WO2021052465A1 - 抗人cd38抗体及其应用 - Google Patents

抗人cd38抗体及其应用 Download PDF

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WO2021052465A1
WO2021052465A1 PCT/CN2020/116191 CN2020116191W WO2021052465A1 WO 2021052465 A1 WO2021052465 A1 WO 2021052465A1 CN 2020116191 W CN2020116191 W CN 2020116191W WO 2021052465 A1 WO2021052465 A1 WO 2021052465A1
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seq
amino acid
acid sequence
cdr1
cdr3
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PCT/CN2020/116191
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English (en)
French (fr)
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任红媛
高攀
林鉴
王骊淳
徐晓红
吴建
邓小芳
毕建军
王晋
陶春艳
王雪
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上海普铭生物科技有限公司
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Publication of WO2021052465A1 publication Critical patent/WO2021052465A1/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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • the present invention belongs to the field of biomedicine, and relates to a new anti-CD38 antibody or functional fragment thereof.
  • the present invention also relates to the application of the antibody or its functional fragment.
  • CD38 is a type II transmembrane glycoprotein with a molecular weight of 46KD, including C-terminal extracellular region (258 amino acids), transmembrane region (21 amino acids) and N-terminal (21 amino acids). Its function is receptor-mediated Adhesion and signal transduction, and through its extracellular enzyme activity mediate calcium mobilization, catalyze the formation of cyclic ADP ribose (cADPR) and ADPR.
  • cADPR cyclic ADP ribose
  • CD38 is uniformly highly expressed on multiple myeloma cells (MM), but is low on normal lymphocytes and bone marrow cells and some tissues of non-hematopoietic origin. Since CD38 is a transmembrane glycoprotein, it has in vitro enzymatic activity, as well as the role of receptors and adhesion molecules, so it is an ideal target for MM therapy; based on its unique mechanism of action, low toxicity and single agent activity, CD38 antibody is also Applies to joint programs. Currently, many studies are exploring the efficacy of CD38 antibody-based therapies applied to newly diagnosed high-risk MM patients. In addition, the therapeutic effect of CD38 antibody on other hematological malignancies (acute lymphocytic leukemia, NK/T cell lymphoma and acute myeloid leukemia) is also under study.
  • MM myeloma cells
  • CD38 antibodies use antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis ( ADPC) and directly inhibit the enzymatic activity of CD38 to achieve the anti-MM mechanism.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • ADPC antibody-dependent cellular phagocytosis
  • CD38 antibodies that are in clinical trials or have been approved include Daratumumab (Darzalex), Isatuximab, MOR-202, and TAK-079.
  • CD38 a transmembrane extracellular enzyme highly expressed on the surface of solid tumor cells, induces rapid tumor cell death through a variety of immune-mediated mechanisms, including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated Cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) and through apoptosis (apoptosis).
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated Cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • Darzalex has also been shown to be able to target immunosuppressive cells in the tumor microenvironment to exhibit immunomodulatory activity.
  • Darzalex also has the potential to treat other types of tumors that highly express CD38 molecules, including diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), plasma cell Leukemia (PCL), acute myeloid leukemia (AML), follicular lymphoma (FL) and mantle cell lymphoma (MCL), etc.
  • DLBCL diffuse large B-cell lymphoma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • PCL plasma cell Leukemia
  • AML acute myeloid leukemia
  • FL follicular lymphoma
  • MCL mantle cell lymphoma
  • the technical problem to be solved by the present invention is to obtain a high-affinity antibody that specifically binds to CD38, especially human CD38, through hybridoma screening and humanization technology.
  • the purpose of the present invention is to provide an antibody or fragment thereof that specifically binds to CD38, especially human CD38, and to provide its use based on the antibody or fragment thereof.
  • the "fragment" of the antibody molecule of the present invention encompasses various functional fragments of the antibody, for example, its antigen-binding portion, such as Fab, F(ab') 2 or scFv fragments.
  • the present invention provides the following technical solutions.
  • the present invention provides an antibody molecule or a fragment thereof, which has the following heavy chain CDRs regions:
  • VH-CDR1 has the amino acid sequence shown in SEQ ID NO: 16 or has one or several amino acid mutations based on the amino acid sequence shown in SEQ ID NO: 16;
  • VH-CDR2 has the amino acid sequence shown in SEQ ID NO: 17 or has one or several amino acid mutations based on the amino acid sequence shown in SEQ ID NO: 17;
  • VH-CDR3 has the amino acid sequence shown in SEQ ID NO: 18 or has one or several amino acid mutations based on the amino acid sequence shown in SEQ ID NO: 18;
  • VL-CDR1 has the amino acid sequence shown in SEQ ID NO: 4 or has one or several amino acid mutations based on the amino acid sequence shown in SEQ ID NO: 4;
  • VL-CDR2 has the amino acid sequence shown in SEQ ID NO: 5 or has one or several amino acid mutations based on the amino acid sequence shown in SEQ ID NO: 5;
  • VL-CDR3 has the amino acid sequence shown in SEQ ID NO: 19 or has one or several amino acid mutations based on the amino acid sequence shown in SEQ ID NO: 19;
  • the amino acid mutation in the CDRs region of the antibody enables the combination of the 6 CDRs of the antibody to form an antigen binding site after mutation, and at least part of the biological activity of the antibody molecule or fragments before the mutation of the CDRs region is retained.
  • the antibody molecule or fragment thereof of the present invention wherein the antibody molecule or fragment thereof before the mutation of the CDRs region includes the VH-CDR1 shown in SEQ ID NO: 16 and the VH-CDR2 shown in SEQ ID NO: 17
  • the antibodies or fragments of the present invention specifically bind to CD38 on the cell membrane surface, and can induce CD38+ cell apoptosis by binding to CD38 on the cell membrane surface.
  • the antibody molecule or fragment thereof of the present invention has antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for CD38+ cells.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the antibody molecule or fragment thereof of the present invention can compete with at least one antibody molecule selected from the following group to bind to CD38: Daratumumab, Isatuximab, and Mor202.
  • the antibody molecule or fragment thereof of the present invention comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises a variable region selected from SEQ ID NO:1 , SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 41, and SEQ ID NO: 147
  • the CDR1 (VH-CDR1) is selected from SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 32.
  • VH-CDR2 shown in SEQ ID NO: 37 and SEQ ID NO: 42, and selected from, for example, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID CDR3 represented by NO: 137 (VH-CDR3); and
  • the antibody molecule or fragment thereof comprises a light chain variable region (VL), wherein the light chain variable region comprises a variable region selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 23, SEQ ID NO: 29.
  • CDR1 VL-CDR1 shown in SEQ ID NO: 34, SEQ ID NO: 39, SEQ ID NO: 44, SEQ ID NO: 49, SEQ ID NO: 136, and SEQ ID NO: 148, selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 39, SEQ ID NO: 44, SEQ ID NO: 49, SEQ ID NO: 136, and SEQ ID NO: 148.
  • the CDR2 (VL-CDR2) shown in ID NO: 5, SEQ ID NO: 24 and SEQ ID NO: 35 is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 19. SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 138 and SEQ ID NO: CDR3 (VL- CDR3).
  • the heavy chain variable region may comprise a combination of CDRs selected from the following (VH-CDR1, VH-CDR2, VH-CDR3):
  • VH-CDR1 shown in SEQ ID NO: 7 VH-CDR2 shown in SEQ ID NO: 8
  • VH-CDR3 shown in SEQ ID NO: 9;
  • VH-CDR1 shown in SEQ ID NO: 11 VH-CDR2 shown in SEQ ID NO: 12, VH-CDR3 shown in SEQ ID NO: 13;
  • VH-CDR1 as shown in SEQ ID NO: 16, VH-CDR2 as shown in SEQ ID NO: 17 and VH-CDR3 as shown in SEQ ID NO: 18;
  • VH-CDR1 shown in SEQ ID NO: 20 VH-CDR2 shown in SEQ ID NO: 21, VH-CDR3 shown in SEQ ID NO: 22;
  • VH-CDR1 as shown in SEQ ID NO: 26
  • VH-CDR2 as shown in SEQ ID NO: 27
  • VH-CDR3 as shown in SEQ ID NO: 28;
  • VH-CDR1 as shown in SEQ ID NO: 31, VH-CDR2 as shown in SEQ ID NO: 32, VH-CDR3 as shown in SEQ ID NO: 33;
  • VH-CDR1 as shown in SEQ ID NO: 26
  • VH-CDR2 as shown in SEQ ID NO: 37
  • VH-CDR3 as shown in SEQ ID NO: 38;
  • VH-CDR1 as shown in SEQ ID NO: 41
  • VH-CDR2 as shown in SEQ ID NO: 42
  • VH-CDR3 as shown in SEQ ID NO: 43;
  • VH-CDR1 as shown in SEQ ID NO: 11
  • VH-CDR2 as shown in SEQ ID NO: 12
  • VH-CDR3 as shown in SEQ ID NO: 46;
  • VH-CDR1 as shown in SEQ ID NO: 11
  • VH-CDR2 as shown in SEQ ID NO: 12
  • VH-CDR3 as shown in SEQ ID NO: 47;
  • VH-CDR1 as shown in SEQ ID NO: 11
  • VH-CDR2 as shown in SEQ ID NO: 12
  • VH-CDR3 as shown in SEQ ID NO: 48;
  • VH-CDR1 as shown in SEQ ID NO:16
  • VH-CDR2 as shown in SEQ ID NO:17
  • VH-CDR3 as shown in SEQ ID NO:137;
  • VH-CDR1 as shown in SEQ ID NO: 147
  • VH-CDR2 as shown in SEQ ID NO: 42
  • VH-CDR3 as shown in SEQ ID NO: 43;
  • the antibody molecule or fragment thereof comprises a light chain variable region (VL), wherein the light chain variable region may comprise a combination of CDRs selected from the group consisting of (VL-CDR1, VL-CDR2, VL-CDR3) :
  • VL-CDR1 shown in SEQ ID NO: 4 VL-CDR2 shown in SEQ ID NO: 5
  • VL-CDR3 shown in SEQ ID NO: 6;
  • VL-CDR1 shown in SEQ ID NO: 4 VL-CDR2 shown in SEQ ID NO: 5
  • VL-CDR3 shown in SEQ ID NO: 10;
  • VL-CDR1 as shown in SEQ ID NO:14
  • VL-CDR2 as shown in SEQ ID NO:5
  • VL-CDR3 as shown in SEQ ID NO:15;
  • VL-CDR1 shown in SEQ ID NO: 4 VL-CDR2 shown in SEQ ID NO: 5, VL-CDR3 shown in SEQ ID NO: 19;
  • VL-CDR1 shown in SEQ ID NO: 23 VL-CDR2 shown in SEQ ID NO: 24, VL-CDR3 shown in SEQ ID NO: 25;
  • VL-CDR1 shown in SEQ ID NO: 34 VL-CDR2 shown in SEQ ID NO: 35, VL-CDR3 shown in SEQ ID NO: 36;
  • VL-CDR1 as shown in SEQ ID NO: 39
  • VL-CDR2 as shown in SEQ ID NO: 24
  • VL-CDR3 as shown in SEQ ID NO: 40;
  • VL-CDR1 shown in SEQ ID NO: 44 VL-CDR2 shown in SEQ ID NO: 24, VL-CDR3 shown in SEQ ID NO: 45;
  • VL-CDR1 shown in SEQ ID NO: 49 VL-CDR2 shown in SEQ ID NO: 24, VL-CDR3 shown in SEQ ID NO: 40;
  • VL-CDR1 shown in SEQ ID NO: 4 VL-CDR2 shown in SEQ ID NO: 5
  • VL-CDR3 shown in SEQ ID NO: 138;
  • VL-CDR1 shown in SEQ ID NO: 148 VL-CDR2 shown in SEQ ID NO: 24, VL-CDR3 shown in SEQ ID NO: 45.
