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WO2023046037A1 - 抗cd40抗体及其用途 - Google Patents

抗cd40抗体及其用途 Download PDF

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Publication number
WO2023046037A1
WO2023046037A1 PCT/CN2022/120689 CN2022120689W WO2023046037A1 WO 2023046037 A1 WO2023046037 A1 WO 2023046037A1 CN 2022120689 W CN2022120689 W CN 2022120689W WO 2023046037 A1 WO2023046037 A1 WO 2023046037A1
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amino acid
seq
acid sequence
heavy chain
light chain
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PCT/CN2022/120689
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English (en)
French (fr)
Inventor
陆臻桢
王玉洁
张正平
徐宏江
赵伟
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正大天晴药业集团股份有限公司
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Priority to CA3232171A priority Critical patent/CA3232171A1/en
Priority to CN202280060361.8A priority patent/CN117940456A/zh
Priority to AU2022350419A priority patent/AU2022350419A1/en
Priority to EP22872081.9A priority patent/EP4406971A1/en
Priority to KR1020247013602A priority patent/KR20240067115A/ko
Priority to MX2024003278A priority patent/MX2024003278A/es
Priority to JP2024516611A priority patent/JP2024535834A/ja
Publication of WO2023046037A1 publication Critical patent/WO2023046037A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • C07K2317/24—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
    • C07K2317/70—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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • 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 disclosure belongs to the field of biotechnology, and in particular relates to antibodies specifically binding to CD40 and antigen-binding fragments thereof, and to methods for using such antibodies and antigen-binding fragments.
  • CD40 is a type I transmembrane glycoprotein that belongs to the tumor necrosis factor receptor (TNFR) superfamily member.
  • CD40 is constitutively expressed in antigen-presenting cells (including dendritic cells, B cells, and macrophages), and is also expressed in small amounts in non-hematopoietic cells such as epithelial cells, endothelial cells, smooth muscle cells, fibroblasts, keratinocytes, etc. .
  • CD40 is also expressed in various tumor cells, such as B lymphoma cells.
  • CD40L CD154
  • CD40L CD40L
  • CD4 + T lymphocytes activated B cells, memory T cells, activated NK cells and activated platelets .
  • the CD40/CD40L signaling pathway participates in the body's humoral immune response and cellular immune response, and plays a key regulatory role in T cell-dependent antibody immune responses and inflammatory responses such as the activation, proliferation and differentiation of B cells, antibody production, and antibody class switching.
  • T cell-dependent antibody immune responses and inflammatory responses such as the activation, proliferation and differentiation of B cells, antibody production, and antibody class switching.
  • Studies have shown that a large number of T cells and B cells targeting self-antigens are infiltrated in the tissues and organs of patients with autoimmune diseases, and they show persistent high expression of CD40 and/or CD40L.
  • the results of IHC staining in patients with primary Sjogren's syndrome showed that CD40 was enriched in salivary gland epithelial cells and accompanied by increased serum autoantibodies such as anti-SSA.
  • the key role of CD40/CD40L signaling pathway in inflammatory bowel disease has been confirmed in animal models such as mice.
  • antibodies that specifically bind to CD40 and inhibit the CD40/CD40L signaling pathway have potential clinical value for the treatment of immune diseases such as inflammatory diseases and autoimmune diseases.
  • the anti-CD40 antibody Iscalimab (CFZ533) developed by Novartis is currently in clinical trials for the treatment of CD40-mediated immune diseases.
  • the results show that Iscalimab is administered at a dose of 10 mg/kg intravenously to patients with primary Sjogren's syndrome (Enrollment condition is positive serum autoantibodies), 62% (13/21) patients ESSDAI disease activity score is less than 5 points, the treatment effect is significantly better than 36% (4/11) of the placebo group.
  • the number of patients with immune diseases worldwide is huge, therefore, there is an urgent need to develop more anti-CD40 antibodies with better drug properties.
  • the disclosure provides isolated CD40-binding antibodies or antigen-binding fragments thereof.
  • the antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.
  • the antibody or antigen-binding fragment thereof is a monoclonal antibody, monospecific antibody, bispecific antibody, trispecific antibody, multispecific antibody, Fab fragment, F(ab')2 fragment, Fd fragment , Fv fragments, dAbs, isolated CDRs, single chain Fv molecules, recombinant polypeptides, fusion proteins, bispecific molecules or combinations thereof.
  • an antibody of the disclosure, or an antigen-binding fragment thereof binds human CD40. In other embodiments, an antibody of the disclosure, or antigen-binding fragment thereof, binds CD40 (eg, human CD40 and cynomolgous CD40). In some embodiments, an antibody of the disclosure, or antigen-binding fragment thereof, blocks the interaction of CD40 with CD40L. In some embodiments, an antibody of the disclosure, or antigen-binding fragment thereof, inhibits CD40 activity.
  • the anti-CD40 antibody or antigen-binding fragment thereof provided by the present disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), wherein, (1) heavy chain CDR1 comprises SEQ The amino acid sequence of the heavy chain CDR1 in ID NO: 9, the heavy chain CDR2 comprises the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 9 and the heavy chain CDR3 comprises the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 9; (2) heavy Chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 10, heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 10, and heavy chain CDR3 comprises the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 10; (3) Heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 11, heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 11 and
  • the anti-CD40 antibody or antigen-binding fragment thereof provided by the present disclosure comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, wherein, (1) heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively comprise SEQ The amino acid sequences shown in ID NOs: 1, 2 and 5 or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86% of the amino acid sequences shown in SEQ ID NOs: 1, 2 and 5 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences;( 2) The heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 1, 3 and 5 or have at least 80% of the amino acid sequences shown in SEQ ID NOs: 1, 3 and 5, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 8
  • the anti-CD40 antibody or antigen-binding fragment thereof provided by the present disclosure comprises light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3), wherein, (1) light chain CDR1 comprises SEQ The amino acid sequence of the light chain CDR1 in ID NO:14, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO:14 and the light chain CDR3 comprises the amino acid sequence of the light chain CDR3 in SEQ ID NO:14; (2) light chain Chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 15, light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 15 and light chain CDR3 comprises the amino acid sequence of light chain CDR3 in SEQ ID NO: 15; (3) The light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 16, the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ
  • the anti-CD40 antibody or antigen-binding fragment thereof provided by the present disclosure comprises light chain CDR1, light chain CDR2 and light chain CDR3, wherein, (1) light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise SEQ The amino acid sequence shown in ID NOs: 6, 7 and 8 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86% of the amino acid sequence shown in SEQ ID NOs: 6, 7 and 8 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences;( 2) light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 21, 7 and 8 or have at least 80% of the amino acid sequence shown in SEQ ID NOs: 21, 7 and 8, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
  • the anti-CD40 antibody or antigen-binding fragment thereof provided by the present disclosure comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3, wherein, (1) heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 9, heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 9, heavy chain CDR3 comprises the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 9, light Chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 14, light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 14, and light chain CDR3 comprises the amino acid sequence of light chain CDR3 in SEQ ID NO: 14 (2) heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO:10, heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO:10, and heavy chain CDR3 comprises
  • the anti-CD40 antibody or antigen-binding fragment thereof provided by the present disclosure comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3, wherein, (1) heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 1, 2, 5, 6, 7 and 8 or with SEQ ID NOs: 1 , 2, 5, 6, 7 and 8 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence; (2) heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, Light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 1, 3, 5, 6, 7 and 8 or with SEQ ID
  • an anti-CD40 antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region comprising SEQ ID NOs: 9, 10, 11, 12, 13, 22, 30 , 38, 44 or 52 amino acid sequence shown, or with SEQ ID NOs: 9, 10, 11, 12, 13, 22, 30, 38, 44 or 52 amino acid sequence shown in at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% identity amino acid sequences.
  • an anti-CD40 antibody or antigen-binding fragment thereof provided herein comprises a light chain variable region comprising SEQ ID NOs: 14, 15, 16, 17, 23, 31, 39 , 45 or 53 of the amino acid sequence, or at least 80%, 81%, 82%, 83 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity amino acid sequence.
  • the anti-CD40 antibody or antigen-binding fragment thereof provided by the present disclosure comprises a heavy chain variable region and a light chain variable region, wherein (1) the heavy chain variable region and the light chain variable region respectively comprise The amino acid sequence shown in SEQ ID NOs: 9 and 14 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% of the amino acid sequence shown in SEQ ID NOs: 9 and 14 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence; (2) heavy The chain variable region and the light chain variable region comprise the amino acid sequence shown in SEQ ID NOs: 10 and 15 respectively or have at least 80%, 81%, 82%, 83% of the amino acid sequence shown in SEQ ID NOs: 10 and 15 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
  • an anti-CD40 antibody or antigen-binding fragment thereof provided herein comprises a heavy chain comprising a heavy chain variable region and a heavy chain constant region, and a light chain comprising a light chain variable region. region and light chain constant region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of the above-mentioned heavy chain variable region and light chain variable region respectively, and the heavy chain constant region comprises human IgG1, IgG2 or IgG4 constant Regions, preferably IgG1 and IgG4 constant regions, the light chain constant region comprises a human kappa constant region or a human lambda constant region.
  • the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NOs: 54, 55 or 56 or has 1, 2, The amino acid sequence of 3, 4 or 5 amino acid substitutions, deletions and additions, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 57 or has 1, 2, Amino acid sequences of 3, 4 or 5 amino acid substitutions, deletions and additions.
  • the anti-CD40 antibody or antigen-binding fragment thereof provided by the present disclosure comprises a heavy chain and a light chain, wherein (1) the heavy chain comprises the amino acid sequences shown in SEQ ID NOs: 9 and 54, and the light chain Comprising the amino acid sequence shown in SEQ ID NOs: 14 and 57; (2) the heavy chain includes the amino acid sequence shown in SEQ ID NOs: 10 and 54, and the light chain includes the amino acid sequence shown in SEQ ID NOs: 15 and 57; (3) the heavy chain comprises the amino acid sequence shown in SEQ ID NOs: 11 and 54, and the light chain comprises the amino acid sequence shown in SEQ ID NOs: 15 and 57; (4) the heavy chain comprises the amino acid sequence shown in SEQ ID NOs: 10 and 54 The amino acid sequence shown, and the light chain comprises the amino acid sequence shown in SEQ ID NOs: 16 and 57; (5) the heavy chain comprises the amino acid sequence shown in SEQ ID NOs: 11 and 54, and the light chain comprises the amino acid sequence shown in SEQ ID NO
  • the anti-CD40 antibody or antigen-binding fragment thereof provided herein comprises two heavy chains (H) and two light chains (L), or consists of two heavy chains and two light chains, the heavy chain and the light chain are connected to each other by disulfide bonds, wherein each heavy chain comprises the above-mentioned heavy chain variable region (VH) and the heavy chain constant region, wherein the heavy chain variable region (VH) comprises the framework region (FR) and the above-mentioned Heavy chain complementarity determining region (HCDR), each light chain comprises the above-mentioned light chain variable region (VL) and light chain constant region, wherein the light chain variable region (VL) comprises FR and the above-mentioned light chain complementarity determining region ( LCDR), wherein the C-terminus of the variable region of the heavy chain is linked to the N-terminus of the constant region of the heavy chain, and the C-terminus of the variable region of the light chain is linked to the N-terminus of the constant region of the light chain.
  • H
  • Antibodies of the present disclosure may be, for example, full-length antibodies of the IgGl, IgG2 or IgG4 isotype.
  • an antibody of the present disclosure may be a single chain antibody (scFv), or an antibody fragment, such as a Fab, F(ab')2 fragment, Fd fragment, Fv fragment, dAb, or isolated CDRs.
  • the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds CD40 produced by a hybridoma selected from the group consisting of A01, A02, A03, Panel consisting of hybridomas of B01, B02 and B03. Accordingly, the present disclosure also includes antibodies or antigen-binding fragments thereof produced by hybridomas A01, A02, A03, B01, B02, and B03, as well as any hybridoma that produces an antibody disclosed herein.
  • the disclosure provides an antibody or antigen-binding fragment thereof that binds to the same epitope on CD40 as any exemplary anti-CD40 antibody or antigen-binding fragment thereof of the disclosure.
  • the present disclosure provides antibodies or antigen-binding fragments thereof that compete for binding to CD40 with any of the exemplary anti-CD40 antibodies or antigen-binding fragments thereof of the disclosure.
  • the present disclosure also provides recombinant polypeptides or fusion proteins comprising one or more anti-CD40 antibodies of the present disclosure or antigen-binding fragments thereof and at least one other functional fragment, including but not limited to another A peptide, protein, cytokine, or receptor ligand.
  • recombinant polypeptides or fusion proteins can be prepared by genetic modification, chemical methods and the like.
  • fusion protein generally refers to a new polypeptide sequence obtained by linking two or more identical or different polypeptide sequences, especially recombinant one or more polypeptide sequences that are not linked in nature. Polypeptide sequences of the same or different polypeptide sequences.
  • the present disclosure also provides a bispecific molecule comprising one or more anti-CD40 antibodies or antigen-binding fragments thereof of the present disclosure and at least some specificities different from those of the antibodies or antigen-binding fragments of the present disclosure.
  • Other functional moieties eg, another antibody or antigen-binding fragment thereof
  • the bispecific molecule is capable of binding to at least two different binding sites or targets.
  • "bispecific molecule” encompasses molecules having two (ie, bispecific molecules), three (ie, trispecific molecules), four (ie, tetraspecific molecules) or more specificities. These bispecific molecules can be prepared by genetic modification, chemical methods and the like.
  • the present disclosure also provides an immunoconjugate comprising an anti-CD40 antibody of the present disclosure or an antigen-binding portion thereof, wherein the anti-CD40 antibody of the present disclosure or an antigen-binding portion thereof is combined with a therapeutic agent (such as a cytotoxic agent) or imaging agents, etc.), such as antibody-drug conjugates (ADCs).
  • a therapeutic agent such as a cytotoxic agent
  • ADCs antibody-drug conjugates
  • An anti-CD40 antibody of the disclosure, or an antigen-binding portion thereof may be part of a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the present disclosure also provides immune cells, such as T cells (ie, CAR-T cells), comprising the chimeric antigen receptor.
  • the present disclosure also provides a gene vector, which comprises a gene encoding the anti-CD40 antibody of the present disclosure or an antigen-binding portion thereof, and enables the gene to enter and express in mammalian cells (preferably human cells) .
  • gene vectors include, but are not limited to, naked plasmid vectors, yeast vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, poxvirus vectors, rhabdovirus vectors, or baculovirus vectors. Techniques for inserting DNA into these gene vectors are well known to those of ordinary skill in the art.
  • the present disclosure also provides a pharmaceutical composition, which comprises the anti-CD40 antibody or antigen-binding fragment thereof of the present disclosure, and one or more pharmaceutically acceptable carriers.
  • the present disclosure also provides a drug comprising the recombinant polypeptide, fusion protein, bispecific molecule, immunoconjugate, chimeric antigen receptor or gene carrier of the present disclosure, and a pharmaceutically acceptable carrier combination.
  • the disclosure also provides an isolated nucleic acid encoding an anti-CD40 antibody of the disclosure, or an antigen-binding fragment thereof.
  • the present disclosure also provides an expression vector comprising the nucleic acid, and a host cell comprising the expression vector.
  • the present disclosure also provides a method for preparing an anti-CD40 antibody or an antigen-binding fragment thereof, comprising the steps of: (i) expressing an anti-CD40 antibody or an antigen-binding fragment thereof in a host cell, and (ii) obtaining an anti-CD40 antibody or an antigen-binding fragment thereof from Anti-CD40 antibodies or antigen-binding fragments thereof are isolated from host cells or cell culture thereof.
  • the present disclosure provides a method for treating or preventing an immune disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CD40 antibody of the present disclosure, or an antigen-binding portion thereof, an encoding nucleic acid, Pharmaceutical composition, recombinant polypeptide, fusion protein, bispecific molecule, immunoconjugate, chimeric antigen receptor or gene carrier.
  • the subject is a human.
  • the immune disease includes, but is not limited to, inflammatory disease, allergic reaction, autoimmune disease, or transplant-related disease.
  • the immune diseases include, but are not limited to: allergic reactions, Addison's disease, ankylosing spondylitis, spondyloarthritis, asthma, atherosclerosis, coronary heart disease, autoimmunity hepatitis, autoimmune mumps, type 1 diabetes, epididymitis, nephritis, Reiter's syndrome, thyroiditis, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease , hemolytic anemia, idiopathic thrombocytopenia, systemic lupus erythematosus, subacute cutaneous lupus erythematosus, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, arthritis, sarcoidosis, dryness syndrome, dry eye
  • the method further comprises administering a second therapeutic agent including nonsteroidal anti-inflammatory drugs (NSAIDs), salicylates, hydroxychloroquine, sulfasalazine, corticosteroids Steroids, cytotoxic drugs, or immunosuppressive drugs and/or antibodies.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • salicylates hydroxychloroquine
  • sulfasalazine hydroxychloroquine
  • corticosteroids Steroids
  • cytotoxic drugs or immunosuppressive drugs and/or antibodies.
  • Figures 1A-1B show the activity of purified mouse anti-CD40 antibodies blocking the binding of 293T-hCD40-NF ⁇ B cells to CD40L protein;
  • Figure 2 shows the inhibitory activity of purified mouse anti-CD40 antibodies to the apoptosis of Ramos cells induced by CD40L and IL-4;
  • Figure 3 shows the binding activity of chimeric anti-CD40 antibodies Chi-A01, Chi-A02 and Chi-A03 to CHO-K1-hCD40 cells;
  • Figure 4 shows the agonistic activity of chimeric anti-CD40 antibodies Chi-A01, Chi-A02, Chi-A03, Chi-B01, Chi-B02 and Chi-B03 on Ramos cell apoptosis, and the effect of CP-870893 on Ramos cell apoptosis Agonistic activity is shown as a positive control;
  • Figure 5 shows the binding activity of humanized anti-CD40 antibodies hzA01-3.1, hzA01-3.3 and hzA01-3.4 to 293T-hCD40-NF ⁇ B cells;
  • Figure 6 shows the activity of humanized anti-CD40 antibodies hzA01-1.1, hzA01-2.1, hzA01-3.1, hzA01-3.3 and hzA01-3.4 blocking the binding of 293T-hCD40-NF ⁇ B cells to CHO-K1-hCD40L cells;
  • Figure 7 shows the inhibitory activity of humanized anti-CD40 antibodies hzA01-1.1, hzA01-2.1, hzA01-3.1, hzA01-3.3 and hzA01-3.4 on the apoptosis of Ramos cells induced by CD40L and IL-4;
  • Figure 8 shows the agonistic activity of humanized anti-CD40 antibodies hzA01-1.1, hzA01-2.1, hzA01-3.1, hzA01-3.3 and hzA01-3.4 on Ramos cell apoptosis, and the agonistic activity of CP-870893 on Ramos cell apoptosis as Positive controls are shown;
  • Figure 9 shows the inhibitory activity of humanized anti-CD40 antibodies hzA01-3.1, hzA01-3.3 and hzA01-3.4 on CD40L-induced expression of costimulatory molecule CD86 in human peripheral blood B lymphocytes;
  • Figure 10 shows the inhibitory activity of humanized anti-CD40 antibodies hzA01-3.1, hzA01-3.3 and hzA01-3.4 on the proliferation of human peripheral blood B lymphocytes induced by CD40L and IL-4;
  • Figure 11 shows the agonistic activity of humanized anti-CD40 antibodies hzA01-3.1, hzA01-3.3 and hzA01-3.4 inducing the proliferation of human peripheral blood B lymphocytes, and the agonistic activity of CP-870893 inducing the proliferation of human peripheral blood B lymphocytes as a positive control Shows;
  • Figure 12 shows the IFN ⁇ release in response to the humanized anti-CD40 antibody hzA01-3.3 in the MLR reaction, wherein the mDC and DC (undifferentiated culture) groups did not add anti-CD40 antibody and IgG4 isotype control antibody, as a negative control out;
  • Figure 13 shows the agonistic activity of humanized anti-CD40 antibody hzA01-3.3 on imDC cell maturation, and the agonistic activity of CP-870893 on imDC cell maturation is shown as a positive control;
  • Figure 14 shows the ADCC effect mediated by the humanized anti-CD40 antibody hzA01-3.3, and the ADCC activity mediated by Rituximab is shown as a positive control;
  • Figure 15 shows the CDC effect mediated by the humanized anti-CD40 antibody hzA01-3.3, and the CDC activity mediated by Rituximab is shown as a positive control;
  • Figure 16A shows the concentration of human anti-mouse IgM antibody in NDG mouse serum after treatment with humanized anti-CD40 antibody hzA01-3.3
  • Figure 16B shows the concentration of human anti-mouse IgM antibody in NDG mouse serum after treatment with humanized anti-CD40 antibody hzA01-3.3 Concentration of anti-mouse IgG antibody;
  • Figure 17A shows the content of human CD4 + T cells infiltrated in mouse spleen after treatment with humanized anti-CD40 antibody hzA01-3.3
  • Figure 17B shows the amount of human CD19 infiltrated in mouse spleen after treatment with humanized anti-CD40 antibody hzA01-3.3 + B cell content, where compared with the IgG4 isotype control group by t-test, * indicates P ⁇ 0.05, ** indicates P ⁇ 0.01, *** indicates P ⁇ 0.001;
  • Figure 18 shows the effect of humanized anti-CD40 antibody hzA01-3.3 on the body weight of Sjogren's syndrome model mice
  • Figure 19 shows the effect of humanized anti-CD40 antibody hzA01-3.3 on the saliva flow rate of Sjogren's syndrome model mice
  • Figure 20A shows the submandibular gland index of the Sjogren's syndrome model mouse after the treatment of the humanized anti-CD40 antibody hzA01-3.3
  • Figure 20B shows the IL- The content of 6
  • Figure 20C shows the submandibular gland pathological score of the Sjögren's syndrome model mice after the treatment of the humanized anti-CD40 antibody hzA01-3.3, wherein, by t test (the blank control group is compared with the model group, hzA01-3.3 administration group and model group), * means P ⁇ 0.05, ** means P ⁇ 0.01, *** means P ⁇ 0.001;
  • FIGS. 21A-21D show that the live cells of CD3 + T cells, CD4 + T cells, CD8 + T cells and CD19 + B cells accounted for CD45 + The percentage of viable cells,
  • Figure 21E-21H shows the percentage of CD3 + T cells, CD4 + T cells, CD8 + T cells and CD19 + B cells in the spleen of Sjogren's syndrome model mice after treatment with humanized anti-CD40 antibody hzA01-3.3 Live cells as a percentage of CD45 + live cells.
  • CD40 includes CD40 variants, homologs, orthologs and paralogs.
  • antibodies specific for human CD40 protein may, under certain circumstances, cross-react with CD40 protein of another species (eg, monkey).
  • antibodies specific for human CD40 protein may be completely specific for human CD40 protein without cross-reacting with other species or types of proteins, or with CD40 only of certain but not all other species. Proteins cross-react.
  • CD40 is known and may be referred to as B cell surface antigen CD40, Bp50, CD40L receptor, CDW40, MGC9013, p50 and tumor necrosis factor receptor superfamily member 5 (TNFRSF5).
  • Human CD40 or “hCD40” are interchangeable herein and refer to a protein having the amino acid sequence of human CD40, such as a CD40 protein comprising the amino acid sequence shown in SEQ ID NO:62.
  • the terms “monkey CD40” or “cyno CD40” etc. are interchangeable herein and refer to a protein having an amino acid sequence of monkey CD40, such as a CD40 protein comprising the amino acid sequence shown in SEQ ID NO:63.
  • antibody refers to a binding protein having at least one antigen (eg, CD40) binding domain.
  • An antibody or antigen-binding fragment thereof of the present disclosure may be a whole antibody or any fragment thereof, including monoclonal antibodies or fragments thereof and antibody variants or fragments thereof.
  • Examples of antibodies or antigen-binding fragments thereof include monospecific, bispecific, trispecific or multispecific antibodies, Fab fragments, F(ab')2 fragments, Fv fragments, isolated CDR regions, single chain Fv (scFv ) and any other antibody fragment known in the art.
  • the anti-CD40 antibodies or antigen-binding fragments thereof disclosed herein may be of the IgGl, IgG2, IgG3 or IgG4 isotype.
  • the term "isotype" refers to the antibody class encoded by the heavy chain constant region genes.
  • the anti-CD40 antibodies or antigen-binding fragments thereof disclosed herein are of the IgGl and IgG4 isotypes.
  • Anti-CD40 antibodies or antigen-binding fragments thereof of the present disclosure may be derived from any species including, but not limited to, mouse, rat, rabbit, primate, llama, and human.
  • an anti-CD40 antibody or antigen-binding fragment thereof of the present disclosure may be a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.
  • an "antibody” of the present disclosure includes a full-length antibody and any antigen-binding portion (ie, "antigen-binding fragment") or single chain thereof.
  • full-length antibodies are glycoproteins comprising two heavy (H) chains and two light (L) chains, wherein the heavy and light chains are linked by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can also be divided into hypervariable regions (ie, complementarity determining regions (CDRs)) and framework regions (FRs) with relatively conserved sequences.
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminal to the carboxyl terminal.
  • the variable regions of antibodies comprise the binding domains that interact with the antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • various cells of the immune system eg, effector cells
  • Clq first component
  • special “full-length antibodies”, such as Nanobodies have only heavy (H) chains and no light (L) chains, as understood by those skilled in the art.
  • an “antigen-binding fragment” or “antibody-binding portion” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, CD40 protein). It has been demonstrated that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.
  • Examples encompassed by the term "antigen-binding portion/fragment" of an antibody include: (i) Fab fragments: monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, comprising A bivalent fragment of two Fab fragments connected by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of an antibody single arm; (v) dAb fragments consisting of VH domains (see Ward et al., Nature.
  • VH and VH of the Fv fragment are encoded by different genes, the VH and VL can be connected into a single protein chain through a linker using a recombination method, wherein VL and VH are paired to form a monovalent molecule called single-chain Fv (scFv ); see Bird et al., Science.242:423-426 (1988); Huston et al., Proc.Natl.Acad.Sci.85:5879-5883 (1988), these single-chain antibodies are also encompassed in the term antigen Combine parts/fragments.
  • antigen-binding portion/fragment recombinant polypeptides, fusion proteins and bispecific molecules comprising the antigen-binding portion/fragment are also encompassed within the term antigen-binding portion/fragment.
  • antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments can be functionally screened by the same methods as full-length antibodies.
  • Mouse antibody or “murine antibody” refers to an antibody in which both the framework and CDR regions of the variable regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from mouse germline immunoglobulin sequences.
  • Mouse antibodies of the disclosure may include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or point mutations in vitro or by somatic mutations in vivo), but “mouse antibodies” do not Included are antibodies with mouse framework sequences inserted into CDR sequences derived from other mammalian germlines.
  • Chimeric antibody refers to an antibody formed by combining genetic material from a non-human source and genetic material from a human.
  • chimeric antibodies are antibodies that have genetic material from one species and genetic material from another species.
  • the variable regions of the light and heavy chains can both be derived from the variable regions of an antibody from one animal species (e.g., mouse, rat, etc.), while the constant portions are identical to the sequence of an antibody from another species (e.g., human). source.
  • B cells or hybridoma cells of non-human origin can be used to produce variable regions combined with constant regions of human origin. Chimeric antibodies are also denoted "Chi" in this disclosure.
  • a “humanized antibody” is an antibody that contains complementarity determining regions (CDRs) derived from a non-human antibody and framework regions and constant regions derived from a human antibody.
  • CDRs complementarity determining regions
  • a humanized CD40-binding antibody provided herein can comprise CDRs derived from one or more murine antibodies as well as human framework regions and human constant regions. Accordingly, in some embodiments, the humanized antibodies provided herein bind to the same epitope on CD40 as the murine antibody from which the CDRs of the antibody are derived. Exemplary humanized antibodies are provided herein. Additional CD40-binding humanized antibodies or variants thereof comprising the heavy and light chain CDRs provided herein can be generated using any human framework sequence and are also encompassed by the present disclosure.
  • suitable framework sequences for use in the present disclosure include those framework sequences that are structurally similar to the framework sequences provided herein. Additional modifications can be made in the framework regions to alter the properties of the antibodies provided herein. Such additional backbone modifications may include chemical modifications, point mutations to reduce immunogenicity or remove T cell epitopes, or back mutations to residues in the original germline sequence. In some embodiments, such modifications include those corresponding to mutations exemplified herein, including back mutations to germline sequences. For example, in some embodiments, one or more amino acids in the human framework regions of the VH and/or VL of the humanized antibodies provided herein are backmutated to the corresponding amino acid in the parental murine antibody. In this disclosure, humanized antibodies are also denoted "hz".
  • Fc domain or “Fc region” refers to an antibody sequence comprising CH2 and CH3 constant domains as defined according to the Kabat numbering system.
  • the Fc region can be from human IgG.
  • the Fc region can be from a human IgG1 or human IgG4 Fc region.
  • derived when used with reference to a molecule or polypeptide relative to a reference antibody or other binding protein means a molecule or polypeptide capable of specifically binding to the same epitope as the reference antibody or other binding protein.
  • isolated refers to a compound of interest (eg, antibody, antigen-binding fragment, or nucleic acid) that has been separated from its natural environment.
  • compound of interest eg, antibody, antigen-binding fragment, or nucleic acid
  • Antibody that specifically binds an antigen and "antibody specific for an antigen” are used interchangeably herein with the term “antibody that specifically binds an antigen”.
