZIAGEN)、恩曲他滨(EMTRIVA)等。
术语“抗真菌剂”包括抑制或消除真菌生长、致病和/或存活的任何物质。这包括例如那他霉素、龟裂杀菌素、非律平、制霉菌素、两性霉素B、坎底辛、绿叶刺蕊草(patchouli)、印度楝树种子油(neem seed oil)、椰子油(Coconut Oil)等。
术语“抗原生动物剂”包括抑制或消除原生动物生物体(例如寄生虫)生长、发病和/或存活的任何物质。抗原生动物剂的实例包括抗疟疾试剂例如，奎宁、奎尼丁等。
本文使用的术语“免疫调节剂”指抑制或调节免疫应答的天然或合成活性剂或者药物。免疫应答可以是体液应答或细胞应答。免疫调节剂包含免疫抑制剂。
术语“免疫检查点分子”意指免疫系统中存在的一类抑制性信号分子，通过调节外周组织中免疫反应的持续性和强度避免组织损伤，并参与维持对于自身抗原的耐受(Pardoll DM.，The blockade of immune checkpoints in cancer immunotherapy.Nat Rev Cancer，2012，12(4)：252-264)。研究发现，肿瘤细胞能够逃避体内免疫系统而失控增殖的原因之一是利用了免疫检查点分子的抑制性信号通路，由此抑制了T淋巴细胞活性，使得T淋巴细胞不能有效发挥对肿瘤的杀伤效应(Yao S，Zhu Y和Chen L.，Advances in targeting cell surface signaling molecules for immune modulation.Nat Rev Drug Discov，2013，12(2)：130-146)。免疫检查点分子包括但不限于程序性死亡1(PD-1)、PD-L1、PD-L2、细胞毒T淋巴细胞抗原4(CTLA-4)、LAG-3和TIM-3。
术语“共刺激分子”是指T细胞上的与共刺激配体特异性结合从而介导T细胞的共刺激反应(例如但不限于增殖)的相应结合配偶体。共刺激分子是除抗原受体或其配体之外的有助于有效免疫应答的细胞表面分子。共刺激分子包括但不限于MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白、细胞因子受体、整联蛋白、信号传导淋巴细胞活化分子(SLAM蛋白)、激活NK细胞受体、OX40、CD40、GITR、4-1BB(即CD137)、CD27和CD28。在一些实施方案中，“共刺激分子”是OX40、GITR、4-1BB(即CD137)、CD27和/或CD28。
术语“细胞因子”是由一种细胞群释放，作为细胞间介质作用于另一细胞的蛋白质的通称。此类细胞因子的例子有淋巴因子、单核因子、白介素(IL)，诸如IL-1，IL-1α，IL-2，IL-3，IL-4，IL-5，IL-6，IL-7，IL-8，IL-9，IL-11，IL-12，IL-15；肿瘤坏死因子，诸如TNF-α或TNF-β；及其它多肽因子，包括LIF和kit配体(KL)和γ-干扰素。如本文中使用的，术语细胞因子包括来自天然来源或来自重组细胞培养物的蛋白质及天然序列细胞因子的生物学活性等效物，包括通过人工合成产生的小分子实体，及其药剂学可接受的衍生物和盐。
术语“抑制剂”或“拮抗剂”包括使所给出分子(例如，免疫检查点分子)的某些参数(例如，活性)降低的物质。例如，这个术语包括使得所给出的分子被抑制至少5％、10％、20％、30％、40％或更多的活性(例如，PD-L1活性)的物质。因此，抑制作用不必是100％。
术语“激活剂”包括使所给出分子(例如，共刺激分子)的某些参数(例如，活性)增加的物质。例如，这个术语包括使得所给出的分子被增加至少5％、10％、20％、30％、40％或更多的活性(例如，OX40活性)的物质。因此，激活作用不必是100％。
术语“药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂、载体或稳定剂等。
术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。
术语“组合产品”是指一种剂量单位形式的固定组合或非固定组合或用于组合施用的部分的试剂盒，其中两种或更多种治疗剂可以独立地在同一时间或在时间间隔内分开施用，尤其是在这些时间间隔允许组合伴侣展示协作，例如，协同效应时。术语“固定组合”是指本发明抗体和组合伴侣(例如其他治疗剂，例如免疫调节剂，例如免疫抑制剂或抗炎剂)以单一实体或剂量的形式同时施用于患者。术语“非固定组合”意指本发明抗体和组合伴侣(例如其他治疗剂，例如免疫调节剂，例如免疫抑制剂或抗炎剂)作为分开的实体同时、并行或依次施用于患者，没有特定的时间限制，其中这样的施用提供了患者体内两种化合物的治疗有效水平。后者也适用于鸡尾酒疗法，例如施用三种或更多种治疗剂。在一个优选的实施方案中，药物组合是非固定组合。
“免疫原性”指特定物质引发免疫应答的能力。肿瘤是免疫原性的，并且增强肿瘤免疫原性有助于通过免疫应答清除肿瘤细胞。
“免疫应答”指脊椎动物中针对外源物的生物应答，所述应答保护生物体免于这些物质和由其引起的疾病。免疫应答由免疫系统的细胞(例如，T淋巴细胞、B淋巴细胞、天然杀伤(NK)细胞、巨噬细胞、嗜酸性粒细胞、肥大细胞、树突细胞或嗜中性粒细胞)和由这些细胞或肝脏产生的可溶性大分子(包括抗体、细胞因子和补体)的作用介导，其导致从脊椎动物机体选择性靶向、结合、损伤、破坏和/或消除入侵病原体、细胞或感染有病原体的组织、癌性或其他异常细胞、或者(在自身免疫或病理性炎症的情况下)正常人细胞或组织。免疫反应包括例如活化或抑制T细胞，例如，效应T细胞或Th细胞，诸如CD4+或CD8+T细胞，或者抑制或耗尽Treg细胞。“T效应”(“T eff”)细胞指具有细胞溶解活性的T细胞(例如CD4+和CD8+T细胞)以及T辅助(Th)细胞(其分泌细胞因子并活化和引导其他免疫细胞)，但不包括调节性T细胞(Treg细胞)。
术语“有效量”指本发明的抗体或片段或缀合物或组合物的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定：诸如哺乳动物的物种；它的大小、年龄和一般健康；涉及的具体疾病；疾病的程度或严重性；个体患者的应答；施用的具体抗体；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。抗体或抗体片段或其缀合物或组合物的治疗有效量可以根据多种因素如疾病状态、个体的年龄、性别和重量和抗体或抗体部分在个体中激发所需反应的能力而变动。治疗有效量也是这样的一个量，其中抗体或抗体片段或其缀合物或组合物的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数(例如肿胀率等)至少约20％、更优选地至少约40％、甚至更优选地至少约50％、60％或70％和仍更优选地至少约80％或90％。可以在预示人自身免疫疾病或炎症中的功效的动物模型系统中评价化合物抑制可度量参数(例如，肿胀率)的能力。
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。
本文所述的“个体”或“受试者”可以互换使用，包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。在一些实施方案中，个体或受试者可以是哺乳动物，例如，灵长类，优选地，高级灵长类，例如，人类(例如，患有本文所述疾病或具有患有本文所述疾病的风险的患者)。在一个实施方案中，受试者患有本文所述疾病(例如，如本文所述的肿瘤或感染)或具有患有本文所述疾病的风险。在某些实施方案中，受试者接受或已经接受过其它治疗，例如化疗治疗和/或放射疗法。备选地或组合下，受试者因感染而免疫受损或具有因感染而免疫受损的风险。