  • the heavy chain variable region in the antibody molecule or fragment thereof comprises a variable region selected from SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68 to SEQ ID NO: 88, SEQ ID NO: 140 to SEQ ID NO: 142
  • the light chain variable region comprises selected from SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, Any one of SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 89 to SEQ ID NO: 105, SEQ ID NO: 134 and SEQ ID NO: 135, SEQ ID NO: 143 to SEQ ID NO: 146
  • the antibody molecule or fragment thereof comprises a combination of CDRs selected from:
  • VH-CDR1 as shown in SEQ ID NO:1, VH-CDR2 as shown in SEQ ID NO: 2, VH-CDR3 as shown in SEQ ID NO: 3; as shown in SEQ ID NO: 4 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 5, VL-CDR3 as shown in SEQ ID NO: 6;
  • VH-CDR1 as shown in SEQ ID NO: 7
  • VH-CDR2 as shown in SEQ ID NO: 8
  • VH-CDR3 as shown in SEQ ID NO: 9
  • VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 5
  • VL-CDR3 as shown in SEQ ID NO: 10;
  • VH-CDR1 as shown in SEQ ID NO: 11
  • VH-CDR2 as shown in SEQ ID NO: 12
  • VH-CDR3 as shown in SEQ ID NO: 13
  • VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 5
  • VL-CDR3 as shown in SEQ ID NO: 15;
  • VH-CDR1 as shown in SEQ ID NO: 16
  • VH-CDR2 as shown in SEQ ID NO: 17
  • VH-CDR3 as shown in SEQ ID NO: 18
  • VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 5
  • VL-CDR3 as shown in SEQ ID NO: 19;
  • VH-CDR1 as shown in SEQ ID NO: 20, VH-CDR2 as shown in SEQ ID NO: 21, VH-CDR3 as shown in SEQ ID NO: 22; as shown in SEQ ID NO: 23 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 24, VL-CDR3 as shown in SEQ ID NO: 25;
  • VH-CDR1 as shown in SEQ ID NO: 26, VH-CDR2 as shown in SEQ ID NO: 27, VH-CDR3 as shown in SEQ ID NO: 28; as shown in SEQ ID NO: 29 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 24, VL-CDR3 as shown in SEQ ID NO: 30;
  • VH-CDR1 as shown in SEQ ID NO: 31, VH-CDR2 as shown in SEQ ID NO: 32, VH-CDR3 as shown in SEQ ID NO: 33; as shown in SEQ ID NO: 34 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 35, VL-CDR3 as shown in SEQ ID NO: 36;
  • VH-CDR1 as shown in SEQ ID NO: 26, VH-CDR2 as shown in SEQ ID NO: 37, VH-CDR3 as shown in SEQ ID NO: 38; as shown in SEQ ID NO: 39 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 24, VL-CDR3 as shown in SEQ ID NO: 40;
  • VH-CDR1 as shown in SEQ ID NO: 41
  • VH-CDR2 as shown in SEQ ID NO: 42
  • VH-CDR3 as shown in SEQ ID NO: 43
  • VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 24, VL-CDR3 as shown in SEQ ID NO: 45;
  • VH-CDR1 as shown in SEQ ID NO: 11
  • VH-CDR2 as shown in SEQ ID NO: 12
  • VH-CDR3 as shown in SEQ ID NO: 47
  • VL-CDR1 VL-CDR2 as shown in SEQ ID NO: 5
  • VL-CDR3 as shown in SEQ ID NO: 15;
  • VH-CDR1 as shown in SEQ ID NO: 16
  • VH-CDR2 as shown in SEQ ID NO: 17
  • VH-CDR3 as shown in SEQ ID NO: 137
  • VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 5, VL-CDR3 as shown in SEQ ID NO: 19;
  • VH-CDR1 as shown in SEQ ID NO: 16, VH-CDR2 as shown in SEQ ID NO: 17, VH-CDR3 as shown in SEQ ID NO: 137; as shown in SEQ ID NO: 4 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 5, VL-CDR3 as shown in SEQ ID NO: 138;
  • VH-CDR1 as shown in SEQ ID NO: 31, VH-CDR2 as shown in SEQ ID NO: 32, VH-CDR3 as shown in SEQ ID NO: 33; as shown in SEQ ID NO: 136 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 35, VL-CDR3 as shown in SEQ ID NO: 36;
  • VH-CDR1 as shown in SEQ ID NO: 147, VH-CDR2 as shown in SEQ ID NO: 42 and VH-CDR3 as shown in SEQ ID NO: 43; as shown in SEQ ID NO: 44 VL-CDR1, VL-CDR2 as shown in SEQ ID NO: 24, VL-CDR3 as shown in SEQ ID NO: 45;
  • variable region of the heavy chain and the variable region of the light chain contained in the antibody molecule or a fragment thereof may be selected from the following combinations:
  • amino acid sequence shown in SEQ ID NO: 50 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 50; and, the amino acid sequence shown in SEQ ID NO: 51 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 51;
  • amino acid sequence shown in SEQ ID NO: 54 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 54; and, the amino acid sequence shown in SEQ ID NO: 55 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 55;
  • amino acid sequence shown in SEQ ID NO: 58 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 58; and, the amino acid sequence shown in SEQ ID NO: 59 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 59;
  • amino acid sequence shown in SEQ ID NO: 64 or the amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 64; and, the amino acid sequence shown in SEQ ID NO: 65 Or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 65;
  • the antibody molecules or fragments of the present invention can be monoclonal antibodies, single-chain antibodies, bifunctional antibodies, single-domain antibodies, nanobodies, fully or partially humanized antibodies or chimeric antibodies, etc., or the antibody Molecules or fragments thereof can be half-antibodies or antigen-binding fragments of half-antibodies, such as scFv, BsFv, dsFv, (dsFv) 2 , Fab, Fab', F(ab') 2 or Fv; for antibody fragments, antibodies are particularly preferred The antigen-binding fragment.
  • the antibody molecule or fragment thereof further comprises a human or murine constant region, preferably a human or murine light chain constant region (CL) and/or a heavy chain constant region (CH);
  • a human or murine constant region preferably a human or murine light chain constant region (CL) and/or a heavy chain constant region (CH);
  • the antibody molecule or fragment thereof comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and/or a kappa or lambda light chain constant region.
  • the antibody molecule is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is IgG1 or IgG4 Subtype, the light chain constant region is ⁇ type;
  • the heavy chain constant region of the monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 106 or an amino acid sequence having at least 75% identity with the amino acid sequence;
  • the light chain constant region of the monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 107 or an amino acid sequence having at least 75% identity with the amino acid sequence.
  • the "at least 75% identity” mentioned herein is, for example, at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or even 99% identity, etc. Any percentage of identity ⁇ 75%.
  • the present invention also provides a conjugate or fusion protein comprising the antibody or fragment thereof of the present invention.
  • the conjugate or fusion protein may include other parts that are bound to the antibody or fragments of the present invention by chemical or physical methods, such as cell surface receptors, small molecule compounds such as amino acids and carbohydrates, small molecule polymers, or other parts of the present invention. Any other part of the antibody that is modified, or even an active protein or polypeptide.
  • the present invention also provides a nucleic acid molecule that encodes the heavy chain CDR, light chain CDR, light chain variable region, heavy chain variable region, heavy chain, or light chain in any antibody or fragment thereof of the present invention.
  • the present invention provides a vector comprising the nucleic acid molecule of the present invention.
  • the vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, and the like.
  • the vector or nucleic acid molecule of the present invention can be used to transform or transfect a host cell or enter the host cell in any manner for the purpose of preservation or expression of antibodies.
  • the present invention provides a host cell comprising the nucleic acid molecule and/or vector of the present invention, or the host cell is transformed or transfected by the nucleic acid molecule and/or vector of the present invention.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.
  • the antibody molecules or fragments thereof and corresponding conjugates or fusion proteins, nucleic acid molecules, vectors and/or host cells provided by the present invention can be obtained by using any conventional technical methods known in the art.
  • the antibody molecules or fragments thereof, conjugates or fusion proteins, nucleic acid molecules, vectors and/or host cells can be included in pharmaceutical compositions, more particularly in pharmaceutical preparations, so as to be used for each according to actual needs. kind of purpose.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody molecule or fragments, conjugates or fusion proteins, nucleic acid molecules, vectors and/or host cells of the present invention. , And optional pharmaceutically acceptable excipients.
  • the present invention provides the use of the above-mentioned antibody molecules or fragments, conjugates or fusion proteins, nucleic acid molecules, vectors, host cells, and/or pharmaceutical compositions in the preparation of medicines, which are used for treatment and CD38 expression.
  • the disease is a hematological malignancy, preferably multiple myeloma or non-Hodgkin's lymphoma.
  • the present invention provides a method for preventing or treating diseases related to CD38 expression or CD38-mediated, the method comprising administering to a subject in need the antibody molecule of the present invention or a fragment, conjugate, or Fusion proteins, nucleic acid molecules, vectors, host cells and/or pharmaceutical compositions, and optionally other drugs or means.
  • the optional other drugs or means refer to other immune enhancing drugs or means that can be administered in combination with the antibody molecule of the present invention or its fragments, conjugates or fusion proteins, nucleic acid molecules, vectors, host cells and/or pharmaceutical compositions,
  • small molecule chemicals, targeted drugs, antibodies and other recombinant protein drugs, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapy drugs, and radiotherapy can be carried out in any form, for example, simultaneously, continuously or at intervals.
  • the disease is a hematological malignancy, preferably multiple myeloma or non-Hodgkin's lymphoma.
  • the subject is a mammal, preferably, the subject is a human.
  • the present invention provides a kit comprising the antibody molecule or fragments thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell and/or pharmaceutical composition of the present invention.
  • CD38 also known as cyclic adenosine diphosphate ribose hydrolase, is a type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain.
  • CD38-mediated signal transduction including lymphocyte proliferation, cytokine release, regulation of B cell and bone marrow cell development and survival, and induction of dendritic cell maturation.
  • CD38 is dysregulated in many hematopoietic malignancies and cell lines derived from various hematopoietic malignancies.
  • Hematopoietic malignancies include non-Hodgkin's lymphoma (NHL) and Burkitt’s lymphoma.
  • Burkitt's lymphoma BL
  • multiple myeloma MM
  • B-CLL B chronic lymphocytic leukemia
  • ALL T acute Lymphoma
  • T cell lymphoma TCL
  • acute myelogenous Leukemia AML
  • HCL Hairy Cell Leukemia
  • HCL Hodgkin's Lymphoma
  • CML Chronic Myelogenous Leukemia
  • CD38- the most primitive pluripotent stem cell of the hematopoietic system is CD38-.
  • amino acid mutation means amino acid substitutions, insertions and/or deletions in the polypeptide sequence, or changes to a part that is chemically linked to a protein.
  • the mutation can be a change in the carbohydrate or PEG structure attached to the protein.
  • Amino acid mutation means herein an amino acid substitution, insertion and/or deletion in a polypeptide sequence.
  • amino acid mutations are generally for amino acids encoded by DNA, such as the 20 amino acids that have codons in DNA and RNA.
  • amino acid substitution or “substitution” means herein that an amino acid at a specific position in the parent polypeptide sequence is replaced by a different amino acid. Specifically, in some embodiments, the substitution is for a non-naturally occurring amino acid at a specific position, and these amino acids are not naturally occurring in the organism or in any organism.
  • substitution E272Y refers to a variant polypeptide in which the glutamic acid at position 272 is replaced by tyrosine, in this case an Fc variant.
  • the protein is engineered to change the nucleic acid coding sequence without changing the starting amino acid (e.g.
  • CGG encoding arginine
  • CGA still encoding arginine to increase the expression level of the host organism
  • Amino acid substitution that is to say, although a new gene encoding the same protein is produced, if the protein has the same amino acid at a specific position at its beginning, it is not an amino acid substitution.
  • amino acid insertion or “insertion” means the addition of an amino acid sequence at a specific position in the parent polypeptide sequence.
  • -233E or 233E indicates that glutamic acid is inserted after position 233 and before position 234.
  • -233ADE or A233ADE indicates that AlaAspGlu is inserted after position 233 and before position 234.
  • amino acid deletion or “deletion” means the removal of the amino acid sequence at a specific position in the parent polypeptide sequence.
  • G236- or G236# or G236del indicates the deletion of glycine at position 236.
  • EDA233- or EDA233# indicates that the sequence GluAspAla is deleted from position 233.
  • residue means a position in a protein and the identity of its related amino acid.
  • Asparagine 297 also known as Asn297 or N297
  • residue 297 in the human antibody IgG1 is residue 297 in the human antibody IgG1.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complementary-dependent cytotoxicity
  • IgG and IgM antibodies which when combined with surface antigens trigger a typical complement pathway, including the formation of membrane attack complexes and target cell lysis.
  • the antibody of the present invention binds to CD38, it induces CDC against CD38+ cancer cells.
  • antibody should be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (ie, "complete antibody molecules") and antigen-binding fragments thereof.
  • antigen-binding portion of an antibody, “antigen-binding fragment” of an antibody, and similar terms include any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide that specifically binds to an antigen to form a complex Or glycoprotein.
  • the term “antigen-binding fragment” or “antibody fragment” of an antibody refers to one or more fragments of the antibody that retain the ability to specifically bind to hemolysin A.
  • Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, CDR-containing fragments, or isolated CDRs.
  • Antigen-binding fragments of antibodies can be derived from, for example, complete antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombination involving manipulation and expression of the variable and (optionally) constant domains of the DNA-encoding antibody Genetic engineering technology. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including e.g. phage-antibody libraries), or may be synthetic.