  • An antibody that "specifically binds to human CD40” refers to an antibody that binds to human CD40 (and possibly CD40 of other non-human species) but does not substantially bind to non-CD40.
  • the antibody binds human CD40 with "high affinity", that is, with a KD of 5.0 ⁇ 10 -8 M or less, 1.0 ⁇ 10 -8 M or less, preferably 5.0 ⁇ 10 -9 M or less, more It is preferably 1.0 ⁇ 10 -9 M or less.
  • substantially does not bind to proteins or cells means not to bind to proteins or cells, or not to bind to them with high affinity, that is, to bind proteins or cells with a KD of 1.0 ⁇ 10 -6 M or higher, preferably 1.0 ⁇ 10 -5 M or higher, 1.0 ⁇ 10 -4 M or higher , more preferably 1.0 ⁇ 10 -3 M or higher, 1.0 ⁇ 10 -2 M or higher.
  • high affinity for IgG means that the antigen-binding K D is 5.0 ⁇ 10 -8 M or less, 1.0 ⁇ 10 -8 M or less, preferably 5.0 ⁇ 10 -9 M or less, More preferably, it is 1.0 ⁇ 10 -9 M or less. But for other antibody isotypes, "high affinity” binding may be different.
  • "high affinity" binding of an IgM isotype refers to binding to the antigen with a KD of 1.0 ⁇ 10 -6 M or less, preferably 1.0 ⁇ 10 -7 M or less, more preferably 1.0 ⁇ 10 -8 M or smaller.
  • Identity refers to the similarity between two or more nucleic acid sequences or between two or more polypeptide sequences.
  • sequence identity of the present disclosure is at least 85%, 90% or 95%, preferably at least 95%.
  • Non-limiting examples include: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% %, 100%.
  • Sequence comparison and determination of percent identity between two sequences can be performed with the default settings of the BLASTN/BLASTP algorithm on the website of the National Center For Biotechnology Institute.
  • an antibody that "competes for binding” refers to an antibody that partially or completely blocks the binding of other antibodies to the target. Whether two antibodies compete with each other for target binding, i.e., whether and to what extent one antibody blocks binding of the other antibody to the target, can be determined using competition assays known in the art, such as solid-phase direct or indirect radioimmunoassays (RIA ), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay, etc.
  • the antibody competes with another antibody for binding to the target and blocks binding of the other antibody to the target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90% or 100%.
  • Two or more antibodies "bind to the same epitope” means that the antibodies bind to the same stretch of amino acid residues, as determined by a given method.
  • Techniques for determining whether an antibody binds "the same epitope on CD40" as an antibody described herein include, for example, epitope mapping methods such as x-ray analysis of crystals of antigen:antibody complexes and hydrogen/deuterium exchange mass spectrometry ( HDX-MS).
  • EC 50 also known as the half effective concentration
  • IC 50 also known as the half inhibitory concentration
  • EC50 and IC50 can be measured by ELISA or FACS analysis or any other method known in the art.
  • KD refers to the equilibrium dissociation constant, which is obtained from the ratio of Kd to Ka (ie, Kd / Ka ) and is expressed as molarity (M).
  • M molarity
  • the KD value of an antibody can be determined using methods well established in the art.
  • a preferred method for determining the K of an antibody is by using surface plasmon resonance techniques, preferably using a biosensor system such as Surface plasmon resonance system analysis.
  • Cross-linking refers to higher order multimerization of CD40 on cells induced by binding of an anti-CD40 antibody to an Fc ⁇ R (eg, Fc ⁇ RIIb in cis or trans), resulting in the induction of agonistic activity of CD40.
  • Fc ⁇ R eg, Fc ⁇ RIIb in cis or trans
  • Non-human animal includes all vertebrates such as mammals and non-mammals, preferably mammals such as non-human primates, sheep, dogs, cats, cows and horses.
  • Effective dose means an amount sufficient to achieve, or at least in part, the desired effect.
  • a therapeutically “effective amount” or “effective dose” of a drug or therapeutic agent is an amount sufficient to prevent or ameliorate symptoms associated with a disease or disorder, preferably resulting in a reduction in the severity of disease symptoms, when used alone or in combination with another therapeutic agent, The amount by which the frequency and duration of the asymptomatic period is increased, or the impairment or incapacity caused by the disease is prevented.
  • the therapeutically effective amount is related to the disease being treated, wherein the actual effective amount can be easily determined by those skilled in the art.
  • a pH value of about 5.5 means a pH of 5.5 ⁇ 5%, preferably a pH of 5.5 ⁇ 2%, more preferably a pH of 5.5 ⁇ 1%.
  • a pH value of about 5.5 means a pH of 5.5 ⁇ 5%, preferably a pH of 5.5 ⁇ 2%, more preferably a pH of 5.5 ⁇ 1%.
  • the use of the singular includes the plural unless specifically stated otherwise.
  • any percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, unless otherwise indicated.
  • the amino acid sequence IDs (SEQ ID NO.) of the heavy chain variable regions, light chain variable regions, and CDRs of exemplary antibodies of the disclosure, or antigen-binding fragments thereof, are provided in Table 1 below. Some antibodies have the same CDRs, and some antibodies have the same VH or VL.
  • the heavy chain constant region of the antibody may be a human IgG1, IgG2 or IgG4 constant region, preferably IgG1 and IgG4 constant region, for example comprising the amino acid sequence shown in SEQ ID NOs: 54, 55 or 56 or the same as SEQ ID NOs: 54, 55 or 56
  • the amino acid sequences shown are compared to amino acid sequences having 1, 2, 3, 4 or 5 amino acid substitutions, deletions and additions.
  • the light chain constant region of the antibody can be a human kappa constant region or a human lambda constant region, for example comprising the amino acid sequence shown in SEQ ID NO: 57 or having 1, 2, 3, 4 compared with the amino acid sequence shown in SEQ ID NO: 57 Or the amino acid sequence of 5 amino acid substitutions, deletions and additions.
  • These antibodies may also comprise a mouse IgGl or IgG4 heavy chain constant region and/or a mouse kappa or lambda constant region.
  • the CDRs of an antibody can be defined in various ways, for example by numbering systems/methods such as Kabat, Chothia, IMGT, AbM or Contact; or by numbering systems such as Kabat, Chothia, IMGT, AbM and Contact Two or more combination definitions in the / method (for example, HCDR1 is defined by AbM, HCDR2 is defined by Kabat or AbM, HCDR3 is defined by IMGT or Kabat, and LCDR1-3 is defined by Kabat); it can also be defined with Kabat and Chothia
  • the Combined numbering system is defined, where the Combined numbering system combines the ranges defined by Kabat and Chothia, based on which a larger range is taken (for example, HCDR1 defined by Kabat is H31-H35, and HCDR1 defined by Chothia is H26- H32, then
  • CDR complementarity determining region
  • amino acid sequences corresponding to other CDR definition rules should also fall within the protection scope of the present disclosure.
  • the amino acid sequence of HCDR1 of mouse, chimeric and humanized A01 is: TSGVH (SEQ ID NO:79), HCDR2 of mouse, chimeric A01 and hzA01-3.4
  • the amino acid sequence of the amino acid sequence is: VIWAGGDTNYNSALMS (SEQ ID NO: 2), the amino acid sequence of the HCDR2 of hzA01-1.1, hzA01-2.1 and hzA01-3.1 is: VIWAGGDTNYNPSLKS (SEQ ID NO: 80), the HCDR2 of hzA01-1.2 and hzA01-2.2
  • the amino acid sequence of the amino acid sequence is: VIWAGGDTNYADSVKG (SEQ ID NO: 81), the amino acid sequence of the HCDR
  • VH and/or VL sequences (or CDR sequences) of other anti-CD40 antibodies that bind to human CD40 can be "mixed and paired" with the VH and/or VL sequences (or CDR sequences) of the disclosed anti-CD40 antibodies or antigen-binding portions thereof ".
  • VH and VL chains (or CDRs thereof) are mixed and paired, the VH sequence in a particular VH/VL pair can be replaced by a structurally similar VH sequence.
  • an antibody or antigen-binding fragment thereof of the present disclosure comprises:
  • an antibody or antigen-binding fragment thereof of the present disclosure comprises:
  • an antibody of the present disclosure or an antigen-binding fragment thereof comprises HCDR2 listed in Tables 1 and 10 and the CDRs of other anti-CD40 antibodies, such as HCDR1 and/or HCDR3 of other anti-CD40 antibodies, and/or other Anti-CD40 antibodies LCDR1, LCDR2 and/or LCDR3.
  • the CDR3 domain is independent of the CDR1 and/or CDR2 domain
  • the binding specificity of an antibody to the same antigen can be determined independently, and it can be predicted that multiple antibodies with the same binding specificity can be generated based on the CDR3 sequence. kinds of antibodies.
  • an antibody or antigen-binding fragment thereof of the present disclosure comprises HCDR2 listed in Table 1 and Table 10, and HCDR3 listed in Table 1 and Table 10 and/or LCDR3 listed in Table 1, or another HCDR3 and/or LCDR3 of an anti-CD40 antibody, wherein the antibody or antigen-binding portion thereof specifically binds human CD40.
  • These antibodies preferably (a) compete for CD40 binding with the anti-CD40 antibodies of the disclosure; (b) retain functional characteristics; (c) bind the same epitope; and/or (d) have similar binding affinities.
  • an antibody of the present disclosure, or an antigen-binding portion thereof further comprises an LCDR2 listed in Tables 1 and 10, or an LCDR2 of another anti-CD40 antibody, wherein the antibody or antigen-binding portion thereof specifically binds to a human CD40.
  • an antibody of the present disclosure, or an antigen-binding portion thereof further comprises the HCDR1 listed in Table 1 and Table 10 and/or the LCDR1 listed in Table 1 and Table 10, or the HCDR1 and and/or LCDR1, wherein the antibody or antigen-binding portion thereof specifically binds human CD40.
  • the heavy chain variable region and/or light chain variable region or CDR1, CDR2, and CDR3 sequences of an antibody of the disclosure, or an antigen-binding portion thereof may comprise one or more conservative modifications. It is understood in the art that certain conservative sequence modifications do not abolish antigen binding specificity.
  • an antibody of the present disclosure comprises a heavy chain variable region and/or a light chain variable region comprising CDR1, CDR2 and CDR3, respectively ,in:
  • the HCDR1 sequence comprises the sequences listed in Table 1 and Table 10, and/or conservative modifications thereof;
  • the HCDR2 sequence comprises the sequences listed in Table 1 and Table 10, and/or conservative modifications thereof;
  • the HCDR3 sequence comprises the sequences listed in Table 1 and Table 10, and/or conservative modifications thereof; and/or
  • LCDR1 and/or LCDR2 and/or LCDR3 sequences comprise the sequences listed in Table 1 and Table 10; and/or conservative modifications thereof;
  • the antibody or antigen-binding portion thereof specifically binds human CD40.
  • conservative sequence modification refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the disclosure by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions refer to substitution of amino acid residues with amino acid residues having similar structural or chemical properties (eg, similar side chains). Families of amino acid residues having similar side chains are known in the art.
  • amino acid residues include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine acid, isoleucine, proline, phenylalanine, methionine), beta branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine
  • one or more amino acid residues in the CDR regions of the disclosed anti-CD40 antibodies, or antigen-binding portions thereof, can be substituted with other amino acid residues from the same side chain family, and the resulting antibodies can be paired using the functional assays described herein. It performs functional testing.
  • the antibodies of the present disclosure or antigen-binding fragments thereof contain modifications to the variable regions (including CDR regions and/or framework regions), or, the antibodies of the disclosure or antigen-binding portions thereof may also contain Fc modifications, such as altering the effector functions of the antibodies .
  • one embodiment of the present disclosure provides an isolated anti-CD40 monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and/or a light chain variable region comprising HCDR1 of the above sequence of the disclosure , HCDR2 and HCDR3, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the above sequence of the present disclosure, but contains a framework sequence framework different from the above sequence of the present disclosure.
  • Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. Such framework sequences are preferably those structurally similar to the framework sequences used by the anti-CD40 antibodies of the present disclosure.
  • the CDR1, CDR2 and CDR3 sequences can be grafted into a framework region having the same sequence as the germline immunoglobulin gene for that framework sequence, or the CDR sequences can be grafted into a framework with one or more mutations compared to the germline sequence in the district. For example, in some cases it may be beneficial to mutate residues in the framework regions to maintain or enhance the antigen-binding ability of the antibody (see, e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).
  • variable region modification is to mutate amino acid residues within the CDR1, CDR2 and/or CDR3 regions to improve one or more properties (eg, affinity, physicochemical properties) of the antibody of interest. Mutations can be introduced by point mutations or PCR-mediated mutagenesis, and their effects on antibody binding or other functional properties can be assessed by in vitro or in vivo assays known in the art. Amino acids may be substituted, added or deleted, and substitutions are preferred. In addition, typically no more than one, two, three, four or five residues within each CDR region are altered.
  • an antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region and a light chain variable region comprising: (a) a HCDR1 region, It comprises the disclosed sequence, or has 1, 2, 3, 4 or 5 amino acid substitutions, deleted or added amino acid sequences; (b) HCDR2 region, which comprises the disclosed sequence, or has 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions of amino acid sequences; (c) HCDR3 region comprising the disclosed sequence, or 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions of amino acid sequences; ( d) LCDR1 region, which comprises the sequence of the disclosure, or has 1, 2, 3, 4 or 5 amino acid substitutions, deletions or added amino acid sequences; (e) LCDR2 region, which comprises the sequence of the disclosure, or has 1 , 2, 3, 4 or 5 amino acid substitutions, deletions or additions of amino acid sequences; and (f) an LCDR3 region comprising a sequence of the disclosure, or having 1, 2, 3, 4 or 5
  • Antibodies or antigen-binding fragments thereof of the present disclosure include modifications of the VH and/or VL framework regions to improve antibody properties. Typically, such framework region modifications can reduce the immunogenicity of the antibody. For example, one or more framework residues are "backmutated" to the corresponding germline sequence. These residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody was derived.
  • Another type of framework modification involves mutating one or more residues in the framework region, or even one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach, also known as “de-immunization,” is described in more detail in US Patent Publication No. 20030153043.
  • the antibodies or antigen-binding fragments thereof of the present disclosure include Fc modifications, which can be amino acid insertions, deletions or substitutions, and are usually used to change one or more functional properties of antibodies, such as serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent cytotoxicity.
  • the disclosed antibodies or antigen-binding fragments thereof may also be chemically modified (e.g., attached to one or more chemical functional groups), or modified to alter their glycosylation, to alter one or more functional properties of the antibody .
  • the Fc region is modified by pegylation (eg, by reacting the antibody or fragment thereof with polyethylene glycol (PEG).
  • the glycosylation of an antibody of the disclosure, or antigen-binding fragment thereof is altered.
  • Such glycosylation modifications can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence.
  • one or more amino acid substitutions can be made to eliminate a glycosylation site in the framework region of one or more variable domains, thereby eliminating glycosylation at that site.
  • deglycosylation can increase the affinity of the antibody for the antigen. See, eg, US Patents 5,714,350 and 6,350,861.
  • CD40 activity includes, but is not limited to, activation of B cells, such as proliferation of B cells, production of antibodies, antibody isotype switching, or differentiation into plasma cells; activation of T cells, such as proliferation of T cells or secretion of cytokines; Activation of dendritic cells, such as proliferation, differentiation and maturation of dendritic cells; and activation of macrophages. It can also inhibit CD40 activity by interacting with other molecules.
  • B cells such as proliferation of B cells, production of antibodies, antibody isotype switching, or differentiation into plasma cells
  • T cells such as proliferation of T cells or secretion of cytokines
  • dendritic cells such as proliferation, differentiation and maturation of dendritic cells
  • macrophages activation of macrophages. It can also inhibit CD40 activity by interacting with other molecules.
  • CD40 activity also includes inhibiting tumor cell growth and/or proliferation, inducing tumor cell apoptosis, and the like.
  • an anti-CD40 antibody or antigen-binding fragment thereof provided herein binds CD40 and results in inhibition of B cell activation. In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof provided herein binds CD40 and inhibits the proliferation of B cells.
  • an anti-CD40 antibody or antigen-binding fragment thereof provided herein binds CD40 and results in inhibition of T cell activation. In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof provided herein binds CD40 and inhibits T cell proliferation and/or cytokine production. In one embodiment, an anti-CD40 antibody or antigen-binding fragment thereof provided herein binds CD40 and inhibits the production of one or more cytokines selected from the group consisting of: IL-2, IFN ⁇ , TNF, IL -1, IL-4, IL-5, IL-6, IL-12, IL-13, IL-17 and GM-CSF.
  • an anti-CD40 antibody or antigen-binding fragment thereof provided herein binds CD40 and results in inhibition of the production of the cytokine IFN ⁇ . In one embodiment, an anti-CD40 antibody or antigen-binding fragment thereof provided herein binds CD40 and results in inhibition of the production of the cytokine IL-6.
  • the present disclosure provides methods of modulating an immune response comprising contacting T cells and antigen presenting cells with an anti-CD40 antibody or antigen-binding fragment thereof of the present disclosure.
  • modulation of an immune response by an anti-CD40 antibody or antigen-binding fragment thereof provided herein can be measured in a mixed lymphocyte reaction (MLR).
  • MLR mixed lymphocyte reaction
  • an anti-CD40 antibody or antigen-binding fragment thereof provided herein inhibits cytokine production by lymphocytes in the MLR.
  • an anti-CD40 antibody or antigen-binding fragment thereof provided herein inhibits the production of the cytokine IFN ⁇ in the MLR.
  • an anti-CD40 antibody or antigen-binding fragment thereof provided herein binds CD40 and inhibits the activation of dendritic cells. In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof provided in the present disclosure binds to CD40 and inhibits the differentiation and maturation of dendritic cells.
  • the anti-CD40 antibody or antigen-binding fragment thereof provided in the present disclosure binds to CD40 and inhibits tumor cell apoptosis. In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof provided in the present disclosure binds CD40 and inhibits apoptosis of Ramos cells.
  • the anti-CD40 antibody or antigen-binding fragment thereof provided in the present disclosure has no obvious agonistic activity when binding to CD40. In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof provided in the present disclosure has no obvious agonistic activity on tumor cell apoptosis. In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof provided by the present disclosure has no obvious agonistic activity on the apoptosis of Ramos cells. In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof provided by the present disclosure has no significant agonistic activity on the activation of B cells. In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof provided by the present disclosure has no significant agonistic activity on the proliferation of B cells.
  • the anti-CD40 antibody or antigen-binding fragment thereof provided by the present disclosure has no significant agonistic activity on the activation of dendritic cells. In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof provided in the present disclosure has no obvious agonistic activity on the differentiation and maturation of dendritic cells.
  • a substance "without appreciable agonistic activity” exhibits an agonistic activity detected in the assay not higher than about 25%, preferably not higher than about 20%, 15%, 10%, 5%, 1% of the agonistic activity induced by the natural substance or negative control %, 0.5%, or even not more than about 0.1%, or the agonistic activity detected in the assay is at least 30%, 40%, 50%, 60%, 70%, 80%, 85% lower than that induced by the positive control , 90%, 95%, or 100%.
  • a non-specific immunoglobulin that does not bind CD40 such as an IgG4 isotype control antibody, serves as a negative control.
  • an agonistic anti-CD40 antibody such as CP-870893, serves as a positive control.
  • the anti-CD40 antibody or antigen-binding fragment thereof provided by the present disclosure has good safety.
  • the anti-CD40 antibody or antigen-binding fragment thereof provided by the present disclosure has a good therapeutic effect on Sjogren's syndrome.
  • the present disclosure provides pharmaceutical compositions comprising one or more anti-CD40 antibodies of the present disclosure, or antigen-binding fragments thereof, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial agents, isotonic agents, and combinations thereof that are physiologically compatible. The selection and use of suitable "pharmaceutically acceptable carriers” is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).
  • Antibodies or pharmaceutical compositions can be administered by any suitable method or route.
  • Routes of administration include, for example, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).
  • the active ingredient can be coated in a material that protects it from acids and other natural conditions that might inactivate it.
  • parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intrathecal, intraorbital, cardiac Intratracheal, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.
  • the pharmaceutical composition may be administered by a parenteral route, eg, topically, epidermally or mucosally, such as intranasally, orally, vaginally, rectally, sublingually or topically.
  • dosages may range from about 0.0001 to 100 mg/kg body weight of the host.
  • anti-CD40 antibodies or antigen-binding portions thereof, have a variety of in vitro and in vivo applications involving the diagnosis, treatment and/or prevention of immune diseases.
  • Treatment refers to methods of alleviating and/or stabilizing symptoms, disorders, conditions, delaying and/or arresting the progression of a disease, and/or lessening the severity of a disease.
  • Anti-CD40 antibodies or antigen-binding portions thereof, encoding nucleic acids, pharmaceutical compositions, recombinant polypeptides, fusion proteins, bispecific molecules, immunoconjugates, chimeric antigen receptors or gene vectors of the present disclosure can be administered to human subjects , to treat the subject's immune disease.
  • the present disclosure provides a method for treating or preventing an immune disease in a subject, comprising administering to the subject a therapeutically effective amount of the anti-CD40 antibody of the present disclosure or an antigen-binding portion thereof, an encoding nucleic acid , a pharmaceutical composition, a recombinant polypeptide, a fusion protein, a bispecific molecule, an immunoconjugate, a chimeric antigen receptor, or a gene vector.
  • the subject is a human.
  • Immunode disease refers to any disease associated with the progression of an immune response in an individual, including cellular and/or humoral immune responses.
  • the immune disease includes, but is not limited to, inflammatory disease, allergic reaction, autoimmune disease or transplantation-related disease.
  • Inflammatory disease refers to any disease, disorder, or condition in which excessive inflammatory symptoms, host tissue damage, or loss of function result from an excessive or uncontrolled inflammatory response, including allergic inflammation of the skin, kidneys, gastrointestinal tract, and respiratory tract , psoriasis, nephritis, epididymitis, inflammatory bowel disease and asthma.
  • An autoimmune disease is any disease, disorder or condition in which the body attacks and damages its own tissues caused by an overactive immune system, for example, multiple sclerosis, arthritis, myasthenia gravis, psoriasis, scleroderma, autoimmune Immune hepatitis, autoimmune mumps and type I diabetes etc.
  • the immune diseases include, but are not limited to: allergic reactions, Addison's disease, ankylosing spondylitis, spondyloarthritis, asthma, atherosclerosis sclerosis, coronary heart disease, autoimmune hepatitis, autoimmune mumps, type 1 diabetes, epididymitis, nephritis, Reiter's syndrome, thyroiditis, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenia, systemic lupus erythematosus, subacute cutaneous lupus erythematosus, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, Arthritis, sarcoidosis, Sjogren's syndrome, dry eye, hidradenitis suppurativa, transplant-related disease, vascu
  • transplant-related diseases include, but are not limited to, transplant immune rejection and graft-versus-host disease (GVHD).
  • GVHD graft-versus-host disease
  • examples of inflammatory bowel disease include, but are not limited to, Crohn's disease and ulcerative colitis.
  • examples of arthritis include, but are not limited to, rheumatoid arthritis, juvenile arthritis, and psoriatic arthritis.
  • examples of nephritis include, but are not limited to: lupus nephritis.
  • examples of psoriasis include, but are not limited to: psoriasis vulgaris, pustular psoriasis (e.g., palmoplantar psoriasis, generalized pustular psoriasis), erythroderma type psoriasis and arthropathic psoriasis.
  • an anti-CD40 antibody of the present disclosure can be administered alone, or in combination with a second therapeutic agent.
  • the second therapeutic agent comprises a non-steroidal anti-inflammatory drug (NSAID), a salicylate, hydroxychloroquine, sulfasalazine, a corticosteroid, a cytotoxic drug, or an immunosuppressive drug and/or or antibodies.
  • NSAIDs include, but are not limited to, ibuprofen, naproxen, diclofenac, indomethacin, ketotrolac, meloxicam, nexicam, tetraprofen, and sulindac.
  • Immunosuppressive drugs and/or antibodies including, but not limited to, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, CTLA4-Ig fusions, anti-B lymphocyte stimulating antibodies, and anti-T cells Antibodies (eg anti-CD-3).
  • Cytotoxic drugs include, but are not limited to, methotrexate and cyclophosphamide.
  • the combinations of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions wherein each agent is in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents may be administered sequentially.
  • Embodiment 1 An isolated anti-CD40 antibody or antigen-binding fragment thereof comprising:
  • Heavy chain CDR1 includes the amino acid sequence of heavy chain CDR1 in SEQ ID NO:9
  • heavy chain CDR2 includes the amino acid sequence of heavy chain CDR2 in SEQ ID NO:9
  • heavy chain CDR3 includes the amino acid sequence of heavy chain CDR3 in SEQ ID NO:9 amino acid sequence
  • Heavy chain CDR1 includes the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 10
  • heavy chain CDR2 includes the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 10
  • heavy chain CDR3 includes the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 10 amino acid sequence
  • Heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 11
  • heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 11
  • heavy chain CDR3 comprises the heavy chain CDR3 in SEQ ID NO: 11 amino acid sequence
  • Heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 12
  • heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 12
  • heavy chain CDR3 comprises the heavy chain CDR3 in SEQ ID NO: 12 amino acid sequence
  • Heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 13
  • heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 13
  • heavy chain CDR3 comprises the heavy chain CDR3 in SEQ ID NO: 13 amino acid sequence
  • Heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 22
  • heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 22
  • heavy chain CDR3 comprises the heavy chain CDR3 in SEQ ID NO: 22 amino acid sequence
  • Heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 30
  • heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 30
  • heavy chain CDR3 comprises the heavy chain CDR3 in SEQ ID NO: 30 amino acid sequence
  • Heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 38
  • heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 38
  • heavy chain CDR3 comprises the heavy chain CDR3 in SEQ ID NO: 38 amino acid sequence
  • Heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 44
  • heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 44
  • heavy chain CDR3 comprises the heavy chain CDR3 in SEQ ID NO: 44 the amino acid sequence of;
  • Heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 52
  • heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 52
  • heavy chain CDR3 comprises the heavy chain CDR3 in SEQ ID NO: 52 the amino acid sequence of;
  • the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 14
  • the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 14
  • the light chain CDR3 comprises the light chain CDR3 in SEQ ID NO: 14 amino acid sequence
  • the light chain CDR1 includes the amino acid sequence of the light chain CDR1 in SEQ ID NO: 15
  • the light chain CDR2 includes the amino acid sequence of the light chain CDR2 in SEQ ID NO: 15
  • the light chain CDR3 includes the light chain CDR3 in SEQ ID NO: 15 amino acid sequence
  • the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 16
  • the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 16
  • the light chain CDR3 comprises the light chain CDR3 in SEQ ID NO: 16 amino acid sequence
  • the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 17
  • the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 17
  • the light chain CDR3 comprises the light chain CDR3 in SEQ ID NO: 17 amino acid sequence
  • Light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 23
  • light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 23
  • light chain CDR3 comprises the light chain CDR3 in SEQ ID NO: 23 amino acid sequence
  • the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 31
  • the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 31
  • the light chain CDR3 comprises the light chain CDR3 in SEQ ID NO: 31 amino acid sequence
  • Light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 39
  • light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 39
  • light chain CDR3 comprises the light chain CDR3 in SEQ ID NO: 39 amino acid sequence
  • Light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 45
  • light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 45
  • light chain CDR3 comprises the light chain CDR3 in SEQ ID NO: 45 the amino acid sequence of;
  • Light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 53
  • light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 53
  • light chain CDR3 comprises the light chain CDR3 in SEQ ID NO: 53 amino acid sequence.
  • Embodiment 2 The antibody or antigen-binding fragment thereof of embodiment 1 comprising:
  • Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 1, 2 and 5 or have at least 80% of the amino acid sequences shown in SEQ ID NOs: 1, 2 and 5 the identity of the amino acid sequence;
  • Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 1, 3 and 5 or have at least 80% of the amino acid sequences shown in SEQ ID NOs: 1, 3 and 5 the identity of the amino acid sequence;
  • Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 1, 4 and 5 or have at least 80% of the amino acid sequences shown in SEQ ID NOs: 1, 4 and 5 the identity of the amino acid sequence;
  • Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 18, 19 and 20 or have at least 80% of the amino acid sequences shown in SEQ ID NOs: 18, 19 and 20 the identity of the amino acid sequence;
  • Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 24, 25 and 26 or have at least 80% of the amino acid sequences shown in SEQ ID NOs: 24, 25 and 26 the identity of the amino acid sequence;
  • Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 32, 33 and 34 or have at least 80% of the amino acid sequences shown in SEQ ID NOs: 32, 33 and 34 the identity of the amino acid sequence;
  • Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 40, 41 and 42 or have at least 80% of the amino acid sequences shown in SEQ ID NOs: 40, 41 and 42 identical amino acid sequences; or
  • Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 46, 47 and 48 or have at least 80% of the amino acid sequences shown in SEQ ID NOs: 46, 47 and 48 an amino acid sequence of identity; and/or
  • Light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8 or have at least 80% of the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8 the identity of the amino acid sequence;
  • Light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 21, 7 and 8 or have at least 80% of the amino acid sequence shown in SEQ ID NOs: 21, 7 and 8 the identity of the amino acid sequence;
  • Light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 27, 28 and 29 or have at least 80% of the amino acid sequences shown in SEQ ID NOs: 27, 28 and 29 the identity of the amino acid sequence;
  • Light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 35, 36 and 37 or have at least 80% of the amino acid sequence shown in SEQ ID NOs: 35, 36 and 37 the identity of the amino acid sequence;
  • Light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 43, 36 and 37 or have at least 80% of the amino acid sequences shown in SEQ ID NOs: 43, 36 and 37 identical amino acid sequences; or
  • Light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 49, 50 and 51 or have at least 80% of the amino acid sequence shown in SEQ ID NOs: 49, 50 and 51 identical amino acid sequences.