术语“组合疗法”是指施用两种或更多种治疗剂或治疗方式(例如放射疗法或手术)以治疗如本公开所述的IL-23相关疾病。这种施用包括以基本上同时的方式共同施用这些治疗剂，例如以具有固定比例的活性成分的单一胶囊。或者，这种施用包括对于各个活性成分在多种或在分开的容器(例如片剂、胶囊、粉末和液体)中的共同施用。粉末和/或液体可以在施用前重构或稀释至所需剂量。此外，这种施用还包括以大致相同的时间或在不同的时间以顺序的 方式使用每种类型的治疗剂。在任一情况下，治疗方案将提供药物组合在治疗本文所述的病症或病状中的有益作用。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有免疫系统疾病(自身免疫疾病或炎症)家族病史的受试者是预防性方案的候选。通常，在免疫系统疾病(自身免疫疾病或炎症)的背景中，术语“预防”是指在免疫系统疾病(自身免疫疾病或炎症)的病征或症状发生前，特别是在具有免疫系统疾病(自身免疫疾病或炎症)风险的受试者中发生前的药物施用。
“受试者/患者样品”指从患者或受试者得到的细胞或流体的集合。组织或细胞样品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细胞。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。
本发明的这些以及其它方面和实施方案在附图(附图简述紧随其后)和以下的发明详述中得到描述并且示例于以下实施例中。上文以及整个本申请中所论述的任何或所有特征可以在本发明的各种实施方案中组合。以下实施例进一步说明本发明，然而，应理解实施例以说明而非限定的方式来描述，并且本领域技术人员可以进行多种修改。
实施例
实施例1.构建报告细胞系Jurkat/NF-κB-GFP+hCD40
NF-κB-GFP报告基因慢病毒系统(QIAGEN，CCS-013G)的启动子中有多个NF-κB转录因子结合元件，该启动子的激活可以驱动GFP的表达。根据试剂盒的说明书，用NF-κB-GFP报告基因慢病毒感染Jurkat细胞(ATCC，TIB-152)，并用嘌呤霉素(InvivoGen，ant-pr-1)筛选感染慢病毒的Jurkat细胞。加入10ng/ml TNFα蛋白(Sino Biological，10602-HNAE)刺激利用嘌呤霉素筛选到的细胞，24h后流式细胞仪(BD，FACSAria III)分选GFP荧光的细胞亚群，获得Jurkat/NF-κB-GFP细胞。
将人CD40 CDS(Sino Biological，HG10774-M)构建至慢病毒核心质粒pCDH(System Biosciences)中，使用标准步骤用PEI(polyscience，24885-2)向293FT细胞(Thermo Fisher Scientific，R70007)中按1∶1∶1∶1共转染所获得的慢病毒核心质粒和辅助质粒pPACKH1-GAG、pPACKH1-REV、pVSVG(均来自System Biosciences)，转染6h后换液，37℃培养于含10％胎牛血清(Biological Industries，04-001-1A)的DMEM培养基(Lifetechnologies，C11995500CP)中，继续培养48h收集含有人CD40慢病毒的上清。
本文所用阳性对照CD40L序列来自专利WO 2016/177771(SEQ ID NO：15)，阴性对照HEL序列如表10所示。CD40L和HEL的DNA序列在金唯智公司进行全基因合成。对于CD40L和HEL纯化，将DNA序列插入真核表达载体pFUSE(InvivoGen，pfuse-hg1fc1)中，使用标准步骤用PEI转染至293F细胞(Thermo Fisher Scientific，R79007)；利用Freestyle培养基(Lifetechnologies，12338026)在37℃摇床培养细胞。培养7天后收集上清液，并在AKTA系统(GE)上使用Superdex TM 200 Increase预装柱(GE，28-9909-44)纯化。
CD40信号转导可以激活NF-κB通路，如果CD40成功表达于Jurkat/NF-κB-GFP细胞膜表面，CD40L刺激将会诱导GFP的表达。将如上所述分选获得的构建好的Jurkat/NF-κB-GFP细胞用上述获得的人CD40慢病毒上清感染，16h后加入100nM上述获得CD40L蛋白(作为阳性对照)和HEL(作为阴性对照)刺激，24h后流式细胞仪单细胞分选GFP荧光最强的细胞至96孔板中。
将上述96孔板中的细胞在含有10％胎牛血清(Biological Industries，04-001-1A)的RPMI1640培养基(Lifetechnologies，C11875500CP)中培养2周，待单细胞长起来后分别加入100nM上述获得三聚化CD40L蛋白(作为阳性对照)和HEL(作为阴性对照)，挑选CD40L强阳性、HEL阴性的单克隆，作为Jurkat/NF-κB-GFP+hCD40(简称NF-κB-GFP+hCD40)细胞，用于后续实验。结果如图1，CD40L刺激后Jurkat/NF-κB-GFP+hCD40单克隆GFP阳性率为94.7％，HEL刺激无激活。
实施例2.从噬菌体展示抗体库中筛选CD40结合抗体
从天津市血液中心及上海秒通生物公司购买30例健康成人外周血，利用ficoll分离液(灏洋，LDS1075)离心获取PBMC，离心条件：20℃，2000rpm，升5降0模式30分钟。使用如上所述获得的PBMC构建人天然抗体库，库的构建为常规方法(phage display，Tim Clackson and Henry B.Lowman)。将获得抗体重链和轻链可变区随机组合以单链抗体scFv的形式展示于噬菌体衣壳蛋白pIII的N端，获得噬菌体展示抗体库，库容达到10 10。
噬菌体筛选为常规技术(Phage Display：A Laboratory Manual，Carlos F Barbas III)。首先，将生物素化CD40蛋白(acrobiosystems，TN5-H82F9)与如上所述获得的噬菌体展示抗体库室温孵育2h。孵育结束后直接加入150ul链霉亲和素磁珠Dynabeads M280(Lifetechnologies，11205D)，在室温混匀仪上孵育30min。用0.05％PBS-tween(PBS：Lifetechnologies，70011044；Tween：Sigma-Aldrich，9005-64-5)洗去不与抗原结合的噬菌体，最后用0.2M甘氨酸-盐酸(PH2.2)洗脱与抗原结合的噬菌体。应用洗脱的噬菌体感染大肠杆菌XL1-blue(Agilent，200236)，加入辅助噬菌体VCSM13(Agilent，200251)扩增后用于下一轮筛选。共进行三轮筛选。三轮后，富集与CD40结合的抗体噬菌体，筛选结果如表1。
表1 利用噬菌体展示技术从人天然抗体库中筛选CD40结合抗体
Figure PCTCN2020084186-appb-000057
为了初步评估筛选到CD40结合抗体的阳性率，我们分别挑取第二、三轮29个噬菌体单克隆，进行噬菌体ELISA分析。噬菌体ELISA为常规技术，具体如下：从深孔板中培养的噬菌体单克隆中各挑取第二、三轮29个噬菌体单克隆，37℃ 300rpm摇至OD＝0.5～0.8，加入辅助噬菌体VCSM13扩增后30℃过夜诱导噬菌体，用于抗体表达。在ELISA板(Corning，3690)上包被1μg/ml的人源CD40(Acrobiosystems，CD0-H5228)，4℃过夜孵育。向过夜孵育后的ELISA板上加入过夜诱导的噬菌体上清，室温孵育1h，0.05％PBST洗8次。利用BSA(Solarbio，A8020-100)作为阴性对照。加入HRP偶联的抗-M13(GE，27-9421-01，M13为噬菌体衣壳蛋白)检测结合的抗体噬菌体。将CD40结合信号是BSA对照的3倍以上定义为阳性克隆。结果如图2，其中第二轮筛选、第三轮筛选的阳性率分别为6.9％、27.6％。
实施例3.共刺激分子CD40的激动剂抗体筛选