  • DNA can be sequenced and manipulated chemically or by using molecular biology techniques, such as arranging one or more variable and/or constant domains into a suitable configuration, or introducing codons; forming cysteine residues; Modification, addition or removal of amino acids, etc.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragment; and (vii) the smallest recognition unit (for example, isolated complementarity determining region (CDR), such as CDR3 peptide) composed of amino acid residues in the hypervariable region of the mimic antibody, or restricted FR3-CDR3- FR4 peptide.
  • CDR complementarity determining region
  • engineered molecules such as the following are also encompassed in the expression "antigen-binding fragment” as used herein: domain-specific antibodies, single-domain antibodies, domain-deletion antibodies, chimeric antibodies, CDR-grafted antibodies , Bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, mini-antibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIP) and shark variable IgNAR domains.
  • SMIP small modular immunopharmaceuticals
  • the antigen-binding fragment of an antibody will typically contain at least one variable domain.
  • the variable domain can be of any size or amino acid composition, and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences.
  • the VH and VL domains can be positioned relative to each other in any suitable arrangement.
  • the variable region can be dimeric and contain VH-VH, VH-VL, or VL-VL dimers.
  • the antigen-binding fragment of the antibody may contain a monomeric VH or VL domain.
  • the antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
  • variable and constant domains that can be found in the antigen-binding fragments of the antibodies of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; ( iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; ( x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL.
  • variable and constant domains may be directly linked to each other or may be linked through a full or partial hinge or connector region .
  • the hinge region can be composed of at least 2 (for example, 5, 10, 15, 20, 40, 60 or more) amino acids, which produce flexible or semi-flexible bonds between adjacent variable and/or constant domains in a single polypeptide molecule United.
  • the antigen-binding fragment of the antibody of the present invention may comprise any of the variable and constant domain configurations listed above non-covalently associated with each other and/or with one or more monomeric VH or VL domains (e.g. Through disulfide bonds) homodimers or heterodimers (or other multimers).
  • the antigen-binding fragments can be monospecific or multispecific (e.g., bispecific).
  • Multispecific antigen-binding fragments of antibodies will typically contain at least two different variable domains, where each variable domain is capable of specifically binding to a separate antigen or different epitopes on the same antigen.
  • Any multispecific antibody format (including the exemplary bispecific antibody format disclosed herein) may be suitable for use in the context of the antigen-binding fragments of the antibodies of the present invention using conventional techniques available in the art.
  • murine antibodies are obtained by hybridoma technology, and candidate murine antibodies are obtained by antibody activity analysis (ELISA binding, epitope competition with control antibodies, affinity kinetics); then, the variable regions of the light and heavy chains of the candidate murine antibodies are encoded
  • the sequence was cloned to the upstream of the sequence encoding the constant region of the light and heavy chains of human antibodies, and expressed in mammalian cells to prepare chimeric antibodies.
  • the complementary dependent cytotoxicity (CDC) of the chimeric antibodies on effector cells was verified , Antibody-dependent cell-mediated cytotoxicity (ADCC), and verification of the apoptotic activity of Duadi-luc; then select the humanized template according to the Germline database, carry out the humanized design of the antibody sequence, and obtain the human source
  • the antibody activity analysis was performed again to verify the complementation-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) of the humanized modified antibody on the effector cells, and the cell-mediated cytotoxicity of Duadi-luc.
  • Apoptosis activity and other in vitro cytological experiments are verified; then the in vitro physical and chemical properties of the humanized antibody are analyzed, and finally a new humanized anti-CD38 antibody sequence with affinity and specificity equal to or better than that of the control antibody is obtained.
  • the antibody molecule provided by the present invention has close or consistent antigen binding recognition epitopes with known antibodies, and at the same time has the same or better functional activities as known antibodies, including in vitro ADCC, CDC and Daudi cell apoptosis activities Wait.
  • Figure 1 shows the results of flow cytometry identification of CD38 expressing cell lines, where Figure 1A: cell line CHO blank; Figure 1B: cell line 1-T-12; Figure 1C: cell line 1-T-14.
  • Figure 2 shows the results of the first round (Figure 2A) and second round (Figure 2B) fusion screening of hybridoma cells.
  • Figure 3 shows the binding activity of the antibody of the present invention to the antigen CD38 expressed on the cell surface.
  • Figure 4 shows the antibody-dependent cytotoxicity of the antibodies of the present invention, where Figure 4A: 8B7 humanized antibody; Figure 4B: 20M19 humanized antibody; Figure 4C: 32A7 humanized antibody; Figure 4D: 5E19 humanized antibody Antibody.
  • Figure 5 shows the results of epitope analysis of the antibody of the present invention, in which Figure 5A: relative to Daratumumab; Figure 5B: relative to Mor202; Figure 5C: relative to Isatuximab.
  • Figure 6 shows the serum stability results of the antibody of the present invention, in which Figure 6A: Daratumumab; Figure 6B: 8B7-H3L3; Figure 6C: 17H13-H2L2; Figure 6D: 20M19-H1L1.
  • the heavy chain constant region sequence of the human monoclonal antibody IgG1 subclass is shown in SEQ ID NO: 106 and SEQ ID NO: 126
  • the light chain constant region sequence of the human monoclonal antibody ⁇ subclass is shown in SEQ ID NO: 107 and SEQ ID NO: 127.
  • Recombinant human CD38 protein purchased from ACRO biosystem, Cat No: CD8-H5224.
  • the fully synthesized Daratumumab, Isatuximab and Mor202 antibody light chain variable region and heavy chain variable region genes were cloned into eukaryotic expression vectors containing the human-kappa light chain constant region and human IgG1 heavy chain constant region coding genes upstream, respectively.
  • the light and heavy chain expression plasmids of Daratumumab, Isatuximab and Mor202 were obtained respectively.
  • the obtained plasmid was transferred to E. coli for amplification, and a large number of plasmids containing the light and heavy chains of Daratumumab, Isatuximab or Mor202 antibody were isolated and then mixed with PEI and co-transfected into HEK293 cells.
  • the cells were transfected for 5-6 days, the culture supernatant was taken, and the expression supernatant was purified by Mabselect affinity chromatography column to obtain Daratumumab, Isatuximab and Mor202 antibody recombinant proteins.
  • Daratumumab See SEQ ID NO: 128 to SEQ ID NO: 129 for the sequence.
  • Isatuximab See SEQ ID NO: 130 to SEQ ID NO: 131 for the sequence.
  • Mor202 For the sequence, see SEQ ID NO: 132 to SEQ ID NO: 133.
  • the human CD38 high expressing CHO cell line 1-T-14 and the human CD38 low expressing CHO cell line 1-T-12 were prepared.
  • the CD38 gene reading frame was cloned from the vector containing CD38 cDNA (Beijing Yiqiao Shenzhou, Cat:HG10818-UT), and the CD38 gene reading frame (which has been sequenced to verify the correct sequence) was digested by restriction enzymes It was cloned into a stable expression vector containing the screening gene for glutamine synthetase (GS), electrotransfected (Nucleofector IIb, Lonza) suspension cultured CHO-K1 cells, and the transfected cells were placed in a 50 ⁇ M MSX (Sigma , Cat: M5379) CD CHO AGTTM medium (Gibco, Cat: 12490-025), seeded in 96-well cell culture plate, 37°C, 5% CO 2 static culture for 2-3 weeks, screening by MSX pressure , Pre-screening under the microscope, magnified culture, and finally selected 2 clones (1-T-12 and 1-T-14) by flow cytometry (FACS), which were magnified culture and
  • Balb/c mice aged 8-10 weeks were immunized with Freund’s adjuvant and water-soluble adjuvant.
  • Freund’s adjuvant was injected twice into the intraperitoneal cavity on day 0 and day 14, and water-soluble adjuvant On day 0 and day 21, 8-10 week-old Balb/c mice were injected intramuscularly twice.
  • the immune antigen was: human CD38 recombinant protein, which was recombinantly expressed in HEK293 cells by Acro Biosytem Biotechnology Co., Ltd. .
  • the dose for the first immunization was 50 ⁇ g, and the dose for the second immunization was 25 ⁇ g. Before immunization, the mouse serum was taken as a negative control for the test.
  • Freund’s adjuvant was taken on the 28th day after the initial immunization, and the water-soluble adjuvant was taken from the tail vein on the 35th day after the initial immunization.
  • 96 wells coated with recombinant human CD38 protein were used.
  • the ELISA method was used to detect the serum titer on the enzyme-labeled plate; mice whose serum titer reached the fusion requirement were boosted, and 25 ⁇ g of antigen was diluted with D-PBS into 500 ⁇ l, and injected into the abdominal cavity. Spleen cells from mice with high serum titers were taken from 18 to 38 days after the initial immunization for the next cell fusion.
  • mice with the required titer were selected, the eyeballs were removed, and the blood was collected, and the serum was separated as the positive control serum for antibody detection; and the mouse spleen was aseptically taken, and the B lymphocyte suspension was prepared according to the conventional method.
  • ECF Electro Cell Fusion
  • the myeloma cells P3X63Ag8.653 and B lymphocytes were mixed at a ratio of 4:1 and then fused by electric shock. After the electric shock, place the cells in a 37°C CO 2 incubator, stand still for 30 minutes, 1000RPM, centrifuge at room temperature for 10 minutes, resuspend the cells in 360ml hybridoma selection medium, and plate them into a 384-well plate, ensuring that the plating density is 8000 ⁇ 20,000 cells/well. The medium was changed once after 2-3 days, and positive hybridomas were screened on 7-10 days.
  • the fused hybridoma cells were cultured in a 384-well plate, and the supernatant was used to analyze the antibodies secreted by the hybridoma cells by FACS.
  • Several clones were screened. These clones can bind to the human CD38 high-expressing strain 1-T-14 but not. CHO-blank cells. The selected clones were single-celled by the limiting dilution method, and each hybridoma clone obtained after 3 rounds secreted only one antibody.
  • the total RNA of the cells is extracted according to the instructions of the RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.); the total RNA of the hybridomas is obtained by using 5 ⁇ PrimeScript RT Master Mix (Takara) Reverse transcription into cDNA; use degenerate primers (Anke Krebber.
  • the heavy chain variable region sequence of the murine anti-human CD38 monoclonal antibody and the published heavy chain constant region sequence of the human monoclonal antibody IgG1 subclass were spliced together to construct the mammalian cell expression vector pCDNA3.4;
  • the light chain variable region sequence of the murine anti-human CD38 monoclonal antibody and the published light chain constant region sequence of the human monoclonal antibody ⁇ subclass are spliced together and constructed into the mammalian cell expression vector pCDNA3.1.
  • the heavy chain vector and light chain vector of the constructed anti-human CD38 chimeric antibody were paired and mixed, and HEK293 cells were transfected with polyethyleneimine (PEI). After about 7 days, the cell supernatant was collected and purified by Mabselect to obtain the anti-human CD38 chimeric antibody. ⁇ Antibody protein.
  • murine antibody named-xiIgG The resulting chimeric antibody is referred to herein as "murine antibody named-xiIgG”.
  • VH and VL The light and heavy chain variable region (VH and VL) sequences from part of the murine antibody are shown below, and the antigen complementarity determinants (CDR) of the heavy chain and light chain are underlined.
  • the results show that all murine antibodies with the above light and heavy chains have a better binding effect with human CD38 recombinant protein than other murine antibodies.
  • the cell binding experiment has obvious effects and has stronger complement-dependent cytotoxicity and cytotoxicity. Antibody-dependent cytotoxicity; and, after being converted to chimeric antibodies, the effects of murine antibodies are retained, and the binding activity is similar to or higher than that of the control antibodies Daratumumab or Isatuximab.
  • Integrating the antibody coding schemes of Kabat and Chothia the amino acid sequence regions of the six antigen complementarity determinants (CDR) of the heavy and light chains of the murine antibody and the framework region supporting the conservative three-dimensional conformation of the antibody are determined.
  • CDR complementarity determinants
  • N asparagine glycosylation site
  • N deamidation site N deamidation site
  • D aspartic acid
  • the humanized antibody heavy chain variable region gene was constructed into a mammalian cell expression vector containing the heavy chain constant region gene of the human monoclonal antibody IgG1 subclass; the light chain variable region gene was constructed into the human monoclonal antibody ⁇ subclass Class of light chain constant region genes in mammalian cell expression vectors.
  • the constructed anti-human CD38 humanized antibody heavy chain vector and light chain vector are paired and mixed, and HEK293 cells are transfected with polyethyleneimine (PEI). After about 7 days, the cell supernatant is collected and purified by Mabselect to obtain anti-human CD38 human Sourced antibody protein.