  • Embodiment 3 The antibody or antigen-binding fragment thereof of embodiment 1 comprising:
  • Heavy chain CDR1 includes the amino acid sequence of heavy chain CDR1 in SEQ ID NO:9
  • heavy chain CDR2 includes the amino acid sequence of heavy chain CDR2 in SEQ ID NO:9
  • heavy chain CDR3 includes the amino acid sequence of heavy chain CDR3 in SEQ ID NO:9
  • the amino acid sequence of light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 14
  • the light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 14
  • light chain CDR3 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 14 Amino acid sequence of chain CDR3;
  • Heavy chain CDR1 includes the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 10
  • heavy chain CDR2 includes the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 10
  • heavy chain CDR3 includes the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 10
  • the amino acid sequence of the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 15
  • the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 15
  • the light chain CDR3 comprises the light chain CDR3 in SEQ ID NO: 15 Amino acid sequence of chain CDR3;
  • Heavy chain CDR1 includes the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 11
  • heavy chain CDR2 includes the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 11
  • heavy chain CDR3 includes the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 11
  • the amino acid sequence of the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 15
  • the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 15
  • the light chain CDR3 comprises the light chain CDR3 in SEQ ID NO: 15 Amino acid sequence of chain CDR3;
  • Heavy chain CDR1 includes the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 10
  • heavy chain CDR2 includes the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 10
  • heavy chain CDR3 includes the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 10
  • the amino acid sequence of light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 16
  • the light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 16
  • light chain CDR3 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 16 Amino acid sequence of chain CDR3;
  • Heavy chain CDR1 includes the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 11
  • heavy chain CDR2 includes the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 11
  • heavy chain CDR3 includes the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 11
  • the amino acid sequence of light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 16
  • the light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 16
  • light chain CDR3 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 16 Amino acid sequence of chain CDR3;
  • Heavy chain CDR1 includes the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 10
  • heavy chain CDR2 includes the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 10
  • heavy chain CDR3 includes the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 10
  • the amino acid sequence of light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 17
  • light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 17
  • light chain CDR3 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 17 Amino acid sequence of chain CDR3;
  • Heavy chain CDR1 includes the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 12
  • heavy chain CDR2 includes the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 12
  • heavy chain CDR3 includes the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 12
  • the amino acid sequence of light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 17
  • the light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 17
  • light chain CDR3 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 17 Amino acid sequence of chain CDR3;
  • Heavy chain CDR1 includes the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 13
  • heavy chain CDR2 includes the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 13
  • heavy chain CDR3 includes the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 13
  • the amino acid sequence of light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 17
  • the light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 17
  • light chain CDR3 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 17 Amino acid sequence of chain CDR3;
  • Heavy chain CDR1 includes the amino acid sequence of heavy chain CDR1 in SEQ ID NO:22
  • heavy chain CDR2 includes the amino acid sequence of heavy chain CDR2 in SEQ ID NO:22
  • heavy chain CDR3 includes the amino acid sequence of heavy chain CDR3 in SEQ ID NO:22
  • the amino acid sequence of the light chain CDR1 comprises the amino acid sequence of the light chain CDR1 in SEQ ID NO: 23
  • the light chain CDR2 comprises the amino acid sequence of the light chain CDR2 in SEQ ID NO: 23
  • the light chain CDR3 comprises the light chain CDR3 in SEQ ID NO: 23 Amino acid sequence of chain CDR3;
  • Heavy chain CDR1 includes the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 30
  • heavy chain CDR2 includes the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 30
  • heavy chain CDR3 includes the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 30
  • the amino acid sequence of light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 31
  • the light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO:
  • light chain CDR3 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 31 Amino acid sequence of chain CDR3;
  • Heavy chain CDR1 includes the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 38
  • heavy chain CDR2 includes the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 38
  • heavy chain CDR3 includes the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 38
  • the amino acid sequence of light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 39
  • the light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 39
  • light chain CDR3 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 39 Amino acid sequence of chain CDR3;
  • Heavy chain CDR1 includes the amino acid sequence of heavy chain CDR1 in SEQ ID NO:44
  • heavy chain CDR2 includes the amino acid sequence of heavy chain CDR2 in SEQ ID NO:44
  • heavy chain CDR3 includes the amino acid sequence of heavy chain CDR3 in SEQ ID NO:44
  • the amino acid sequence of light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 45
  • the light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 45
  • light chain CDR3 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 45 the amino acid sequence of chain CDR3;
  • Heavy chain CDR1 includes the amino acid sequence of heavy chain CDR1 in SEQ ID NO:52
  • heavy chain CDR2 includes the amino acid sequence of heavy chain CDR2 in SEQ ID NO:52
  • heavy chain CDR3 includes the amino acid sequence of heavy chain CDR3 in SEQ ID NO:52
  • the amino acid sequence of light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO:53
  • the light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO:53
  • light chain CDR3 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO:53 Amino acid sequence of chain CDR3.
  • Embodiment 4 The antibody or antigen-binding fragment thereof of any one of embodiments 1-3, comprising:
  • heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 1, 2, 5, 6, 7 and 8 or with Amino acid sequences having at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 1, 2, 5, 6, 7 and 8;
  • heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 1, 3, 5, 6, 7 and 8 or with Amino acid sequences having at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 1, 3, 5, 6, 7 and 8;
  • heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 1, 4, 5, 6, 7 and 8 or with Amino acid sequences having at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 1, 4, 5, 6, 7 and 8;
  • heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 18, 19, 20, 21, 7 and 8 or with Amino acid sequences having at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 18, 19, 20, 21, 7 and 8;
  • Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 24, 25, 26, 27, 28 and 29 or with Amino acid sequences having at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 24, 25, 26, 27, 28 and 29;
  • heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 32, 33, 34, 35, 36 and 37 or with Amino acid sequences having at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 32, 33, 34, 35, 36 and 37;
  • heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 40, 41, 42, 43, 36 and 37 or with Amino acid sequences having at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 40, 41, 42, 43, 36 and 37; or
  • Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 46, 47, 48, 49, 50 and 51 or with Amino acid sequences having at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 46, 47, 48, 49, 50 and 51.
  • Embodiment 5 The antibody or antigen-binding fragment thereof of any one of embodiments 1-4, comprising:
  • heavy chain variable region comprises the amino acid sequence shown in 9, 10, 11, 12, 13, 22, 30, 38, 44 or 52, or with SEQ ID NOs: 9, An amino acid sequence having at least 80% identity to the amino acid sequence shown in 10, 11, 12, 13, 22, 30, 38, 44 or 52; and/or
  • a light chain variable region comprising the amino acid sequence shown in SEQ ID NOs: 14, 15, 16, 17, 23, 31, 39, 45 or 53, or with SEQ ID NOs: An amino acid sequence having at least 80% identity to the amino acid sequence indicated by the amino acid sequence indicated by 14, 15, 16, 17, 23, 31, 39, 45 or 53.
  • Embodiment 6 The antibody or antigen-binding fragment thereof of any one of embodiments 1-5, comprising a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region and the light chain variable region comprise the amino acid sequences shown in SEQ ID NOs: 9 and 14 or amino acids with at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 9 and 14 sequence;
  • the heavy chain variable region and the light chain variable region comprise the amino acid sequences shown in SEQ ID NOs: 10 and 15 or amino acids with at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 10 and 15 sequence;
  • the heavy chain variable region and the light chain variable region respectively comprise the amino acid sequences shown in SEQ ID NOs: 11 and 15 or amino acids with at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 11 and 15 sequence;
  • the heavy chain variable region and the light chain variable region respectively comprise the amino acid sequences shown in SEQ ID NOs: 10 and 16 or amino acids having at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 10 and 16 sequence;
  • the heavy chain variable region and the light chain variable region respectively comprise the amino acid sequences shown in SEQ ID NOs: 11 and 16 or amino acids having at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 11 and 16 sequence;
  • the heavy chain variable region and the light chain variable region respectively comprise the amino acid sequences shown in SEQ ID NOs: 10 and 17 or amino acids having at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 10 and 17 sequence;
  • the heavy chain variable region and the light chain variable region comprise the amino acid sequences shown in SEQ ID NOs: 12 and 17 or amino acids with at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 12 and 17 sequence;
  • the heavy chain variable region and the light chain variable region respectively comprise the amino acid sequences shown in SEQ ID NOs: 13 and 17 or amino acids having at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 13 and 17 sequence;
  • the heavy chain variable region and the light chain variable region respectively comprise the amino acid sequences shown in SEQ ID NOs: 22 and 23 or amino acids having at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 22 and 23 sequence;
  • the heavy chain variable region and the light chain variable region comprise the amino acid sequences shown in SEQ ID NOs: 30 and 31 or amino acids with at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 30 and 31 sequence;
  • the heavy chain variable region and the light chain variable region comprise the amino acid sequences shown in SEQ ID NOs: 38 and 39 or amino acids with at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 38 and 39 sequence;
  • the heavy chain variable region and the light chain variable region comprise the amino acid sequences shown in SEQ ID NOs: 44 and 45 or amino acids with at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 44 and 45 sequence; or
  • the heavy chain variable region and the light chain variable region comprise the amino acid sequences shown in SEQ ID NOs: 52 and 53 or amino acids with at least 80% identity to the amino acid sequences shown in SEQ ID NOs: 52 and 53 sequence.
  • Embodiment 7 The antibody or antigen-binding fragment thereof of any one of embodiments 1-6, further comprising a heavy chain constant region and a light chain constant region, the heavy chain constant region Comprising the amino acid sequence shown in SEQ ID NOs: 54, 55 or 56 or having 1, 2, 3, 4 or 5 amino acid substitutions, deletions and additions compared to the amino acid sequence shown in SEQ ID NOs: 54, 55 or 56 Amino acid sequence, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 57 or has 1, 2, 3, 4 or 5 amino acid substitutions, deletions and additions compared with the amino acid sequence shown in SEQ ID NO: 57 amino acid sequence.
  • Embodiment 8 The antibody or antigen-binding fragment thereof of any one of embodiments 1-7, wherein the antibody or antigen-binding fragment thereof: (a) binds human CD40; (b) binds monkey CD40; (c ) blocking CD40 interaction with CD40L; and/or (d) inhibiting CD40 activity.
  • Embodiment 9 The antibody or antigen-binding fragment thereof of any one of embodiments 1-8, wherein said antibody or antigen-binding fragment thereof is chimeric or humanized.
  • Embodiment 10 The antibody or antigen-binding fragment thereof of any one of embodiments 1-9, wherein said antibody or antigen-binding fragment thereof is of the IgGl, IgG2 or IgG4 isotype.
  • Embodiment 11 The antibody or antigen-binding fragment thereof according to any one of embodiments 1-10, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of monoclonal antibodies, monospecific antibodies, bispecific antibodies, trispecific antibodies, Specific antibodies, multispecific antibodies, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAb fragments, isolated CDR regions, single chain Fv molecules, or combinations thereof.
  • Embodiment 12 An isolated antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof binds to the same epitope as the antibody or antigen-binding fragment thereof of any one of embodiments 1-11.
  • Embodiment 13 An isolated antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof competes for binding to CD40 with the antibody or antigen-binding fragment thereof of any one of embodiments 1-11.
  • Embodiment 14 An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of embodiments 1-11.
  • Embodiment 15 An expression vector comprising the nucleic acid molecule of embodiment 14.
  • Embodiment 16 A host cell comprising the nucleic acid molecule of embodiment 14 or the expression vector of embodiment 15.
  • Embodiment 17 A recombinant polypeptide or fusion protein comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-11.
  • Embodiment 18 A bispecific molecule comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-11.
  • Embodiment 19 An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-11 and a therapeutic agent, such as a cytotoxic agent or an imaging agent, linked thereto.
  • a therapeutic agent such as a cytotoxic agent or an imaging agent
  • Embodiment 20 A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-11 and one or more pharmaceutically acceptable carriers.
  • Embodiment 21 A method of treating or preventing an immune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody of any one of embodiments 1-11 or an antigen-binding fragment thereof, or the pharmaceutical composition of embodiment 20.
  • Embodiment 22 The method of embodiment 21, wherein the immune disease includes, but is not limited to, an inflammatory disease, an allergic reaction, an autoimmune disease, or a transplant-related disease.
  • Embodiment 23 The method according to embodiment 21, wherein the immune disease includes, but is not limited to, allergic reactions, Addison's disease, ankylosing spondylitis, spondyloarthritis, asthma, atherosclerosis, coronary heart disease, autoimmune Immune hepatitis, autoimmune mumps, type 1 diabetes, epididymitis, nephritis, Reiter's syndrome, thyroiditis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenia, systemic lupus erythematosus, subacute cutaneous lupus erythematosus, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, arthritis, sarcoidosis, Sjogren's syndrome, dry eye, Hidradenitis suppurativa, transplant-related disease, vasculitis, and/or
  • Embodiment 24 The method according to any one of embodiments 21-23, wherein the method further comprises administering a therapeutically effective amount of a second therapeutic agent.
  • Embodiment 25 The method of embodiment 24, wherein the second therapeutic agent comprises a non-steroidal anti-inflammatory drug, a salicylate, hydroxychloroquine, sulfasalazine, a corticosteroid, a cytotoxic drug, or Immunosuppressant drugs and/or antibodies.
  • the second therapeutic agent comprises a non-steroidal anti-inflammatory drug, a salicylate, hydroxychloroquine, sulfasalazine, a corticosteroid, a cytotoxic drug, or Immunosuppressant drugs and/or antibodies.
  • the cDNAs encoding human CD40 recombinant protein (hCD40-mFc, SEQ ID NO:64) and monkey CD40 recombinant protein (cynoCD40-mFc, SEQ ID NO:65) containing mouse antibody heavy chain Fc were obtained by gene synthesis, respectively Cloned into the expression vector pcDNA3.1(+), transfected into CHO cells for transient expression. The cell culture supernatant was collected, and the recombinant proteins hCD40-mFc and cynoCD40-mFc were purified using MabSelect Sure LX purification column (GE).
  • cDNAs encoding full-length hCD40 SEQ ID NO:62
  • cynoCD40 SEQ ID NO:63
  • Thermo, Cat: L3000015 the two recombinant plasmids were respectively transfected into CHO-K1 cells to obtain stable cell lines CHO-K1-hCD40 and CHO-K1-cynoCD40.
  • cDNAs encoding full-length hCD40L (SEQ ID NO:66) and cynoCD40L (SEQ ID NO:67) were obtained by gene synthesis, and then subcloned into the expression vector pcDNA3.1(+) to obtain pcDNA3.1-hCD40L and pcDNA3.1-cynoCD40L recombinant plasmid. Then, according to the instructions of Lipofectamin3000 transfection reagent (Thermo, Cat: L3000015), the two recombinant plasmids were transfected into CHO-K1 cells, respectively, to obtain stable cell lines CHO-K1-hCD40L and CHO-K1-cynoCD40L.
  • the plasmid pGL4.32[luc2P/NF- ⁇ B-RE/Hygro] Vector was transfected into HEK293T cells to obtain a stable cell line 293T-NF ⁇ B. Then the BamHI and XhoI sites of the pcDNA3.1/Zeo(+) vector were inserted into the coding sequence of the full-length hCD40 (SEQ ID NO:62) to obtain the recombinant plasmid pcDNA3.1/Zeo(+)-hCD40.
  • the recombinant plasmid pcDNA3.1/Zeo(+)-hCD40 was transfected into 293T-NF ⁇ B cells to obtain 293T-hCD40-NF ⁇ B stable cell line.
  • hCD40-mFc Purified recombinant protein hCD40-mFc (SEQ ID NO:64) was used as antigen, hCD40-mFc was mixed with complete Freund's adjuvant (Sigma, Cat: F5881-10X10mL) according to volume ratio 1:1 and emulsified, and injected subcutaneously Immunize mice. 2-4 weeks after the initial immunization, hCD40-mFc or cynoCD40-mFc and Alum adjuvant (Thermo, Cat: 77161) were thoroughly mixed and emulsified at a ratio of 1:1 by volume, and injected subcutaneously and intramuscularly alternately. A booster immunization was performed every 2 weeks for a total of 6 weeks. After the immunization, the serum of each mouse was collected to detect the serum titer of anti-CD40 specific antibody. Mice with higher serum titers were selected for subsequent splenocyte fusion.
  • mice 3-4 days before the fusion, the mice were injected with hCD40-mFc intraperitoneally, and the mice were immunized before fusion.
  • the mouse spleen was aseptically removed, ground and red blood cell lysed, and then resuspended in electrofusion buffer (BTX, Cat: 47-0001) to obtain a single cell suspension.
  • BTX electrofusion buffer
  • a few myeloma cells in the growth phase SP2/0 were mixed, and the cell fusion was completed by using an electric fusion instrument (BTX).
  • the fused cells were mixed with hybridoma medium (Gibco, Cat: 12045-076) containing 1 ⁇ HAT, and hybridoma cells were obtained after culturing at 37° C. and 5% CO 2 for 7 days.
  • the culture supernatant of hybridoma cells was collected for screening of mouse antibodies.
  • hybridoma cells with hCD40 binding activity detected by ELISA and FACS were selected for expansion and culture, and after the cells reached a certain number, they were subcloned by limiting dilution. Then the subcloned hybridoma cells were incubated and cultured at 37° C., 5% CO 2 for 7 days, and then the culture supernatant of the subcloned hybridoma cells was collected for further screening of mouse antibodies.
  • the cell culture supernatant was collected by centrifugation, and then the cell culture supernatant was loaded onto a protein G column (Genscript, Cat: L00209-10), and the protein G column was rinsed with PBS buffer, The antibody bound to the protein G column was then eluted with 100mM glycine (pH 2.8), and the eluate was immediately neutralized with 1M Tris-HC. Then, the mouse antibody was replaced into PBS buffer by ultrafiltration.
  • the hCD40-His protein (Acro, Cat: CD0-H5228) was diluted with PBS buffer (pH 7.4) to a concentration of 0.1 ⁇ g/mL, coated on a 96-well plate at 100 ⁇ L/well, and incubated overnight at 4°C.
  • the 96-well plate was washed with PBST (PBS containing 0.5% Tween-20), and then 200 ⁇ L/well of blocking solution (PBST containing 1% BSA) was added to block at room temperature for 2 hours. Discard the blocking solution, add 100 ⁇ L/well hybridoma cell culture supernatant, and incubate at room temperature for 2 hours.
  • the cells were washed with PBS containing 2% FBS, and then the cells were resuspended in PBS containing 2% FBS, and the mean fluorescence intensity (MFI) of staining was detected by flow cytometry (BD AccuriC6) to analyze the association of anti-CD40 antibody with CHO- Binding of K1-hCD40 cells.
  • MFI mean fluorescence intensity
  • Anti-CD40 Antibody Blocks the Binding Activity of 293T-hCD40-NF ⁇ B Cells to CD40L
  • Blocking activity of antibodies against CD40L-mediated upregulation of fluorescence signal in 293T-hCD40-NF ⁇ B cells, and IC 50 values were calculated by Graphpad Prism. As shown in Figures 1A and 1B, several purified mouse anti-CD40 antibodies had the activity of blocking the binding of 293T-hCD40-NF ⁇ B cells to CD40L.
  • the MAPK activation pathway mediated by CD40/CD40L is a potential mechanism for inhibiting tumor cell proliferation and inducing tumor cell apoptosis.
  • the human B lymphoma cell Ramos (Burkitt lymphoma cell) endogenously expressing CD40 was used as a model to detect the inhibitory activity of anti-CD40 antibody on Ramos cell apoptosis. Specifically, the Ramos cell suspension with a cell concentration of 1 ⁇ 10 6 cells/mL was added to a 96-well U-bottom plate at 50 ⁇ L/well, and a gradiently diluted purified mouse anti-CD40 antibody was added (the final concentration range was 0.02 ng/well).
  • the hybridoma cells screened in Example 2 were separated from the total RNA in the cells according to the operating instructions of the RNA extraction kit (Takara, Cat: 9767), and were separated by reverse
  • the first-strand cDNA was synthesized using a recording kit (Thermo, Cat: K1652).
  • the first-strand cDNA was used as a template, mixed with mouse IgG primer and Kappa primer respectively, cloned and sequenced using polymerase chain reaction (PCR) technology, and the variable region sequence of mouse IgG antibody was obtained.
  • the DNA sequences of the VH and VL of the mouse antibody were linked to the DNA sequences of the human IgG4 (SEQ ID NO:54) heavy chain constant region and the Kappa light chain constant region (SEQ ID NO:57), respectively, and then Cloned into pcDNA3.1(+) vector to construct recombinant human-mouse chimeric antibody.
  • These chimeric antibodies were named: Chi-A01, Chi-A02, Chi-A03, Chi-B01, Chi-B02 and Chi-B03, respectively.
  • Iscalimab (CFZ533) was selected as the reference antibody.
  • the DNA sequences of the heavy chain and light chain of the antibody were obtained by chemical synthesis, and cloned into the pcDNA3.1 (+) vector to construct the reference antibody (heavy chain and light chain).
  • the amino acid sequence of the chain is shown in SEQ ID NOs: 58 and 59 of the present disclosure, the heavy chain variable region and the light chain variable region are underlined), and the reference antibody is also referred to herein as BM (the abbreviation of Benchmark).
  • the chimeric antibody and reference antibody were transiently transfected and expressed using the expiCHO system (Gibco, Cat: A29129) according to the operating instructions of the transfection kit. After transfection, the cells were shaken at 37°C with 8% CO 2 Cultured for 6 days. The cell culture supernatant was collected by centrifugation, and then the cell culture supernatant was loaded onto a protein A column (GE Healthcare, Cat: 17-5474), and the protein A column was washed with 10 column volumes of PBS buffer, and then washed with acetate buffer ( 300 mM acetic acid, pH 3.6) to elute the antibody bound to the protein A column, and immediately neutralize the eluate with 1M Tris-HCl. Then, the chimeric antibody was replaced by ultrafiltration into PBS buffer.
  • acetate buffer 300 mM acetic acid, pH 3.6
  • Example 2 For the specific method, but some changes are made slightly: replace hybridoma cell culture with serially diluted chimeric anti-CD40 antibody and BM (final concentration ranging from 0.064ng/mL to 5000ng/mL, diluted in a 5-fold gradient) supernatant; and PE-conjugated goat anti-mouse IgG (H+L) antibody was replaced by PE-conjugated donkey anti-human IgG (H+L) (Abcam, Cat: ab102439).
  • Figure 3 shows the binding activity of chimeric antibodies to CHO-K1-hCD40 cells, wherein the binding EC 50 of Chi-A01 and Chi-A02 to CHO-K1-hCD40 cells is comparable to that of BM.
  • Table 2 and Table 3 show the inhibitory activity of chimeric antibodies on the apoptosis of Ramos cells induced by CD40L and IL-4, wherein Chi-A01 and Chi-A02 inhibit 50% of tumor cell apoptosis induced by CD40L and IL-4
  • concentration (IC 50 ) of BM is about 5pM, while the IC 50 of BM is 18.32pM (about 3-4 times that of Chi-A01 and Chi-A02), indicating that Chi-A01 and Chi-A02 can inhibit the apoptosis of Ramos cells Activity is better than BM.
  • Inhibition % (Ramos induced apoptosis after incubation with CD40L and IL-4-Ramos induced apoptosis after incubation with CD40L and IL-4 and anti-CD40 antibody MFI)/(Ramos induced apoptosis after incubation with CD40L and IL-4
  • the apoptotic MFI-Ramos was only incubated with the culture medium and the natural apoptotic MFI) ⁇ 100%. According to the formula calculation, the average maximum inhibition rate of Chi-A01 and Chi-A02 was equivalent to that of BM.
  • NA means no IC50 value was fitted.
  • the agonistic anti-CD40 antibody CP-870893 (prepared in-house, see SEQ ID NOs: 60 and 61 in this disclosure for the heavy chain and light chain sequences) combined with Ramos cells can generate stimulation signals to induce Ramos apoptosis. Use Ramos cells to detect the agonistic activity of chimeric anti-CD40 antibodies.
  • CDR-graft method the heavy chain variable region sequence and the light chain variable region sequence of the mouse antibody A01 were compared and analyzed by the protein database, and the human germline antibody sequence with the highest sequence homology was screened out.
  • the complementarity-determining regions (CDRs) of mouse antibodies are grafted into the framework of the screened human germline antibody sequences, and the amino acid residues in the CDRs and/or frameworks are further mutated to obtain more candidate variable region sequences.
  • Light chain VL The combination of human germline framework sequence IGKV1-39*01 and IGKJ1*01 is used for humanized sequence design to construct light chain variant hzA01L1 (SEQ ID NO:15); human germline framework sequence IGKV3-20 The combination of *01 and IGKJ1*01 was used for humanized sequence design, and the light chain variant hzA01L2 (SEQ ID NO:16) was constructed; the combination of human germline framework sequence IGKV2-30*02 and IGKJ2*02 was used for human source According to the sequence design, the light chain variant hzA01L3 (SEQ ID NO: 17) was constructed.
  • Heavy chain VH The combination of human germline framework sequences IGHV4-4*08 and IGHJ3*01 was used for humanized sequence design to construct heavy chain variants hzA01H1, hzA01H3 and hzA01H4 (see SEQ ID NO: 10, SEQ ID NO :12 and SEQ ID NO:13); the combination of human germline framework sequences IGHV3-33*01 and IGHJ3*01 was used for humanized sequence design to construct the heavy chain variant hzA01H2 (SEQ ID NO:11).
  • the DNA sequences of the VH and VL of the above humanized antibody were linked to the DNA sequences of the human IgG4 (SEQ ID NO:54) heavy chain constant region and the Kappa light chain constant region (SEQ ID NO:57) respectively by chemical synthesis method, Then clone into pcDNA3.1(+) vector to construct recombinant humanized antibody.
  • Table 4 shows the alignment of the VH and VL sequences of the humanized antibodies.
  • the humanized antibody was transiently transfected using the expiCHO expression system (Gibco, Cat: A29129) according to the operation instructions of its transfection kit. After transfection, the cells were cultured with shaking at 37°C and 8% CO 2 6 days. Collect the cell culture supernatant by centrifugation, then incubate the cell culture supernatant with protein A magnetic beads (GenScript, Cat: L00273) at room temperature for 2 hours with shaking, wash with PBS solution, and then wash with acetate buffer (300mM acetic acid, pH 3.6) Elute the antibody bound to the protein A magnetic beads and immediately neutralize the eluate with 1M Tris-HCl. Then, the humanized antibody was replaced into PBS buffer by ultrafiltration.
  • the expiCHO expression system Gibco, Cat: A29129
  • the AHC biosensor Formebio, Cat: 18-5060
  • the AHC biosensor was immersed in an anti-CD40 antibody solution at a final concentration of 5 ⁇ g/mL to capture the antibody.
  • the AHC biosensor was used to combine the hCD40-His protein and CynoCD40-His protein which were diluted in a gradient (the final concentration range was 1.56nM-100nM, according to 2-fold gradient dilution).
  • the Fortebio molecular interaction instrument (Fortebio Octet RED96e) was used to detect the reaction signal in real time to obtain the association and dissociation curves. After the dissociation of each cycle is completed, the AHC biosensor is regenerated, and then the next capture is performed, and the determination of the affinity of different antibodies to CD40 is completed repeatedly.
  • K D K d /K a D.
  • the binding affinity K D of humanized antibodies hzA01-3.1, hzA01-3.3 and hzA01-3.4 to human or cynomolgus monkey CD40 antigen is between 0.1 nM and 1 nM, which is comparable to the affinity of chimeric Chi-A01 .
  • ND Indicates not detected.
  • Anti-CD40 antibody blocks CHO-K1-hCD40L cells and hCD40-mFc, CHO-K1-CynoCD40L cells and cynoCD40-mFc binding activity
  • the cells were washed with PBS containing 2% FBS, PE-coupled goat anti-mouse IgG (H+L) antibody (Abcam, Cat: ab97041) diluted 1:500 by volume was added, and incubated at 4°C for 0.5 hours.
  • H+L PE-coupled goat anti-mouse IgG
  • Anti-CD40 Antibody Blocks the Binding Activity of 293T-hCD40-NF ⁇ B Cells to CHO-K1-hCD40L Cells
  • the detection reagent was added according to the instructions of the firefly luciferase kit (vazyme, Cat: DD1201-03), the fluorescence signal of the cells was detected by a microplate reader (Thermo Varioskan Flash), and the anti-CD40 antibody was analyzed by relative fluorescence intensity (RLU). Blocking activity against CD40L-mediated upregulation of fluorescence signaling in 293T-hCD40-NF ⁇ B cells. The results are shown in Figure 6. In the concentration range of 0-2 ⁇ g/mL, the humanized antibodies hzA01-1.1, hzA01-2.1, hzA01-3.1, hzA01-3.3 and hzA01-3.4 all showed significant blocking activity.
  • the results are shown in Figure 8.