首先，我们将第三轮噬菌体展示富集到的抗体亚克隆到分泌型慢病毒载体pCDH中，同时将N27 ScFv克隆到分泌型慢病毒载体pCDH中作为阴性对照，N27 ScFv序列见表11。向293FT细胞中按1∶1∶1∶1共转染慢病毒核心质粒和辅助质粒pPACKH1-GAG、pPACKH1-REV、pVSVG，转染6h后换液，37℃培养于含10％胎牛血清(Biological Industries，04-001-1A)的DMEM培养基中，继续培养48h收集含有CD40结合抗体慢病毒文库的上清；用50000pg所获得的CD40结合抗体的慢病毒文库感染HEK293细胞，感染16h后，离心去除含有慢病毒的培养基，添加新鲜培养基，利用流式分选技术将感染的HEK293细胞分选到96孔板中，使每个孔仅含有1个感染HEK293细胞，培养细胞3周，待细胞达到一定数量后取上清。上清加入如上所述获得的Jurkat/NF-κB-GFP+hCD40报告细胞3×10 5个，同时加入2.5μg/ml山羊抗人Fc(SouthernBiotech，SBA-2048-01)的二抗，交联细胞分泌的抗体以增强激动剂抗体的激活强度；培养24h后测试细胞上清的活性，阳性抗体的检测结果如图3；从细胞上清活性为阳性的HEK293细胞中提取抗体基因，构建至pFUSE载体进行测序，获得阳性抗体VH及VL序列，获得的两个阳性抗体编号为NK003和NK004，其序列(包括CDR、VH/VL和重链、轻链)参见序列表的表4至表8中的序列。
实施例4.全长抗体IgG的表达和纯化
抗体的表达与纯化为常规方法，具体如下：
根据抗体序列，合成筛选出的激动剂抗体NK003、NK004重链和轻链DNA(金唯智)，将其分别克隆至载体pFUSE中，使用标准步骤用PEI将分别含有重链和轻链的质粒二者按1∶1瞬时共转染至293F悬浮细胞，以表达全长抗体。培养1周后在AKTA系统上使用Superdex TM200Increase预装柱纯化。
SDS-PAGE法鉴定纯化抗体，应用分子排阻预装柱Superdex 200分析抗体的聚集。结果如图4，其中NK003洗脱峰形对称且洗脱时的保留时间与单个分子量的单克隆抗体保持一致，同时抗体中存在少量聚集体。
实施例5.人源CD40抗体NK003、NK004的体外表征
5.1 NK003、NK004与CD40的结合
通过与TNFR家族成员OX40、4-1BB和GITR的直接ELISA，评价NK003的结合选择性。在ELISA板上包被含有1μg/ml人源OX40(Acrobiosystems，OX0-H5224)、4-1BB(Acrobiosystems，41B-H5227)、GITR(Acrobiosystems，GIR-H5228)和CD40(Acrobiosystems，CD0-H5228)的PBS溶液，4℃过夜孵育。加入如实施例4获得的用PBS稀释至不同浓度的NK003：0.625μg/ml、1.25μg/ml、2.5μg/ml和5μg/ml，室温孵育30min后，0.05％PBS-Tween洗8次。加入羊抗人HRP偶联的Fc抗体(SouthernBiotech，2048-05)检测结合的NK003。如图5A所示，NK003选择性与人CD40结合，与其他测试的TNFR家族蛋白低结合或不结合。
为了评价NK004与CD40的结合，在ELISA板上包被1μg/ml的人源CD40(Acrobiosystems，CD0-H5228)的PB溶液，4℃过夜孵育。加入如实施例4获得的PBS稀释至不同浓度NK004：0.002nM、0.016nM、0.13nM、1nM、8nM、64nM和500nM，HEL为阴性对照抗体，室温孵育30min后，0.05％PBS-Tween洗8次。加入羊抗人HRP偶联的Fc抗体检测结合的NK004。如图5B所示，NK004可以与人CD40结合。
5.2 NK003阻断CD40L与CD40结合
用ELISA评价NK003对CD40L与CD40结合的作用。首先，使用蛋白生物素化试剂盒(GeneCopoeia，BI001)，根据说明书生物素化CD40L。在ELISA板上包被1μg/ml的人源CD40，4℃过夜孵育。加入如实施例4获得的PBS稀释至不同浓度NK003：0.156μg/ml、0.313μg/ml、0.625μg/ml、1.25μg/ml、2.5μg/ml、5μg/ml、10μg/ml和20μg/ml，HEL为对照抗体，室温孵育1h后，加入2.5μg/ml生物素化的CD40L，室温孵育30min后，0.05％PBS-Tween洗8次。加入Streptavidin-HRP使用酶标仪(SpectraMax i3x)读取OD405数值，检测结合CD40L与CD40的结合量。结果如图所示6，NK003阻断CD40与CD40L的结合。
5.3 NK003、NK004以交联形式激活NF-κB-GFP+hCD40报告细胞系
本实施例中通过流式细胞术检测NK003、NK004抗体激活NF-κB-GFP+hCD40报告细胞系的情况，并且测定两种抗体的EC50，具体步骤如下：
分别取3×10 5个/管如实施例1中获得NF-κB-GFP+hCD40报告细胞，分别加入如实施例4中获得的稀释至不同浓度的NK003或NK004：0.001μg/ml、0.005μg/ml、0.01μg/ml、0.05μg/ml、0.1μg/ml、0.5μg/ml、1μg/ml、5μg/ml和10μg/ml，10μg/ml，利用HEL抗体做阴性对照；为了交联，各组同时加入2.5μg/ml浓度的二抗，即山羊抗人Fc。在含有10％胎牛血清(Biological Industries，04-001-1A)的RPMI 1640培养基(Lifetechnologies，C11875500CP)中37℃共同培养24h后，PBS洗三次，应用流式细胞仪进行分析。获得的数据使用GraphPad Prism 6.0拟合曲线并计算EC50。结果如图7，NK003对CD40的激活依赖二抗交联，以交联形式激活NF-κB-GFP+hCD40报告细胞系，EC50为2nM(图7A)。NK004对CD40的激活不依赖二抗交联，以组成型模式激活NF-κB-GFP+hCD40报告细胞系，加入二抗时EC50为4Nm(图7B)。
5.4表面等离子共振对NK003动力学和亲和力定量分析
Biacore T200(GE Healthcare)用于检测抗体NK003、NK004的亲和力。将NK003、NK004以10μL/min流过Protein A芯片，捕获在芯片上。用Running buffer(HBS-EP+，GE)梯度稀释待测抗原人CD40重组蛋白(Acrobiosystems，CD0-H5228)，浓度设置为2μM、1μM、500nM、250nM、125nM、62.5nM、31.25nM。将上述不同浓度CD40按30μL/min流速流过捕获有抗体的芯片，结合120s，然后芯片以30μL/min的流速流过Running buffer，240s，抗原逐渐从捕获抗体的芯片上解离。数据处理使用Biacore T200仪器配套软件BIAevaluation software S200进行，计算结合常数(Ka)、解离常数(Kd)和平衡解离常数(KD)。结果如表2所示。
表2 应用表面等离子共振技术平台测量抗体与人CD40的亲和力
传感器 抗体 抗原 ka(1/Ms) kd(1/s) K D(M)
Protein A NK003 CD40 3.67E+04 1.21E-02 3.31E-07
Protein A NK004 CD40 8.87E+04 3.90E-02 4.40E-07
5.5 NK003交叉反应
通过流式细胞术检测表达恒河猴和人的CD40的293FT细胞与NK003的结合，测定NK003与恒河猴和人CD40的交叉反应。
将恒河猴CD40(NCBI，NM_001265862.1)与人CD40(NCBI，NM_001250.5)的CDS区(金唯智合成)克隆到pCDH载体中，使用标准步骤用PEI瞬时转染到293FT细胞中，转染6h后换液，37℃培养于含10％胎牛血清(Biological Industries，04-001-1A)的DMEM培养基中，表达48h后获得分别表达恒河猴CD40的细胞和表达人CD40的细胞。