  • PEI polyethyleneimine
  • the partial light and heavy chain variable region sequences obtained by humanization are shown below, in which the antigenic complementarity determinants (CDR) of the heavy and light chains are underlined.
  • the humanized antibody is named "murine antibody named-HmLn", where m and n are the numbers of the humanized (hz) engineered sequences (VH_hz and VL_hz) of VH and VL, respectively.
  • the anti-human antibody capture method was used to determine the antibody affinity.
  • the capture antibody (AHC) biological probe of the Fc segment of the anti-human antibody in PBS for 10 minutes; 200 ⁇ l of the diluted antibody sample (including the chimeric antibody of the present invention, humanized antibody and control antibody Daratumumab; antibody working concentration 15 ⁇ g/mL) was loaded onto the AHC bioprobe, and then equilibrated in PBS for 100s, and then the AHC probe was combined with human CD38 protein (ACRO biosystem, Cat No. CD8-H5224) of different dilution concentrations for binding reaction.
  • ACRO biosystem Cat No. CD8-H5224
  • the time is 600s, after which the AHC probe is transferred to PBS for dissociation reaction, and the time is 600s.
  • the blank control response value was subtracted, and the software was used to perform 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding.
  • the cells were mixed with the final concentration of 50 ⁇ g/ml to 7.6ng/ml antibody and control antibody, and then 6.25% human serum complete complement dissolved in cell culture medium RPMI-1640 was added to the cells, and incubated at 37°C3 hour. Afterwards, the cytotoxicity test was performed by the CCK-8 kit, and finally the absorbance at 450 nm was detected by the MD microplate reader. Use softmax pro7 software to fit a 4-parameter curve by absorbance value to calculate the EC50 of the sample.
  • ADCC Antibody Dependent Cytotoxicity
  • the antibody of the present invention and the control antibody Daratumumab (Dara) were tested to induce antibody-dependent cytotoxicity (ADCC) against the CHO cell line 1-T-14 stably expressing human CD38.
  • the engineered Jurkat cells are used as effector cells, which stably express the Fc ⁇ RIIIa-Fc ⁇ RIa ⁇ hybrid receptor and are driven by the NFAT response element to express firefly luciferase.
  • the biological activity of the antibody in the ADCC mechanism of action is quantified by luciferase produced by the activation of the NFAT pathway.
  • the 1.5E5 effector cells were mixed with the antibody of the present invention and the control antibody Daratumumab at a final concentration of 33 ⁇ g/ml to 85pg/ml, and then 2.5E4 target cells, namely the CHO cell line 1-T-14 stably expressing human CD38 (E:T ratio of effector cells to target cells is 6:1) was added and incubated at 37°C for 16 hours. Afterwards, it was detected by the Promega Kit Bio-Glo TM Luciferase Assay System, and finally the LUM value was detected by the MD microplate reader, and the induction factor and EC50 were calculated using the following formula.
  • Induction factor (reading value of tested well-background value)/(reading value of negative control well-background value)
  • the induction multiple was fitted with a 4-parameter curve using prism7 software, and the EC50 of the sample was calculated.
  • a GE BIAcore instrument S200 was used to determine the antibody-antigen interaction force.
  • Kit Alex FluorTM 488 Annexin V/Dead Cell Apoptosis Kit (invitrogen, Cat: V13245, Lot: 1923636)
  • Cross-linked and “non-cross-linked” in the table refer to the form of bridged Fab and non-bridged Fab, respectively.
  • the monoclonal supernatant containing the murine antibody of the present invention is first used to bind the antigen cell, ie, the human CD38 highly expressing CHO cell line 1-T-14, and then the control antibody is added to compete with the murine antibody to compete with the epitope. Combine. The binding signal of the labeled control antibody is detected. The lower the binding signal of the control antibody is, the more it can be proved that the murine antibody occupies the epitope recognized by the control antibody, and that the antigen binding epitope of the antibody of the present invention is close to or consistent with that of the control antibody.
  • Analytical method Inject 50 ⁇ l of antibody with a concentration of 1mg/ml into the pre-equilibrated chromatography column, flow at room temperature and flow rate of 0.7ml/min for 35 minutes, and detect the absorption values of the machines A280 and A214 at the same time, and enter according to the peak output. Time and peak volume are used to determine the monomer content and ratio of the antibody.
  • Table 11 The retention time and monomer ratio of the main peak of the antibody of the present invention
  • Analysis method Analyze the hydrophobic nature of the antibody according to the instructions for the hydrophobic chromatography column.
  • the antibody to be tested is diluted to 2 ⁇ g/ml with the antibody diluent, and diluted 3 times in 8 gradients;
  • mice Female Balb/C mice, 3 mice/group, respectively in the tail vein or abdominal cavity, 200 ⁇ g antibody to be tested/mouse;
  • the antibody diluent dilutes the antibody standard, and the dilution of the standard is still adjusted according to the preliminary experiment, so that the standard is fitted to a linear curve (if appropriate software is available, an S-shaped curve can also be fitted).

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Abstract

提供了一种结合人CD38的抗体分子或其片段及其在预防或治疗疾病中的应用。该抗体分子是经过免疫小鼠，与骨髓瘤细胞融合产生杂交瘤细胞后，筛选得到的抗人CD38抗体分子。

Description

抗人CD38抗体及其应用
本专利申请要求于2019年9月20日提交的申请号为CN201910889707.5的中国发明专利申请的优先权权益，在此将其全部内容引入作为参考。
技术领域
本发明属于生物医药领域，涉及一种新的抗CD38抗体或其功能性片段。本发明还涉及所述抗体或其功能性片段的应用。
背景技术
CD38为一种II型跨膜糖蛋白，分子量46KD，包括C端胞外区(258个氨基酸)、跨膜区(21个氨基酸)和N端(21个氨基酸)，功能表现为受体介导的粘附和信号传导，以及经由其胞外酶活性介导钙动员，催化环状ADP核糖(cADPR)和ADPR的形成。
CD38在多发性骨髓瘤细胞(MM)上均一性地高表达，而在正常淋巴细胞和骨髓细胞以及一些非造血起源的组织上低表达。由于CD38属于跨膜糖蛋白，具有体外酶活性，同时还具有受体和粘连分子的作用，因而是MM治疗的理想靶点；基于其独特的作用机制、低毒性和单剂活性，CD38抗体也适用于联合方案。目前，有多个研究正在探索以CD38抗体为基础的疗法应用于新确诊的高危的MM患者的疗效。除此之外，CD38抗体用于其他血液性恶性肿瘤(急性淋巴细胞白血病、NK/T细胞淋巴瘤和急性髓性白血病)的治疗效果也在研究当中。
目前有多家制药公司在研发针对CD38的单克隆抗体，此类抗体通过抗体依赖性细胞介导的细胞毒效应(ADCC)、补体依赖的细胞毒效应(CDC)、抗体依赖的细胞吞噬作用(ADPC)以及直接抑制CD38的酶活性来达到抗MM的机制。目前，处于临床试验阶段或已获批的CD38抗体包括Daratumumab(Darzalex)、Isatuximab、MOR-202和TAK-079等。
在目前针对CD38开发的单克隆抗体药物中，强生的Darzalex是全球获批的首个CD38介导性、溶细胞性的抗体药物，具有广谱杀伤活性，可靶向结合多发性骨髓瘤及多种实体瘤细胞表面高度表达的跨膜胞外酶CD38分子，通过多种免疫介导的作用机制诱导肿瘤细胞的快速死亡，包括互补依赖 性细胞毒作用(CDC)、抗体依赖性细胞介导的细胞毒作用(ADCC)和抗体依赖性细胞吞噬作用(ADCP)以及通过细胞凋亡(apoptosis)。此外，Darzalex也已被证明能够靶向肿瘤微环境中的免疫抑制细胞从而表现出免疫调节活性。除了多发性骨髓瘤，Darzalex也有潜力治疗高表达CD38分子的其他类型肿瘤，包括弥漫性大B细胞淋巴癌(DLBCL)、慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL)、浆细胞性白血病(PCL)、急性髓性白血病(AML)、滤泡性淋巴瘤(FL)和套细胞淋巴瘤(MCL)等。
此外，新的抗CD38抗体的开发和应用是本领域所需要的。
发明内容
本发明要解决的技术问题是，通过杂交瘤筛选和人源化技术，获得特异性结合CD38、特别是人CD38的高亲和力抗体。
针对上述技术问题，本发明的目的是提供一种特异性结合CD38、特别是人CD38的抗体或其片段，并基于该抗体或其片段，提供其用途。本发明所述抗体分子的“片段”涵盖抗体的各种功能性片段，例如其抗原结合部分，如Fab、F(ab’) 2或scFv片段。