  • the humanized antibodies hzA01-1.1 and hzA01-2.1 have weak agonistic activity, and the humanized antibodies hzA01-3.1, hzA01-3.3 and hzA01-3.4 do not produce significantly higher activity than BM in the concentration range of 0.01nM to 2000nM. stimulant activity.
  • CD40 expressed on human peripheral blood-derived B lymphocytes can bind to CD40L and induce B cell activation, thereby causing upregulation of cell surface activation signals (such as CD86).
  • Human B lymphocyte purification kit (Stemcell, Cat: 17954) was used to separate and purify B lymphocytes from healthy human PBMC, and the peripheral blood B lymphocyte suspension with a cell concentration of 1 ⁇ 106 /mL was added at 50 ⁇ L/well.
  • Into a 96-well U-bottom plate and add anti-CD40 antibody in serial dilution (final concentration range is 0.002ng/mL ⁇ 2000ng/mL, according to 10-fold serial dilution), and then add CHO-K1- hCD40L cell suspension.
  • CD40 expressed on human peripheral blood-derived B lymphocytes can combine with CD40L to induce B cell proliferation.
  • the IC 50 of the humanized antibodies hzA01-3.1, hzA01-3.3 and hzA01-3.4 in inhibiting the proliferation of human peripheral blood B lymphocytes induced by CD40L and IL-4 was about 3-6 times lower than that of BM.
  • Mononuclear cells were isolated from healthy human PBMC according to the operating instructions of the monocyte isolation kit (Stemcell, Cat: 19359), and then the cell concentration was adjusted to 1 ⁇ 10 6 cells/mL. According to the operating instructions of the DC cell culture kit (Stemcell, Cat: 10985), DC cell differentiation inducer was added, and cultured at 37°C, containing 5% CO 2 for 5 days, and then DC cell maturation stimulator was added, The culture was continued for 2 days to differentiate the monocytes into fully mature DC cells (mDC).
  • mDC fully mature DC cells
  • CD4 + T cells were isolated from healthy human PBMC according to the operating instructions of EasySep TM CD4 Positive T Cell Isolation Kit (Stemcell, Cat: 17952), and then the cell concentration was adjusted to 2 ⁇ 10 6 cells/mL. Add mDC cells at 1 ⁇ 104 cells/well to 96-well U-bottom plate, and add serially diluted anti-CD40 antibody or IgG4 isotype control antibody (final concentration range is 10ng/mL ⁇ 10000ng/mL, diluted by 10-fold gradient) ), mix well and incubate at room temperature for 30 minutes, then add CD4 + T cell suspension according to the ratio of mDC to CD4 + T cell number 1:20, mix well and incubate at 37°C with 5% CO 2 for 96 Hours, the culture supernatant was collected by centrifugation.
  • the IFN ⁇ in the culture supernatant was detected by ELISA, the OD450 absorbance value was read using a microplate reader (Bio-radiMark), and the IFN ⁇ concentration was calculated and analyzed by Graphpad Prism . The results are shown in Figure 12.
  • the humanized antibody hzA01-3.3 inhibited the mixed lymphocyte reaction in a concentration-dependent manner within the concentration range of 10ng/mL-10000ng/mL, and at the saturation concentration (1000ng/mL-10000ng/mL)
  • the release of IFN ⁇ was lower than that of BM, which indicated that hzA01-3.3 had a better inhibitory ability to mixed lymphocyte reaction than BM at saturating concentration.
  • Monocytes were isolated from PBMCs according to the operating instructions of the monocyte isolation kit (Stemcell, Cat: 19359), induced and cultured for 5 days by a DC cell differentiation inducer (Stemcell, Cat: 10988), and immature DC cells were collected ( imDC), and adjust the cell concentration to 1 ⁇ 10 6 cells/mL.
  • ImDC cell suspension was added to a 96-well plate at 50 ⁇ L/well, and then anti-CD40 antibody or IgG4 isotype control antibody (final concentration range of 100 ng/mL-10000 ng/mL, diluted by 10-fold gradient) was added in a gradient dilution. °C, containing 5% CO 2 and incubated overnight.
  • Fc receptors Human B cells, DC cells, and PBMCs express Fc receptors (FcR) on the surface of cells.
  • FcR Fc receptors
  • an anti-CD40 antibody binds to FcR, it may mediate antibody Fc crosslinking (Fc Crosslinking) to generate agonistic activity, or mediate B cell depletion by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the cells were collected by centrifugation, washed and resuspended with PBS containing 2% FBS, and the fluorescence signal of the cells was detected by a flow cytometer (Sartorius IQue3), and the apoptosis molecule CD95 on the Ramos cells was analyzed by the mean fluorescence intensity (MFI) of the staining.
  • MFI mean fluorescence intensity
  • the ADCC effect mediated by anti-CD40 antibody was evaluated. Specifically, the Ramos cell suspension with a cell concentration of 1 ⁇ 10 6 cells/mL was added to a 96-well plate at 50 ⁇ L/well, and a gradiently diluted anti-CD40 antibody or Rituximab was added (the final concentration range was 0.46 ng/mL to 1000 ng /mL, according to 3-fold serial dilution), mix well and incubate at room temperature for 20 minutes.
  • Example 8 In vivo efficacy of humanized anti-CD40 antibody
  • human PBMCs will produce human anti-mouse IgG antibodies and human anti-mouse IgM antibodies in response to mouse antigens; on the other hand, human PBMC cells in mice will produce anti-KLH after being stimulated by exogenous proteins such as KLH specific antibody.
  • the method for constructing a mouse model that produces human antibodies is as follows.
  • mice Select 6-8 week-old female NDG immunodeficient mice (purchased from Biocytogen), and inject 1 ⁇ 10 7 into each mouse’s tail vein on day 0 healthy human PBMC cells; on day 0-5, each mouse was intraperitoneally injected with 10 ⁇ g recombinant human IL-4 (Acro, Cat: IL4-H4218); and on day 0 and day 7, each mouse was given intraperitoneally Rats were intraperitoneally injected with 50 ⁇ g KLH protein (Sigma, Cat: H7017-20MG). The mice were grouped according to Table 8. Starting from the 0th day, each mouse was injected with the antibody drug through the intraperitoneal cavity for 3 consecutive times at a frequency of once a week.
  • the titer of anti-KLH antibody in mouse serum was detected by the following method: 100 ⁇ L/well of KLH protein (Sigma, Cat: H7017-20MG) with a concentration of 2 ⁇ g/mL was added to a 96-well plate, and incubated at 4 °C overnight. After washing the 96-well plate with PBST, 200 ⁇ L/well of blocking solution (PBST containing 1% BSA) was added and blocked at room temperature for 2 hours. Discard the blocking solution, then add 100 ⁇ L/well of serially diluted mouse serum, and incubate at room temperature for 2 hours.
  • KLH protein Sigma, Cat: H7017-20MG
  • hzA01-3.3 decreased the spleen coefficient of mice in a dose-dependent manner; the spleen coefficients of mice in hzA01-3.3 (5 mg/kg) group and BM (5 mg/kg) group were similar.
  • the blank control is NDG mice without immune reconstitution and administration;
  • the IgG4 isotype control is Anti-HEL-Human IgG4 (S228P L235E) Isotype-control (Biointron, Cat: B109805)
  • flow cytometry was used to analyze the content of human T lymphocytes and B lymphocytes infiltrated in the spleens of mice in each group. Specifically, the spleens of mice were taken to prepare a single cell suspension, and the cell concentration was adjusted to 1 ⁇ 106 Add PE-conjugated mouse anti-human CD4 antibody (Biolegend, Cat: 980804) and APC-conjugated mouse anti-human CD19 antibody (BD, Cat: 555415) to 96-well U-bottom plate at 100 ⁇ L/well.
  • PE-conjugated mouse anti-human CD4 antibody Biolegend, Cat: 980804
  • APC-conjugated mouse anti-human CD19 antibody BD, Cat: 555415
  • mice that on day 22, the spleens of IgG4 isotype control mice had a large number of human CD4 + T cells and human CD19 + B cells infiltrated; while hzA01-3.3 (0.5 mg/kg) and hzA01-3.3 ( 5mg/kg) group mice spleen infiltrating T cells and B cells were significantly lower than the IgG4 isotype group, hzA01-3.3 dose-dependent reduction of human T cells and B cell infiltration content.
  • hzA01-3.3 0.5 mg/kg
  • hzA01-3.3 5mg/kg
  • the antigen was prepared according to the following method: 20 C57BL/6 mice (6-8 weeks old, female; Biocytogen Pharmaceutical Technology Co., Ltd.) were taken, the submandibular glands were collected, the capsule and connective tissue were separated, and they were washed with PBS (Servicebio , Cat: G4202-500ML) wash. Under low temperature environment, mechanically homogenate, then centrifuge to get the supernatant to obtain the submandibular gland protein, and use the submandibular gland protein as the antigen.
  • PBS Servicebio , Cat: G4202-500ML
  • the hCD40/hCD40L mouse Sjogren's syndrome model was constructed as follows: on day 0 and day 7, the antigen was mixed with complete Freund's adjuvant (Sigma, Cat: F5881) 1:1 (v/v) to make the submandibular gland Protein emulsion was injected subcutaneously at two points on the back of hCD40/hCD40L mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), with a total amount of 200 ⁇ L; :F5506) 1:1 (v/v) to make a submandibular gland protein emulsion, and injected subcutaneously at two points on the back of hCD40/hCD40L mice, with a total volume of 200 ⁇ L.
  • mice were divided into 5 groups according to the average initial saliva volume. From the 0th day, they were administered twice a week for a total of 10 times (respectively on the 0th, 4th, 7th, 11th, 14th, and 18th day). , 21, 25, 28 and 32 days administration), the end of the test on the 35th day; weighing once every 2 weeks during this period.
  • the grouping and dosing regimen are shown in Table 9.
  • the blank control is hCD40/hCD40L mice without antigen immunization; N/A, not applicable; i.v., intravenous injection; BIW, administered twice a week.
  • the drug efficacy of hzA01-3.3 was evaluated by salivary flow rate measurement, submandibular gland index calculation, submandibular gland IL-6 detection, blood and spleen flow cytometry and submandibular gland HE pathological staining examination.
  • the specific methods are as follows:
  • the spleen of the mouse was taken out to make a single cell suspension, and the cell density was adjusted to 1 ⁇ 108 cells/mL, and 100 ⁇ L was added to erythrocyte lysate (Biolegend, Cat: 420301) for lysis in the dark for 30 minutes.
  • PE-labeled anti-mouse CD45 antibody Biolegend, Cat: 103106
  • Brilliant Violet 510 TM -labeled anti-mouse CD3 antibody Biolegend, Cat: 100234
  • FITC-labeled anti-mouse CD4 antibody Biolegend, Cat: 100510
  • PE/Cyanine7-labeled anti-mouse CD8a antibody Biolegend, Cat: 100722
  • APC-labeled anti-mouse CD19 antibody Biolegend, Cat: 115512
  • the mouse IL-6ELISA kit Abcam, Cat: ab222503

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Abstract

提供了抗CD40抗体及其用途，例如特异性结合CD40的小鼠、人、嵌合或人源化的抗体或其抗原结合片段，还提供了编码所述抗体及其抗原结合片段的核酸分子，以及用于表达所述抗体或其抗原结合片段的表达载体和宿主细胞。进一步提供了所述抗体及其抗原结合片段的制备方法和使用方法，包括治疗和预防免疫疾病。

Description

抗CD40抗体及其用途 技术领域
本公开属于生物技术领域，具体涉及特异性结合CD40的抗体和其抗原结合片段，并且涉及使用这类抗体和抗原结合片段的方法。
背景技术
CD40是一种I型跨膜糖蛋白，属于肿瘤坏死因子受体(TNFR)超家族成员。CD40组成性地表达于抗原递呈细胞(包括树突状细胞、B细胞和巨噬细胞)，还少量表达于在非造血细胞如上皮细胞、内皮细胞、平滑肌细胞、成纤维细胞、角质细胞等。此外，CD40还表达于多种肿瘤细胞，例如B淋巴瘤细胞。CD40的主要配体CD40L(CD154)，是一种II型跨膜糖蛋白，CD40L主要表达于活化的CD4 +T淋巴细胞、活化的B细胞、记忆T细胞、激活的NK细胞和活化的血小板中。
CD40/CD40L信号通路参与机体的体液免疫反应和细胞免疫反应，在B细胞的活化、增殖与分化，抗体的产生、抗体类别转换等T细胞依赖的抗体免疫反应以及炎症反应中起关键调节作用。研究表明，自身免疫性疾病患者的组织和器官中浸润大量针对自身抗原的T细胞和B细胞，且表现为CD40和/或CD40L持续性高表达。原发性干燥综合征患者IHC染色结果显示，CD40在唾液腺上皮细胞富集并伴随着血清中的抗SSA等自身抗体增高。此外，CD40/CD40L信号通路在炎性肠病中的关键作用已在小鼠等动物模型中证实。
因此，特异性结合CD40并抑制CD40/CD40L信号通路的抗体，对免疫疾病例如炎性疾病和自身免疫病的治疗具有潜在的临床价值。例如，Novartis公司开发的抗CD40抗体Iscalimab(CFZ533)，目前正在用于治疗CD40介导的免疫疾病的临床试验中，结果显示，Iscalimab以10mg/kg静脉注射剂量施用于原发性干燥综合征患者(入组条件为血清自身抗体阳性)时，62％(13/21)患者ESSDAI疾病活动评分低于5分，治疗效果显著优于安慰剂组的36％(4/11)。目前全球免疫疾病患者的数量庞大，因此，亟需开发更多具有更好药物特性的抗CD40抗体。
发明内容
一方面，本公开提供了分离的结合CD40的抗体或其抗原结合片段。在一些实施方案中，抗体或其抗原结合片段是鼠类抗体、嵌合抗体、人源化抗体或人抗体。在一些实施方案中，抗体或其抗原结合片段是单克隆抗体、单特异性抗体、双特异性抗体、三特异性抗体、多特异性抗体、Fab片段、F(ab')2片段、Fd片段、Fv片段、dAb、分离的CDR、单链Fv分子、重组多肽、融合蛋白、双特异性分子或其组合。在一些实施方案中，本公开的抗体或其抗原结合片段结合人CD40。在另一些实施方案中，本公开的抗体或其抗原结合片段结合CD40(例如人CD40和食蟹猴(cynomolgous)CD40)。在一些实施方案中，本公开的抗体或其抗原结合片段阻断CD40与CD40L相互作用。在一些实施方案中，本公开的抗体或其抗原结合片段抑制CD40活性。
在一些实施方案中，本公开提供的抗CD40抗体或其抗原结合片段包含重链CDR1(HCDR1)、重链CDR2(HCDR2)和重链CDR3(HCDR3)，其中，(1)重链CDR1包含SEQ ID NO：9中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：9中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：9中重链CDR3的氨基酸序列；(2)重链CDR1包含SEQ ID NO：10中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：10中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：10中重链CDR3的氨基酸序列；(3)重链CDR1包含SEQ ID NO：11中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：11中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：11中重链CDR3的氨基酸序列；(4)重链CDR1包含SEQ ID NO：12中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：12中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：12中重链CDR3的氨基酸序列；(5)重链CDR1包含SEQ ID NO：13中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：13中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：13中重链CDR3的氨基酸序列；(6)重链CDR1包含SEQ ID NO：22中重 链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：22中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：22中重链CDR3的氨基酸序列；(7)重链CDR1包含SEQ ID NO：30中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：30中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：30中重链CDR3的氨基酸序列；(8)重链CDR1包含SEQ ID NO：38中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：38中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：38中重链CDR3的氨基酸序列；(9)重链CDR1包含SEQ ID NO：44中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：44中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：44中重链CDR3的氨基酸序列；或(10)重链CDR1包含SEQ ID NO：52中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：52中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：52中重链CDR3的氨基酸序列。
在一些实施方案中，本公开提供的抗CD40抗体或其抗原结合片段包含重链CDR1、重链CDR2和重链CDR3，其中，(1)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：1、2和5所示的氨基酸序列或与SEQ ID NOs：1、2和5所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(2)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：1、3和5所示的氨基酸序列或与SEQ ID NOs：1、3和5所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(3)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：1、4和5所示的氨基酸序列或与SEQ ID NOs：1、4和5所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(4)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：18、19和20所示的氨基酸序列或与SEQ ID NOs：18、19和20所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(5)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：24、25和26所示的氨基酸序列或与SEQ ID NOs：24、25和26所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(6)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：32、33和34所示的氨基酸序列或与SEQ ID NOs：32、33和34所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(7)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：40、41和42所示的氨基酸序列或与SEQ ID NOs：40、41和42所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；或(8)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：46、47和48所示的氨基酸序列或与SEQ ID NOs：46、47和48所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段包含轻链CDR1(LCDR1)、轻链CDR2(LCDR2)和轻链CDR3(LCDR3)，其中，(1)轻链CDR1包含SEQ ID NO：14中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：14中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：14中轻链CDR3的氨基酸序列；(2)轻链CDR1包含SEQ ID NO：15中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：15中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：15中轻链CDR3的氨基酸序列；(3)轻链CDR1包含SEQ ID NO：16中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：16中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：16中轻链CDR3的氨基酸序列；(4)轻链CDR1包含SEQ ID NO：17中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：17中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：17中轻链CDR3的氨基酸序列；(5)轻链CDR1包含SEQ ID NO：23中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：23中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：23中轻链CDR3的氨基酸序列；(6)轻链CDR1包含SEQ ID NO：31中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：31中轻链CDR2的氨基酸序列和轻链CDR3 包含SEQ ID NO：31中轻链CDR3的氨基酸序列；(7)轻链CDR1包含SEQ ID NO：39中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：39中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：39中轻链CDR3的氨基酸序列；(8)轻链CDR1包含SEQ ID NO：45中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：45中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：45中轻链CDR3的氨基酸序列；或(9)轻链CDR1包含SEQ ID NO：53中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：53中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：53中轻链CDR3的氨基酸序列。