分别取3×10 5个/管如上所述获得的表达恒河猴和人CD40的293FT细胞，加入如实施例4所述制备的用PBS稀释至不同浓度的NK003：0.001μg/ml、0.005μg/ml、0.01μg/ml、0.05μg/ml、0.1μg/ml、0.5μg/ml、1μg/ml、5μg/ml和10μg/ml，HEL 10μg/ml作为阴性对照，将细胞和不同浓度NK003 4℃共同孵育30min，PBS洗三次，按1∶100加入Alexa Fluro 488山羊抗人Fc荧光二抗(Lifetechnologies，A11013)，避光4℃孵育30min后采用流式细胞仪进行分析。使用GraphPad Prism 6.0拟合曲线并计算EC50，结果如图8所示，可见NK003与恒河猴CD40(图8B)、人CD40(图8A)均有交叉反应，与恒河猴CD40结合EC50为8nM，与人CD40结合EC50为10nM。
5.6 NK003诱导肿瘤细胞凋亡
淋巴瘤细胞Raji和Ramos膜表面有CD40的表达，我们使用Raji细胞(ATCC，CRL-7936)和Ramos细胞(ATCC，CRL-1596)，检测NK003促进肿瘤细胞凋亡的能力。
将Raji和Ramos细胞以3×10 5/mL的密度接种于24孔板，在含有10％胎牛血清(Lifetechnologies，10091148)的RPMI 1640培养基中培养，细胞接种同时加入如上文实施例4中所述制备的PBS稀释至不同浓度的NK003：0.03μg/ml、0.06μg/ml、0.09μg/ml、0.3μg/ml、0.6μg/ml、0.9μg/ml、3μg/ml、6μg/ml和9μg/ml，HEL9μg/ml作为阴性对照，同时每孔按2.5μg/ml的浓度加入山羊抗人Fc做交联剂。共同培养24h后，PBS洗三次，1∶100加入PE-CD95(Biolegend，305611)，避光4℃孵育30min。采用流式细胞仪分析细胞凋亡标志分子CD95的表达(以MFI表示)，检测细胞凋亡情况。使用GraphPad Prism 6.0拟合曲线并计算EC50。结果如图9，NK003促进Raji的凋亡，EC50为2.6nM(图9A)；NK003促进Ramos细胞的凋亡，EC50为3nM(图9B)。
实施例6.原代T细胞活性实验
6.1 DC细胞激活
使用人外周血单核细胞(PBMC)检测NK003激活树突状细胞(DC)的能力。DC细胞的分离为常规方法，具体如下：PBMC(Hemacare，PB009C-3)在37℃含有10％胎牛血清(Lifetechnologies，10091148)的RPMI 1640培养基中培养，6小时后收获贴壁的单核细胞；除去上清，添加含有100ng/mL GM-CSF(R&D Systems，204-IL)和10ng/mL IL4(R&D Systems，215-GM)的无血清RPMI1640培养基培养，37℃；三天后将其中一半的培养基更换，将GM-CSF和IL4补充至100ng/mL和10ng/mL；培养的第六天取悬浮的细胞换至新的培养瓶，应用同样的含有100ng/mL GM-CSF和10ng/mL IL4的无血清RPMI1640培养基继续培养24h，获得DC细胞。
在第七天时，将上述诱导获得的DC细胞以1×10 5个/孔的数量接种于96孔板，加入不同浓度的NK003：0.01μg/ml、0.05μg/ml、0.1μg/ml、0.5μg/ml、1μg/ml、10μg/ml和100μg/ml，100μg/ml HEL做阴性对照；为了交联，各组同时加入2.5μg/ml浓度的山羊抗人Fc。
将上述DC细胞和抗体共同孵育24h后，PBS洗三次，1∶100加入PE-CD11c(DC细胞标志分子，Biolegend，117309)和APC-CD86(DC细胞激活标志分子，Biolegend，105007)，避光4℃孵育30min。应用流式细胞仪检测DC细胞激活情况，以APC-CD86的MFI表示。 DC细胞的激活可导致更强的抗肿瘤T细胞应答。使用GraphPad Prism 6.0拟合曲线并计算EC50。结果如图10，NK003可以显著激活人DC细胞，EC50为2nM(图10A)，加交联剂(山羊抗人Fc，anti-Fc)后EC50为8nM(图10B)
Figure PCTCN2020084186-appb-000058
且交联形式的NK003激活DC的强度比未交联明显增强(MFI显著更高)。
6.2 B细胞增殖
使用人外周血单核细胞(PBMC)检测NK003诱导B细胞增殖的能力。使用CD19免疫磁珠试剂盒(Miltenyi Biotec，130-050-301)，根据试剂盒说明书从人PBMC(Hemacare，PB009C-3)中分选CD19 +B细胞。
将上述分选获得的B细胞培养于含有10％胎牛血清(Lifetechnologies，10091148)的RPMI1640培养基中，添加10ng/mL IL4。将B细胞以1×10 5个/孔的数量接种于96孔板，加入不同浓度的NK003：0.01μg/ml、0.05μg/ml、0.1μg/ml、0.5μg/ml、1μg/ml、10μg/ml和100μg/ml，100μg/ml HEL做阴性对照对照。37℃共同孵育48h后，使用CellTiter-Glo试剂盒(Promega，G7570)检测细胞的增殖情况：根据试剂盒说明书取70μl的细胞加入100μl
Figure PCTCN2020084186-appb-000059
Reagent，混匀避光静置10min，使用酶标仪读取荧光强度，强度越高表示细胞增殖越强。使用GraphPad Prism 6.0拟合曲线并计算EC50。结果如图11，NK003可以促进人B细胞的增殖，EC50为4nM。
6.3 B细胞激活
使用人外周血单核细胞(PBMC)检测NK003激活B细胞的能力。
如6.2所示，从人PBMC中用CD19免疫磁珠试剂盒分选CD19 +B细胞。将上述分选获得的B细胞培养于含有10％胎牛血清(Lifetechnologies，10091148)的RPMI 1640培养基中。将B细胞以1×10 5个/孔的数量接种于96孔板，加入不同浓度的NK003：0.01μg/ml、0.05μg/ml、0.1μg/ml、0.5μg/ml、1μg/ml、10μg/ml和100μg/ml，100μg/ml HEL做阴性对照。为了交联，还进一步将上述各组加入2.5μg/ml浓度的山羊抗人Fc。共同孵育48h后，PBS洗三次，1∶100加入PE-CD86(B细胞激活标志分子，Biolegend，105007)，避光4℃孵育30min。应用流式细胞仪检测在加入抗体后(交联和不交联情况下)B细胞激活情况，以APC-CD86的MFI表示。使用GraphPad Prism 6.0拟合曲线并计算EC50。结果如图12A和B所示，NK003可以激活人B细胞，不加交联剂(山羊抗人Fc，anti-Fc)时EC50为70Nm(图12A)，加交联剂后EC50为4nM(图12B)，且交联形式的NK003激活B细胞的强度比未交联明显增强(CD86表达显著更高)。
实施例7.人源CD40抗体NK003的体内表征
进行不同的体内实验以进一步表征NK003人源抗体的作用。
7.1 SCID小鼠中NK003抑制肿瘤的生长
抗体依赖的细胞介导的细胞毒性作用(ADCC)是指抗体的Fab段结合肿瘤细胞的抗原表位，其Fc段与杀伤细胞(NK细胞、巨噬细胞等)表面的FcR结合，介导杀伤细胞直接杀伤靶细胞。为了评价NK003介导的ADCC杀伤肿瘤细胞的作用，我们使用SCID小鼠接种表达有CD40的肿瘤细胞。
八只五周龄的雄性SCID小鼠购于维通利华，饲养于SPF级动物房。饲养一周后随机分为两组，背部按2.5×10 6个/只的数量皮下接种Raji细胞，每组接种四只，接种当天记为Day 0。第一组给药NK003抗体，第二组给药HEL对照抗体。
Day 1起，将NK003以7.5mg/kg的剂量腹腔注射，每7天给药一次，共三次，HEL为对照抗体，给药剂量和次数同NK003。每三天称量小鼠体重并用游标卡尺测量肿瘤的垂直尺寸，肿瘤的体积＝(长×宽×宽)/2，绘制肿瘤生长曲线。
结果显示，与对照组相比，NK003组小鼠未见明显肿瘤(图13A)，且NK003组小鼠存活率为100％(图13B)。NK003可以通过介导ADCC作用显著抑制肿瘤的生长，提高小鼠生存率。
7.2 CD40人源化小鼠中NK003激活免疫系统