具体而言，本发明提供以下技术方案。
一方面，本发明提供一种抗体分子或其片段，具有以下重链CDRs区：
VH-CDR1：具有SEQ ID NO:16所示氨基酸序列或在SEQ ID NO:16所示氨基酸序列的基础上进行一个或几个氨基酸突变；
VH-CDR2：具有SEQ ID NO:17所示氨基酸序列或在SEQ ID NO:17所示氨基酸序列的基础上进行一个或几个氨基酸突变；
VH-CDR3：具有SEQ ID NO:18所示氨基酸序列或在SEQ ID NO:18所示氨基酸序列的基础上进行一个或几个氨基酸突变；
以及，以下轻链CDRs区：
VL-CDR1：具有SEQ ID NO:4所示氨基酸序列或在SEQ ID NO:4所示氨基酸序列的基础上进行一个或几个氨基酸突变；
VL-CDR2：具有SEQ ID NO:5所示氨基酸序列或在SEQ ID NO:5所示氨基酸序列的基础上进行一个或几个氨基酸突变；
VL-CDR3：具有SEQ ID NO:19所示氨基酸序列或在SEQ ID NO:19所示氨基酸序列的基础上进行一个或几个氨基酸突变；
其中，所述抗体CDRs区的氨基酸突变使突变后抗体6个CDRs组合能够形成抗原结合位点，且至少部分的保留了CDRs区突变前抗体分子或其片段的生物活性。
优选的，本发明所述抗体分子或其片段，其中所述CDRs区突变前抗体分子或其片段包括SEQ ID NO:16所示的VH-CDR1、SEQ ID NO:17所示的VH-CDR2、SEQ ID NO:18所示的VH-CDR3、SEQ ID NO:4所示的VL-CDR1、SEQ ID NO:5所示的VL-CDR2、SEQ ID NO:19所示的VL-CDR3。
优选的，本发明所述的抗体或其片段，所述抗体分子或其片段特异性结合细胞膜表面CD38，且能够通过结合细胞膜表面CD38诱导CD38+细胞凋亡。
优选的，本发明所述的抗体分子或其片段，所述抗体分子或其片段对于CD38+细胞具有抗体依赖性细胞介导的细胞毒作用(ADCC)、补体依赖性细胞毒作用(CDC)。
优选的，本发明所述的抗体分子或其片段，所述抗体分子或其片段能够与至少一种选自下组的抗体分子竞争结合CD38：Daratumumab、Isatuximab、Mor202。
在一个实施方式中，本发明所述的抗体分子或其片段，所述抗体分子或其片段包含重链可变区(VH)，其中所述重链可变区包含选自如SEQ ID NO:1、SEQ ID NO:7、SEQ ID NO:11、SEQ ID NO:16、SEQ ID NO:20、SEQ ID NO:26、SEQ ID NO:31、SEQ ID NO:41和SEQ ID NO:147所示的CDR1(VH-CDR1)，选自如SEQ ID NO:2、SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:17、SEQ ID NO:21、SEQ ID NO:27、SEQ ID NO:32、SEQ ID NO:37和SEQ ID NO:42所示的CDR2(VH-CDR2)，和选自如SEQ ID NO:3、SEQ ID NO:9、SEQ ID NO:13、SEQ ID NO:18、SEQ ID NO:22、SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:38、SEQ ID NO:43、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48和SEQ ID NO:137所示的CDR3(VH-CDR3)；和
所述抗体分子或其片段包含轻链可变区(VL)，其中所述轻链可变区包含选自如SEQ ID NO:4、SEQ ID NO:14、SEQ ID NO:23、SEQ ID NO:29、SEQ ID NO:34、SEQ ID NO:39、SEQ ID NO:44、SEQ ID NO:49、SEQ ID NO:136和SEQ ID NO:148所示的CDR1(VL-CDR1)，选自如SEQ ID NO:5、SEQ ID NO:24和SEQ ID NO:35所示的CDR2(VL-CDR2)，选自如SEQ  ID NO:6、SEQ ID NO:10、SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:25、SEQ ID NO:30、SEQ ID NO:36、SEQ ID NO:40、SEQ ID NO:45、SEQ ID NO:138和SEQ ID NO:139所示的CDR3(VL-CDR3)。
根据本发明的具体实施方式，所述重链可变区可包含选自以下的CDR组合(VH-CDR1、VH-CDR2、VH-CDR3)：
(1)如SEQ ID NO:1所示的VH-CDR1、如SEQ ID NO:2所示的VH-CDR2、如SEQ ID NO:3所示的VH-CDR3；
(2)如SEQ ID NO:7所示的VH-CDR1、如SEQ ID NO:8所示的VH-CDR2、如SEQ ID NO:9所示的VH-CDR3；
(3)如SEQ ID NO:11所示的VH-CDR1、如SEQ ID NO:12所示的VH-CDR2、如SEQ ID NO:13所示的VH-CDR3；
(4)如SEQ ID NO:16所示的VH-CDR1、如SEQ ID NO:17所示的VH-CDR2、如SEQ ID NO:18所示的VH-CDR3；
(5)如SEQ ID NO:20所示的VH-CDR1、如SEQ ID NO:21所示的VH-CDR2、如SEQ ID NO:22所示的VH-CDR3；
(6)如SEQ ID NO:26所示的VH-CDR1、如SEQ ID NO:27所示的VH-CDR2、如SEQ ID NO:28所示的VH-CDR3；
(7)如SEQ ID NO:31所示的VH-CDR1、如SEQ ID NO:32所示的VH-CDR2、如SEQ ID NO:33所示的VH-CDR3；
(8)如SEQ ID NO:26所示的VH-CDR1、如SEQ ID NO:37所示的VH-CDR2、如SEQ ID NO:38所示的VH-CDR3；
(9)如SEQ ID NO:41所示的VH-CDR1、如SEQ ID NO:42所示的VH-CDR2、如SEQ ID NO:43所示的VH-CDR3；
(10)如SEQ ID NO:11所示的VH-CDR1、如SEQ ID NO:12所示的VH-CDR2、如SEQ ID NO:46所示的VH-CDR3；
(11)如SEQ ID NO:11所示的VH-CDR1、如SEQ ID NO:12所示的VH-CDR2、如SEQ ID NO:47所示的VH-CDR3；
(12)如SEQ ID NO:11所示的VH-CDR1、如SEQ ID NO:12所示的VH-CDR2、如SEQ ID NO:48所示的VH-CDR3；
(13)如SEQ ID NO:16所示的VH-CDR1、如SEQ ID NO:17所示的VH-CDR2、如SEQ ID NO:137所示的VH-CDR3；
(14)如SEQ ID NO:147所示的VH-CDR1、如SEQ ID NO:42所示的VH-CDR2、如SEQ ID NO:43所示的VH-CDR3；
和/或，所述抗体分子或其片段包含轻链可变区(VL)，其中所述轻链可变区可包含选自以下的CDR组合(VL-CDR1、VL-CDR2、VL-CDR3)：
(1)如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:6所示的VL-CDR3；
(2)如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:10所示的VL-CDR3；
(3)如SEQ ID NO:14所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:15所示的VL-CDR3；
(4)如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:19所示的VL-CDR3；
(5)如SEQ ID NO:23所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:25所示的VL-CDR3；
(6)如SEQ ID NO:29所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:30所示的VL-CDR3；
(7)如SEQ ID NO:34所示的VL-CDR1、如SEQ ID NO:35所示的VL-CDR2、如SEQ ID NO:36所示的VL-CDR3；
(8)如SEQ ID NO:39所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:40所示的VL-CDR3；
(9)如SEQ ID NO:44所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:45所示的VL-CDR3；
(10)如SEQ ID NO:49所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:40所示的VL-CDR3；
(11)如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:138所示的VL-CDR3；
(12)如SEQ ID NO:136所示的VL-CDR1、如SEQ ID NO:35所示的VL-CDR2、如SEQ ID NO:36所示的VL-CDR3；
(13)如SEQ ID NO:136所示的VL-CDR1、如SEQ ID NO:35所示的VL-CDR2、如SEQ ID NO:139所示的VL-CDR3；
(14)如SEQ ID NO:148所示的VL-CDR1、如SEQ ID NO:24所示的 VL-CDR2、如SEQ ID NO:45所示的VL-CDR3。
优选地，所述抗体分子或其片段中的重链可变区包含选自SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68至SEQ ID NO:88、SEQ ID NO:140至SEQ ID NO:142中任一个所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的氨基酸序列；和/或
轻链可变区包含选自SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:89至SEQ ID NO:105、SEQ ID NO:134和SEQ ID NO:135、SEQ ID NO:143至SEQ ID NO:146中任一个所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的氨基酸序列。
根据本发明的具体实施方式，所述抗体分子或其片段包含选自以下的CDR组合：
(1)如SEQ ID NO:1所示的VH-CDR1、如SEQ ID NO:2所示的VH-CDR2、如SEQ ID NO:3所示的VH-CDR3；如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:6所示的VL-CDR3；
(2)如SEQ ID NO:7所示的VH-CDR1、如SEQ ID NO:8所示的VH-CDR2、如SEQ ID NO:9所示的VH-CDR3；如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:10所示的VL-CDR3；
(3)如SEQ ID NO:11所示的VH-CDR1、如SEQ ID NO:12所示的VH-CDR2、如SEQ ID NO:13所示的VH-CDR3；如SEQ ID NO:14所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:15所示的VL-CDR3；
(4)如SEQ ID NO:16所示的VH-CDR1、如SEQ ID NO:17所示的VH-CDR2、如SEQ ID NO:18所示的VH-CDR3；如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:19所示的VL-CDR3；
(5)如SEQ ID NO:20所示的VH-CDR1、如SEQ ID NO:21所示的VH-CDR2、如SEQ ID NO:22所示的VH-CDR3；如SEQ ID NO:23所示的 VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:25所示的VL-CDR3；
(6)如SEQ ID NO:26所示的VH-CDR1、如SEQ ID NO:27所示的VH-CDR2、如SEQ ID NO:28所示的VH-CDR3；如SEQ ID NO:29所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:30所示的VL-CDR3；
(7)如SEQ ID NO:31所示的VH-CDR1、如SEQ ID NO:32所示的VH-CDR2、如SEQ ID NO:33所示的VH-CDR3；如SEQ ID NO:34所示的VL-CDR1、如SEQ ID NO:35所示的VL-CDR2、如SEQ ID NO:36所示的VL-CDR3；
(8)如SEQ ID NO:26所示的VH-CDR1、如SEQ ID NO:37所示的VH-CDR2、如SEQ ID NO:38所示的VH-CDR3；如SEQ ID NO:39所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:40所示的VL-CDR3；
(9)如SEQ ID NO:41所示的VH-CDR1、如SEQ ID NO:42所示的VH-CDR2、如SEQ ID NO:43所示的VH-CDR3；如SEQ ID NO:44所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:45所示的VL-CDR3；
(10)如SEQ ID NO:11所示的VH-CDR1、如SEQ ID NO:12所示的VH-CDR2、如SEQ ID NO:47所示的VH-CDR3；如SEQ ID NO:14所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:15所示的VL-CDR3；
(11)如SEQ ID NO:16所示的VH-CDR1、如SEQ ID NO:17所示的VH-CDR2、如SEQ ID NO:137所示的VH-CDR3；如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:19所示的VL-CDR3；
(12)如SEQ ID NO:16所示的VH-CDR1、如SEQ ID NO:17所示的VH-CDR2、如SEQ ID NO:137所示的VH-CDR3；如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:138所示的VL-CDR3；
(13)如SEQ ID NO:31所示的VH-CDR1、如SEQ ID NO:32所示的 VH-CDR2、如SEQ ID NO:33所示的VH-CDR3；如SEQ ID NO:136所示的VL-CDR1、如SEQ ID NO:35所示的VL-CDR2、如SEQ ID NO:36所示的VL-CDR3；
(14)如SEQ ID NO:147所示的VH-CDR1、如SEQ ID NO:42所示的VH-CDR2、如SEQ ID NO:43所示的VH-CDR3；如SEQ ID NO:44所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:45所示的VL-CDR3；
优选地，所述抗体分子或其片段包含的重链可变区和轻链可变区可选自以下组合：