在一些实施方案中，本公开提供的抗CD40抗体或其抗原结合片段包含轻链CDR1，轻链CDR2和轻链CDR3，其中，(1)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：6、7和8所示的氨基酸序列或与SEQ ID NOs：6、7和8所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(2)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：21、7和8所示的氨基酸序列或与SEQ ID NOs：21、7和8所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(3)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：27、28和29所示的氨基酸序列或与SEQ ID NOs：27、28和29所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(4)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：35、36和37所示的氨基酸序列或与SEQ ID NOs：35、36和37所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(5)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：43、36和37所示的氨基酸序列或与SEQ ID NOs：43、36和37所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；或(6)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：49、50和51所示的氨基酸序列或与SEQ ID NOs：49、50和51所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，本公开提供的抗CD40抗体或其抗原结合片段包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，其中，(1)重链CDR1包含SEQ ID NO：9中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：9中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：9中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：14中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：14中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：14中轻链CDR3的氨基酸序列；(2)重链CDR1包含SEQ ID NO：10中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：10中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：10中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：15中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：15中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：15中轻链CDR3的氨基酸序列；(3)重链CDR1包含SEQ ID NO：11中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：11中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：11中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：15中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：15中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：15中轻链CDR3的氨基酸序列；(4)重链CDR1包含SEQ ID NO：10中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：10中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：10中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：16中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：16中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：16中轻链CDR3的氨基酸序列；(5)重链CDR1包含SEQ ID NO：11中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：11中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：11中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：16中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：16中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：16中轻链CDR3的氨基酸序列；(6)重链CDR1包含SEQ ID NO：10中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：10中重链CDR2的氨基酸序列，重链CDR3包 含SEQ ID NO：10中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：17中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：17中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：17中轻链CDR3的氨基酸序列；(7)重链CDR1包含SEQ ID NO：12中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：12中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：12中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：17中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：17中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：17中轻链CDR3的氨基酸序列；(8)重链CDR1包含SEQ ID NO：13中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：13中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：13中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：17中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：17中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：17中轻链CDR3的氨基酸序列；(9)重链CDR1包含SEQ ID NO：22中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：22中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：22中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：23中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：23中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：23中轻链CDR3的氨基酸序列；(10)重链CDR1包含SEQ ID NO：30中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：30中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：30中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：31中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：31中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：31中轻链CDR3的氨基酸序列；(11)重链CDR1包含SEQ ID NO：38中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：38中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：38中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：39中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：39中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：39中轻链CDR3的氨基酸序列；(12)重链CDR1包含SEQ ID NO：44中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：44中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：44中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：45中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：45中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：45中轻链CDR3的氨基酸序列；或(13)重链CDR1包含SEQ ID NO：52中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：52中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：52中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：53中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：53中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：53中轻链CDR3的氨基酸序列。
在一些实施方案中，本公开提供的抗CD40抗体或其抗原结合片段包含重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，其中，(1)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：1、2、5、6、7和8所示的氨基酸序列或与SEQ ID NOs：1、2、5、6、7和8所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(2)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：1、3、5、6、7和8所示的氨基酸序列或与SEQ ID NOs：1、3、5、6、7和8所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(3)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：1、4、5、6、7和8所示的氨基酸序列或与SEQ ID NOs：1、4、5、6、7和8所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(4)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：18、19、20、21、7和8所示的氨基酸序列或与SEQ ID NOs：18、19、20、21、7和8所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(5)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：24、25、26、27、28和29所示的氨基酸序列或与SEQ ID NOs：24、25、26、27、28和29所示的氨基酸序列具有至少80％、81％、82％、83％、84％、 85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(6)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：32、33、34、35、36和37所示的氨基酸序列或与SEQ ID NOs：32、33、34、35、36和37所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(7)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：40、41、42、43、36和37所示的氨基酸序列或与SEQ ID NOs：40、41、42、43、36和37所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；或(8)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：46、47、48、49、50和51所示的氨基酸序列或与SEQ ID NOs：46、47、48、49、50和51所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，本公开提供的抗CD40抗体或其抗原结合片段包含重链可变区，所述重链可变区包含SEQ ID NOs：9、10、11、12、13、22、30、38、44或52所示的氨基酸序列，或与SEQ ID NOs：9、10、11、12、13、22、30、38、44或52所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，本公开提供的抗CD40抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含SEQ ID NOs：14、15、16、17、23、31、39、45或53所示的氨基酸序列，或与SEQ ID NOs：14、15、16、17、23、31、39、45或53所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，本公开提供的抗CD40抗体或其抗原结合片段结包含重链可变区和轻链可变区，其中，(1)重链可变区和轻链可变区分别包含SEQ ID NOs：9和14所示的氨基酸序列或与SEQ ID NOs：9和14所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(2)重链可变区和轻链可变区分别包含SEQ ID NOs：10和15所示的氨基酸序列或与SEQ ID NOs：10和15所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(3)重链可变区和轻链可变区分别包含SEQ ID NOs：11和15所示的氨基酸序列或与SEQ ID NOs：11和15所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(4)重链可变区和轻链可变区分别包含SEQ ID NOs：10和16所示的氨基酸序列或与SEQ ID NOs：10和16所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(5)重链可变区和轻链可变区分别包含SEQ ID NOs：11和16所示的氨基酸序列或与SEQ ID NOs：11和16所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(6)重链可变区和轻链可变区分别包含SEQ ID NOs：10和17所示的氨基酸序列或与SEQ ID NOs：10和17所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(7)重链可变区和轻链可变区分别包含SEQ ID NOs：12和17所示的氨基酸序列或与SEQ ID NOs：12和17所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(8)重链可变区和轻链可变区分别包含SEQ ID NOs：13和17所示的氨基酸序列或与SEQ ID NOs：13和17所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(9)重链可变区和轻链可变区分别包含SEQ ID NOs：22和23所示的氨基酸序列或与 SEQ ID NOs：22和23所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(10)重链可变区和轻链可变区分别包含SEQ ID NOs：30和31所示的氨基酸序列或与SEQ ID NOs：30和31所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(11)重链可变区和轻链可变区分别包含SEQ ID NOs：38和39所示的氨基酸序列或与SEQ ID NOs：38和39所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(12)重链可变区和轻链可变区分别包含SEQ ID NOs：44和45所示的氨基酸序列或与SEQ ID NOs：44和45所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；或(13)重链可变区和轻链可变区分别包含SEQ ID NOs：52和53所示的氨基酸序列或与SEQ ID NOs：52和53所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，本公开提供的抗CD40抗体或其抗原结合片段包含重链和轻链，所述重链包含重链可变区和重链恒定区，所述轻链包含轻链可变区和轻链恒定区，其中重链可变区和轻链可变区分别包含上述的重链可变区和轻链可变区的氨基酸序列，重链恒定区包含人IgG1，IgG2或IgG4恒定区，优选IgG1和IgG4恒定区，轻链恒定区包含人κ恒定区或人λ恒定区。在一些具体的实施方案中，所述重链恒定区包含SEQ ID NOs：54、55或56所示氨基酸序列或与SEQ ID NOs：54、55或56所示氨基酸序列相比具有1、2、3、4或5个氨基酸取代、删除和添加的氨基酸序列，所述轻链恒定区包含SEQ ID NO：57所示氨基酸序列或者与SEQ ID NO：57所示氨基酸序列相比具有1、2、3、4或5个氨基酸取代、删除和添加的氨基酸序列。
在一些实施方案中，本公开提供的抗CD40抗体或其抗原结合片段结包含重链和轻链，其中，(1)重链包含SEQ ID NOs：9和54所示的氨基酸序列，和轻链包含SEQ ID NOs：14和57所示的氨基酸序列；(2)重链包含SEQ ID NOs：10和54所示的氨基酸序列，和轻链包含SEQ ID NOs：15和57所示的氨基酸序列；(3)重链包含SEQ ID NOs：11和54所示的氨基酸序列，和轻链包含SEQ ID NOs：15和57所示的氨基酸序列；(4)重链包含SEQ ID NOs：10和54所示的氨基酸序列，和轻链包含SEQ ID NOs：16和57所示的氨基酸序列；(5)重链包含SEQ ID NOs：11和54所示的氨基酸序列，和轻链包含SEQ ID NOs：16和57所示的氨基酸序列；(6)重链包含SEQ ID NOs：10和54所示的氨基酸序列，和轻链包含SEQ ID NOs：17和57所示的氨基酸序列；(7)重链包含SEQ ID NOs：12和54所示的氨基酸序列，和轻链包含SEQ ID NOs：17和57所示的氨基酸序列；(8)重链包含SEQ ID NOs：13和54所示的氨基酸序列，和轻链包含SEQ ID NOs：17和57所示的氨基酸序列；(9)重链包含SEQ ID NOs：22和54所示的氨基酸序列，和轻链包含SEQ ID NOs：23和57所示的氨基酸序列；(10)重链包含SEQ ID NOs：30和54所示的氨基酸序列，和轻链包含SEQ ID NOs：31和57所示的氨基酸序列；(11)重链包含SEQ ID NOs：38和54所示的氨基酸序列，和轻链包含SEQ ID NOs：39和57所示的氨基酸序列；(12)重链包含SEQ ID NOs：44和54所示的氨基酸序列，和轻链包含SEQ ID NOs：45和57所示的氨基酸序列；或(13)重链包含SEQ ID NOs：52和54所示的氨基酸序列，和轻链包含SEQ ID NOs：53和57所示的氨基酸序列。
在一些实施方案中，本公开提供的抗CD40抗体或其抗原结合片段包含两条重链(H)和两条轻链(L)，或由两条重链和两条轻链组成，重链和轻链通过二硫键相互连接，其中每条重链包含上述的重链可变区(VH)和重链恒定区，其中重链可变区(VH)包含骨架区(FR)和上述的重链互补决定区(HCDR)，每条轻链包含上述的轻链可变区(VL)和轻链恒定区，其中轻链可变区(VL)包含FR和上述的轻链互补决定区(LCDR)，其中重链可变区的C-末端连接至重链恒定区的N-末端，轻链可变区的C-末端连接至轻链恒定区的N-末端。本公开的抗体可以是，例如IgG1，IgG2或IgG4同种型的全长抗体。在另一些实施方案中，本公开的抗体可以是单链抗体(scFv)、或抗体片段，例如Fab、F(ab')2片段、Fd片段、Fv片段、dAb或分离的CDR。
一方面，本公开提供一种结合CD40的分离的抗体或其抗原结合片段，所述的抗体或其抗原结合片段 由杂交瘤产生，所述的杂交瘤选自本文称为A01、A02、A03、B01、B02和B03的杂交瘤组成的组。因此，本公开还包括杂交瘤A01、A02、A03、B01、B02和B03以及产生本文公开的抗体的任何杂交瘤产生的抗体或其抗原结合片段。
一方面，本公开提供与本公开任何示例性抗CD40抗体或其抗原结合片段结合CD40上相同表位的抗体或其抗原结合片段。在一些实施方案中，本公开提供与本公开任何示例性抗CD40抗体或其抗原结合片段竞争结合CD40的抗体或其抗原结合片段。
另一方面，本公开还提供重组多肽或融合蛋白，其包含一种或多种本公开的抗CD40抗体或其抗原结合片段与至少一种其他功能片段，所述其他功能片段包括但不限于另一种肽、蛋白质、细胞因子或受体配体。这些重组多肽或融合蛋白可以通过基因改造、化学法等进行制备。在本公开中，术语“融合蛋白”通常是指由两个或多个相同或不同的多肽序列连接得到的新的多肽序列，尤其是指重组得到的，包含天然并不连接的一个或多个相同或不同的多肽序列的多肽序列。
在另一方面，本公开还提供一种双特异性分子，其包含一种或多种本公开的抗CD40抗体或其抗原结合片段与至少一些不同于本公开的抗体或抗原结合片段特异性的其他功能部分(例如，另一种抗体或其抗原结合片段)，所述双特异性分子能够与至少两个不同结合位点或靶点结合。本文所用的“双特异性分子”涵盖具有两种(即双特异性分子)、三种(即三特异性分子)、四种(即四特异性分子)或更多种的特异性的分子。这些双特异性分子可以通过基因改造、化学法等进行制备。
在另一方面，本公开还提供一种免疫缀合物，其包含本公开的抗CD40抗体或其抗原结合部分，其中本公开的抗CD40抗体或其抗原结合部分与治疗剂(例如细胞毒素剂或成像剂等)相连接，例如抗体-药物偶联物(ADC)。本公开的抗CD40抗体或其抗原结合部分可以是嵌合抗原受体(CAR)的一部分。本公开还提供了包含所述嵌合抗原受体的免疫细胞，例如T细胞(即，CAR-T细胞)。此外，本公开还提供了基因载体，所述基因载体包含编码本公开的抗CD40抗体或其抗原结合部分的基因，并且能使所述基因进入哺乳动物细胞(优选人细胞)，并在其中表达。这些基因载体包括但不限于，裸质粒载体、酵母运载体、腺病毒载体、腺相关病毒载体、逆转录病毒载体、痘病毒载体、棒状病毒载体或杆状病毒载体。用于将DNA插入这些基因载体中的技术是本领域普通技术人员所熟知的。
在另一方面，本公开还提供一种药物组合物，其包含本公开的抗CD40抗体或其抗原结合片段，和一种或多种药学上可接受的载体。在另一些实施方案中，本公开还提供了包含本公开的重组多肽、融合蛋白、双特异性分子、免疫缀合物、嵌合抗原受体或基因载体，以及药学上可接受的载体的药物组合物。
在另一方面，本公开还提供编码本公开的抗CD40抗体或其抗原结合片段的分离的核酸。本公开还提供包括所述核酸的表达载体，以及包含所述表达载体的宿主细胞。
在另一方面，本公开还提供一种制备抗CD40抗体或其抗原结合片段的方法，其包括以下步骤：(i)在宿主细胞中表达抗CD40抗体或其抗原结合片段，和(ii)从宿主细胞或其细胞培养物中分离抗CD40抗体或其抗原结合片段。
在其他方面，本公开提供用于在受试者中治疗或预防免疫疾病的方法，所述方法包括向受试者施用治疗有效量的本公开的抗CD40抗体或其抗原结合部分、编码核酸、药物组合物、重组多肽、融合蛋白、双特异性分子、免疫缀合物、嵌合抗原受体或基因载体。在某些实施方式中，受试者是人。
在一些实施方案中，所述免疫疾病包括但不限于炎性疾病、过敏反应、自身免疫性疾病或移植相关疾病。在一些具体的实施方案中，所述免疫疾病包括但不限于：过敏反应、艾迪生氏病(Addison’s disease)、强直性脊柱炎、脊柱关节炎、哮喘、动脉粥样硬化、冠心病、自身免疫性肝炎、自身免疫性腮腺炎、I型糖尿病、附睾炎、肾炎、赖特综合征、甲状腺炎、格雷夫斯病(Graves’disease)、格林-巴利综合征(GBS)、桥本氏病、溶血性贫血、特发性血小板减少性症、系统性红斑狼疮、亚急性皮肤型红斑狼疮、多发性硬化症、重症肌无力、银屑病、硬皮病、关节炎、结节病、干燥综合征、干眼症、化脓性汗腺炎、移植相关疾病、血管炎和/或炎性肠病。
在一些实施方案中，在所述方法还包括施用第二治疗剂，所述第二治疗剂包括非甾体抗炎药(NSAID)、水杨酸盐、羟化氯喹、柳氮磺吡啶、皮质类固醇、细胞毒性药物、或免疫抑制药物和/或抗体。
基于以下具体描述和实施例，当前公开内容的其他特征和优势之处将会更加明晰，具体描述和实施例不应解读为限制性的。在本公开中引用的所有文献、Genbank记录、专利和已公开专利申请的内容通过引 用的方式明确地包含在本文中。
附图说明
图1A-1B示出纯化的小鼠抗CD40抗体阻断293T-hCD40-NFκB细胞与CD40L蛋白结合的活性；
图2示出纯化的小鼠抗CD40抗体对CD40L和IL-4所诱导的Ramos细胞凋亡的抑制活性；
图3示出嵌合抗CD40抗体Chi-A01、Chi-A02和Chi-A03与CHO-K1-hCD40细胞的结合活性；
图4示出嵌合抗CD40抗体Chi-A01、Chi-A02、Chi-A03、Chi-B01、Chi-B02和Chi-B03对Ramos细胞凋亡的激动活性，CP-870893对Ramos细胞凋亡的激动活性作为阳性对照示出；
图5示出人源化抗CD40抗体hzA01-3.1、hzA01-3.3和hzA01-3.4与293T-hCD40-NFκB细胞的结合活性；
图6示出人源化抗CD40抗体hzA01-1.1、hzA01-2.1、hzA01-3.1、hzA01-3.3和hzA01-3.4阻断293T-hCD40-NFκB细胞与CHO-K1-hCD40L细胞结合的活性；
图7示出人源化抗CD40抗体hzA01-1.1、hzA01-2.1、hzA01-3.1、hzA01-3.3和hzA01-3.4对CD40L和IL-4所诱导的Ramos细胞凋亡的抑制活性；
图8示出人源化抗CD40抗体hzA01-1.1、hzA01-2.1、hzA01-3.1、hzA01-3.3和hzA01-3.4对Ramos细胞凋亡的激动活性，CP-870893对Ramos细胞凋亡的激动活性作为阳性对照示出；
图9示出人源化抗CD40抗体hzA01-3.1、hzA01-3.3和hzA01-3.4对CD40L诱导的人外周血B淋巴细胞共刺激分子CD86表达的抑制活性；
图10示出人源化抗CD40抗体hzA01-3.1、hzA01-3.3和hzA01-3.4对CD40L和IL-4诱导的人外周血B淋巴细胞增殖的抑制活性；
图11示出人源化抗CD40抗体hzA01-3.1、hzA01-3.3和hzA01-3.4诱导人外周血B淋巴细胞增殖的激动活性，CP-870893诱导人外周血B淋巴细胞增殖的激动活性作为阳性对照示出；
图12示出在MLR反应中响应于人源化抗CD40抗体hzA01-3.3的IFNγ释放量，其中mDC和DC(未经分化培养)组未加入抗CD40抗体和IgG4同型对照抗体，作为阴性对照示出；
图13示出人源化抗CD40抗体hzA01-3.3对imDC细胞成熟的激动活性，CP-870893对imDC细胞成熟的激动活性作为阳性对照示出；
图14示出人源化抗CD40抗体hzA01-3.3介导的ADCC效应，Rituximab介导的ADCC活性作为阳性对照示出；
图15示出人源化抗CD40抗体hzA01-3.3介导的CDC效应，Rituximab介导的CDC活性作为阳性对照示出；
图16A示出人源化抗CD40抗体hzA01-3.3治疗后NDG小鼠血清中人抗小鼠IgM抗体的浓度，图16B示出人源化抗CD40抗体hzA01-3.3治疗后NDG小鼠血清中人抗小鼠IgG抗体的浓度；
图17A示出人源化抗CD40抗体hzA01-3.3治疗后小鼠脾脏内浸润的人CD4 +T细胞含量，17B示出人源化抗CD40抗体hzA01-3.3治疗后小鼠脾脏内浸润的人CD19 +B细胞含量，其中，通过t检验与IgG4同型对照组进行比较，*表示P≤0.05，**表示P<0.01，***表示P<0.001；
图18示出人源化抗CD40抗体hzA01-3.3对干燥综合征模型小鼠体重的影响；
图19示出人源化抗CD40抗体hzA01-3.3对干燥综合征模型小鼠唾液流率的影响；
图20A示出人源化抗CD40抗体hzA01-3.3治疗后干燥综合征模型小鼠的颌下腺指数，20B示出人源化抗CD40抗体hzA01-3.3治疗后干燥综合征模型小鼠的颌下腺中IL-6的含量，图20C示出人源化抗CD40抗体hzA01-3.3治疗后干燥综合征模型小鼠的颌下腺病理评分，其中，通过t检验(空白对照组与模型组进行比较，hzA01-3.3给药组与模型组进行比较)，*表示P≤0.05，**表示P<0.01，***表示P<0.001；
图21A-21D示出人源化抗CD40抗体hzA01-3.3治疗后干燥综合征模型小鼠血液中CD3 +T细胞、CD4 +T细胞、CD8 +T细胞和CD19 +B细胞的活细胞占CD45 +活细胞的百分比，图21E-21H示出人源化抗CD40抗体hzA01-3.3治疗后干燥综合征模型小鼠脾脏中CD3 +T细胞、CD4 +T细胞、CD8 +T细胞和CD19 +B细胞的活细胞占CD45 +活细胞的百分比。
发明详述
应当理解，本文所用的术语只是为了描述具体实施例，并非旨在进行限制。除非另有定义，否则本文使用的所有技术和科学术语的含义与本公开所属领域的普通技术人员通常所理解的含义相同。
术语“CD40”包括CD40变体，同源物，直系同源物和旁系同源物。例如，在一些实施例中，对人CD40蛋白特异的抗体可以在某些情况下与另一物种(例如猴)的CD40蛋白发生交叉反应。在另一些实施方案中，对人CD40蛋白特异的抗体可以完全地对人CD40蛋白特异而不与其他物种或其他类型的蛋白发生交叉反应，或者仅与某些其他物种但并非所有其他物种的CD40蛋白发生交叉反应。
CD40是已知的并且可以将其称为B细胞表面抗原CD40、Bp50、CD40L受体、CDW40、MGC9013、p50和肿瘤坏死因子受体超家族成员5(TNFRSF5)。“人CD40”或“hCD40”在本文中可以互换，是指具有人CD40氨基酸序列的蛋白，例如包含SEQ ID NO:62所示的氨基酸序列的CD40蛋白。术语“猴CD40”或“cyno CD40”等在本文中可以互换，是指具有猴CD40氨基酸序列的蛋白，例如包含SEQ ID NO:63所示的氨基酸序列的CD40蛋白。
如本文所用的术语“抗体”是指具有至少一个抗原(例如CD40)结合结构域的结合蛋白。本公开的抗体或其抗原结合片段可以是整个抗体或其任何片段，包括单克隆抗体或其片段和抗体变体或其片段。抗体或其抗原结合片段的实例包括单特异性、双特异性、三特异性或多特异性抗体、Fab片段、F(ab’)2片段、Fv片段、分离的CDR区、单链Fv(scFv)和本领域任何已知的其他抗体片段。本文公开的抗CD40抗体或其抗原结合片段可以是IgG1、IgG2、IgG3或IgG4同种型。术语“同种型”是指由重链恒定区基因编码的抗体种类。在一些实施方案中，本文公开的抗CD40抗体或其抗原结合片段是IgG1和IgG4同种型。本公开的抗CD40抗体或其抗原结合片段可以衍生自任何物种，包括但不限于小鼠、大鼠、兔、灵长类动物、美洲驼和人。本公开的抗CD40抗体或其抗原结合片段可以是鼠类抗体、嵌合抗体、人源化抗体或人抗体。除非另有说明，否则本公开的“抗体”包括全长抗体及其任何抗原结合部分(即“抗原结合片段”)或单链。通常，全长抗体是包含两条重(H)链和两条轻(L)链的糖蛋白，其中重链和轻链通过二硫键连接。每条重链由重链可变区(VH)和重链恒定区组成。重链恒定区由三个结构域组成，即CH1，CH2和CH3。每条轻链由轻链可变区(VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区还可以划分为高变区(即，互补决定区(CDR))和序列较为保守的骨架区(FR)。每个VH和VL由三个CDR和四个FR组成，从氨基端到羧基端以FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4的顺序排布。抗体的可变区包含与抗原相互作用的结合结构域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括多种免疫系统细胞(例如，效应细胞)和经典补体系统的第一成分(C1q)。同时，如本领域技术人员所了解的，特殊的“全长抗体”，例如纳米抗体，其仅只有重(H)链而没有轻(L)链。
抗体的“抗原结合片段”或“抗体结合部分”是指抗体的一个或多个片段，其保留特异性结合抗原(例如，CD40蛋白)的功能。已证实，抗体的抗原结合功能可以通过全长抗体的片段来实施。涵盖在术语抗体的“抗原结合部分/片段”中的实例包括：(i)Fab片段：由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab')2片段，包含在铰链区通过二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体单臂的VL和VH结构域组成的Fv片段；(v)由VH结构域组成的dAb片段(参见Ward et al.,Nature.341:544-546(1989))；(vi)分离的互补决定区(CDR)；以及(vii)纳米抗体，一种包含单个可变结构域和两个恒定结构域的重链可变区。此外，尽管Fv片段的两个结构域VL和VH由不同基因编码，但可以采用重组的方法通过接头将VH和VL连接成单蛋白链，其中VL和VH配对形成单价分子称单链Fv(scFv)；参见Bird et al.,Science.242:423-426(1988)；Huston et al.,Proc.Natl.Acad.Sci.85:5879-5883(1988)，这些单链抗体也涵盖在术语抗原结合部分/片段中。此外，包含该抗原结合部分/片段的重组多肽、融合蛋白和双特异性分子也涵盖在术语抗原结合部分/片段中。这些抗体片段可以由本领域技术人员已知的常规技术获得，且片段可以通过与全长抗体相同的方法进行功能筛选。
“小鼠抗体”或“鼠源抗体”是指可变区中骨架区和CDR区均来自小鼠种系免疫球蛋白序列的抗体。此外，如果抗体包含恒定区，则恒定区也来自小鼠种系免疫球蛋白序列。本公开的小鼠抗体可包括不由小鼠种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机突变或点突变或通过体内体细胞突变引入的突变)，但“小鼠抗体”不包括在小鼠骨架序列中插入源自其他哺乳动物种系CDR序列的抗体。
“嵌合抗体”是指通过将来自非人来源的遗传物质和来自人的遗传物质组合而成的抗体。或更笼统地说，嵌合抗体是指具有来自一种物种的遗传物质和来自另一物种的遗传物质的抗体。例如，轻链和重链的可变 区可以均来自一个动物物种(如小鼠、大鼠等)的抗体的可变区，而恒定部分则与来自另一物种(如人)的抗体序列同源。例如，为获得嵌合抗体，可利用非人源的B细胞或杂交瘤细胞产生可变区，而与其组合的恒定区则来自人。在本公开中，嵌合抗体也被表示为“Chi”。
“人源化抗体”是下述抗体：所述抗体含有衍生自非人抗体的互补决定区(CDR)和衍生自人抗体的骨架区以及恒定区。例如，本文提供的结合CD40的人源化抗体可以包含衍生自一种或多种鼠类抗体的CDR以及人骨架区和人恒定区。因此，在一些实施方案中，本文提供的人源化抗体与所述抗体的CDR所衍生自的鼠类抗体结合CD40上的相同表位。本文提供了示例性人源化抗体。包含本文提供的重链CDR和轻链CDR的另外的结合CD40的人源化抗体或其变体可以使用任何人骨架序列产生，并且也包括在本公开中。在一些实施方案中，适用于在本公开中使用的骨架序列包括在结构上与本文提供的骨架序列类似的那些骨架序列。可以在骨架区中进行另外修饰以改变本文提供的抗体的特性。此类另外的骨架修饰可以包括化学修饰，点突变以降低免疫原性或去除T细胞表位，或回复突变为原始种系序列中的残基。在一些实施方案中，此类修饰包括对应于本文示例的突变的那些修饰，包括对种系序列的回复突变。例如，在一些实施方案中，本文提供的人源化抗体的VH和/或VL的人骨架区中的一个或多个氨基酸被回复突变为亲本鼠类抗体中对应的氨基酸。在本公开中，人源化抗体也被表示为“hz”。
术语“Fc结构域”或“Fc区”是指包含根据Kabat编号系统定义的CH2和CH3恒定结构域的抗体序列。Fc区可以来自人IgG。例如，Fc区可以来自人IgG1或人IgG4Fc区。
本文所述的“衍生的”，在用于相对于参考抗体或其他结合蛋白的分子或多肽时，意指能够与参考抗体或其他结合蛋白特异性地结合相同表位的分子或多肽。
“分离的”是指已经从其天然环境中分离的目标化合物(例如，抗体、抗原结合片段或核酸)。
“特异性结合抗原的抗体”和“对抗原特异的抗体”在本文中与术语“与抗原特异性结合的抗体”可互换使用。“特异性结合人CD40”的抗体是指与人CD40(还可能是其他非人物种的CD40)结合但基本上不结合非CD40的抗体。优选地，抗体以“高亲和力”结合人CD40，即K D为5.0×10 -8M或更小，1.0×10 -8M或更小，优选为5.0×10 -9M或更小，更优选为1.0×10 -9M或更小。
术语“基本上不结合”蛋白质或细胞是指不与蛋白质或细胞结合，或不以高亲和力与其结合，即结合蛋白质或细胞的K D为1.0×10 -6M或更高，优选为1.0×10 -5M或更高，1.0×10 -4M或更高，更优选为1.0×10 - 3M或更高，1.0×10 -2M或更高。
术语“高亲和力”对IgG而言，是指结合抗原的K D为5.0×10 -8M或更小，1.0×10 -8M或更小，优选为5.0×10 -9M或更小，更优选为1.0×10 -9M或更小。但对于其他抗体同种型，“高亲和力”结合可能有所不同。例如，IgM同种型的“高亲和力”结合，是指结合抗原的K D为1.0×10 -6M或更小，优选1.0×10 -7M或更小，更优选1.0×10 -8M或更小。
“同一性”是指两个或多个核酸序列之间或两个或多个多肽序列之间的相似性。本公开的序列同一性至少为85％，90％或95％，优选为至少95％。非限制性实施例包括：85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Institute网站上的BLASTN/BLASTP算法的默认设置来进行。
“竞争结合”的抗体指部分或完全阻断其他抗体与靶标结合的抗体。两种抗体是否彼此竞争结合靶标，即一种抗体是否阻断另一抗体结合靶标且至何种程度，可以使用本领域已知的竞争实验来确定，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)，夹心式竞争测定等。在某些实施方案中，抗体与另一抗体竞争结合靶标并使另一抗体与靶标的结合阻断至少10％、20％、30％、40％、50％、60％、70％、80％、90％或100％。
两种或多种抗体“结合相同表位”意指抗体结合氨基酸残基的相同区段，如通过给定方法测定。用于确定抗体是否与本文所述抗体结合“CD40上相同的表位”的技术包括例如表位作图方法，诸如，抗原：抗体复合物的晶体的x射线分析和氢/氘交换质谱法(HDX-MS)。
术语“EC 50”，又称半数有效浓度，是指在特定暴露时间后，能达到50％最大效应的抗体浓度。术语“IC 50”，又称半数抑制浓度，是指相对于不存在抗体的情况，将特异性生物学或生化功能抑制50％的抗体浓度。EC 50和IC 50两者均可以通过ELISA或FACS分析或本领域已知的任何其他方法进行测量。
“K D”指平衡解离常数，其获自K d比K a(即，K d/K a)的比率并且表示为摩尔浓度(M)。抗体的K D值可以使用本领域充分建立的方法来测定。用于测定抗体的K D的优选方法是通过使用表面等离子共振技术，优选使用生物传感器系统诸如
Figure PCTCN2022120689-appb-000001
表面等离子共振系统分析。
“交联”是指在由抗CD40的抗体结合到FcγR(例如顺式或反式FcγRIIb)所诱导的细胞上CD40的高阶多聚化，从而导致诱导CD40的激动活性。
“患者”或“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳动物及非哺乳动物，优选哺乳动物，例如非人的灵长类动物、绵羊、狗、猫、牛和马。
“有效剂量”或“有效量”是指足以实现或至少部分实现期望效果的量。药物或治疗剂的治疗“有效量”或“有效剂量”是指当单独或与另一治疗剂组合使用时，足以防止或改善疾病或病症相关症状的量，优选引起疾病症状的严重程度降低、无症状期频率和持续时间增加、或防止由疾病引起的损伤或无能的量。治疗有效量与被治疗的疾病相关，其中本领域技术人员可以方便地判别出实际的有效量。
除特别声明外，本公开中的“约”是指在所给定的具体数值范围±5％范围内波动，优选在±2％范围内波动，更优选在±1％范围内波动。例如pH值为约5.5表示pH为5.5±5％，优选pH为5.5±2％，更优选pH为5.5±1％。除非另外特别说明，否则单数的使用包括复数。
除非另外特别说明，否则词语“一个”或“一种”意指“至少一个”或“至少一种”，短语“至少一个”的含义等同于“一个或多个”的含义，“和/或”的使用意指“和”或者“或”。
如本文中所述，任何百分比范围、比率范围或整数范围应当理解为包括在列举的范围内的任意整数的值，除非另外指出。
在本文中，除非上下文另有规定，词语“包含”、“包括”和“含有”将被理解为表示包括所述的步骤或要素或一组步骤或要素，但不排除任何其他步骤或要素或一组步骤或要素。“由……组成”所表示的是包括并且限于短语“由……组成”之后的内容。因此，短语“由……组成”表示所列出的要素是需要的或必需的，并且没有其他要素可存在。“基本由……组成”所表示的是包括列于此短语之后的任意要素，并且限于不妨碍或有助于所列的要素的如在本公开详述的活性或作用的其他要素。因此，短语“基本由……组成”表示所列出的要素是需要的或必需的，但其他要素是可选的并可取决于其是否影响所列出的要素的活性或作用而存在或不存在。
本公开的多个方面在以下更加具体地加以描述。
本公开的示例性抗体或其抗原结合片段的重链可变区、轻链可变区和CDR的氨基酸序列ID(SEQ ID NO.)提供于下表1中。一些抗体具有相同的CDR，一些抗体具有相同的VH或VL。抗体的重链恒定区可以是人IgG1，IgG2或IgG4恒定区，优选IgG1和IgG4恒定区，例如包含SEQ ID NOs：54、55或56所示氨基酸序列或与SEQ ID NOs：54、55或56所示氨基酸序列相比具有1、2、3、4或5个氨基酸取代、删除和添加的氨基酸序列。抗体的轻链恒定区可以是人κ恒定区或人λ恒定区，例如包含SEQ ID NO：57所示氨基酸序列或者与SEQ ID NO：57所示氨基酸序列相比具有1、2、3、4或5个氨基酸取代、删除和添加的氨基酸序列。这些抗体也可以包含小鼠IgG1或IgG4重链恒定区和/或小鼠κ恒定区或λ恒定区。
表1 可变区和CDR的氨基酸序列ID(SEQ ID NO.)