在C57BL/6遗传背景下，将人源CD40及Fc受体FcγRIIA、FcγRIIB、FcγRIIIA插入小鼠基因组，在表达人源CD40和Fc受体蛋白的同时取代小鼠內源CD40和Fc受体的表达。CD40人源化小鼠模型为上海交通大学李福彬构建并提供，饲养于SPF级动物房。OT-1小鼠为上海交通大学李福彬提供，饲养于SPF级动物房。颈椎脱臼处死OT-1小鼠并摘取脾脏，挤压破碎脾脏以收集脾细胞。使用免疫磁珠试剂盒(R&D Systems，MAGM203)，按照试剂盒说明书分离CD8 +T细胞，即为OT-1CD8 +T细胞。
选取八周龄雄性CD40人源化小鼠按2×10 6个/只的数量尾静脉注射OT-1 CD8 +T细胞，同时腹腔注射0.1mg/kg DEC-OVA(Sigma-Aldrich，SAB4700735)以及5mg/kg NK003(相同剂量的HEL为对照抗体)。接种细胞七天后，颈椎法处死小鼠分离脾脏。研磨脾脏获得单细胞悬液，裂解法去除红细胞后，使用抗-CD4(ebioscience，RM4-5)、抗-CD8(Biolegend，53-6.7)、抗-CD45.1(Biolegend，A20)和抗-TCR-Vα2(Biolegend，B20.1)染色，流式细胞仪检测OVA特异性OT-1CD8 +T细胞的增殖。CD45.1 +CD8 +TCR-Vα2 +亚群即为OT-1CD8 +T细胞(OT1细胞)。CD45.1 +CD8 +亚群为CD8细胞，CD45.1 +CD4 +亚群为CD4细胞。结果如图14，NK003组OT-1CD8 +T细胞比例显著升高，细胞数显著增加，CD8与CD4比值也相应增高。NK003可以激活CD40人源化小鼠免疫系统。
7.3 CD40人源化小鼠中NK003抑制肿瘤的生长
为了评价NK003激活免疫系统抑制肿瘤的作用，我们使用如7.2中所述的上海交通大学李福彬构建并提供的CD40人源化小鼠进行肿瘤细胞MC38(中国医学科学院基础医学研究所基础医学细胞中心，3111C0001CCC000523)的接种。
本文所用阳性对照抗CD40抗体CP870893序列来自专利US 7338660(轻链和重链序列参见其中的SEQ ID NO：46和SEQ ID NO：48)。将金唯智合成的CP870893轻链和重链转染到pFUSE载体中，按1∶1瞬时共转染293F悬浮细胞，表达全长抗体，表达1周后在AKTA系统上使用Superdex TM 200 Increase预装柱纯化(具体步骤同实施例4所述)。
将八周龄的雄性CD40人源化小鼠背部按2×10 6个/只的数量皮下接种MC38细胞，当肿瘤长至约100mm 3随机分为三组：HEL对照组8只，NK003及CP870893(阳性对照)组各五只，将抗体以3mg/kg的剂量腹腔注射，每3天给药一次，共两次。每三天称量小鼠体重并用游标卡尺测量肿瘤的垂直尺寸，肿瘤的体积＝(长×宽×宽)/2，绘制肿瘤生长曲线及小鼠体重曲线。抑瘤率＝(对照组平均体积一实验组平均体积)/对照组平均体积×100％。
结果如图15A，其中在第18天时，NK003的肿瘤抑制率为75％，而CP870893的肿瘤抑制率仅为60％。同时我们对小鼠体重进行检测，如图15B所示，应用NK003抗体的小鼠体重无明显降低。因此，NK003可以显著抑制肿瘤的生长，并且对小鼠体重没有显著影响。
实施例8.NK003-V12、NK003-S267E/L328F Fc区突变体增加与FcγRIIB的结合和CD40激 动剂活性
为了增加与FcγR的结合和增加NK003抗体的激动剂活性，NK003 Fc区(IgG1，EU编号)的第233位残基谷氨酸(E)突变为天冬氨酸(D)，第237位残基甘氨酸(G)突变为天冬氨酸(D)，第268位残基组氨酸(H)突变为天冬氨酸(D)，第271位残基脯氨酸(P)突变为甘氨酸(G)，第330位残基丙氨酸(A)突变为精氨酸(R)，产生NK003-V12突变体。NK003Fc区(IgG1，EU编号)的第267位残基丝氨酸(S)突变为谷氨酸(E)，第328位残基亮氨酸(L)突变为苯丙氨酸(F)，产生NK003-S267E/L328F突变体(序列见序列表)。NK003-V12和NK003-S267E/L328F突变体构建以后通过测序确认。
表3 NK003Fc区突变体突变位点概括
Figure PCTCN2020084186-appb-000060
为了评价NK003-V12和NK003-S267E/L328F突变体的激动剂活性，将金唯智合成的NK003-V12和NK003-S267E/L328F突变体的轻链和重链转染到pFUSE载体中，按1∶1瞬时共转染293F悬浮细胞，表达全长抗体，表达1周后在AKTA系统上使用Superdex TM 200Increase预装柱纯化(具体步骤同实施例4所述)。
将金唯智合成的FcγRIIA(NCBI，NM_001136219.1)与FcγRIIB(NCBI，NM_004001.4)CDS区克隆到pCDH载体中，使用标准步骤用PEI瞬时转染到293FT细胞中，转染6h后换液，37℃培养于含10％胎牛血清(Biological Industries，04-001-1A)的DMEM培养基中，表达48h后获得分别表达FcγRIIA的细胞和表达FcγRIIB的293FT细胞。
分别取如上所述2×10 5个/管293FT-FcγRIIA或293FT-FcγRIIB细胞，每管中分别加入2×10 5个/管NF-κB-GFP+hCD40报告细胞(实施例1所述)。向每管中加入制备的PBS稀释的不同浓度NK003、NK003-V12和NK003-S267E/L328F：0.01μg/ml、0.05μg/ml、0.1μg/ml、0.5μg/ml、1μg/ml、5μg/ml和10μg/ml，10μg/ml HEL做阴性对照；37℃共同培养24h后，PBS洗三次，应用流式细胞仪分析，检测GFP的MFI。结果如图16，NK003-V12、NK003-S267E/L328F和野生型NK003相比，激动剂活性显著增强。
实施例9.人源CD40抗体NK003的亲和力成熟
针对NK003 VH、VL的互补决定区和骨架区分别构建突变库，以Fab形式(图17)插入pcomb3噬菌体载体(Biovector Inc.108925)，使用噬菌体亲和力成熟方法完成NK003的优化。
9.1 NK003 VH、VL互补决定区CDR3亲和力成熟
9.1.1 NK003 VH、VL互补决定区CDR3突变库的构建
以CDR3插入终止密码子的NK003轻链DNA(SEQ ID NO：72)为模板，分别使用引物对VL1/VL2、CL1/CL2扩增轻链VL、CL片段。反应条件：95℃2min，1cycle；95℃30s，65℃30s，72℃10s，35cycle；72℃5min，1cycle。使用天根回收试剂盒回收PCR目的 片段。引物如下：
VL1：5’-GCGGCCGAGCTCGATGTTGTGATGACTCAG-3’
VL2：5’-GGTCCCCTGGCCAAA  AGT GTA CGG AGT TCA TAG ACC TTG CAT
GCAGTAATAAAG-3’(横线处随意两处突变为MNN，由金唯智公司高通量合成)
CL1：5’-TTTGGCCAGGGGACCAAGCTGGAGATC-3’
CL2：5’-TATCTAGATTAATTAAATCACTCTCCCCTGTTGAAGCTC-3’
将上述两部分PCR产物进行overlap PCR扩增获得NK003轻链突变库。反应条件：95℃2min，1cycle；95℃30s，65℃30s，72℃20s，8cycle；加入VL1/CL2引物，95℃30s，65℃30s，72℃20s，27cycle；72℃5min，1cycle。用SacI(NEB，R3156L)和PacI(NEB，R0547L)分别对NK003轻链突变库、pcomb3载体双酶切，使用天根回收试剂盒回收PCR目的片段。突变库基因与载体按摩尔比3∶1经T4连接酶(NEB，M0202L)25℃连接3h。将连接产物使用电转法转至XL1-Blue电转感受态中，构建库容量为5×10 5轻链突变库，命名为pcomb3-NK003-LCDR3。