(1)如SEQ ID NO:50所示的氨基酸序列或与如SEQ ID NO:50所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:51所示的氨基酸序列或与如SEQ ID NO:51所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(2)如SEQ ID NO:52所示的氨基酸序列或与如SEQ ID NO:52所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:53所示的氨基酸序列或与如SEQ ID NO:53所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(3)如SEQ ID NO:54所示的氨基酸序列或与如SEQ ID NO:54所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:55所示的氨基酸序列或与如SEQ ID NO:55所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(4)如SEQ ID NO:56所示的氨基酸序列或与如SEQ ID NO:56所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:57所示的氨基酸序列或与如SEQ ID NO:57所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(5)如SEQ ID NO:58所示的氨基酸序列或与如SEQ ID NO:58所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:59所示的氨基酸序列或与如SEQ ID NO:59所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(6)如SEQ ID NO:60所示的氨基酸序列或与如SEQ ID NO:60所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:61所示 的氨基酸序列或与如SEQ ID NO:61所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(7)如SEQ ID NO:62所示的氨基酸序列或与如SEQ ID NO:62所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:63所示的氨基酸序列或与如SEQ ID NO:63所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(8)如SEQ ID NO:64所示的氨基酸序列或与如SEQ ID NO:64所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:65所示的氨基酸序列或与如SEQ ID NO:65所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(9)如SEQ ID NO:66所示的氨基酸序列或与如SEQ ID NO:66所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:67所示的氨基酸序列或与如SEQ ID NO:67所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(10)如SEQ ID NO:69所示的氨基酸序列或与如SEQ ID NO:69所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:90所示的氨基酸序列或与如SEQ ID NO:90所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(11)如SEQ ID NO:71所示的氨基酸序列或与如SEQ ID NO:71所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:90所示的氨基酸序列或与如SEQ ID NO:90所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(12)如SEQ ID NO:74所示的氨基酸序列或与如SEQ ID NO:74所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:92所示的氨基酸序列或与如SEQ ID NO:92所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(13)如SEQ ID NO:75所示的氨基酸序列或与如SEQ ID NO:75所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:94所示的氨基酸序列或与如SEQ ID NO:94所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(14)如SEQ ID NO:76所示的氨基酸序列或与如SEQ ID NO:76所示的 氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:94所示的氨基酸序列或与如SEQ ID NO:94所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(15)如SEQ ID NO:77所示的氨基酸序列或与如SEQ ID NO:77所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:95所示的氨基酸序列或与如SEQ ID NO:95所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(16)如SEQ ID NO:83所示的氨基酸序列或与如SEQ ID NO:83所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:102所示的氨基酸序列或与如SEQ ID NO:102所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(17)如SEQ ID NO:84所示的氨基酸序列或与如SEQ ID NO:84所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:102所示的氨基酸序列或与如SEQ ID NO:102所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(18)如SEQ ID NO:85所示的氨基酸序列或与如SEQ ID NO:85所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:134所示的氨基酸序列或与如SEQ ID NO:134所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(19)如SEQ ID NO:88所示的氨基酸序列或与如SEQ ID NO:88所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:103所示的氨基酸序列或与如SEQ ID NO:103所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(20)如SEQ ID NO:88所示的氨基酸序列或与如SEQ ID NO:88所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:104所示的氨基酸序列或与如SEQ ID NO:104所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(21)如SEQ ID NO:140所示的氨基酸序列或与如SEQ ID NO:140所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:143所示的氨基酸序列或与如SEQ ID NO:143所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(22)如SEQ ID NO:141所示的氨基酸序列或与如SEQ ID NO:141所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:144所示的氨基酸序列或与如SEQ ID NO:144所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(23)如SEQ ID NO:142所示的氨基酸序列或与如SEQ ID NO:142所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:144所示的氨基酸序列或与如SEQ ID NO:144所示的氨基酸序列具有至少75％同一性的氨基酸序列。
本发明的抗体分子或其片段可以是单克隆抗体、单链抗体、双功能抗体、单域抗体、纳米抗体、完全或部分人源化的抗体或者嵌合抗体等任意形式，或者，所述抗体分子或其片段可以是半抗体或半抗体的抗原结合片段，例scFv、BsFv、dsFv、(dsFv) 2、Fab、Fab'、F(ab') 2或Fv；关于抗体的片段，特别优选抗体的抗原结合片段。
优选地，所述抗体分子或其片段还包含人或鼠的恒定区，优选包含人或鼠的轻链恒定区(CL)和/或重链恒定区(CH)；
更优选地，所述抗体分子或其片段包含选自IgG、IgA、IgM、IgD或IgE的重链恒定区和/或κ或λ型轻链恒定区。
根据本发明的具体实施方式，所述抗体分子为单克隆抗体，优选为鼠源、嵌合或人源化的单克隆抗体；优选地，所述单克隆抗体的重链恒定区为IgG1或IgG4亚型，轻链恒定区为κ型；
优选地，所述单克隆抗体的重链恒定区包含如SEQ ID NO:106所示的氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列；
优选地，所述单克隆抗体的轻链恒定区包含如SEQ ID NO:107所示氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列。
本文所提出的“至少75％同一性”为例如至少80％、优选至少85％、更优选至少90％、进一步优选至少91％、92％、93％、94％、95％、96％、97％、98％或甚至99％同一性等≥75％的任何百分比的同一性。
基于本发明的抗体分子或其片段，本发明还提供包含本发明的抗体或其片段的缀合物或融合蛋白。该缀合物或融合蛋白可包含通过化学或物理方法结合于本发明所述抗体或其片段的其他部分，例如细胞表面受体、小分子化合物如氨基酸和糖类、小分子聚合物或对本发明所述抗体进行修饰的任何其 它部分，或者甚至是活性蛋白或多肽。
另一方面，本发明还提供一种核酸分子，其编码本发明任意抗体或其片段中的重链CDR、轻链CDR、轻链可变区、重链可变区、重链或轻链。
还一方面，本发明提供一种载体，其包含本发明的核酸分子。所述载体可以为真核表达载体、原核表达载体、人工染色体及噬菌体载体等。本发明的载体或核酸分子可以用于转化或转染宿主细胞或以任何方式进入宿主细胞内，用于保存或表达抗体等目的。
因此，另一方面，本发明提供一种宿主细胞，所述宿主细胞包含本发明的核酸分子和/或载体，或者所述宿主细胞被本发明的核酸分子和/或载体转化或转染。宿主细胞可以是任何原核或真核细胞，例如细菌或昆虫、真菌、植物或动物细胞。
基于本发明的公开内容，本发明提供的抗体分子或其片段以及相应的缀合物或融合蛋白、核酸分子、载体和/或宿主细胞可以通过使用本领域已知的任何常规技术方法获得。所述抗体分子或其片段、缀合物或融合蛋白、核酸分子、载体和/或宿主细胞可以被包含在药物组合物中，更特别地被包含在药物制剂中，从而根据实际需要用于各种目的。
因此，在又一方面，本发明还提供一种药物组合物，所述药物组合物包含本发明所述的抗体分子或其片段、缀合物或融合蛋白、核酸分子、载体和/或宿主细胞，以及任选的药学上可接受的辅料。
还一方面，本发明提供上述抗体分子或其片段、缀合物或融合蛋白、核酸分子、载体、宿主细胞和/或药物组合物在制备药物中的用途，所述药物用于治疗与CD38表达相关或由CD38介导的疾病；
优选地，所述疾病为恶性血液肿瘤，优选为多发性骨髓瘤或非霍奇金淋巴瘤。
另外，本发明提供一种预防或治疗与CD38表达相关或由CD38介导的疾病的方法，所述方法包括给有此需要的受试者施用本发明的抗体分子或其片段、缀合物或融合蛋白、核酸分子、载体、宿主细胞和/或药物组合物，以及任选的其他药物或手段。该任选的其他药物或手段是指可以与本发明抗体分子或其片段、缀合物或融合蛋白、核酸分子、载体、宿主细胞和/或药物组合物联合施用的其他免疫增强药物或手段，例如小分子化药、靶向药、抗体等重组蛋白药、疫苗、ADC、溶瘤病毒、基因和核酸治疗药物和放射疗法。二 者的联合施用可以采取任意形式进行，例如同时、连续或间隔一定时间进行。
优选地，所述疾病为恶性血液肿瘤，优选为多发性骨髓瘤或非霍奇金淋巴瘤。所述受试者为哺乳类动物，优选地，所述受试者为人。
再一方面，本发明提供一种试剂盒，所述试剂盒包括本发明的抗体分子或其片段、缀合物或融合蛋白、核酸分子、载体、宿主细胞和/或药物组合物。
术语“CD38”，又称环腺苷二磷酸核糖水解酶，是具有长C末端细胞外结构域和短N末端细胞质结构域的II型跨膜糖蛋白。在造血细胞中，功能作用的分类被认为是CD38介导的信号传导的结果，包括淋巴细胞增殖、细胞因子释放、B细胞和骨髓细胞发育和存活的调控，以及树突状细胞成熟的诱导。CD38在许多造血系统恶性疾病中以及来源于各种造血系统恶性疾病的细胞系中失调，造血系统恶性疾病包括非霍奇金氏淋巴瘤(non-Hodgkin’slymphoma，NHL)、伯基特氏淋巴瘤(Burkitt’s lymphoma，BL)、多发性骨髓瘤(MM)、B慢性淋巴细胞白血病(B-CLL)、B和T急性礼拜那细胞白血病(ALL)、T细胞淋巴瘤(TCL)、急性骨髓性白血病(AML)、毛细胞白血病(HCL)、霍奇金氏淋巴瘤(HL)及慢性骨髓性白血病(CML)。另一方面，造血系统的最原始的多能干细胞是CD38-。尽管近期在抗癌剂的发现和研发方面取得了进展，但涉及CD38表达性肿瘤的许多癌症形式仍具有不良预后。
术语“氨基酸突变”在本文中意思指多肽序列中的氨基酸取代、插入和/或缺失，或针对以化学方式连接至蛋白质的部分的改变。举例来说，突变可以是附接至蛋白质的碳水化合物或PEG结构的改变。“氨基酸突变”在本文中意思指多肽序列中的氨基酸取代、插入和/或缺失。为清楚起见，除非另外说明，否则氨基酸突变通常是针对DNA编码的氨基酸，例如在DNA和RNA中具有密码子的20种氨基酸。
如本文所使用，“氨基酸取代”或“取代”在本文中意思指亲本多肽序列中特定位置处的氨基酸被不同氨基酸置换。确切地说，在一些实施方案中，取代是针对特定位置处非天然存在的氨基酸，这些氨基酸不是生物体内天然存在的或是任何生物体中的。举例来说，取代E272Y是指272位的谷氨酸被酪氨酸置换的变体多肽，在此情形中是Fc变体。为清楚起见，蛋白质被工程改造成改变核酸编码序列但不改变起始氨基酸(例如CGG(编码精氨酸)变为CGA(仍编码精氨酸)，用以增加宿主生物体表达水平)不是“氨基酸取代”；也就是说，尽管产生了编码同一蛋白质的新基因，但如果该蛋白质在 其起始的特定位置处具有相同氨基酸，就不是氨基酸取代。
如本文所使用，“氨基酸插入”或“插入”意思指在亲本多肽序列中的特定位置处添加氨基酸序列。举例来说，-233E或233E指示在233位后并且在234位前插入谷氨酸。另外，-233ADE或A233ADE指示在233位后并且在234位前插入AlaAspGlu。
如本文所使用，“氨基酸缺失”或“缺失”意思指去除亲本多肽序列中特定位置处的氨基酸序列。举例来说，G236-或G236#或G236del指示在236位处的甘氨酸缺失。另外，EDA233-或EDA233#表示自233位开始缺失序列GluAspAla。
如本文所使用，“残基”意思指蛋白质中的位置及其相关氨基酸的身份。举例来说，天冬酰胺297(又称为Asn297或N297)是在人类抗体IgG1中297位的残基。