Figure PCTCN2022120689-appb-000002
Figure PCTCN2022120689-appb-000003
鉴于抗体可变区的氨基酸序列，本领域技术人员可以常规的方法确定哪些残基构成特定的CDR。如本领域技术人员公知，可以通过多种方法来定义抗体的CDR，例如用Kabat、Chothia、IMGT、AbM或Contact等编号系统/方法定义；或用Kabat、Chothia、IMGT、AbM和Contact等编号系统/方法中的二种或多种组合定义(例如，HCDR1用AbM定义、HCDR2用Kabat或AbM定义、HCDR3用IMGT或Kabat定义、和LCDR1-3用Kabat定义)；还可以用包含了Kabat和Chothia的Combined编号系统来定义，其中Combined编号系统是将Kabat和Chothia定义的范围相结合，基于此取了一个更大的范围(例如，Kabat定义的HCDR1为H31-H35，Chothia定义的HCDR1为H26-H32，则Combined定义的HCDR1为H26-H35)。在不同的编号系统中，CDR的精确编号和放置有所不同。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应了解为涵盖通过任何已知方案界定的互补决定区。
虽然本公开中请求保护的CDR是基于表1和表10示出的序列，但是根据其它CDR的定义规则所对应的氨基酸序列也应当落在本公开的保护范围中。例如，通过Kabat的定义规则来定义CDR时，小鼠、嵌合和人源化A01的HCDR1的氨基酸序列为：TSGVH(SEQ ID NO:79)，小鼠、嵌合A01和hzA01-3.4的HCDR2的氨基酸序列为：VIWAGGDTNYNSALMS(SEQ ID NO:2)，hzA01-1.1、hzA01-2.1和hzA01-3.1的HCDR2的氨基酸序列为：VIWAGGDTNYNPSLKS(SEQ ID NO:80)，hzA01-1.2和hzA01-2.2的HCDR2的氨基酸序列为：VIWAGGDTNYADSVKG(SEQ ID NO:81)，hzA01-3.3的HCDR2的氨基酸序列为：VIWAGGDTNYNSALKS(SEQ ID NO:4)，小鼠、嵌合和人源化A01的HCDR3的氨基酸序列为：HGHFDV(SEQ ID NO:82)，小鼠、嵌合和人源化A01的LCDR1的氨基酸序列为：RSSQSLVHSSGNTYLQ(SEQ ID NO:6)，小鼠、嵌合和人源化A01的LCDR2的氨基酸序列为：KVSNRFS(SEQ ID NO:7)，小鼠、嵌合和人源化A01的LCDR3的氨基酸序列为：SQTTHVPWT(SEQ ID NO:8)。
与人CD40结合的其他抗CD40抗体的VH和/或VL序列(或CDR序列)可以与本公开的抗CD40抗体或其抗原结合部分的VH和/或VL序列(或CDR序列)“混合并配对”。优选地，当VH和VL链(或其CDR)混合并配对时，特定VH/VL对中的VH序列可以由结构相似的VH序列取代。同样，优选地将特定VH/VL对中的VL序列取代为结构相似的VL序列。
因此，在一个实施方案中，本公开的抗体或其抗原结合片段包括：
(a)重链可变区，其包含列于表1和表10中的氨基酸序列；和
(b)轻链可变区，其包含列于表1和表10中的氨基酸序列，或另一种抗CD40抗体的VL，其中该抗体或其抗原结合部分特异性结合人CD40。
在另一个实施方案中，本公开的抗体或其抗原结合片段包括：
(a)列于表1和表10的HCDR1，HCDR2和HCDR3；和
(b)列于表1和表10的LCDR1，LCDR2和LCDR3，或另一种抗CD40抗体的轻链可变区CDR，其中该抗体或其抗原结合部分特异性结合人CD40。
在另一个实施方案中，本公开的抗体或其抗原结合片段包括列于表1和表10的HCDR2以及其他抗CD40抗体的CDR，例如其他抗CD40抗体的HCDR1和/或HCDR3，和/或其他抗CD40抗体LCDR1，LCDR2和/或LCDR3。
此外，本领域内公知，CDR3结构域独立于CDR1和/或CDR2结构域，可以单独确定抗体对同种抗原的结合特异性，并且可以预测到基于该CDR3序列可产生具有相同结合特异性的多种抗体。
在另一个实施方案中，本公开的抗体或其抗原结合片段包含列于表1和表10的HCDR2，以及列于表1和表10的HCDR3和/或列于表1的LCDR3，或另一种抗CD40抗体的HCDR3和/或LCDR3，其中该抗体或其抗原结合部分特异性结合人CD40。这些抗体优选与本公开的抗CD40抗体(a)竞争结合CD40；(b)保留功能特征；(c)结合相同的表位；和/或(d)具有具有相似的结合亲和力。在另一个实施方案中，本公开的抗体或其抗原结合部分还包含列于表1和表10的LCDR2，或另一种抗CD40抗体的LCDR2，其中该抗体或其抗原结合部分特异性结合人CD40。在另一个实施方案中，本公开的抗体或其抗原结合部分还包括列于表1和表10的HCDR1和/或列于表1和表10的LCDR1，或另一种抗CD40抗体的HCDR1和/或LCDR1，其中该抗体或其抗原结合部分特异性结合人CD40。
在另一个实施方案中，本公开的抗体或其抗原结合部分的重链可变区和/或轻链可变区或CDR1、CDR2和CDR3序列可以包含一个或多个保守修饰。本领域应理解，一些保守序列修饰不会使抗原结合特异性消失。
因此，在一个实施方式中，本公开的抗体或其抗原结合部分包含重链可变区和/或轻链可变区，重链可变区和轻链可变区分别包含CDR1、CDR2和CDR3，其中：
(a)HCDR1序列包含表1和表10列出的序列，和/或其保守修饰；和/或
(b)HCDR2序列包含表1和表10列出的序列，和/或其保守修饰；和/或
(c)HCDR3序列包含表1和表10列出的序列，和/或及其保守修饰；和/或
(d)LCDR1和/或LCDR2和/或LCDR3序列包含表1和表10列出的序列；和/或其保守修改；且
(e)抗体或其抗原结合部分特异性结合人CD40。
本文所用的术语“保守序列修饰”是指不显著影响或改变抗体结合特性的氨基酸修饰。这样的保守修饰包括氨基酸取代、添加和删除。可以通过本领域已知的标准技术将修饰引入本公开的抗体中，例如点突变和PCR介导的突变。保守氨基酸取代是指氨基酸残基被具有类似结构或化学特性(例如，相似侧链)的氨基酸残基取代。具有相似侧链的氨基酸残基家族在本领域中已知。这些氨基酸残基家族包括具有碱性侧链(例如，赖氨酸，精氨酸，组氨酸)，酸性侧链(例如，天冬氨酸，谷氨酸)，不带电极性侧链(例如，甘氨酸，天冬酰胺，谷氨酰胺，丝氨酸，苏氨酸，酪氨酸，半胱氨酸，色氨酸)，非极性侧链(例如，丙氨酸，缬氨酸，亮氨酸，异亮氨酸，脯氨酸，苯丙氨酸，甲硫氨酸)，β支链侧链(例如，苏氨酸，缬氨酸，异亮氨酸)和芳香族侧链(例如，酪氨酸，苯丙氨酸，色氨酸，组氨酸)的氨基酸。因此，本公开的抗CD40抗体或其抗原结合部分的CDR区中一个或多个氨基酸残基可以被同一侧链家族的其他氨基酸残基取代，且得到的抗体可以使用本文所述的功能检测对其进行功能测试。
本公开的抗体或其抗原结合片段包含的可变区(包括CDR区和/或骨架骨架区)修饰，或者，本公开的抗体或其抗原结合部分还可以包含Fc修饰，例如改变抗体的效应功能。因此，本公开的一个实施方案提供分离的抗CD40单克隆抗体或其抗原结合片段，其包含重链可变区和/或轻链可变区，重链可变区包含本公开上述序列的HCDR1、HCDR2和HCDR3，轻链可变区包含本公开上述序列的LCDR1、LCDR2和LCDR3，但包含不同于本公开上述序列的骨架序列骨架。这样的骨架序列可以从包括种系抗体基因序列的公开DNA数据库或公开参考文献中获得。这样的骨架序列优选地是与本公开的抗CD40抗体所用的骨架序列在结构上相似的那些。可以将CDR1、CDR2和CDR3序列移植到与该骨架序列的种系免疫球蛋白基因具有相同序列的骨架区中，或者可以将CDR序列移植到与种系序列相比具有一个或多个突变的骨架区中。例如，在一些情况下，对骨架区中的残基进行突变是有益的，可以维持或增强抗体的抗原结合能力(参见，例如，U.S.Pat.Nos.5,530,101；5,585,089；5,693,762和6,180,370)。
另一种类型的可变区修饰是将CDR1、CDR2和/或CDR3区内的氨基酸残基进行突变，从而改善目标抗体的一种或多种特性(例如，亲和力，理化性质)。可以通过点突变或PCR介导的突变引入突变，并可通过本领域已知的体外或体内测定评估突变对抗体结合或其他功能特性的影响。可以是对氨基酸进行取代、添加或删除，优选为取代。此外，通常改变每个CDR区内不多于一个、两个、三个、四个或五个的残基。
在一个实施方案中，本公开提供的抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区和轻链可变区包含：(a)HCDR1区，其包含本公开的序列，或具有1、2、3、4或5个氨基酸取代，删除或添加的氨基酸序列；(b)HCDR2区，其包含本公开的序列，或具有1、2、3、4或5个氨基酸取代，删除或添加的氨基酸序列；(c)HCDR3区，其包含本公开的序列，或具有1、2、3、4或5个氨基酸取代，删除或添加的氨基酸序列；(d)LCDR1区，其包含本公开的序列，或具有1、2、3、4或5个氨基酸取代，删除或添加的氨基酸序列；(e)LCDR2区，其包含本公开的序列，或具有1、2、3、4或5个氨基酸取代，删除或添加的氨基酸序列；和(f)LCDR3区，其包含本公开的序列，或具有1、2、3、4或5个氨基酸取代，删除或添加的氨基酸序列。
本公开的抗体或其抗原结合片段包括VH和/或VL的骨架区修饰，以改善抗体特性。通常，此类骨架区修饰可以降低抗体的免疫原性。例如，将一个或多个骨架残基“回复突变”为相应的种系序列。这些残基可以通过将抗体骨架序列与得到抗体的种系序列相比较而识别出来。
另一种类型的骨架修饰包括对骨架区、或者甚至一个或多个CDR区的一个或多个残基进行突变，以 去除T细胞表位，从而减少抗体可能导致的免疫原性。该方法也称为“去免疫原性”，在美国专利公开号20030153043中有更详细描述。
此外，本公开的抗体或其抗原结合片段包括Fc修饰，Fc修饰可以是氨基酸插入、缺失或取代，通常用来改变抗体一种或多种功能特性，例如血清半衰期、补体结合、Fc受体结合和/或抗原依赖性细胞毒性。
此外，本公开的抗体或其抗原结合片段还可以进行化学修饰(例如，连接一个或多个化学功能基团)，或修饰以改变其糖基化，来改变抗体的一种或多种功能特性。在其他实施方案中，通过聚乙二醇化(例如，通过使抗体或其片段与聚乙二醇(PEG)反应来修饰Fc区。
在另一个实施方案中，改变本公开的抗体或其抗原结合片段的糖基化。这样的糖化修饰可以通过例如改变抗体序列中的一个或多个糖基化位点来实现。例如，可以进行一个或多个氨基酸替换，以消除一个或多个可变区的骨架区的糖基化位点，从而消除该位点的糖基化。这样的去糖基化可以增加抗体对抗原的亲和性。参见，例如美国专利5,714,350和6,350,861。
本公开提供的抗体或其抗原结合片段结合CD40，从而抑制CD40活性。“CD40活性”包括但不限于B细胞的活化，例如B细胞的增殖、抗体的产生、抗体同种型转换或分化为浆细胞；T细胞的活化，例如T细胞的增殖或细胞因子分泌；树突状细胞的活化，例如树突状细胞的增殖、分化成熟；和巨噬细胞的活化。还可以通过与其他分子相互作用抑制CD40活性。此外，“CD40活性”还包括抑制肿瘤细胞生长和/或增殖、诱导肿瘤细胞凋亡等。
在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段结合CD40并使得B细胞的活化受到抑制。在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段结合CD40并使得B细胞的增殖受到抑制。
在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段结合CD40并使得T细胞活化受到抑制。在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段结合CD40并使得T细胞的增殖和/或细胞因子的产生受到抑制。在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段结合CD40并使得选自由以下组成的组的一种或多种细胞因子的产生受到抑制：IL-2、IFNγ、TNF、IL-l、IL-4、IL-5、IL-6、IL-12、IL-13、IL-17和GM-CSF。在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段结合CD40并使得细胞因子IFNγ的产生受到抑制。在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段结合CD40并使得细胞因子IL-6的产生受到抑制。
因此，一方面，本公开提供了调节免疫应答的方法，其包括使T细胞和抗原呈递细胞与本公开的抗CD40抗体或其抗原结合片段接触。在一个实施方案中，可以在混合淋巴细胞反应(MLR)中测量本公开提供的抗CD40抗体或其抗原结合片段对免疫应答的调节。在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段抑制MLR中淋巴细胞的细胞因子的产生。在另一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段抑制MLR中细胞因子IFNγ的产生。
在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段结合CD40并使得树突状细胞的活化受到抑制。在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段结合CD40并使得树突状细胞的分化成熟受到抑制。
在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段结合CD40并使得肿瘤细胞凋亡受到抑制。在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段结合CD40并使得Ramos细胞凋亡受到抑制。
本公开提供的抗CD40抗体或其抗原结合片段结合CD40时无明显的激动活性。在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段对肿瘤细胞的凋亡无明显的激动活性。在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段对Ramos细胞的凋亡无明显的激动活性。在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段对B细胞的活化无明显的激动活性。在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段对B细胞的增殖无明显的激动活性。在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段对树突状细胞的活化无明显的激动活性。在一个实施方案中，本公开提供的抗CD40抗体或其抗原结合片段对树突状细胞的分化成熟无明显的激动活性。“无明显激动活性”的物质显示在试验中检测到的激动活性不高于天然物质或阴性对照诱导的激动活性约25％，优选不高约20％、15％、10％、5％、1％、0.5％或甚至不高约0.1％，或者在试验中检测到的激动活性比阳性对照 诱导的激动活性低至少30％、40％、50％、60％、70％、80％、85％、90％、95％或100％。在一个实施方案中，不结合CD40的非特异性免疫球蛋白，例如IgG4同型对照抗体作为阴性对照。在一个实施方案中，激动性抗CD40抗体，例如CP-870893作为阳性对照。
本公开提供的抗CD40抗体或其抗原结合片段具有良好的安全性。本公开提供的抗CD40抗体或其抗原结合片段对干燥综合征具有良好的治疗效果。
另一方面，本公开提供了药物组合物，其包含一种或多种本公开的抗CD40抗体或其抗原结合片段和药学上可接受的载体。如本文中所用，“药学上可接受的载体”包括生理上相容的任一和所有溶剂、分散介质、涂料、抗菌剂、等渗剂及其组合。合适的“药学上可接受的载体”的选择和使用在Gennaro，ed.,Remington:The Science and Practice of Pharmacy，20th Ed.(Lippincott Williams& Wilkins 2003)中有教导。
可以通过任何合适的方法或途径施用抗体或药物组合物。施用途径包括例如静脉内、肌内、皮下、肠道外、脊柱或表皮施用(例如通过注射或输注)。基于给药途径，可以将活性成分包被在材料中，以保护其不受酸和可能使其失活的其他自然条件的影响。本文所用的短语“肠道外施用”是指除肠内和局部施用以外的施用方式，通常采用注射，并且包括但不限于静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注。或者，药物组合物可以通过非肠道外途径施用，例如局部、表皮或粘膜途径施用，如鼻内、口服、阴道、直肠、舌下或局部施用。
对于抗体的施用，剂量可以为约0.0001至100mg/kg宿主体重的范围内。
本公开的抗CD40抗体或其抗原结合部分具有多种体外和体内应用，涉及免疫疾病的诊断、治疗和/或预防。“治疗”是指减轻和/或稳定症状、病症、病况，延缓和/或阻止疾病的进展，和/或减轻疾病的严重程度的方法。本公开的抗CD40抗体或其抗原结合部分、编码核酸、药物组合物、重组多肽、融合蛋白、双特异性分子、免疫缀合物、嵌合抗原受体或基因载体可以施用至人受试者，以治疗所述受试者的免疫疾病。
另一方面，本公开提供了一种在受试者中治疗或预防免疫疾病的方法，其包括向所述受试者施用治疗有效量的本公开的抗CD40抗体或其抗原结合部分、编码核酸、药物组合物、重组多肽、融合蛋白、双特异性分子、免疫缀合物、嵌合抗原受体或基因载体。在一些实施方案中，所述受试者是人。
“免疫疾病”指与个体中的免疫反应进展相关的任何疾病，包括细胞和/或体液免疫反应。在一些具体的实施方案中，在所述应用和方法中，所述免疫疾病包括但不限于炎性疾病、过敏反应、自身免疫性疾病或移植相关疾病。炎性疾病是指由于过度或失控的炎性响应所导致的过度的炎性症状、宿主组织损害或功能丧失的任何疾病、紊乱或症状，包括皮肤、肾、胃肠道和呼吸道的过敏性炎症、银屑病、肾炎、附睾炎、炎性肠病和哮喘等。自身免疫性疾病是指由免疫系统过度活化引起的机体攻击和损害自身组织的任何疾病、紊乱或症状，例如，发性硬化症、关节炎、重症肌无力、银屑病、硬皮病、自身免疫性肝炎、自身免疫性腮腺炎和I型糖尿病等。
在一些具体的实施方案中，在所述应用和方法中，所述免疫疾病包括但不限于：过敏反应、艾迪生氏病(Addison’s disease)、强直性脊柱炎、脊柱关节炎、哮喘、动脉粥样硬化、冠心病、自身免疫性肝炎、自身免疫性腮腺炎、I型糖尿病、附睾炎、肾炎、赖特综合征、甲状腺炎、格雷夫斯病(Graves’disease)、格林-巴利综合征(GBS)、桥本氏病、溶血性贫血、特发性血小板减少性症、系统性红斑狼疮、亚急性皮肤型红斑狼疮、多发性硬化症、重症肌无力、银屑病、硬皮病、关节炎、结节病、干燥综合征、干眼症、化脓性汗腺炎、移植相关疾病、血管炎和/或炎性肠病。
在一些实施方案中，移植相关疾病的实例包括但不限于：移植免疫排斥和移植抗宿主病(GVHD)。
在一些实施方案中，炎性肠病的实例包括但不限于：克罗恩病和溃疡性结肠炎。
在一些实施方案中，关节炎的实例包括但不限于：类风湿性关节炎、幼年型关节炎和银屑病关节炎。
在一些实施方案中，肾炎的实例包括但不限于：狼疮肾炎。
在一些实施方案中，银屑病的实例包括但不限于：寻常型银屑病、脓疱型银屑病(如掌跖银屑病、泛发性脓疱性银屑病)、红皮病型银屑病和关节病型银屑病。
在所述应用和方法中，本公开的抗CD40抗体或其抗原结合部分可以单独施用，或者与第二治疗剂联合施用。在一些实施方案中，所述第二治疗剂包括非甾体抗炎药(NSAID)、水杨酸盐、羟化氯喹、柳氮磺吡啶、皮质类固醇、细胞毒性药物、或免疫抑制药物和/或抗体。非甾体抗炎药包括但不限于布洛芬、萘普 生、双氯芬酸、吲哚美星、酮托洛酸、美洛昔康、萘西康、四洛芬酸和苏林达。免疫抑制药物和/或抗体包括但不限于环孢霉素、他克莫司(Tacrolimus)、雷帕霉素、霉酚酸酯、CTLA4-Ig融合体、抗B淋巴细胞刺激抗体和抗T细胞抗体(例如抗CD-3)。胞毒性药物包括但不限于甲氨蝶呤和环磷酰胺。本文讨论的治疗剂的联合可以作为在药学可接受载体中的单一组合物同时施用，或者作为分开的组合物同时施用，其中各药剂处于药学可接受载体中。在另一个实施方案中，治疗剂的联合可以按序施用。
此外，如果进行多次联合疗法施用，且药剂按序施用，则在各时间点的按序施用的次序可以反转或保持相同，按序施用可以与同时施用或其任何组合相结合。
尽管为了清楚理解的目的，已经通过举例说明和实施例相当详细地描述了前述发明，但是根据本公开的教义，本领域的普通技术人员将显而易见的是，可另外对本公开进行某些改变和修改而不背离所附权利要求的精神和范围。本公开通过以下实施例进一步说明，并非旨在进行限制。本领域的技术人员将容易地识别多种非关键性参数，所述参数可发生改变或修改以产生基本上类似的结果。
除非另外指出，否则本公开的实践将采用本领域技术范围内的蛋白质化学、生物化学、重组DNA技术和药理学的常规方法。
本公开还提供了以下一些具体的实施方案，但本公开的保护范围不限于此：
实施方案1.一种分离的抗CD40抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
(i)重链CDR1、重链CDR2和重链CDR3，其中，
(1)重链CDR1包含SEQ ID NO：9中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：9中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：9中重链CDR3的氨基酸序列；
(2)重链CDR1包含SEQ ID NO：10中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：10中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：10中重链CDR3的氨基酸序列；
(3)重链CDR1包含SEQ ID NO：11中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：11中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：11中重链CDR3的氨基酸序列；
(4)重链CDR1包含SEQ ID NO：12中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：12中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：12中重链CDR3的氨基酸序列；
(5)重链CDR1包含SEQ ID NO：13中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：13中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：13中重链CDR3的氨基酸序列；
(6)重链CDR1包含SEQ ID NO：22中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：22中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：22中重链CDR3的氨基酸序列；
(7)重链CDR1包含SEQ ID NO：30中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：30中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：30中重链CDR3的氨基酸序列；
(8)重链CDR1包含SEQ ID NO：38中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：38中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：38中重链CDR3的氨基酸序列；
(9)重链CDR1包含SEQ ID NO：44中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：44中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：44中重链CDR3的氨基酸序列；或
(10)重链CDR1包含SEQ ID NO：52中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：52中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：52中重链CDR3的氨基酸序列；和/或
(ii)轻链CDR1、轻链CDR2和轻链CDR3，其中，
(1)轻链CDR1包含SEQ ID NO：14中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：14中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：14中轻链CDR3的氨基酸序列；
(2)轻链CDR1包含SEQ ID NO：15中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：15中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：15中轻链CDR3的氨基酸序列；
(3)轻链CDR1包含SEQ ID NO：16中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：16中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：16中轻链CDR3的氨基酸序列；
(4)轻链CDR1包含SEQ ID NO：17中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：17中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：17中轻链CDR3的氨基酸序列；
(5)轻链CDR1包含SEQ ID NO：23中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：23中轻 链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：23中轻链CDR3的氨基酸序列；
(6)轻链CDR1包含SEQ ID NO：31中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：31中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：31中轻链CDR3的氨基酸序列；
(7)轻链CDR1包含SEQ ID NO：39中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：39中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：39中轻链CDR3的氨基酸序列；
(8)轻链CDR1包含SEQ ID NO：45中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：45中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：45中轻链CDR3的氨基酸序列；或
(9)轻链CDR1包含SEQ ID NO：53中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：53中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：53中轻链CDR3的氨基酸序列。
实施方案2.根据实施方案1所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
(i)重链CDR1、重链CDR2和重链CDR3，其中，
(1)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：1、2和5所示的氨基酸序列或与SEQ ID NOs：1、2和5所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(2)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：1、3和5所示的氨基酸序列或与SEQ ID NOs：1、3和5所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(3)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：1、4和5所示的氨基酸序列或与SEQ ID NOs：1、4和5所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(4)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：18、19和20所示的氨基酸序列或与SEQ ID NOs：18、19和20所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(5)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：24、25和26所示的氨基酸序列或与SEQ ID NOs：24、25和26所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(6)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：32、33和34所示的氨基酸序列或与SEQ ID NOs：32、33和34所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(7)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：40、41和42所示的氨基酸序列或与SEQ ID NOs：40、41和42所示的氨基酸序列具有至少80％同一性的氨基酸序列；或
(8)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：46、47和48所示的氨基酸序列或与SEQ ID NOs：46、47和48所示的氨基酸序列具有至少80％同一性的氨基酸序列；和/或
(ii)轻链CDR1、轻链CDR2和轻链CDR3，其中，
(1)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：6、7和8所示的氨基酸序列或与SEQ ID NOs：6、7和8所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(2)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：21、7和8所示的氨基酸序列或与SEQ ID NOs：21、7和8所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(3)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：27、28和29所示的氨基酸序列或与SEQ ID NOs：27、28和29所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(4)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：35、36和37所示的氨基酸序列或与SEQ ID NOs：35、36和37所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(5)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：43、36和37所示的氨基酸序列或与SEQ ID NOs：43、36和37所示的氨基酸序列具有至少80％同一性的氨基酸序列；或
(6)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：49、50和51所示的氨基酸序列或与SEQ ID NOs：49、50和51所示的氨基酸序列具有至少80％同一性的氨基酸序列。
实施方案3.根据实施方案1所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，其中，
(1)重链CDR1包含SEQ ID NO：9中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：9中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：9中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：14中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：14中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：14中轻链CDR3的氨基酸序列；
(2)重链CDR1包含SEQ ID NO：10中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：10中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：10中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：15中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：15中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：15中轻链CDR3的氨基酸序列；
(3)重链CDR1包含SEQ ID NO：11中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：11中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：11中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：15中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：15中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：15中轻链CDR3的氨基酸序列；
(4)重链CDR1包含SEQ ID NO：10中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：10中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：10中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：16中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：16中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：16中轻链CDR3的氨基酸序列；
(5)重链CDR1包含SEQ ID NO：11中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：11中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：11中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：16中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：16中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：16中轻链CDR3的氨基酸序列；
(6)重链CDR1包含SEQ ID NO：10中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：10中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：10中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：17中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：17中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：17中轻链CDR3的氨基酸序列；
(7)重链CDR1包含SEQ ID NO：12中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：12中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：12中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：17中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：17中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：17中轻链CDR3的氨基酸序列；
(8)重链CDR1包含SEQ ID NO：13中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：13中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：13中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：17中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：17中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：17中轻链CDR3的氨基酸序列；
(9)重链CDR1包含SEQ ID NO：22中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：22中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：22中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：23中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：23中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：23中轻链CDR3的氨基酸序列；
(10)重链CDR1包含SEQ ID NO：30中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：30中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：30中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：31中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：31中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：31中轻链CDR3的氨基酸序列；
(11)重链CDR1包含SEQ ID NO：38中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：38中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：38中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：39中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：39中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：39中轻链CDR3的氨基酸序列；
(12)重链CDR1包含SEQ ID NO：44中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：44中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：44中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：45中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：45中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：45中轻链CDR3的氨基酸序列；或
(13)重链CDR1包含SEQ ID NO：52中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：52中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：52中重链CDR3的氨基酸序列，轻链CDR1包含 SEQ ID NO：53中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：53中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：53中轻链CDR3的氨基酸序列。
实施方案4.根据实施方案1-3中任一项所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，其中，