以CDR3插入终止密码子的NK003重链DNA(SEQ ID NO：73)为模板，分别使用引物对VH1/VH2、CH1-1/CH1-2扩增重链VH、CH1片段。反应条件：95℃2min，1cycle；95℃30s，65℃30s，72℃10s，35cycle；72℃5min，1cycle。引物如下：
VH1：5’-TGCAGCTGCTCGAGCAGGTACAGCTGGTGCAGTC-3’
VH2：5’-CTTTGCCCCAGACGTC  CAT GTA GTA GTA GTA GGT TCA AGT AGC TCC CAC  TCT TTC TCTCGCACAG-3’(横线处随意两处突变为MNN，由金唯智公司高通量合成)
CH1-1：5’-GACGTCTGGGGCAAAGGGACCACGGTC-3’
CH1-2：5’-GCCTGGCCACTAGTTTTGTCAACTTTCTTGTCC-3’
将上述两部分PCR产物进行overlap PCR扩增获得NK003重链突变库，反应条件：95℃2min，1cycle；95℃30s，65℃30s，72℃20s，8cycle；加入VH1/CH1-2引物，95℃30s，65℃30s，72℃20s，27cycle；72℃5min，1cycle。用SpeI(NEB，R3133L)和XhoI(NEB，R0146L)分别对NK003重链突变库、pcomb3-NK003-LCDR3载体双酶切，使用天根回收试剂盒回收PCR目的片段。突变库基因与载体按摩尔比3∶1经T4连接酶25℃连接3h。将连接产物使用电转法转至XL1-Blue电转感受态中，构建库容量为2.4×10 7轻重链双突变库，命名为pcomb3-NK003-AM。
9.1.2噬菌体突变抗体库筛选
使用pcomb3-NK003-AM抗体库，筛选与CD40结合的抗体，具体筛选方法见实施例2。筛选结果如表1。
表12 利用噬菌体展示技术从pcomb3-NK003-AM抗体库中筛选CD40结合抗体
Figure PCTCN2020084186-appb-000061
Figure PCTCN2020084186-appb-000062
为了初步评估筛选到CD40结合抗体的阳性率，我们挑取第三轮32个噬菌体单克隆，进行噬菌体ELISA分析。噬菌体ELISA具体方法见实施例2。将CD40结合信号是BSA对照的3倍以上定义为阳性克隆。结果如图18，第三轮筛选阳性率为96.8％。
将第三轮洗脱的噬菌体感染XL1-blue后涂板，刮板混匀后使用天根质粒小提试剂盒提取NK003-AM质粒。使用SacI和SpeI双酶切后回收1500bp左右的目的片段，送至金唯智进行三代测序。测序结果如图19。依据三代测序结果，合成筛选出的抗体NK003-AM-9、NK003-AM-18重链和轻链DNA(金唯智)，将其分别克隆至载体pFUSE中，纯化表达具体方法见实施例4。NK003-AM-9、NK003-AM-18VH/VL序列参见序列表6至表8。
9.1.3 NK003CDR3亲和力成熟活性鉴定
本实施例中通过流式细胞术检测NK003-AM-9、NK003-AM-18抗体激活NF-κB-GFP+hCD40报告细胞系的情况，具体步骤如下：
分别取3×10 5个/管如实施例1中获得NF-κB-GFP+hCD40报告细胞，分别加入不同浓度的NK003-AM-9、NK003-AM-18及NK003：0.01μg/ml、0.05μg/ml、0.1μg/ml、0.5μg/ml和1μg/ml；为了交联，各组同时加入2.5μg/ml浓度的二抗，即山羊抗人Fc(SouthernBiotech，SBA-2048-01)。在含有10％胎牛血清(Biological Industries，04-001-1A)的RPMI 1640培养基(Lifetechnologies，C11875500CP)中37℃共同培养24h后，PBS洗三次，应用流式细胞仪进行分析。获得的数据使用GraphPad Prism 6.0拟合曲线并计算EC50。结果如图20，NK003-AM-9、NK003-AM-18以交联形式激活NF-κB-GFP+hCD40报告细胞系，EC50分别为0.25nM、0.3nM，二者活性相似，均比野生型NK003提高了近10倍。
9.2 NK003 VH、VL骨架区亲和力成熟
9.2.1 NK003 VH、VL骨架区突变库的构建
以NK003轻链DNA(SEQ ID NO：74)为模板，使用随机突变PCR试剂盒(Agilent Technologies，200550)和引物对VL1/VL2、VL3/VL4进行巢氏PCR扩增轻链VL区域，反应条件：95℃2min，1cycle；95℃30s，65℃30s，72℃30s，28cycle；72℃10min，1cycle。以NK003轻链DNA(SEQ ID NO：11)为模板，使用引物对CL1/CL2扩增轻链CL区域，反应条件：95℃2min，1cycle；95℃30s，65℃30s，72℃30s，30cycle；72℃5min，1cycle。使用天根回收试剂盒回收PCR目的片段。引物如下：
VL1：5’-CTATCGCGATTGCAGTGGCACTGGCTG-3’
VL2：5’-CCAGATTTCAACTGCTCATCAGATGGC-3’
VL3：5’-CTACCGTGGCCCAGGCGGCCGAGCTC-3’
VL4：5’-GAAGACAGATGGTGCAGCCACAGTTCG-3’
CL1：5’-CGAACTGTGGCTGCACCATCTGTCTTC-3’
CL2：5’-TATCTAGATTAATTAAATCACTCTCCCCTGTTGAAGCTC-3’
将上述VL、CL PCR产物进行overlap PCR扩增获得NK003轻链突变库。反应条件：95℃2min，1cycle；95℃30s，65℃30s，72℃40s，8cycle；加入VL3/CL2引物，95℃30s， 65℃30s，72℃40s，27cycle；72℃10min，1cycle。用SacI和PacI分别对NK003轻链突变库、pcomb3载体双酶切，使用天根回收试剂盒回收PCR目的片段。突变库基因与载体按摩尔比3∶1经T4连接酶25℃连接3h。将连接产物使用电转法转至XL1-Blue电转感受态中，构建库容量为1×10 5轻链突变库，命名为pcomb3-NK003-VL。
以NK003重链DNA(SEQ ID NO：75)为模板，使用引物对VH1/VH2、VH3/VH4进行巢氏PCR扩增重链VH区域，反应条件3：95℃2min，1cycle；95℃30s，65℃30s，72℃30s，28cycle；72℃10min，1cycle。以NK003重链DNA(SEQ ID NO：12)为模板，使用引物对CH1-1/CH1-2扩增重链CH1区域，反应条件：95℃2min，1cycle；95℃30s，65℃30s，72℃30s，32cycle；72℃10min，1cycle。使用天根回收试剂盒回收PCR目的片段。引物如下：
VH1：5’-GCCGCTGGATTGTTATTACTCGCTGC-3’
VH2：5’-CAGAGGTGCTCTTGGAGGAGGGTGCC-3’
VH3：5’-GCCATGGCCGAGGTGCAGCTGCTCGAG-3’
VH4：5’-GAAGACCGATGGGCCCTTGGTGGAGGC-3’
CH1-1：5’-GCCTCCACCAAGGGCCCATCGGTCTTC-3’
CH1-2：5’-GCCTGGCCACTAGTTTTGTCAACTTTCTTGTCC-3’
将上述VH、CH1 PCR产物进行overlap PCR扩增获得NK003重链突变库。反应条件：95℃2min，1cycle；95℃30s，65℃30s，72℃40s，8cycle；加入VH3/CH1-2引物，95℃30s，65℃30s，72℃40s，27cycle；72℃10min，1cycle。用SpeI和XhoI分别对NK003重链突变库、pcomb3-NK003-VL载体双酶切，使用天根回收试剂盒回收PCR目的片段。突变库基因与载体按摩尔比3∶1经T4连接酶25℃连接3h。将连接产物使用电转法转至XL1-Blue电转感受态中，构建库容量为2.4×10 7轻重链双突变库，命名为pcomb3-NK003-EP。