术语“ADCC”或“抗体依赖性细胞介导的细胞毒性”意思指表达FcγR的非特异性细胞毒性细胞识别靶细胞上结合的抗体并且随后使靶细胞溶解的细胞介导的反应。ADCC与结合至FcγRIIIa相关；增加与FcγRIIIa的结合引起ADCC活性的增加。
术语“CDC”或“补体依赖的细胞毒性”是指IgG和IgM抗体的效应功能，当与表面抗原结合时引发典型的补体途径，包括形成膜攻击复合体以及靶细胞裂解。本发明的抗体，与CD38结合时，引发对CD38+癌细胞的CDC。
术语“抗体”应理解是涵盖包含两个免疫球蛋白重链和两个免疫球蛋白轻链的抗体分子(即，“完全抗体分子”)以及其抗原结合片段。如本文所用，术语抗体的“抗原结合部分”、抗体的“抗原结合片段”和类似术语，包括特异性结合抗原以形成复合物的任何天然存在、酶促可获得、合成或基因工程改造的多肽或糖蛋白。如本文所用，术语抗体的“抗原结合片段”或“抗体片段”是指抗体中保留特异性结合于溶血素A的能力的一个或多个片段。抗体片段可以包括Fab片段、F(ab′)2片段、Fv片段、dAb片段、含有CDR的片段或经分离的CDR。抗体的抗原结合片段可以使用任何适合的标准技术衍生自例如完全抗体分子，所述适合的标准技术是如蛋白水解消化或涉及DNA编码抗体可变和(视情况)恒定域的操纵和表达的重组基因工程改造技术。这类DNA是已知的和/或是从例如商业来源、DNA文库(包括例如噬菌 体-抗体文库)轻易可获得的或可以是合成的。可以化学方式或通过使用分子生物学技术对DNA进行测序和操纵，例如将一个或多个可变和/或恒定域布置成适合的组态，或引入密码子；形成半胱氨酸残基；修饰、添加或去除氨基酸等。
抗原结合片段的非限制性实例包括：(i)Fab片段；(ii)F(ab′)2片段；(iii)Fd片段；(iv)Fv片段；(v)单链Fv(scFv)分子；(vi)dAb片段；和(vii)由模拟抗体的高变区的氨基酸残基组成的最小识别单位(例如经分离的互补决定区(CDR)，如CDR3肽)，或限制性FR3-CDR3-FR4肽。如以下的其它工程改造分子也涵盖于如本文所用的表述“抗原结合片段”内：结构域-特异性抗体、单-结构域抗体、结构域-缺失抗体、嵌合抗体、CDR-接枝抗体、双功能抗体、三功能抗体、四功能抗体、微型抗体、纳米抗体(nanobody)(例如单价纳米抗体、二价纳米抗体等)、小模块化免疫药物(SMIP)和鲨鱼可变IgNAR结构域。
抗体的抗原结合片段将典型地包含至少一个可变域。可变域可以具有任何尺寸或氨基酸组成，且通常将包含至少一个CDR，其邻近一个或多个构架序列或与其同框。在VH结构域与VL结构域相缔合的抗原结合片段中，VH和VL结构域可以任何适合的排列相对于彼此定位。举例来说，可变区可以是二聚的且含有VH-VH、VH-VL或VL-VL二聚体。或者，抗体的抗原结合片段可以含有单聚VH或VL结构域。
在某些实施例中，抗体的抗原结合片段可以含有共价键联到至少一个恒定域的至少一个可变域。本发明的抗体的抗原结合片段内可以发现的可变和恒定域的非限制性、示例性组态包括：(i)VH-CH1；(ii)VH-CH2；(iii)VH-CH3；(iv)VH-CH1-CH2；(v)VH-CH1-CH2-CH3；(vi)VH-CH2-CH3；(vii)VH-CL；(viii)VL-CH1；(ix)VL-CH2；(x)VL-CH3；(xi)VL-CHl-CH2；(xii)VL-CH1-CH2-CH3；(xiii)VL-CH2-CH3；和(xiv)VL-CL。在可变和恒定域的包括上文所列的示例性组态中的任一个的任何组态中，可变和恒定域可彼此直接键联或可以通过完全或部分铰链或连接符区键联。铰链区可以由至少2(例如5、10、15、20、40、60或更多)个氨基酸组成，其在单一多肽分子中在邻近可变和/或恒定域之间产生柔性或半柔性键联。此外，本发明抗体的抗原结合片段可以包含上文所列的可变和恒定域组态中的任一个彼此非共价缔合和/或与一个或多个单聚VH或VL结构域(例如通过二硫键)的同二聚体或异二聚体(或其它多聚体)。
就完全抗体分子来说，抗原结合片段可以是单特异性或多特异性的(例如双特异性)。抗体的多特异性抗原结合片段将典型地包含至少两个不同的可变域，其中各可变域能够特异性结合于单独的抗原或相同抗原上不同的表位。任何多特异性抗体格式(包括本文所公开的示例性双特异性抗体格式)均可适合于使用所属领域中可用的常规技术在本发明的抗体的抗原结合片段的背景下使用。
在本发明中，利用杂交瘤技术获得鼠抗体，通过抗体活性分析(ELISA结合，与对照抗体的表位竞争，亲和力动力学)获得候选鼠抗体；然后，将编码候选鼠抗体轻重链可变区的序列克隆至编码人抗体轻重链恒定区的序列上游，进行哺乳动物细胞表达，制备得到嵌合抗体，之后通过抗体活性分析，验证嵌合抗体对效应细胞的互补依赖性细胞毒作用(CDC)、抗体依赖性细胞介导的细胞毒作用(ADCC)，以及对Duadi-luc的细胞凋亡活性验证；继而根据Germline数据库选择人源化模板，进行抗体序列的人源化设计，获得的人源化抗体再次通过抗体活性分析，重复验证人源化改造抗体对效应细胞的互补依赖性细胞毒作用(CDC)、抗体依赖性细胞介导的细胞毒作用(ADCC)，以及对Duadi-luc的细胞凋亡活性等体外细胞学实验验证；再进行人源化抗体的体外理化性质分析，最终获得亲和力和特异性与对照抗体相当或者优于对照抗体的抗CD38人源化全新抗体序列。
实验证明，本发明提供的抗体分子与已知抗体具有接近或一致的抗原结合识别表位，同时还与已知抗体具有相同或者更好的功能活性，包括体外ADCC、CDC和Daudi细胞凋亡活性等。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1显示了CD38表达细胞株的流式细胞术鉴定结果，其中，图1A：细胞株CHO blank；图1B：细胞株1-T-12；图1C：细胞株1-T-14。
图2显示了杂交瘤细胞的第一轮(图2A)和第二轮(图2B)融合筛选结果。
图3显示了本发明抗体与细胞表面表达的抗原CD38的结合活性。
图4显示了本发明抗体的抗体依赖性细胞毒性，其中，图4A：8B7人源化抗体；图4B：20M19人源化抗体；图4C：32A7人源化抗体；图4D： 5E19人源化抗体。
图5显示了本发明抗体的抗原表位分析结果，其中，图5A：相对于Daratumumab；图5B：相对于Mor202；图5C：相对于Isatuximab。
图6显示了本发明抗体的血清稳定性结果，其中，图6A：Daratumumab；图6B：8B7-H3L3；图6C：17H13-H2L2；图6D：20M19-H1L1。
实施发明的最佳方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的药材原料、试剂材料等，如无特殊说明，均为市售购买产品。其中：
人单克隆抗体IgG1亚类的重链恒定区序列见SEQ ID NO:106和SEQ ID NO:126，人单克隆抗体κ亚类的轻链恒定区序列见SEQ ID NO:107和SEQ ID NO:127。
重组人CD38蛋白，购自ACRO biosystem，Cat No:CD8-H5224。
实施例1 对照抗体的合成与表达
将全合成的Daratumumab、Isatuximab和Mor202抗体轻链可变区和重链可变区基因分别克隆至装有人-kappa轻链恒定区和人IgG1重链恒定区编码基因上游的真核表达载体中，分别获得Daratumumab、Isatuximab和Mor202轻、重链表达质粒。将获得的质粒转入大肠杆菌扩增，分离获得大量含Daratumumab、Isatuximab或Mor202抗体轻链和重链的质粒，将二者与PEI混合后共转染入HEK293细胞中。
细胞转染5-6天，取培养上清，利用Mabselect亲和层析柱对表达上清进行纯化，获得Daratumumab、Isatuximab和Mor202抗体重组蛋白。
Daratumumab：序列参见SEQ ID NO:128至SEQ ID NO:129。
Isatuximab：序列参见SEQ ID NO:130至SEQ ID NO:131。
Mor202：序列参见SEQ ID NO:132至SEQ ID NO:133。
实施例2 CD38抗原表达细胞株的制备
制备了人CD38高表达CHO细胞株1-T-14和人CD38低表达CHO细胞 株1-T-12。
通过PCR的方法，从含有CD38cDNA的载体(北京义翘神州，Cat:HG10818-UT)中克隆出CD38基因的阅读框，并将CD38基因的阅读框(已经测序验证正确序列)通过酶切的方法克隆入含有谷氨酰氨合成酶(GS)筛选基因的稳定表达载体中，电转染(Nucleofector IIb，Lonza)悬浮培养的CHO-K1细胞，将转染后的细胞置于含有50μM MSX(Sigma，Cat:M5379)的CD CHO AGTTM培养基(Gibco，Cat:12490-025)中，接种于96孔细胞培养板，37℃，5％CO 2静置培养2-3周，通过MSX加压筛选，从镜下预筛、放大培养，最终通过流式细胞分析(FACS)挑选出2个克隆(1-T-12和1-T-14)，进行放大培养和冻存。
两个细胞株与阴性对照CHO细胞株(CHO-blank)的流式细胞术鉴定结果见图1，平均荧光强度(MFI)数值见表1。
表1 CD38抗原表达细胞株的表达(FACS)
细胞株 MFI
CHO blank 391.3
1-T-12 66006.4
1-T-14 133008
实施例3 杂交瘤细胞的筛选和鉴定
1.小鼠免疫
8-10周龄Balb/c小鼠，采用弗氏佐剂和水溶佐剂两种免疫方法，弗氏佐剂在第0天和第14天给小鼠进行两次腹腔免疫注射，水溶佐剂在第0天和第21天给8-10周龄Balb/c小鼠进行两次肌肉免疫注射小鼠，免疫抗原为：人CD38重组蛋白，由Acro biosytem生物科技有限公司在HEK293细胞中重组表达。第一次免疫剂量为50μg，第二次免疫剂量为25μg。免疫前取小鼠血清作为检测时的阴性对照，弗氏佐剂在初次免疫后第28天，水溶佐剂在初次免疫后第35天尾静脉取血，用包被重组人CD38蛋白的96孔酶标板以ELISA法检测血清滴度；血清滴度达到融合要求的小鼠加强免疫，将25μg抗原用D-PBS稀释成500μl，腹腔注射。初次免疫后第18-38天取血清滴度高的小鼠的脾细胞用于下一步的细胞融合。
2.细胞融合及杂交瘤制备
2.1骨髓瘤细胞准备
培养融合所需骨髓瘤细胞P3X63Ag8.653在500ml三角培养瓶至细胞密度0.8-1.0E+6密度时，换液至完全杂交瘤培养基中备用。
2.2淋巴结和B淋巴细胞制备
选取滴度达到要求的小鼠，摘除眼球采血，并分离血清作为抗体检测时的阳性对照血清；并无菌取小鼠脾脏，按照常规方法制备B淋巴细胞悬液。
2.3电融合(Electro Cell Fusion，ECF)
骨髓瘤细胞P3X63Ag8.653和B淋巴细胞以4:1的比例混合后进行电击融合。电击后，将细胞放入37℃CO 2培养箱内，静止30分钟，1000RPM，室温离心10分钟，用360ml杂交瘤选择培养基重悬细胞后，铺板至384孔板内，确保铺板密度为8000～20000细胞/孔。第2-3天后换液一次，第7-10天进行阳性杂交瘤筛选。
2.4阳性杂交瘤筛选(FACS)
将融合后的杂交瘤细胞培养于384孔板中，取上清用FACS分析杂交瘤细胞分泌的抗体，筛选得到若干克隆，该克隆能够结合人CD38高表达株1-T-14而不能够结合CHO-blank细胞。通过有限稀释的方法将筛选到的克隆单细胞化，3轮之后得到的每个杂交瘤细胞克隆只分泌一个抗体。
第一轮和第二轮融合筛选结果见图2。
实施例4 鼠源单克隆抗体的可变区序列鉴定、抗人CD38嵌合抗体的制备以及抗体筛选
将分泌抗人CD38抗体的杂交瘤细胞扩大培养后，按照RNAfast200试剂盒(上海飞捷生物技术有限公司)说明书步骤提取细胞总RNA；利用5×PrimeScript RT Master Mix(Takara)将杂交瘤细胞总RNA反转录成cDNA；使用简并引物(Anke Krebber.1997)和Extaq PCR试剂(Takara)扩增抗体轻链可变区IgVL(κ)和重链可变区VH序列；利用PCR clean-up Gel extraction试剂盒(Macherey-Nagel公司)纯化PCR扩增产物；按照pClone007 Simple Vector Kit试剂盒(擎科生物科技有限公司)说明书将扩增PCR产物连接至T载体并转化大肠杆菌感受态细胞，菌株扩增、抽提质粒后进行DNA测序获得单克隆抗体可变区序列。
将鼠源抗人CD38单克隆抗体的重链可变区序列和公开发表的人单克隆 抗体IgG1亚类的重链恒定区序列拼接在一起，构建到哺乳动物细胞表达载体pCDNA3.4中；将鼠源抗人CD38单克隆抗体的轻链可变区序列和公开发表的人单克隆抗体κ亚类的轻链恒定区序列拼接在一起，构建到哺乳动物细胞表达载体pCDNA3.1中。将构建好的抗人CD38嵌合抗体的重链载体和轻链载体配对混合，使用聚乙烯亚胺(PEI)转染HEK293细胞，约7天后收集细胞上清，使用Mabselect纯化得到抗人CD38嵌合抗体蛋白。
得到的嵌合抗体在本文以“鼠源抗体命名-xiIgG”表示。
对获取的鼠源抗体和嵌合抗体进行杂交瘤细胞培养上清或纯化得到的抗体的抗原ELISA结合实验、Octet结合实验、体外细胞结合实验、体外细胞学实验(补体依赖性细胞毒性(CDC)和抗体依赖性细胞毒性(ADCC))等单克隆抗体的筛选(实验操作见下文)，将鼠源抗体与相应的嵌合抗体彼此验证，综合各个实验的结合，筛选得到部分鼠源抗体及其嵌合抗体。
来自部分鼠源抗体的轻重链可变区(VH和VL)序列如下所示，其中重链和轻链的抗原互补决定簇(CDR)以下划线示出。
鼠源抗体2H17：
>2H17_VH(SEQ ID NO:50)
Figure PCTCN2020116191-appb-000001
>2H17_VL(SEQ ID NO:51)
Figure PCTCN2020116191-appb-000002
鼠源抗体4J1：
>4J1_VH(SEQ ID NO:52)
Figure PCTCN2020116191-appb-000003
>4J1_VL(SEQ ID NO:53)
Figure PCTCN2020116191-appb-000004
Figure PCTCN2020116191-appb-000005
鼠源抗体5E19：
>5E19_VH(SEQ ID NO:54)
Figure PCTCN2020116191-appb-000006
>5E19_VL(SEQ ID NO:55)
Figure PCTCN2020116191-appb-000007
鼠源抗体8B7：
>8B7_VH(SEQ ID NO:56)
Figure PCTCN2020116191-appb-000008
>8B7_VL(SEQ ID NO:57)
Figure PCTCN2020116191-appb-000009
鼠源抗体13D15：
>13D15_VH(SEQ ID NO:58)
Figure PCTCN2020116191-appb-000010
>13D15_VL(SEQ ID NO:59)
Figure PCTCN2020116191-appb-000011
Figure PCTCN2020116191-appb-000012
鼠源抗体15A10：
>15A10_VH(SEQ ID NO:60)
Figure PCTCN2020116191-appb-000013
>15A10_VL(SEQ ID NO:61)
Figure PCTCN2020116191-appb-000014
鼠源抗体17H13：
>17H13_VH(SEQ ID NO:62)
Figure PCTCN2020116191-appb-000015
>17H13_VL(SEQ ID NO:63)
Figure PCTCN2020116191-appb-000016
鼠源抗体20M19：
>20M19_VH(SEQ ID NO:64)
Figure PCTCN2020116191-appb-000017
>20M19_VL(SEQ ID NO:65)
Figure PCTCN2020116191-appb-000018
鼠源抗体32A7：