(1)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：1、2、5、6、7和8所示的氨基酸序列或与SEQ ID NOs：1、2、5、6、7和8所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(2)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：1、3、5、6、7和8所示的氨基酸序列或与SEQ ID NOs：1、3、5、6、7和8所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(3)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：1、4、5、6、7和8所示的氨基酸序列或与SEQ ID NOs：1、4、5、6、7和8所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(4)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：18、19、20、21、7和8所示的氨基酸序列或与SEQ ID NOs：18、19、20、21、7和8所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(5)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：24、25、26、27、28和29所示的氨基酸序列或与SEQ ID NOs：24、25、26、27、28和29所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(6)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：32、33、34、35、36和37所示的氨基酸序列或与SEQ ID NOs：32、33、34、35、36和37所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(7)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：40、41、42、43、36和37所示的氨基酸序列或与SEQ ID NOs：40、41、42、43、36和37所示的氨基酸序列具有至少80％同一性的氨基酸序列；或
(8)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：46、47、48、49、50和51所示的氨基酸序列或与SEQ ID NOs：46、47、48、49、50和51所示的氨基酸序列具有至少80％同一性的氨基酸序列。
实施方案5.根据实施方案1-4中任一项所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
(i)重链可变区，所述重链可变区包含9、10、11、12、13、22、30、38、44或52所示的氨基酸序列，或与SEQ ID NOs：9、10、11、12、13、22、30、38、44或52所示的氨基酸序列具有至少80％同一性的氨基酸序列；和/或
(ii)轻链可变区，所述轻链可变区包含SEQ ID NOs：14、15、16、17、23、31、39、45或53所示的氨基酸序列，或与SEQ ID NOs：14、15、16、17、23、31、39、45或53所示的氨基酸序列所示的氨基酸序列具有至少80％同一性的氨基酸序列。
实施方案6.根据实施方案1-5中任一项所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，
(1)重链可变区和轻链可变区分别包含SEQ ID NOs：9和14所示的氨基酸序列或与SEQ ID NOs：9和14所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(2)重链可变区和轻链可变区分别包含SEQ ID NOs：10和15所示的氨基酸序列或与SEQ ID NOs：10和15所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(3)重链可变区和轻链可变区分别包含SEQ ID NOs：11和15所示的氨基酸序列或与SEQ ID NOs：11和15所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(4)重链可变区和轻链可变区分别包含SEQ ID NOs：10和16所示的氨基酸序列或与SEQ ID NOs：10和 16所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(5)重链可变区和轻链可变区分别包含SEQ ID NOs：11和16所示的氨基酸序列或与SEQ ID NOs：11和16所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(6)重链可变区和轻链可变区分别包含SEQ ID NOs：10和17所示的氨基酸序列或与SEQ ID NOs：10和17所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(7)重链可变区和轻链可变区分别包含SEQ ID NOs：12和17所示的氨基酸序列或与SEQ ID NOs：12和17所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(8)重链可变区和轻链可变区分别包含SEQ ID NOs：13和17所示的氨基酸序列或与SEQ ID NOs：13和17所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(9)重链可变区和轻链可变区分别包含SEQ ID NOs：22和23所示的氨基酸序列或与SEQ ID NOs：22和23所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(10)重链可变区和轻链可变区分别包含SEQ ID NOs：30和31所示的氨基酸序列或与SEQ ID NOs：30和31所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(11)重链可变区和轻链可变区分别包含SEQ ID NOs：38和39所示的氨基酸序列或与SEQ ID NOs：38和39所示的氨基酸序列具有至少80％同一性的氨基酸序列；
(12)重链可变区和轻链可变区分别包含SEQ ID NOs：44和45所示的氨基酸序列或与SEQ ID NOs：44和45所示的氨基酸序列具有至少80％同一性的氨基酸序列；或
(13)重链可变区和轻链可变区分别包含SEQ ID NOs：52和53所示的氨基酸序列或与SEQ ID NOs：52和53所示的氨基酸序列具有至少80％同一性的氨基酸序列。
实施方案7.根据实施方案1-6中任一项所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段还包含重链恒定区和轻链恒定区，所述重链恒定区包含SEQ ID NOs：54、55或56所示的氨基酸序列或与SEQ ID NOs：54、55或56所示氨基酸序列相比具有1、2、3、4或5个氨基酸取代、删除和添加的氨基酸序列，所述轻链恒定区包含SEQ ID NO：57所示氨基酸序列或者与SEQ ID NO：57所示氨基酸序列相比具有1、2、3、4或5个氨基酸取代、删除和添加的氨基酸序列。
实施方案8.根据实施方案1-7中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段：(a)结合人CD40；(b)结合猴CD40；(c)阻断CD40与CD40L相互作用；和/或(d)抑制CD40活性。
实施方案9.根据实施方案1-8中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段是嵌合或人源化的。
实施方案10.根据实施方案1-9中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段是IgG1、IgG2或IgG4同种型。
实施方案11.根据实施方案1-10中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段选自单克隆抗体、单特异性抗体、双特异性抗体、三特异性抗体、多特异性抗体、Fab片段、F(ab')2片段、Fd片段、Fv片段、dAb片段、分离的CDR区、单链Fv分子或其组合。
实施方案12.一种分离的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段与实施方案1-11中任一项所述的抗体或其抗原结合片段结合相同表位。
实施方案13.一种分离的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段与实施方案1-11中任一项所述的抗体或其抗原结合片段竞争结合CD40。
实施方案14.一种分离的核酸分子，其编码实施方案1-11中任一项所述的抗体或其抗原结合片段。
实施方案15.一种表达载体，其包含实施方案14所述的核酸分子。
实施方案16.一种宿主细胞，其包含实施方案14所述的核酸分子或实施方案15所述的表达载体。
实施方案17.一种重组多肽或融合蛋白，其包含实施方案1-11中任一项所述的抗体或其抗原结合片段。
实施方案18.一种双特异性分子，其包含实施方案1-11中任一项所述的抗体或其抗原结合片段。
实施方案19.一种免疫缀合物，其包含实施方案1-11中任一项所述的抗体或其抗原结合片段以及与其连接的治疗剂，例如细胞毒素剂或成像剂。
实施方案20.一种药物组合物，其包含实施方案1-11中任一项所述的抗体或其抗原结合片段和一种或多种药学上可接受的载体。
实施方案21.一种在有需要的受试者中治疗或预防免疫疾病的方法，所述方法包括向所述受试者施用治疗有效量的实施方案1-11中任一项所述的抗体或其抗原结合片段，或者实施方案20所述的药物组合物。
实施方案22.根据实施方案21所述的方法，其中所述免疫疾病包括但不限于炎性疾病、过敏反应、自身免疫性疾病或移植相关疾病。
实施方案23.根据实施方案21所述的方法，其中所述免疫疾病包括但不限于过敏反应、艾迪生氏病、强直性脊柱炎、脊柱关节炎、哮喘、动脉粥样硬化、冠心病、自身免疫性肝炎、自身免疫性腮腺炎、I型糖尿病、附睾炎、肾炎、赖特综合征、甲状腺炎、格雷夫斯病、格林-巴利综合征、桥本氏病、溶血性贫血、特发性血小板减少性症、系统性红斑狼疮、亚急性皮肤型红斑狼疮、多发性硬化症、重症肌无力、银屑病、硬皮病、关节炎、结节病、干燥综合征、干眼症、化脓性汗腺炎、移植相关疾病、血管炎和/或炎性肠病。
实施方案24.根据实施方案21-23中任一项所述的方法，其中所述方法还包括施用治疗有效量的第二治疗剂。
实施方案25.根据实施方案24所述的方法，其中所述第二治疗剂包括非甾体抗炎药、水杨酸盐、羟化氯喹、柳氮磺吡啶、皮质类固醇、细胞毒性药物、或免疫抑制药物和/或抗体。
实施例
虽然，上文中已经用一般性说明及具体实施方案对本公开做了详尽的描述，但在本公开基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本公开精神的基础上所做的这些修改或改进，均属于本公开要求保护的范围。
实施例1小鼠抗CD40单克隆抗体的制备
重组蛋白的制备
将编码含小鼠抗体重链Fc的人CD40重组蛋白(hCD40-mFc，SEQ ID NO:64)和猴CD40重组蛋白(cynoCD40-mFc，SEQ ID NO:65)的cDNA通过基因合成获得，分别亚克隆到表达载体pcDNA3.1(+)中，转染到CHO细胞进行瞬时表达。收集细胞培养上清，使用MabSelect Sure LX纯化柱(GE)纯化重组蛋白hCD40-mFc和cynoCD40-mFc。
稳定细胞系的构建
分别将编码全长hCD40(SEQ ID NO:62)和cynoCD40(SEQ ID NO:63)的cDNA通过基因合成获得，然后亚克隆到表达载体pcDNA3.1(+)中，得到pcDNA3.1-hCD40和pcDNA3.1-cynoCD40重组质粒。然后按照Lipofectamin3000转染试剂(Thermo,Cat:L3000015)的操作说明，将两个重组质粒分别转染到CHO-K1细胞中，获得稳定细胞系CHO-K1-hCD40和CHO-K1-cynoCD40。
分别将编码全长hCD40L(SEQ ID NO:66)和cynoCD40L(SEQ ID NO:67)的cDNA通过基因合成获得，然后亚克隆到表达载体pcDNA3.1(+)中，得到pcDNA3.1-hCD40L和pcDNA3.1-cynoCD40L重组质粒。然后按照Lipofectamin3000转染试剂(Thermo,Cat:L3000015)的操作说明，将两个重组质粒分别转染到CHO-K1细胞中，分别获得稳定细胞系CHO-K1-hCD40L和CHO-K1-cynoCD40L。
按照lipofectamin3000转染试剂(Thermo,Cat:L3000015)的操作说明，将质粒pGL4.32[luc2P/NF-κB-RE/Hygro]Vector(Promega,Cat:E8491)转染到HEK293T细胞，得到稳定细胞系293T-NFκB。然后再将pcDNA3.1/Zeo(+)载体的BamHI和XhoI位点插入全长hCD40(SEQ ID NO:62)的编码序列，得到重组质粒pcDNA3.1/Zeo(+)-hCD40。按照lipofectamin3000转染试剂(Thermo,Cat:L3000015)的操作说明，将重组质粒pcDNA3.1/Zeo(+)-hCD40转染到293T-NFκB细胞中，获得293T-hCD40-NFκB稳定细胞系。
小鼠免疫
纯化的重组蛋白hCD40-mFc(SEQ ID NO:64)作为抗原，将hCD40-mFc与完全弗氏佐剂(Sigma,Cat:F5881-10X10mL)按照体积比1:1充分混合并乳化，通过皮下注射免疫小鼠。初次免疫2-4周后，将hCD40-mFc或cynoCD40-mFc与Alum佐剂(Thermo,Cat:77161)按照体积比1:1的比例充分混合并乳化，以皮下注射和肌肉注射交替的方式每隔2周进行一次加强免疫，共免疫6周。免疫结束后，收集每只小鼠的血清进行抗CD40特异性抗体血清滴度的检测。选取血清滴度较高的小鼠进行后续的脾细胞融合。
杂交瘤的制备和筛选
融合前3-4天，通过腹腔注射hCD40-mFc，对小鼠进行融合前冲击免疫。融合当天，无菌取小鼠脾脏，进行研磨和红细胞裂解，然后以电融合缓冲液(BTX,Cat:47-0001)重悬，获得单细胞悬液，按细胞数目2:1的比例与对数生长期的骨髓瘤细胞SP2/0混合，采用电融合仪(BTX)完成细胞融合。将融合后的细胞与含有1×HAT的杂交瘤培养基(Gibco,Cat:12045-076)混匀，在37℃、5％CO 2条件培养7天后获得杂交瘤细胞。收集杂交瘤细胞培养上清，用于小鼠抗体的筛选。
选取经ELISA和FACS检测具有hCD40结合活性的杂交瘤细胞进行扩增培养，待细胞达到一定数目后通过有限稀释法进行亚克隆。然后将亚克隆的杂交瘤细胞于37℃、5％CO 2孵育培养7天后，收集亚克隆的杂交瘤细胞培养上清，用于小鼠抗体的进一步筛选。
小鼠抗体的表达和纯化
将筛选的杂交瘤细胞培养10天后，离心收集细胞培养上清，然后将细胞培养上清上样至蛋白G柱(Genscript,Cat:L00209-10)中，用PBS缓冲液淋洗蛋白G柱，再用100mM甘氨酸(pH 2.8)洗脱与蛋白G柱结合的抗体，并立即用1M Tris-HC中和洗脱液。然后，采用超滤的方法将小鼠抗体置换到PBS缓冲液中。
实施例2小鼠抗CD40抗体的表征
ELISA测定抗CD40抗体的结合活性
用PBS缓冲液(pH 7.4)稀释hCD40-His蛋白(Acro,Cat:CD0-H5228)至浓度为0.1μg/mL，并按100μL/孔包被于96孔板，4℃孵育过夜。用PBST(含0.5％Tween-20的PBS)洗涤96孔板，然后加入200μL/孔的封闭液(含1％BSA的PBST)，于室温封闭2小时。弃去封闭液，加入100μL/孔的杂交瘤细胞培养上清，于室温孵育2小时。PBST洗涤96孔板后，加入按体积比1:10000稀释的HRP偶联山羊抗鼠IgG(H+L)抗体(Jackson Immuno,Cat:115-035-062)，室温孵育1小时。PBST洗涤96孔板后，加入100μL/孔的TMB溶液，室温避光孵育5分钟，并用0.5M H 2SO 4终止反应。使用Bio-rad iMark酶标仪读取OD450值。
FACS检测抗CD40抗体的结合活性
将细胞浓度为5×10 5个/mL的CHO-K1-hCD40细胞悬液按100μL/孔加到96孔U底板中，然后加入100μL/孔的杂交瘤细胞培养上清，混匀后于4℃孵育1小时。用含2％FBS的PBS洗涤细胞后，加入按体积比1:500稀释的PE偶联山羊抗鼠IgG(H+L)抗体(Abcam,Cat:ab97041)，于4℃孵育1小时。用含2％FBS的PBS洗涤细胞，然后将细胞重悬于含2％FBS的PBS中，使用流式细胞仪(BD AccuriC6)检测染色的平均荧光强度(MFI)来分析抗CD40抗体与CHO-K1-hCD40细胞的结合。
抗CD40抗体阻断293T-hCD40-NFκB细胞与CD40L结合的活性
当293T-hCD40-NFκB细胞上的hCD40与CD40L相互作用后，诱导荧光信号的表达上调。具体地，将对数生长期的293T-hCD40-NFκB细胞重悬于含2％FBS的DMEM培养基中，调节细胞浓度至5×10 5个/mL，然后按10μL/孔加到384孔白板中，并加入杂交瘤细胞培养上清或梯度稀释的纯化的小鼠抗CD40抗体(抗体终浓度范围为0.064ng/mL～5000ng/mL，按5倍梯度稀释)，于37℃振荡孵育20分钟。然后每孔加入10μL终浓度为10μg/mL的人CD40L蛋白(Acro,Cat:CDL-H52Db)，于37℃振荡孵育20分钟后将384孔板转移至37℃、5％CO 2条件下静置孵育5～6小时。孵育后按萤火虫荧光素酶试剂盒(vazyme,Cat:DD1201-03)的操作说明加入检测试剂，使用酶标仪(Thermo Varioskan Flash)读取荧光信号，通过相对荧光强度(RLU)来分析抗CD40抗体对CD40L介导的293T-hCD40-NFκB细胞荧光信号上调的阻断活性，并通过Graphpad Prism计算IC 50值。如图1A和1B所示，多个纯化的小鼠抗CD40抗体具有阻断293T-hCD40-NFκB细胞与CD40L结合的活性。
抗CD40抗体对Ramos细胞凋亡的抑制活性
由CD40/CD40L介导的MAPK活化途径是一种抑制肿瘤细胞增殖和诱导肿瘤细胞凋亡的潜在机制。以内源性表达CD40的人B淋巴瘤细胞Ramos(Burkitt淋巴瘤细胞)为模型，检测抗CD40抗体对Ramos细胞凋亡的抑制活性。具体地，将细胞浓度为1×10 6个/mL的Ramos细胞悬液按50μL/孔加到96孔U底板中，加入梯度稀释的纯化的小鼠抗CD40抗体(终浓度范围是0.02ng/mL～2000ng/mL，按10倍梯度稀 释)，并加入终浓度为2μg/mL的人CD40L蛋白(Acro,Cat:CDL-H52Db)和终浓度为60ng/mL的重组人IL-4(Acro,Cat:IL4-H4218)。混匀后于37℃、5％CO 2条件下孵育过夜，用含2％FBS的PBS洗涤细胞，然后加入PE偶联的小鼠抗人CD95抗体(Biolegend,Cat:305608)，混匀后于4℃孵育30分钟。洗涤细胞后用含2％FBS的PBS重悬细胞，采用流式细胞仪(Sartorius IQue3)检测荧光信号，通过染色的平均荧光强度(MFI)来分析肿瘤细胞凋亡分子CD95的表达情况，并通过Graphpad Prism计算IC 50值。如图2所示，多个纯化的小鼠抗CD40抗体具有抑制CD40L和IL-4诱导的Ramos细胞凋亡的活性。
实施例3嵌合抗CD40抗体的制备
嵌合抗体的构建
将实施例2筛选的杂交瘤细胞(例如，A01、A02、A03、B01、B02和B03等)按照RNA提取试剂盒(Takara,Cat:9767)的操作说明分离细胞中的总RNA，并通过逆转录试剂盒(Thermo,Cat:K1652)合成第一链cDNA。以第一链cDNA作为模板，分别与小鼠lgG引物和Kappa引物混合，使用聚合酶链式反应(PCR)技术克隆并测序，获得小鼠IgG抗体可变区序列。
采用化学合成方法，将小鼠抗体的VH和VL的DNA序列分别与人IgG4(SEQ ID NO:54)重链恒定区和Kappa轻链恒定区(SEQ ID NO:57)的DNA序列连接，然后克隆到pcDNA3.1(+)载体中构建重组人-鼠嵌合抗体。这些嵌合抗体分别命名为：Chi-A01、Chi-A02、Chi-A03、Chi-B01、Chi-B02和Chi-B03。
参照抗体的构建
选择Iscalimab(CFZ533)作为参照抗体，根据美国专利US9221913B2记载的氨基酸序列，采用化学合成方法获得抗体重链和轻链的DNA序列，克隆到pcDNA3.1(+)载体构建参照抗体(重链和轻链的氨基酸序列参见本公开的SEQ ID NOs:58和59，重链可变区和轻链可变区用下划线示出)，参照抗体在本文中也称为BM(即Benchmark的缩写)。
嵌合抗体和参照抗体的表达和纯化
嵌合抗体和参照抗体采用expiCHO系统(Gibco,Cat:A29129)并按照其转染试剂盒的操作说明进行瞬时转染表达，转染后将细胞置于37℃、含8％CO 2条件下振荡培养6天。离心收集细胞培养上清，然后将细胞培养上清上样至蛋白A柱(G.E.Healthcare,Cat:17-5474)中，用10个柱体积的PBS缓冲液洗涤蛋白A柱，再用醋酸缓冲液(300mM醋酸，pH 3.6)洗脱与蛋白A柱结合的抗体，并立即用1M Tris-HCl中和洗脱液。然后，将嵌合抗体超滤置换到PBS缓冲液中。
实施例4嵌合抗CD40抗体的表征
FACS检测抗CD40抗体的结合活性
具体方法参见实施例2，但有些地方稍作改变：用梯度稀释的嵌合抗CD40抗体和BM(终浓度范围为0.064ng/mL～5000ng/mL，按5倍梯度稀释)替代杂交瘤细胞培养上清；并用PE偶联驴抗人IgG(H+L)(Abcam,Cat:ab102439)替代PE偶联山羊抗鼠IgG(H+L)抗体。
图3示出嵌合抗体与CHO-K1-hCD40细胞的结合活性，其中Chi-A01和Chi-A02与CHO-K1-hCD40细胞的结合EC 50与BM相当。
抗CD40抗体对Ramos细胞凋亡的抑制活性
具体方法参见实施例2，但有些地方稍作改变：用梯度稀释的嵌合抗CD40抗体、BM和IgG4同型对照抗体(终浓度范围为0.064ng/mL～1000ng/mL，按5倍梯度稀释)替换梯度稀释的纯化的小鼠抗体(终浓度范围是0.02ng/mL～2000ng/mL，按10倍梯度稀释)。
表2和表3示出嵌合抗体对CD40L和IL-4诱导的Ramos细胞凋亡的抑制活性，其中，Chi-A01和Chi-A02抑制50％由CD40L和IL-4诱导的肿瘤细胞凋亡的浓度(IC 50)约为5pM，而BM的IC 50为18.32pM(约为Chi-A01和Chi-A02的3-4倍)，说明Chi-A01和Chi-A02对Ramos细胞凋亡的抑制活性优于BM。
抑制％＝(Ramos与CD40L和IL-4孵育后诱导凋亡的MFI-Ramos与CD40L和IL-4以及抗CD40抗体孵育后诱导凋亡的MFI)/(Ramos与CD40L和IL-4孵育后诱导凋亡的MFI-Ramos仅与培养基孵育后自然凋亡的MFI)×100％，根据公式计算可知，Chi-A01和Chi-A02的平均最大抑制率与BM相当。
表2嵌合抗体Chi-A01、Chi-A02和Chi-A03对Ramos细胞凋亡的抑制活性
Figure PCTCN2022120689-appb-000004
表3嵌合抗体Chi-B01、Chi-B02和Chi-B03对Ramos细胞凋亡的抑制活性
Figure PCTCN2022120689-appb-000005
注：NA表示未拟合出IC 50值。
抗CD40抗体对Ramos细胞凋亡的激动活性
已知激动性抗CD40抗体CP-870893(内部制备，重链和轻链序列参见本公开的SEQ ID NOs:60和61)与Ramos细胞结合后能产生诱导Ramos凋亡的刺激信号。采用Ramos细胞检测嵌合抗CD40抗体的激动活性，具体地，将细胞浓度为5×10 5个/mL的Ramos细胞悬液按100μL/孔加到96孔U底板，并加入梯度稀释的抗CD40抗体(终浓度范围是0.01ng/mL～10000ng/mL，按10倍梯度稀释)，混匀后于37℃、含5％CO 2的培养条件下孵育过夜。用含2％FBS的PBS洗涤细胞，然后加入PE偶联的小鼠抗人CD95抗体(Biolegend,Cat:305608)，混合后于4℃孵育30分钟。洗涤细胞后用含2％FBS的PBS重悬细胞，采用流式细胞仪(Sartorius IQue3)检测荧光信号，通过染色的平均荧光强度(MFI)来分析肿瘤细胞凋亡分子CD95的表达情况。结果见图4，嵌合抗体Chi-A02、Chi-B02和Chi-B03对Ramos细胞凋亡具有弱激动活性，而嵌合抗体Chi-A01、Chi-A03、Chi-B01和BM对Ramos细胞凋亡不产生激动活性。
实施例5人源化抗CD40抗体的设计和制备
人源化抗体的设计
采用CDR-graft方法，将小鼠抗体A01的重链可变区序列和轻链可变区序列经蛋白质数据库比对分析，筛选出具有最高序列同源性的人胚系抗体序列。将小鼠抗体的互补决定区(CDR)嫁接到筛选出的人胚系抗体序列的骨架中，并进一步突变CDR和/或骨架中的氨基酸残基以获得更多候选的可变区序列。
轻链VL：人胚系骨架序列IGKV1-39*01和IGKJ1*01的组合用于人源化序列设计，构建轻链变体hzA01L1(SEQ ID NO:15)；人胚系骨架序列IGKV3-20*01和IGKJ1*01的组合用于人源化序列设计，构建轻链变体hzA01L2(SEQ ID NO:16)；人胚系骨架序列IGKV2-30*02和IGKJ2*02的组合用于人源化序列设计，构建轻链变体hzA01L3(SEQ ID NO:17)。
重链VH：人胚系骨架序列IGHV4-4*08和IGHJ3*01的组合用于人源化序列设计，构建重链变体hzA01H1、hzA01H3和hzA01H4(分别参见SEQ ID NO:10、SEQ ID NO:12和SEQ ID NO:13)；人胚系骨架序列IGHV3-33*01和IGHJ3*01的组合用于人源化序列设计，构建重链变体hzA01H2(SEQ ID NO:11)。
人源化抗体的构建
采用化学合成方法对上述人源化抗体的VH和VL的DNA序列分别与人IgG4(SEQ ID NO:54)重链恒定区和Kappa轻链恒定区(SEQ ID NO:57)的DNA序列连接，然后克隆到pcDNA3.1(+)载体中构建重 组人源化抗体。表4示出人源化抗体的VH和VL序列的配对。
表4人源化抗体的可变区序列
抗体 VL VH
hzA01-1.1 hzA01L1 hzA01H1
hzA01-1.2 hzA01L1 hzA01H2
hzA01-2.1 hzA01L2 hzA01H1
hzA01-2.2 hzA01L2 hzA01H2
hzA01-3.1 hzA01L3 hzA01H1
hzA01-3.3 hzA01L3 hzA01H3
hzA01-3.4 hzA01L3 hzA01H4
人源化抗体的表达和纯化
人源化抗体采用expiCHO表达系统(Gibco,Cat:A29129)并按照其转染试剂盒的操作说明进行瞬时转染，转染后将细胞置于37℃、含8％CO 2的条件下振荡培养6天。离心收集细胞培养上清，然后将细胞培养上清与蛋白A磁珠(GenScript,Cat:L00273)于室温振荡孵育2小时，用PBS溶液洗涤后，再用醋酸缓冲液(300mM醋酸，pH 3.6)洗脱与蛋白A磁珠结合的抗体，并立即用1M Tris-HCl中和洗脱液。然后，采用超滤的方法将人源化抗体置换到PBS缓冲液中。
实施例6人源化抗CD40抗体的表征
抗CD40抗体的亲和力测定
通过ForteBio分子相互作用仪检测人源化抗CD40抗体与hCD40-His蛋白(Acro,Cat:CD0-H5228)和CynoCD40-His蛋白(Acro,Cat:CD0-C52H6)的亲和力。首先，将AHC生物传感器(Fortebio,Cat:18-5060)活化，然后将AHC生物传感器浸入终浓度为5μg/mL的抗CD40抗体溶液中，以捕获抗体。再用AHC生物传感器分别结合梯度稀释(终浓度范围是1.56nM～100nM，按2倍梯度稀释)的hCD40-His蛋白和CynoCD40-His蛋白。采用Fortebio分子相互作用仪(Fortebio Octet RED96e)实时检测反应信号从而获得结合和解离曲线。每个循环解离完成后，将AHC生物传感器再生，继而进行下一次捕获，循环往复完成不同抗体对CD40亲和力的测定。通过Fortebio Data Analysis 11.0软件以1:1(Langmuir)结合模型分析所得数据，测定K a(K on)和K d(K dis)值，并通过K D＝K d/K a计算解离常数K D，更加详细的检测方法和相关信息参见www.fortebio.com。结果见表5，人源化抗体hzA01-3.1、hzA01-3.3和hzA01-3.4与人或食蟹猴CD40抗原的结合亲和力K D在0.1nM至1nM之间，与嵌合Chi-A01的亲和力相当。
表5 Fortebio检测人源化抗CD40抗体与CD40的亲和力
Figure PCTCN2022120689-appb-000006
ND：表示未检测。
FACS检测抗CD40抗体的结合活性
具体方法参见实施例2，但有些地方稍作改变：用293T-hCD40-NFκB细胞替代CHO-K1-hCD40细胞，用梯度稀释的抗CD40抗体(终浓度范围0.006ng/mL～1000ng/mL，按3倍梯度稀释)替代杂交瘤细胞培 养上清；并用PE偶联驴抗人IgG(H+L)抗体(Abcam,Cat:ab102439)替代PE偶联山羊抗鼠IgG(H+L)抗体，并用流式细胞分析仪(Sartorius IQue3)替代流式细胞仪(BD Accuri C6)。
结果见图5，在10～100ng/mL浓度范围内，人源化抗体hzA01-3.1、hzA01-3.3和hzA01-3.4与293T-hCD40-NFκB细胞的结合达到饱和，且饱和浓度所对应的MFI值与BM具有相同水平。
抗CD40抗体阻断CHO-K1-hCD40L细胞与hCD40-mFc、CHO-K1-CynoCD40L细胞与cynoCD40-mFc 结合的活性
将细胞浓度为3×10 5个/mL的CHO-K1-hCD40L细胞悬液和CHO-K1-CynoCD40L细胞悬液各自按100μL/孔分别加到96孔U底板中，并相应地加入终浓度为2μg/mL的hCD40-mFc蛋白(内部制备，SEQ ID NO:64)或终浓度为2μg/mL的cynoCD40-mFc蛋白(内部制备，SEQ ID NO:65)，然后加入梯度稀释的抗CD40抗体(终浓度范围312.5ng/mL～40000ng/mL，按2倍梯度稀释)，混匀后于4℃孵育1小时。用含2％FBS的PBS洗涤细胞，加入按体积比1:500稀释的PE偶联山羊抗鼠IgG(H+L)抗体(Abcam,Cat:ab97041)，于4℃孵育0.5小时。用含2％FBS的PBS洗涤细胞，然后将细胞重悬于含2％FBS的PBS中，使用流式细胞仪(Sartorius IQue3)检测荧光信号，通过染色的平均荧光强度(MFI)来分析抗CD40抗体阻断CHO-K1-hCD40L细胞与hCD40-mFc蛋白、CHO-K1-cynoCD40L细胞与cynoCD40-mFc蛋白结合的活性，并通过GraphPad Prism软件计算IC 50值。结果见表6，人源化抗体hzA01-3.1和hzA01-3.4在细胞水平的阻断IC 50与BM相当。
表6人源化抗CD40抗体阻断CHO-K1-hCD40L细胞和CHO-K1-CynoCD40L细胞与CD40结合的活性
Figure PCTCN2022120689-appb-000007
抗CD40抗体阻断293T-hCD40-NFκB细胞与CHO-K1-hCD40L细胞结合的活性
将细胞浓度为2.5×10 5个/mL的293T-hCD40-NFκB细胞稀悬液按20μL/孔加到96孔板中，然后加入梯度稀释的抗CD40抗体(终浓度范围是0.007ng/mL～2000ng/mL，按6倍梯度稀释)，同时按细胞数目1:1的比例加入CHO-K1-hCD40L细胞悬液，混匀后于室温孵育20分钟。然后将96孔板转移至37℃、含5％CO 2的条件下静置孵育5～6小时。孵育后按萤火虫荧光素酶试剂盒(vazyme,Cat:DD1201-03)的操作说明加入检测试剂，使用酶标仪(Thermo Varioskan Flash)检测细胞荧光信号，通过相对荧光强度(RLU)分析抗CD40抗体对CD40L介导的293T-hCD40-NFκB细胞荧光信号上调的阻断活性。结果见图6，在0～2μg/mL的浓度范围内，人源化抗体hzA01-1.1、hzA01-2.1、hzA01-3.1、hzA01-3.3和hzA01-3.4均显示出显著的阻断活性。
抗CD40抗体对Ramos细胞凋亡的抑制活性
具体方法参见实施例2，但有些地方稍作修改：用梯度稀释的抗CD40抗体(终浓度范围为0.007ng/mL～2000ng/mL，按6倍梯度稀释)替换梯度稀释的纯化的小鼠抗CD40抗体(终浓度范围是0.02ng/mL～2000ng/mL，按10倍梯度稀释)。
结果见图7，在0～2μg/mL浓度范围内，人源化抗体hzA01-3.1、hzA01-3.3和hzA01-3.4都表现出持续抑制活性，其抑制IC 50比BM低3～4倍。
抗CD40抗体对Ramos细胞凋亡的激动活性
具体方法参见实施例4，但有些地方稍作改变：用梯度稀释的抗CD40抗体(终浓度范围为0.01nM～2000nM，按5倍梯度稀释)替换梯度稀释的抗CD40抗体(终浓度范围为0.01ng/mL～10000ng/mL，按10倍梯度稀释)。
结果见图8，人源化抗体hzA01-1.1和hzA01-2.1具有弱激动活性，人源化抗体hzA01-3.1、hzA01-3.3和hzA01-3.4在0.01nM～2000nM浓度范围内均不产生比BM显著的激动活性。
抗CD40抗体对外周血B淋巴细胞活化的抑制活性
人外周血来源的B淋巴细胞(PBMC-B)上表达的CD40能与CD40L结合，诱导B细胞活化，从而引起细胞表面活化信号上调(例如CD86)。采用人B淋巴细胞纯化试剂盒(Stemcell,Cat:17954)分离纯化来自健康人PBMC的B淋巴细胞，将细胞浓度为1×10 6个/mL的外周血B淋巴细胞悬液按50μL/孔加到96孔U底板中，并加入梯度稀释的抗CD40抗体(终浓度范围为0.002ng/mL～2000ng/mL，按10倍梯度稀释)，然后按细胞数目1:1的比例加入CHO-K1-hCD40L细胞悬液。于37℃、含5％CO 2的条件下孵育过夜。含2％FBS的PBS溶液洗涤细胞，然后加入PE偶联的小鼠抗人CD86抗体(Biolegend,Cat:305438)，与细胞混匀后于4℃孵育30分钟。含2％FBS的PBS溶液洗涤细胞并重悬，采用流式细胞仪(Sartorius IQue3)检测B细胞所表达的共刺激分子CD86的平均荧光强度(MFI)，并通过Graphpad Prism分析计算IC 50值。结果见图9，人源化抗体hzA01-3.1、hzA01-3.3和hzA01-3.4均表现出对CD40L诱导的人外周血B淋巴细胞CD86表达的抑制活性，且IC 50比BM低约2～4倍。
抗CD40抗体对外周血B淋巴细胞增殖的抑制活性
人外周血来源的B淋巴细胞(PBMC-B)上表达的CD40能与CD40L结合，诱导B细胞增殖。将细胞浓度为1×10 6个/mL的人外周血B淋巴细胞悬液按50μL/孔加到96孔U底板，加入梯度稀释的抗CD40抗体(终浓度范围是0.000035nM～35nM，按10倍梯度稀释)，并加入终浓度为2μg/mL的人CD40L蛋白(Acro,Cat:CDL-H52Db)与终浓度为60ng/mL的重组人IL-4蛋白(Acro,Cat:IL4-H4218)。混匀后于37℃、含5％CO 2条件下孵育培养5天。收集细胞，用PBS缓冲液洗涤细胞并重悬，然后加入CellTiter Glo荧光素酶细胞活性检测试剂(Promega,Cat:G7572)，用荧光酶标仪(Thermo Varioskan Flash)检测细胞荧光信号，通过相对荧光强度(RLU)来分析人外周血B淋巴细胞的增殖，并通过Graphpad Prism计算IC 50值。结果见图10，人源化抗体hzA01-3.1、hzA01-3.3和hzA01-3.4抑制CD40L和IL-4诱导的人外周血B淋巴细胞增殖的IC 50比BM低约3～6倍。
抗CD40抗体诱导外周血B淋巴细胞增殖的活性
将细胞浓度为1×10 6个/mL的健康人外周血B淋巴细胞悬液按50μL/孔加到96孔U底板，加入梯度稀释的抗CD40抗体(终浓度范围为0.00035nM～350nM，按10倍梯度稀释)，于37℃、含5％CO 2的条件下孵育培养5天。收集细胞，用PBS缓冲液洗涤细胞并重悬，然后加入CellTiter Glo荧光素酶细胞活性检测试剂(Promega,Cat:G7572)，采用荧光酶标仪(Thermo Varioskan Flash)检测细胞荧光信号，通过相对荧光强度(RLU)来分析外周血B淋巴细胞的增殖。结果见图11，人源化抗体hzA01-3.1在高浓度(350nM)时诱导微弱的增殖，而人源化抗体hzA01-3.3和hzA01-3.4在0～350nM浓度范围内均不产生比BM显著的激动活性。
抗CD40抗体对混合淋巴细胞反应(MLR)的抑制作用
按照单核细胞分离试剂盒(Stemcell,Cat:19359)的操作说明从健康人PBMC中分离单核细胞，然后调节细胞浓度至1×10 6个/mL。按照DC细胞培养试剂盒(Stemcell,Cat:10985)的操作说明加入DC细胞分化诱导剂，并于37℃、含5％CO 2的培养条件下孵育培养5天，再加入DC细胞成熟刺激剂，继续培养2天，使单核细胞分化为完全成熟的DC细胞(mDC)。
按照EasySep TMCD4阳性T细胞分离试剂盒(Stemcell,Cat:17952)的操作说明从健康人PBMC中分离CD4 +T细胞，然后调节细胞浓度到2×10 6个/mL。将mDC细胞按1×10 4个/孔加到96孔U底板中，并加入梯度稀释的抗CD40抗体或IgG4同型对照抗体(终浓度范围是10ng/mL～10000ng/mL，按10倍梯度稀释)，混匀后于室温孵育30分钟，随后按mDC与CD4 +T细胞数目1:20的比例加入CD4 +T细胞悬液，混匀后于37℃、含5％CO 2的条件下培养96小时，离心收集培养上清。
按照IFNγ检测试剂盒(R&D,Cat:DY285B)的操作说明，通过ELISA检测培养上清中的IFNγ，使用酶标仪(Bio-rad iMark)读取OD450吸光值，并通过Graphpad Prism计算分析IFNγ浓度。结果如图12所示，人源化抗体hzA01-3.3在10ng/mL～10000ng/mL浓度范围内呈浓度依赖性抑制混合淋巴细胞反应，且在饱和浓度(1000ng/mL～10000ng/mL)时的IFNγ释放量低于BM，说明在饱和浓度时hzA01-3.3对混合淋巴细胞反应的抑制能力优于BM。
抗CD40抗体对树突状细胞成熟的激动活性
按照单核细胞分离试剂盒(Stemcell,Cat:19359)的操作说明从PBMC中分离单核细胞，经DC细胞分化诱导剂(Stemcell,Cat:10988)诱导培养5天，收集不完全成熟DC细胞(imDC)，并调节细胞浓度至 1×10 6个/mL。imDC细胞悬液按50μL/孔加到96孔板中，然后加入梯度稀释的抗CD40抗体或IgG4同型对照抗体(终浓度范围是100ng/mL～10000ng/mL，按10倍梯度稀释)，于37℃、含5％CO 2的条件孵育过夜。收集细胞，用含2％FBS的PBS洗涤细胞并重悬，加入APC偶联的小鼠抗人CD83抗体(BD,Cat:551073)，混匀后于4℃孵育30分钟，用含2％FBS的PBS洗涤细胞并重悬，采用流式细胞仪(Sartorius IQue3)检测细胞荧光信号，通过染色的平均荧光强度(MFI)来分析细胞表面CD83的表达情况。CD83的表达是成熟DC细胞的特异性标志，结果见图13，CP-870893具有明显刺激imDC细胞成熟的作用，而人源化抗体hzA01-3.3和BM均未针对imDC细胞的成熟产生明显的激动活性。
实施例7抗CD40抗体的Fc效应
人B细胞、DC细胞和PBMC等细胞表面表达Fc受体(FcR)，当抗CD40抗体的Fc端与FcR结合时，可能介导抗体Fc交联(Fc Crosslinking)而产生激动活性，或者介导抗体依赖性细胞介导的细胞毒性作用(ADCC)或补体依赖性细胞毒性作用(CDC)而导致B细胞耗竭。
将细胞浓度为1×10 6个/mL的Ramos细胞悬液按50μL/孔加到96孔U底板中，并加入抗CD40抗体(终浓度范围是1μg/mL～10μg/mL，10倍梯度稀释)，混匀后于室温孵育20分钟。然后加入终浓度为2μg/mL的抗人IgG Fcγ抗体(Jackson Immuno,Cat:109-005-190)，混匀后于37℃、含5％CO 2的条件下孵育过夜。离心收集细胞，用含2％FBS的PBS洗涤并重悬细胞，采用流式分析仪(Sartorius IQue3)检测细胞荧光信号，并通过染色的平均荧光强度(MFI)来分析Ramos细胞上凋亡分子CD95的表达情况。结果如表7所示，在1μg/mL～10μg/mL浓度范围内，人源化抗体hzA01-3.3未产生显著高于BM的交联刺激活性，且交联激动活性远低于CP-870893。
表7抗CD40抗体的Fc交联活性的测定
Figure PCTCN2022120689-appb-000008
以Ramos细胞作为靶细胞，Jurkat-NFAT-Luc细胞(Promega)作为效应细胞，评价抗CD40抗体介导的ADCC效应。具体地，将细胞浓度为1×10 6个/mL的Ramos细胞悬液按50μL/孔加到96孔板中，加入梯度稀释的抗CD40抗体或Rituximab(终浓度范围是0.46ng/mL～1000ng/mL，按3倍梯度稀释)，混匀后于室温孵育20分钟。然后将细胞浓度为5×10 6个/mL的Jurkat-NFAT-Luc细胞悬液按100μL/孔加到96孔板中，其中靶细胞数目：效应细胞数目的比例是1:10，混匀后于37℃孵育5小时。离心收集细胞，PBS洗涤后，加入Luc荧光检测试剂(苏州瑞安,Cat:RA-GL03)，采用荧光酶标仪(Thermo Varioskan Flash)检测细胞荧光信号，并通过相对荧光信号(RLU)值来分析抗CD40抗体介导的ADCC效应。结果见图14，Rituximab具有显著的ADCC效应，而人源化抗体hzA01-3.3与BM均未显示出ADCC活性。