9.2.2噬菌体突变抗体库筛选
使用pcomb3-NK003-EP抗体库，筛选与CD40结合的抗体，具体筛选方法见实施例2。筛选结果如表2。
表13 利用噬菌体展示技术从pcomb3-NK003-EP抗体库中筛选CD40结合抗体
Figure PCTCN2020084186-appb-000063
将第三轮洗脱的噬菌体感染XL1-blue后涂板，刮板混匀后使用天根质粒小提试剂盒提取NK003-EP质粒。使用SacI和SpeI双酶切后回收1500bp左右的目的片段，送至金唯智进行三代测序。测序结果如图21。依据三代测序结果，合成抗体NK003-AM-18-EP1重链DNA(金唯智)，将其克隆至载体pFUSE中。使用标准步骤用PEI将分别含有NK003-AM-18轻链和NK003-AM-18-EP1重链的质粒二者按1∶1瞬时共转染至293F悬浮细胞，以表达全长NK003-AM-18-EP1抗体。纯化表达具体方法见实施例4。NK003-AM-18-EP1 VH序列参见序 列表6。
9.2.3 NK003骨架区亲和力成熟活性鉴定
本实施例中通过流式细胞术检测NK003-AM-18-EP1抗体激活NF-κB-GFP+hCD40报告细胞系的情况，具体步骤如下：
分别取3×10 5个/管如实施例1中获得NF-κB-GFP+hCD40报告细胞，分别加入不同浓度的NK003-AM-18、NK003-AM-18-EP1及NK003：0.0075nM、0.025nM、0.075nM、0.25nM、0.75nM、2.5nM和7.5nM；为了交联，各组同时加入2.5μg/ml浓度的二抗，即山羊抗人Fc。在含有10％胎牛血清(Biological Industries，04-001-1A)的RPMI 1640培养基(Lifetechnologies，C11875500CP)中37℃共同培养24h后，PBS洗三次，应用流式细胞仪进行分析。获得的数据使用GraphPad Prism 6.0拟合曲线并计算EC50。结果如图22，NK003-AM-18-EP1以交联形式激活NF-κB-GFP+hCD40报告细胞系，EC50为0.35nM，与NK003-AM-18活性相似。
Figure PCTCN2020084186-appb-000064
Figure PCTCN2020084186-appb-000065
Figure PCTCN2020084186-appb-000066
Figure PCTCN2020084186-appb-000067

Claims (22)

  1. 结合CD40的抗体或其抗原结合片段，其包含
    (i)如SEQ ID NO：13、58、60或62所示的重链可变区的3个互补决定区HCDR1、HCDR2和HCDR3，以及如SEQ ID NO：15、64或66所示的轻链可变区的3个互补决定区LCDR1、LCDR2和LCDR3；
    (ii)如SEQ ID NO：13所示的重链可变区的3个互补决定区HCDR1、HCDR2和HCDR3，以及如SEQ ID NO：15所示的轻链可变区的3个互补决定区LCDR1、LCDR2和LCDR3；
    (iii)如SEQ ID NO：58所示的重链可变区的3个互补决定区HCDR1、HCDR2和HCDR3，以及如SEQ ID NO：64所示的轻链可变区的3个互补决定区LCDR1、LCDR2和LCDR3；
    (iv)如SEQ ID NO：60或62所示的重链可变区的3个互补决定区HCDR1、HCDR2和HCDR3，以及如SEQ ID NO：66所示的轻链可变区的3个互补决定区LCDR1、LCDR2和LCDR3；
    (v)如SEQ ID NO：14所示的重链可变区的3个互补决定区HCDR1、HCDR2和HCDR3，以及如SEQ ID NO：16所示的轻链可变区的3个互补决定区LCDR1、LCDR2和LCDR3；
    (vi)(i)-(v)中任一项的CDR组合，其中相比HCDR3和/或LCDR3，在所述序列上包含至少一个且不超过3、2或1个氨基酸改变(优选地氨基酸置换，优选地保守置换)。
  2. 结合CD40的抗体或其抗原结合片段，其包含
    (i)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：5；或者
    (ii)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：51；或者
    (iii)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：52；或者
    (iv)(i)-(iii)中任一项的HCDR组合，其中在所述HCDR3包含至少一个且不超过3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)；
    和
    (i)包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：11；或者
    (ii)包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：53；或者
    (iii)包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：54；或者
    (iv)(i)-(iii)中任一项的LCDR组合，其中在所述LCDR3包含至少一个且不超过3、2或1 个氨基酸改变(优选氨基酸置换，优选保守置换)。
  3. 结合CD40的抗体或其抗原结合片段，其包含
    (i)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：5，以及包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：11；或者
    (ii)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：51，以及包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：53；或者
    (iii)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：52，以及包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：54；或者
    (iv)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：2、SEQ ID NO：4和SEQ ID NO：6，以及包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：8、SEQ ID NO：10和SEQ ID NO：12；
    (v)包含或由下述序列组成的HCDR1、HCDR2和HCDR3：SEQ ID NO：1、SEQ ID NO：3和SEQ ID NO：55，以及包含或由下述序列组成的LCDR1、LCDR2和LCDR3：SEQ ID NO：7、SEQ ID NO：9和SEQ ID NO：56；
    (vi)(i)-(iv)中任一项的HCDR和LCDR组合，其中在所述HCDR3和/或LCDR3中包含至少一个且不超过3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)。
  4. 权利要求1至3中任一项的抗体或其抗原结合片段，其包含重链可变区VH和/或轻链可变区VL，其中，
    (a)重链可变区VH
    (i)包含与选自SEQ ID NO：13、58、60、62或14的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者