将细胞浓度为1×10 6个/mL的Ramos细胞悬液按50μL/孔加到96孔板中，并加入50μL/孔的梯度稀释的抗CD40抗体或Rituximab(终浓度范围是2.4ng/mL～40000ng/mL，按4倍梯度稀释)，同时加入25μL/孔的人血清补体蛋白(Quidel,Cat:A112)，混匀后于37℃、含5％CO 2的培养条件下孵育过夜。按CellTiter Glo荧光素酶细胞活性检测试剂盒(Promega,Cat:G7572)的操作说明加入检测试剂，采用荧光酶标仪(Thermo Varioskan Flash)检测细胞荧光信号，并通过相对荧光信号(RLU)来分析存活的细胞。结果表明，Rituximab具有显著的CDC杀伤活性，而人源化抗体hzA01-3.3与BM相同，都未显示出CDC杀伤作用(图15)，CDC杀伤率％＝(靶细胞荧光值-抗体组荧光值)÷(靶细胞荧光值-检测试剂处理的靶细胞荧光值)×100％。
实施例8人源化抗CD40抗体的体内药效
将健康人的PBMC细胞移植于NDG免疫缺陷小鼠体内进行免疫重建，构建产生人源抗体的小鼠模型。一方面，人PBMC响应于小鼠抗原，会产生人抗小鼠IgG抗体和人抗小鼠IgM抗体，另一方面，小鼠体内的人PBMC细胞接受KLH等外源蛋白刺激后会产生抗KLH特异性抗体。产生人源抗体的小鼠模型的 构建方法如下，选择6～8周龄的雌性NDG免疫缺陷小鼠(购买自百奥赛图)，第0天，给每只小鼠尾静脉注射1×10 7个健康人PBMC细胞；第0～5天，每天给每只小鼠腹腔注射10μg重组人IL-4(Acro,Cat:IL4-H4218)；并在第0天和第7天，给每只小鼠腹腔注射50μg KLH蛋白(Sigma,Cat:H7017-20MG)。按照表8进行小鼠分组，从第0天开始，每只小鼠按每周给药1次的频率，连续3次通过腹腔注射抗体药物。
在第8、14和21天，采集每只小鼠的血样并分离获得血清。按照人IgG ELISA检测试剂盒(Thermo,Cat:BMS2091)和人IgM ELISA检测试剂盒(Novus,Cat:NBP2-60477)的操作说明，分别检测小鼠血清中的人抗小鼠IgG抗体和人抗小鼠IgM抗体的浓度。使用酶标仪(Bio-rad iMark)读取OD450吸光值，并通过Graphpad Prism计算分析血清中人抗小鼠IgG抗体和人抗小鼠IgM抗体的浓度。另外，采用如下方法检测小鼠血清中抗KLH抗体的滴度：将浓度为2μg/mL的KLH蛋白(Sigma,Cat:H7017-20MG)按100μL/孔加到96孔板中，于4℃孵育过夜。PBST洗涤96孔板后，加入200μL/孔的封闭液(含1％BSA的PBST)，于室温封闭2小时。弃去封闭液，然后加入100μL/孔的梯度稀释的小鼠血清，于室温孵育2小时。PBST洗涤96孔板后，加入以体积比1:10000稀释的HRP偶联山羊抗人IgG(H+L)抗体(Jackson Immuno,Cat:109-035-088)，室温孵育1小时。PBST洗涤96孔板后，加入100μL/孔的TMB溶液，室温避光孵育5分钟，并用0.5M H 2SO 4终止反应，在Bio-rad iMark酶标仪读取450nM吸光度。结果显示，与IgG4同型组相比，人源化抗体hzA01-3.3能显著抑制人抗小鼠IgG抗体和人抗小鼠IgM抗体的产生(图16A和16B)；且hzA01-3.3治疗组未针对KLH产生特异性抗体(数据未显示)。
在第22天，处死小鼠后摘取脾脏，称重并分析各组小鼠的脾脏系数(脾脏系数＝脾脏重量/小鼠体重×1000)。结果参见表8，IgG4同型对照组小鼠脾脏明显肿大且脾脏系数明显增高；而hzA01-3.3(0.5mg/kg)组和hzA01-3.3(5mg/kg)组小鼠的脾脏肿大有明显缓解，hzA01-3.3呈剂量依赖地降低小鼠的脾脏系数；hzA01-3.3(5mg/kg)组和BM(5mg/kg)组小鼠的脾脏系数相当。
表8NDG小鼠脾脏系数的测定
小鼠分组(n＝只数) 平均脾重(g) 平均体重(g) 脾脏系数
空白对照,n＝3 0.04±0.01 23.33±2.71 1.81±0.40
IgG4同型对照,n＝4 0.24±0.03 22.50±1.27 10.82±1.14
hzA01-3.3(0.5mg/kg),n＝5 0.15±0.03 23.14±1.65 6.31±1.06
hzA01-3.3(5mg/kg),n＝5 0.13±0.04 22.56±2.16 5.50±1.42
BM(5mg/kg),n＝5 0.13±0.03 24.04±0.38 5.58±1.09
注：空白对照是未经免疫重建和给药的NDG小鼠；IgG4同型对照为Anti-HEL-Human IgG4(S228P L235E)Isotype-control(Biointron,Cat:B109805)
另外，采用流式细胞术分析各组小鼠脾脏内浸润的人T淋巴细胞和B淋巴细胞含量，具体地，取小鼠脾脏制备成单细胞悬液，并调节细胞浓度至1×10 6个/mL，按100μL/孔加到96孔U底板中，分别加入PE偶联小鼠抗人CD4抗体(Biolegend,Cat:980804)和APC偶联小鼠抗人CD19抗体(BD,Cat:555415)，混匀后于4℃孵育30分钟，用含2％FBS的PBS洗涤并重悬细胞，采用流式细胞仪(Thermo Attune Nxt)检测细胞荧光信号，并通过染色细胞数目的百分比(％)来分析小鼠脾脏内浸润的人CD4 +T细胞和CD19 +B细胞含量。结果表明(图17)，在第22天，IgG4同型对照组小鼠的脾脏有大量人CD4 +T细胞和人CD19 +B细胞浸润；而hzA01-3.3(0.5mg/kg)和hzA01-3.3(5mg/kg)组小鼠脾脏内浸润的T细胞和B细胞则显著低于IgG4同型组，hzA01-3.3呈剂量依赖性降低人T细胞和B细胞浸润含量。
实施例9抗CD40抗体在颌下腺蛋白诱导的hCD40/hCD40L转基因小鼠干燥综合征模型中的药效学评价
按照如下方法制备抗原：取C57BL/6小鼠20只(6-8周龄，雌性；百奥赛图医药科技股份有限公司)，采集颌下腺，分离包膜及结缔组织，于4℃用PBS(Servicebio，Cat:G4202-500ML)洗净。于低温环境下，机械匀浆，之后离心取上清液，得到颌下腺蛋白，将颌下腺蛋白作为抗原。
按照如下方法构建hCD40/hCD40L小鼠干燥综合征模型：在第0天和第7天，将抗原与完全弗氏佐剂(Sigma，Cat:F5881)1:1(v/v)混合制成颌下腺蛋白乳剂，于hCD40/hCD40L小鼠(北京维通利华实验动物技术有限公司)背部皮下两点注射，总量为200μL；在第14天，将抗原与不完全弗氏佐剂(Sigma，Cat:F5506)1:1(v/v)混合制成颌下腺蛋白乳剂，于hCD40/hCD40L小鼠背部皮下两点注射，总量为200 μL。
将hCD40/hCD40L小鼠按初始唾液量平均值分为5组，从第0天开始，每周给药两次，共给药10次(分别于第0、4、7、11、14、18、21、25、28和32天给药)，第35天结束试验；期间每2周称重一次。分组及给药方案如表9所示。
表9分组及给药方案
Figure PCTCN2022120689-appb-000009
注：空白对照为未经抗原免疫的hCD40/hCD40L小鼠；N/A，不适用；i.v.，静脉注射；BIW，每周给药两次。
通过唾液流率测量、颌下腺指数计算、颌下腺IL-6检测、血液及脾脏流式检测和颌下腺HE病理染色检查对hzA01-3.3进行药效评价，具体方法如下：
在第-3、6、13、19、27和34天，用1％戊巴比妥钠溶液以60mg/kg的剂量将小鼠麻醉，腹腔注射毛果芸香碱(Aladdin，Cat:P129614)1.25mg/kg，收集注射后15分钟内的唾液，计算唾液流率，唾液流率＝[棉球湿重(mg)-棉球干重(mg)]/15分钟；并用GraphPad Prism对唾液流率数据进行T-test分析。
在第35天，每只小鼠采集血样100μL；血样中加入PE标记的抗小鼠CD45抗体(Biolegend，Cat:103106)、Brilliant Violet 510 TM标记的抗小鼠CD3抗体(Biolegend，Cat:100234)、FITC标记的抗小鼠CD4抗体(Biolegend，Cat:100510)、PE/Cyanine7标记的抗小鼠CD8a抗体(Biolegend，Cat:100722)和APC标记的抗小鼠CD19抗体(Biolegend，Cat:115512)，于4℃避光孵育30分钟；然后加入红细胞裂解液(Biolegend，Cat:420301)，于室温避光裂解30分钟；之后用PBS洗涤并重悬细胞，采用流式细胞仪检测荧光信号。
在第35天，取出小鼠的脾脏将其制成单细胞悬液，调节细胞密度至1×10 8个/mL，取100μL加入红细胞裂解液(Biolegend，Cat:420301)避光裂解30分钟，PBS洗涤后加入PE标记的抗小鼠CD45抗体(Biolegend，Cat:103106)、Brilliant Violet 510 TM标记的抗小鼠CD3抗体(Biolegend，Cat:100234)、FITC标记的抗小鼠CD4抗体(Biolegend，Cat:100510)、PE/Cyanine7标记的抗小鼠CD8a抗体(Biolegend，Cat:100722)和APC标记的抗小鼠CD19抗体(Biolegend，Cat:115512)，于4℃避光孵育30分钟；之后用PBS洗涤并重悬细胞，采用流式细胞仪检测荧光信号。
在第35天，取出小鼠的颌下腺，称重，计算颌下腺指数，颌下腺指数＝颌下腺重量(mg)/小鼠体重(g)；取一份颌下腺固定于10％中性福尔马林，进行病理HE染色，并对颌下腺病理评分，用GraphPad Prism对颌下腺病理评分数据进行T-test分析；另取一份颌下腺，低温机械匀浆，低温离心取上清，并按照小鼠IL-6ELISA试剂盒(Abcam，Cat:ab222503)的操作说明检测上清中IL-6的含量。
体重变化结果显示(图18)，各组动物体重增长与空白对照组相当，表明hzA01-3.3在各剂量下均无明显毒副作用。
唾液流率结果显示(图19)，hzA01-3.3(5mg/kg)和hzA01-3.3(25mg/kg)组唾液流率均呈现回升趋势；与模型组相比，hzA01-3.3(25mg/kg)组在第27天和第34天的唾液流率显著升高(P<0.05)，表明hzA01-3.3能够改善小鼠的唾液流率。
颌下腺指数、颌下腺IL-6含量、以及颌下腺病理评分结果显示(图20A-20C)，hzA01-3.3(1mg/kg)、hzA01-3.3(5mg/kg)和hzA01-3.3(25mg/kg)组小鼠的各指标均呈现降低趋势；与模型组相比，hzA01-3.3(5mg/kg)组和hzA01-3.3(25mg/kg)组小鼠的各指标均显著降低，表明hzA01-3.3能够改善颌下腺指数、抑制颌下腺中IL-6的分泌和降低颌下腺病理评分。
血液及脾脏流式检测结果显示(图21A-21H)，hzA01-3.3给药组动物血液和脾脏中CD3 +T细胞、CD4 +T细胞、CD8 +T细胞和CD19 +B细胞的百分比与空白对照组相比均没有显著变化，表明hzA01-3.3对上述细胞数量的变化无特异性作用。预期hzA01-3.3能够在临床上表现出良好的安全性或/和良好的治疗效果。
本公开的序列信息总结于表10中。
表10序列信息
Figure PCTCN2022120689-appb-000010
Figure PCTCN2022120689-appb-000011
Figure PCTCN2022120689-appb-000012
Figure PCTCN2022120689-appb-000013
Figure PCTCN2022120689-appb-000014
Figure PCTCN2022120689-appb-000015

Claims (15)

  1. 一种分离的抗CD40抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (i)重链CDR1、重链CDR2和重链CDR3，其中，
    (1)重链CDR1包含SEQ ID NO：9中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：9中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：9中重链CDR3的氨基酸序列；
    (2)重链CDR1包含SEQ ID NO：10中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：10中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：10中重链CDR3的氨基酸序列；
    (3)重链CDR1包含SEQ ID NO：11中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：11中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：11中重链CDR3的氨基酸序列；
    (4)重链CDR1包含SEQ ID NO：12中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：12中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：12中重链CDR3的氨基酸序列；
    (5)重链CDR1包含SEQ ID NO：13中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：13中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：13中重链CDR3的氨基酸序列；
    (6)重链CDR1包含SEQ ID NO：22中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：22中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：22中重链CDR3的氨基酸序列；
    (7)重链CDR1包含SEQ ID NO：30中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：30中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：30中重链CDR3的氨基酸序列；
    (8)重链CDR1包含SEQ ID NO：38中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：38中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：38中重链CDR3的氨基酸序列；
    (9)重链CDR1包含SEQ ID NO：44中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：44中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：44中重链CDR3的氨基酸序列；或
    (10)重链CDR1包含SEQ ID NO：52中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：52中重链CDR2的氨基酸序列和重链CDR3包含SEQ ID NO：52中重链CDR3的氨基酸序列；和/或
    (ii)轻链CDR1、轻链CDR2和轻链CDR3，其中，
    (1)轻链CDR1包含SEQ ID NO：14中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：14中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：14中轻链CDR3的氨基酸序列；
    (2)轻链CDR1包含SEQ ID NO：15中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：15中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：15中轻链CDR3的氨基酸序列；
    (3)轻链CDR1包含SEQ ID NO：16中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：16中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：16中轻链CDR3的氨基酸序列；
    (4)轻链CDR1包含SEQ ID NO：17中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：17中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：17中轻链CDR3的氨基酸序列；
    (5)轻链CDR1包含SEQ ID NO：23中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：23中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：23中轻链CDR3的氨基酸序列；
    (6)轻链CDR1包含SEQ ID NO：31中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：31中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：31中轻链CDR3的氨基酸序列；
    (7)轻链CDR1包含SEQ ID NO：39中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：39中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：39中轻链CDR3的氨基酸序列；
    (8)轻链CDR1包含SEQ ID NO：45中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：45中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：45中轻链CDR3的氨基酸序列；或
    (9)轻链CDR1包含SEQ ID NO：53中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：53中轻链CDR2的氨基酸序列和轻链CDR3包含SEQ ID NO：53中轻链CDR3的氨基酸序列。
  2. 根据权利要求1所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (i)重链CDR1、重链CDR2和重链CDR3，其中，
    (1)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：1、2和5所示的氨基酸序列或与SEQ  ID NOs：1、2和5所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (2)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：1、3和5所示的氨基酸序列或与SEQ ID NOs：1、3和5所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (3)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：1、4和5所示的氨基酸序列或与SEQ ID NOs：1、4和5所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (4)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：18、19和20所示的氨基酸序列或与SEQ ID NOs：18、19和20所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (5)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：24、25和26所示的氨基酸序列或与SEQ ID NOs：24、25和26所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (6)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：32、33和34所示的氨基酸序列或与SEQ ID NOs：32、33和34所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (7)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：40、41和42所示的氨基酸序列或与SEQ ID NOs：40、41和42所示的氨基酸序列具有至少80％同一性的氨基酸序列；或
    (8)重链CDR1、重链CDR2和重链CDR3分别包含SEQ ID NOs：46、47和48所示的氨基酸序列或与SEQ ID NOs：46、47和48所示的氨基酸序列具有至少80％同一性的氨基酸序列；和/或
    (ii)轻链CDR1、轻链CDR2和轻链CDR3，其中，
    (1)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：6、7和8所示的氨基酸序列或与SEQ ID NOs：6、7和8所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (2)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：21、7和8所示的氨基酸序列或与SEQ ID NOs：21、7和8所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (3)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：27、28和29所示的氨基酸序列或与SEQ ID NOs：27、28和29所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (4)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：35、36和37所示的氨基酸序列或与SEQ ID NOs：35、36和37所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (5)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：43、36和37所示的氨基酸序列或与SEQ ID NOs：43、36和37所示的氨基酸序列具有至少80％同一性的氨基酸序列；或
    (6)轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：49、50和51所示的氨基酸序列或与SEQ ID NOs：49、50和51所示的氨基酸序列具有至少80％同一性的氨基酸序列。
  3. 根据权利要求1所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，其中，
    (1)重链CDR1包含SEQ ID NO：9中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：9中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：9中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：14中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：14中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：14中轻链CDR3的氨基酸序列；
    (2)重链CDR1包含SEQ ID NO：10中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：10中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：10中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：15中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：15中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：15中轻链CDR3的氨基酸序列；
    (3)重链CDR1包含SEQ ID NO：11中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：11中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：11中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：15中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：15中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：15中轻链CDR3的氨基酸序列；
    (4)重链CDR1包含SEQ ID NO：10中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：10中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：10中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：16中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：16中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：16中轻链CDR3的氨基酸序列；
    (5)重链CDR1包含SEQ ID NO：11中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：11中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：11中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：16中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：16中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：16中轻链CDR3的氨基酸序列；
    (6)重链CDR1包含SEQ ID NO：10中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：10中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：10中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：17中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：17中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：17中轻链CDR3的氨基酸序列；
    (7)重链CDR1包含SEQ ID NO：12中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：12中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：12中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：17中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：17中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：17中轻链CDR3的氨基酸序列；
    (8)重链CDR1包含SEQ ID NO：13中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：13中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：13中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：17中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：17中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：17中轻链CDR3的氨基酸序列；
    (9)重链CDR1包含SEQ ID NO：22中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：22中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：22中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：23中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：23中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：23中轻链CDR3的氨基酸序列；
    (10)重链CDR1包含SEQ ID NO：30中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：30中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：30中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：31中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：31中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：31中轻链CDR3的氨基酸序列；
    (11)重链CDR1包含SEQ ID NO：38中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：38中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：38中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：39中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：39中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：39中轻链CDR3的氨基酸序列；
    (12)重链CDR1包含SEQ ID NO：44中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：44中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：44中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：45中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：45中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：45中轻链CDR3的氨基酸序列；或
    (13)重链CDR1包含SEQ ID NO：52中重链CDR1的氨基酸序列，重链CDR2包含SEQ ID NO：52中重链CDR2的氨基酸序列，重链CDR3包含SEQ ID NO：52中重链CDR3的氨基酸序列，轻链CDR1包含SEQ ID NO：53中轻链CDR1的氨基酸序列，轻链CDR2包含SEQ ID NO：53中轻链CDR2的氨基酸序列，和轻链CDR3包含SEQ ID NO：53中轻链CDR3的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3，其中，
    (1)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：1、2、5、6、7和8所示的氨基酸序列或与SEQ ID NOs：1、2、5、6、7和8所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (2)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：1、3、5、6、7和8所示的氨基酸序列或与SEQ ID NOs：1、3、5、6、7和8所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (3)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：1、4、5、6、7和8所示的氨基酸序列或与SEQ ID NOs：1、4、5、6、7和8所示的氨基酸序列具有至少 80％同一性的氨基酸序列；
    (4)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：18、19、20、21、7和8所示的氨基酸序列或与SEQ ID NOs：18、19、20、21、7和8所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (5)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：24、25、26、27、28和29所示的氨基酸序列或与SEQ ID NOs：24、25、26、27、28和29所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (6)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：32、33、34、35、36和37所示的氨基酸序列或与SEQ ID NOs：32、33、34、35、36和37所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (7)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：40、41、42、43、36和37所示的氨基酸序列或与SEQ ID NOs：40、41、42、43、36和37所示的氨基酸序列具有至少80％同一性的氨基酸序列；或
    (8)重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3分别包含SEQ ID NOs：46、47、48、49、50和51所示的氨基酸序列或与SEQ ID NOs：46、47、48、49、50和51所示的氨基酸序列具有至少80％同一性的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (i)重链可变区，所述重链可变区包含9、10、11、12、13、22、30、38、44或52所示的氨基酸序列，或与SEQ ID NOs：9、10、11、12、13、22、30、38、44或52所示的氨基酸序列具有至少80％同一性的氨基酸序列；和/或
    (ii)轻链可变区，所述轻链可变区包含SEQ ID NOs：14、15、16、17、23、31、39、45或53所示的氨基酸序列，或与SEQ ID NOs：14、15、16、17、23、31、39、45或53所示的氨基酸序列所示的氨基酸序列具有至少80％同一性的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，
    (1)重链可变区和轻链可变区分别包含SEQ ID NOs：9和14所示的氨基酸序列或与SEQ ID NOs：9和14所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (2)重链可变区和轻链可变区分别包含SEQ ID NOs：10和15所示的氨基酸序列或与SEQ ID NOs：10和15所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (3)重链可变区和轻链可变区分别包含SEQ ID NOs：11和15所示的氨基酸序列或与SEQ ID NOs：11和15所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (4)重链可变区和轻链可变区分别包含SEQ ID NOs：10和16所示的氨基酸序列或与SEQ ID NOs：10和16所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (5)重链可变区和轻链可变区分别包含SEQ ID NOs：11和16所示的氨基酸序列或与SEQ ID NOs：11和16所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (6)重链可变区和轻链可变区分别包含SEQ ID NOs：10和17所示的氨基酸序列或与SEQ ID NOs：10和17所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (7)重链可变区和轻链可变区分别包含SEQ ID NOs：12和17所示的氨基酸序列或与SEQ ID NOs：12和17所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (8)重链可变区和轻链可变区分别包含SEQ ID NOs：13和17所示的氨基酸序列或与SEQ ID NOs：13和17所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (9)重链可变区和轻链可变区分别包含SEQ ID NOs：22和23所示的氨基酸序列或与SEQ ID NOs：22和23所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (10)重链可变区和轻链可变区分别包含SEQ ID NOs：30和31所示的氨基酸序列或与SEQ ID NOs：30和31所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (11)重链可变区和轻链可变区分别包含SEQ ID NOs：38和39所示的氨基酸序列或与SEQ ID NOs：38和 39所示的氨基酸序列具有至少80％同一性的氨基酸序列；
    (12)重链可变区和轻链可变区分别包含SEQ ID NOs：44和45所示的氨基酸序列或与SEQ ID NOs：44和45所示的氨基酸序列具有至少80％同一性的氨基酸序列；或
    (13)重链可变区和轻链可变区分别包含SEQ ID NOs：52和53所示的氨基酸序列或与SEQ ID NOs：52和53所示的氨基酸序列具有至少80％同一性的氨基酸序列。
  7. 根据权利要求1-6中任一项所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段还包含重链恒定区和轻链恒定区，所述重链恒定区包含SEQ ID NOs：54、55或56所示的氨基酸序列或与SEQ ID NOs：54、55或56所示氨基酸序列相比具有1、2、3、4或5个氨基酸取代、删除和添加的氨基酸序列，所述轻链恒定区包含SEQ ID NO：57所示氨基酸序列或者与SEQ ID NO：57所示氨基酸序列相比具有1、2、3、4或5个氨基酸取代、删除和添加的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段：(a)结合人CD40；(b)结合猴CD40；(c)阻断CD40与CD40L相互作用；和/或(d)抑制CD40活性。
  9. 根据权利要求1-8中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段是嵌合或人源化的；
    任选地，所述抗体或其抗原结合片段是IgG1、IgG2或IgG4同种型；
    任选地，所述抗体或其抗原结合片段选自单克隆抗体、单特异性抗体、双特异性抗体、三特异性抗体、多特异性抗体、Fab片段、F(ab')2片段、Fd片段、Fv片段、dAb片段、分离的CDR区、单链Fv分子或其组合。
  10. 一种分离的核酸分子，其编码权利要求1-9中任一项所述的抗体或其抗原结合片段。
  11. 一种表达载体，其包含权利要求10所述的核酸分子。
  12. 一种宿主细胞，其包含权利要求10所述的核酸分子或权利要求11所述的表达载体。
  13. 一种重组多肽或融合蛋白，其包含权利要求1-9中任一项所述的抗体或其抗原结合片段。
  14. 一种药物组合物，其包含权利要求1-9中任一项所述的抗体或其抗原结合片段和一种或多种药学上可接受的载体。
  15. 一种在有需要的受试者中治疗或预防免疫疾病的方法，所述方法包括向所述受试者施用治疗有效量的权利要求1-9中任一项所述的抗体或其抗原结合片段，或者权利要求14所述的药物组合物；
    任选地，所述免疫疾病包括但不限于炎性疾病、过敏反应、自身免疫性疾病或移植相关疾病；
    任选地，所述免疫疾病包括但不限于过敏反应、艾迪生氏病、强直性脊柱炎、脊柱关节炎、哮喘、动脉粥样硬化、冠心病、自身免疫性肝炎、自身免疫性腮腺炎、I型糖尿病、附睾炎、肾炎、赖特综合症、甲状腺炎、格雷夫斯病、格林-巴利综合症、桥本氏病、溶血性贫血、特发性血小板减少性症、系统性红斑狼疮、亚急性皮肤型红斑狼疮、多发性硬化症、重症肌无力、银屑病、硬皮病、关节炎、结节病、干燥综合症、干眼症、化脓性汗腺炎、移植相关疾病、血管炎和/或炎性肠病；
    任选地，所述方法还包括施用治疗有效量的第二治疗剂；
    任选地，所述第二治疗剂包括非甾体抗炎药、水杨酸盐、羟化氯喹、柳氮磺吡啶、皮质类固醇、细胞毒性药物、或免疫抑制药物和/或抗体。
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