    (ii)包含选自SEQ ID NO：13、58、60、62或14的氨基酸序列或由其组成；或者
    (iii)包含与选自SEQ ID NO：13、58、60、62或14的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中，优选地，所述氨基酸改变发生在FR区中；
    和/或
    (b)轻链可变区VL
    (i)包含与选自SEQ ID NO：15、64、66或16的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
    (ii)包含选自SEQ ID NO：15、64、66或16的氨基酸序列或由其组成；或者
    (iii)包含与选自SEQ ID NO：15、64、66或16的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中，优选地，所述 氨基酸改变发生在FR区中。
  5. 权利要求4的抗体或其抗原结合片段，其中
    (i)重链可变区VH包含SEQ ID NO：13、58、60或62的氨基酸序列或由其组成，和轻链可变区VL包含选自SEQ ID NO：15、64或66的氨基酸序列或由其组成；
    (ii)重链可变区VH包含SEQ ID NO：13的氨基酸序列或由其组成，和轻链可变区VL包含SEQ ID NO：15的氨基酸序列或由其组成；
    (iii)重链可变区VH包含SEQ ID NO：58的氨基酸序列或由其组成，和轻链可变区VL包含SEQ ID NO：64的氨基酸序列或由其组成；
    (iv)重链可变区VH包含SEQ ID NO：60的氨基酸序列或由其组成，和轻链可变区VL包含SEQ ID NO：66的氨基酸序列或由其组成；
    (v)重链可变区VH包含SEQ ID NO：62的氨基酸序列或由其组成，和轻链可变区VL包含SEQ ID NO：66的氨基酸序列或由其组成；
    (vi)重链可变区VH包含SEQ ID NO：14的氨基酸序列或由其组成，和轻链可变区VL包含SEQ ID NO：16的氨基酸序列或由其组成。
  6. 权利要求1至5中任一项的抗体或其抗原结合片段，其包含重链和/或轻链，其中
    (a)重链
    (i)包含与选自SEQ ID NO：17、18、19、67、69、70或20的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
    (ii)包含选自SEQ ID NO：17、18、19、67、69、70或20的氨基酸序列或由其组成；或者
    (iii)包含与选自SEQ ID NO：17、18、19、67、69、70或20的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在重链的CDR区中，更优选地，所述氨基酸改变不发生在重链可变区中，最优选地，所述氨基酸改变发生在重链恒定区中；
    和/或
    (b)轻链
    (i)包含与选自SEQ ID NO：21、68、71或22的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；
    (ii)包含选自SEQ ID NO：21、68、71或22的氨基酸序列或由其组成；或者
    (iii)包含与选自SEQ ID NO：21、68、71或22的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在轻链的CDR区中，更优选地，所述氨基酸改变不发生在轻链可变区中，最优选的，所述重链氨基酸改变发生在轻链恒定区中。
  7. 权利要求6的抗体或其抗原结合片段，其中
    (i)重链包含SEQ ID NO：17、18、19、67、69或70的氨基酸序列或由其组成，和轻链包含SEQ ID NO：21、68或71的氨基酸序列或由其组成，
    (ii)重链包含SEQ ID NO：17、18或19的氨基酸序列或由其组成，和轻链包含SEQ ID NO：21的氨基酸序列或由其组成；
    (iii)重链包含SEQ ID NO：67的氨基酸序列或由其组成，和轻链包含SEQ ID NO：68的氨基酸序列或由其组成；
    (iv)重链包含SEQ ID NO：69或70的氨基酸序列或由其组成，和轻链包含SEQ ID NO：71的氨基酸序列或由其组成；
    (v)重链包含SEQ ID NO：20的氨基酸序列或由其组成，和轻链包含SEQ ID NO：22的氨基酸序列或由其组成。
  8. 权利要求1至7中任一项的结合CD40的抗体或其抗原结合片段，其中所述抗体是单克隆抗体。
  9. 权利要求1至8中任一项的结合CD40的抗体或其抗原结合片段，其中所述抗体是人源化的抗体或人抗体或嵌合抗体。
  10. 权利要求1至9中任一项的抗体或其抗原结合片段，其中所述抗原结合片段是选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体(例如scFv)或(Fab’) 2、单结构域抗体、diabody(dAb)或线性抗体。
  11. 分离的核酸，其编码权利要求1至10中任一项的结合CD40的抗体的任一或多条链或其抗原结合片段。
  12. 载体，其包含权利要求11的核酸，优选地所述载体是表达载体。
  13. 宿主细胞，其包含权利要求11的核酸或权利要求12的载体，优选地，所述宿主细胞是原核的或真核的，更优选的选自酵母细胞、哺乳动物细胞(例如所述宿主细胞是CHO细胞，例如CHOS细胞CHOK1SV细胞或CHOK1SV GS-KO，或所述宿主细胞是293细胞，例如HEK293细胞)或适用于制备抗体或其抗原结合片段的其它细胞。
  14. 制备结合CD40的抗体或其抗原结合片段的方法，所述方法包括在适于表达抗体的情况下，培养包含编码权利要求1-10中任一项的抗体或其抗原结合片段的核酸的宿主细胞，任选地所述方法还包括从所述宿主细胞回收所述抗体或其抗原结合片段。
  15. 免疫缀合物，其包含权利要求1至10中任一项的结合CD40的抗体或其抗原结合片段和其它物质，例如治疗剂或标记。
  16. 药物组合物，其包含权利要求1至10中任一项的结合CD40的抗体或其抗原结合片段或权利要求13的免疫缀合物，以及任选地一种或多种其它治疗剂，以及任选地药用辅料。
  17. 组合产品，其包含权利要求1至10中任一项的结合CD40的抗体或其抗原结合片段 或权利要求15的免疫缀合物，以及一种或多种其它治疗剂。
  18. 权利要求15的免疫缀合物、权利要求16的药物组合物或权利要求17的组合产品，其中所述治疗剂选自化疗剂、其他抗体、细胞毒性剂、疫苗、抗感染活性剂、小分子药物或免疫调节剂。
  19. 在受试者中增强免疫应答(例如抗原特异性T细胞应答)的方法，其包括向受试者施用有效量的权利要求1-10中任一项的抗体或其抗原结合片段、或权利要求16或18的免疫缀合物、或权利要求16或18的药物组合物，或权利要求17或18的组合产品。
  20. 预防或治疗受试者的需要在受试者中调节(例如增强)免疫应答的疾病或CD40相关疾病的方法，所述方法包括向所述受试者施用有效量的权利要求1-10中任一项的抗体或其抗原结合片段、或权利要求15或18的免疫缀合物、或权利要求16或18的药物组合物，或权利要求17或18的组合产品，优选地，所述疾病为肿瘤，例如淋巴瘤、结肠癌、结直肠癌、直肠癌、肺癌(例如非小细胞肺癌)、肝癌、胃癌及其转移性癌症；或感染，例如慢性感染，或病毒感染。
  21. 权利要求20所述的方法，其还包括向所述受试者联合施用一种或多种疗法，例如治疗方式和/或其它治疗剂，优选地，治疗方式包括手术治疗和/或放射疗法，和/或其它治疗剂选自化疗剂、其他抗体、细胞毒性剂、疫苗、抗感染活性剂、小分子药物或免疫调节剂。
  22. 检测试剂盒，其包含权利要求1-10中任一项的抗体或其抗原结合片段或权利要求15的免疫缀合物。
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