>32A7_VH(SEQ ID NO:66)
Figure PCTCN2020116191-appb-000019
>32A7_VL(SEQ ID NO:67)
Figure PCTCN2020116191-appb-000020
结果显示，具有上述轻重链的所有鼠源抗体相对于其他鼠源抗体，具有更为良好的与人CD38重组蛋白的结合作用，细胞结合实验效果明显，并具有较强的补体依赖性细胞毒性和抗体依赖性细胞毒性；并且，在转为嵌合抗体以后，均保留了鼠源抗体的作用，并且结合活性与对照抗体Daratumumab或Isatuximab相近或更高。
实施例5 抗人CD38鼠源抗体的人源化与人源化抗体制备
综合Kabat、Chothia的抗体编码方案，确定鼠源抗体的重链和轻链的6个抗原互补决定簇(CDR)的氨基酸序列区域及支撑抗体保守三维构象的框架区域(framework region)。
随后通过分析搜索已知人源抗体序列，选择与鼠源抗体最为相似接近的人源抗体重链可变区序列，如IGHV1|IGHJ4*01，选择其抗体框架区序列作为模板，将鼠源抗体重链CDR与人源抗体框架区结合，最终生成人源化抗体重链可变区序列。同样过程，生成人源化抗体轻链可变区序列。
上述过程中发现，鼠源抗体CDR直接移植至人框架区的抗体常出现结合活性急剧下降，因此需要将框架区个别氨基酸从人源的改回鼠源的。在确定回复突变位点时，一是对照设计好的人源化抗体序列和原始的鼠源抗体序列，检查有哪些氨基酸的不同；二是检查这些氨基酸是否对支持抗体结构起重要作用或者对与抗原的结合起重要作用。对于人源化设计后的序列，同时还需要检查是否有一些潜在的翻译后修饰位点，如N(天冬酰胺)糖基化位点、N脱酰胺化位点、D(天冬氨酸)异构化位点等。
将人源化抗体重链可变区基因构建到含人单克隆抗体IgG1亚类的重链恒定区基因的哺乳动物细胞表达载体中；轻链可变区基因构建到含人单克隆抗体κ亚类的轻链恒定区基因的哺乳动物细胞表达载体中。构建好的抗人CD38人源化抗体的重链载体和轻链载体配对混合，使用聚乙烯亚胺(PEI)转染HEK293细胞，约7天后收集细胞上清，使用Mabselect纯化得到抗人CD38人源化抗体蛋白。
人源化改造得到的部分轻重链可变区序列如下所示，其中重链和轻链的抗原互补决定簇(CDR)以下划线示出。
>5E19_VH_hz0(SEQ ID NO:68)
Figure PCTCN2020116191-appb-000021
>5E19_VH_hz1(SEQ ID NO:69)
Figure PCTCN2020116191-appb-000022
>5E19_VH_hz2(SEQ ID NO:70)
Figure PCTCN2020116191-appb-000023
>5E19_VH_hz3(SEQ ID NO:71)
Figure PCTCN2020116191-appb-000024
>5E19_VH_hz4(SEQ ID NO:72)
Figure PCTCN2020116191-appb-000025
Figure PCTCN2020116191-appb-000026
>5E19_VL_hz0(SEQ ID NO: 89)
Figure PCTCN2020116191-appb-000027
>5E19_VL_hz1(SEQ ID NO: 90)
Figure PCTCN2020116191-appb-000028
>8B7_VH_hz0(SEQ ID NO: 73)
Figure PCTCN2020116191-appb-000029
>8B7_VH_hz1(SEQ ID NO: 74)
Figure PCTCN2020116191-appb-000030
>8B7_VH_hz3(SEQ ID NO: 75)
Figure PCTCN2020116191-appb-000031
>8B7_VH_hz4(SEQ ID NO: 76)
Figure PCTCN2020116191-appb-000032
>8B7_VH_hz5(SEQ ID NO: 77)
Figure PCTCN2020116191-appb-000033
>8B7_VH_hz6(SEQ ID NO: 78)
Figure PCTCN2020116191-appb-000034
>8B7_VH_hz7(SEQ ID NO: 79)
Figure PCTCN2020116191-appb-000035
>8B7_VH_hz8(SEQ ID NO: 80)
Figure PCTCN2020116191-appb-000036
>8B7_VH_hz9(SEQ ID NO: 81)
Figure PCTCN2020116191-appb-000037
>8B7_VH_hz10(SEQ ID NO: 82)
Figure PCTCN2020116191-appb-000038
>8B7_VL_hz0(SEQ ID NO: 91)
Figure PCTCN2020116191-appb-000039
>8B7_VL_hz1(SEQ ID NO: 92)
Figure PCTCN2020116191-appb-000040
>8B7_VL_hz2(SEQ ID NO: 93)
Figure PCTCN2020116191-appb-000041
>8B7_VL_hz3(SEQ ID NO: 94)
Figure PCTCN2020116191-appb-000042
>8B7_VL_hz4(SEQ ID NO: 95)
Figure PCTCN2020116191-appb-000043
>8B7_VL_hz5(SEQ ID NO: 96)
Figure PCTCN2020116191-appb-000044
>8B7_VL_hz6(SEQ ID NO: 97)
Figure PCTCN2020116191-appb-000045
>8B7_VL_hz7(SEQ ID NO: 98)
Figure PCTCN2020116191-appb-000046
>8B7_VL_hz8(SEQ ID NO: 99)
Figure PCTCN2020116191-appb-000047
>8B7_VL_hz9(SEQ ID NO: 100)
Figure PCTCN2020116191-appb-000048
>17H13_VH_hz0(SEQ ID NO: 83)
Figure PCTCN2020116191-appb-000049
>17H13_VH_hz1(SEQ ID NO: 84)
Figure PCTCN2020116191-appb-000050
>17H13_VH_hz2(SEQ ID NO: 85)
Figure PCTCN2020116191-appb-000051
Figure PCTCN2020116191-appb-000052
>17H13_VH_hz3(SEQ ID NO: 86)
Figure PCTCN2020116191-appb-000053
>17H13_VL_hz0(SEQ ID NO: 101)
Figure PCTCN2020116191-appb-000054
>17H13_VL_hz1(SEQ ID NO: 102)
Figure PCTCN2020116191-appb-000055
>17H13_VL_hz2(SEQ ID NO: 134)
Figure PCTCN2020116191-appb-000056
>17H13_VL_hz3(SEQ ID NO: 135)
Figure PCTCN2020116191-appb-000057
>20M19_VH_hz0(SEQ ID NO: 87)
Figure PCTCN2020116191-appb-000058
Figure PCTCN2020116191-appb-000059
>20M19_VH_hz1(SEQ ID NO: 88)
Figure PCTCN2020116191-appb-000060
>20M19_VL_hz0(SEQ ID NO: 103)
Figure PCTCN2020116191-appb-000061
>20M19_VL_hz1(SEQ ID NO: 104)
Figure PCTCN2020116191-appb-000062
>20M19_VL_hz2(SEQ ID NO: 105)
Figure PCTCN2020116191-appb-000063
>32A7_VH_hz0(SEQ ID NO: 140)
Figure PCTCN2020116191-appb-000064
>32A7_VH_hz1(SEQ ID NO: 141)
Figure PCTCN2020116191-appb-000065
>32A7_VH_hz2(SEQ ID NO:142)
Figure PCTCN2020116191-appb-000066
>32A7_VL_hz0(SEQ ID NO:143)
Figure PCTCN2020116191-appb-000067
>32A7_VL_hz1(SEQ ID NO:144)
Figure PCTCN2020116191-appb-000068
>32A7_VL_hz2(SEQ ID NO:145)
Figure PCTCN2020116191-appb-000069
>32A7_VL_hz3(SEQ ID NO:146)
Figure PCTCN2020116191-appb-000070
将人源化抗体命名为“鼠源抗体命名-HmLn”，其中m和n分别为VH和VL的人源化(hz)改造序列(VH_hz和VL_hz)编号。
实施例6 抗人CD38抗体与抗原的体外结合验证与体外细胞学试验
1.Octet结合实验
进行本发明的抗体和对照抗体Daratumumab(Dara)与重组人CD38蛋白的Octet结合实验。
利用Fortebio(BLITZ pro1.1.0.28)仪器，采用抗人抗体捕获法测定抗体亲和力。测定时将抗人抗体Fc段的捕获抗体(AHC)生物探针浸泡于PBS 10min；将200μl稀释后的抗体样品(包括本发明的嵌合抗体、人源化抗体以及对照抗体Daratumumab；抗体工作浓度为15μg/mL)上样到AHC生物探针上，然后在PBS中平衡100s，进一步将AHC探针与不同稀释浓度的人CD38蛋白(ACRO biosystem，Cat No.CD8-H5224)进行结合反应，结合时间600s，之后将AHC探针转移至PBS中，进行解离反应，时间为600s。实验完毕，扣除空白对照响应值，用软件进行1:1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数。
结果见表2至表5。
表2 抗体与重组人CD38蛋白的体外结合活性
抗体 浓度(nM) 响应 KD(M) kon(1/Ms) kdis(1/s) RMax
4J1-xiIgG 100 0.4598 3.31E-08 2.85E+05 9.44E-03 0.6097
4J1-H0L0 100 -0.01 - 2.32E+05 - 0.0001
4J1-H0L1 100 -0.0089 - 1.09E+06 - 0.0001
Dara 100 0.2098 2.44E-07 4.53E+04 1.11E-02 0.7031
表3 抗体与重组人CD38蛋白的体外结合活性
抗体 浓度(nM) 响应 KD(M) kon(1/Ms) kdis(1/s) RMax
5E19-xiIgG 100 0.3799 2.79E-08 4.70E+05 1.31E-02 0.4941
5E19-H0L0 100 -0.0155 - 8.33E+04 - 0.0032
5E19-H0L1 100 0.0184 - 2.26E+05 5.13E-01 0.5128
5E19-H1L0 100 0.1096 1.30E-06 6.44E+04 8.39E-02 1.6077
5E19-H1L1 100 0.338 7.76E-08 3.17E+05 2.46E-02 0.6181
Dara 100 0.2225 1.60E-07 7.29E+04 1.16E-02 0.5712
表4 抗体与重组人CD38蛋白的体外结合活性
抗体 浓度(nM) 响应 KD(M) kon(1/Ms) kdis(1/s) RMax
8B7-xiIgG 100 0.5302 2.16E-09 3.35E+05 7.23E-04 0.537
8B7-H0L0 100 -0.0161 - 1.53E+06 - 0.0002
8B7-H0L1 100 -0.0019 - 1.65E+07 - 0.0339
8B7-H1L0 100 0.0552 - 5.32E+04 1.43E-01 1.6895
8B7-H1L1 100 0.4727 1.89E-08 2.52E+05 4.76E-03 0.5589
Dara 100 0.2156 1.54E-07 8.16E+04 1.26E-02 0.5496
表5 抗体与重组人CD38蛋白的体外结合活性
抗体 浓度(nM) 响应 KD(M) kon(1/Ms) kdis(1/s) RMax
17H13-xiIgG 100 0.2662 2.29E-08 2.87E+05 6.56E-03 0.3266
17H13-H0L0 100 -0.0063 - 1.96E+06 - 0.0001
17H13-H0L1 100 0.2254 6.16E-08 1.64E+05 1.01E-02 0.3658
17H13-H1L0 100 0.0053 - 8.49E+03 7.90E-01 8.1272
17H13-H1L1 100 0.2275 3.94E-08 3.08E+05 1.21E-02 0.3203
Dara 100 0.2159 1.43E-07 8.83E+04 1.26E-02 0.5239
实验结果显示，部分人源化改造抗体保留了与人CD38重组蛋白的结合活性。
2.体外细胞结合实验
进行本发明的抗体和对照抗体Daratumumab(Dara)与CD38蛋白的体外细胞结合实验。
将抗人CD38抗体从100nM的起始浓度开始做2倍的梯度倍比稀释，共16个浓度点，各个浓度点的抗体取10μl加入384孔板。取细胞表面表达CD38的CHO细胞株1-T-14，用含0.5％BSA的PBS洗涤细胞一次，100g室温离心5分钟，重悬细胞为密度约2x10 6个细胞/ml，取10μl加入到已加抗体的384孔板的孔中。4℃孵育1小时后，加入荧光标记的羊抗人IgG二抗。继续于4℃孵育1小时后，用流式细胞仪分析细胞群的平均荧光强度。
结果见图3和表6。
表6 抗体与抗原的结合活性
Figure PCTCN2020116191-appb-000071
Figure PCTCN2020116191-appb-000072
3.体外细胞学试验
3.1补体依赖性细胞毒性(CDC)
使用Quidel公司的商品化人血清全补体，分析本发明的抗体和对照抗体Daratumumab(Dara)针对稳定表达人CD38的CHO细胞株1-T-14诱导补体依赖性细胞毒性(CDC)的能力。
将细胞分别与终浓度50μg/ml至7.6ng/ml的抗体和对照抗体混匀，然后将溶于细胞培养基RPMI-1640中的6.25％人血清全补体加入细胞中，在37℃下孵育3小时。其后，通过CCK-8试剂盒进行细胞毒性检测，最终通过MD酶标仪检测450nm吸光度。通过吸光值采用softmax pro7软件进行4参数拟合曲线，计算样品的EC50。
结果见表7。
表7 抗体的补体依赖性细胞毒性
板号 抗体 EC50(ng/mL) 相对活性(％)
P1 Dara 154.5 100.0
P1 8B7-H1L1 177.4 87.1
P1 8B7-H3L3 214.5 72.0
P2 Dara 144.5 100.0
P2 20M19-H1L0 234.9 61.5
P2 20M19-H1L1 239.6 60.3
P3 Dara 126.4 100.0
P3 32A7-H1L1 303.2 41.7
P3 32A7-H2L1 203.5 62.1
P4 Dara 125.9 100.0
P4 5E19-H3L1 192.6 65.4
P4 无关抗体 ～ ～
3.2抗体依赖性细胞毒性(ADCC)
检测本发明的抗体和对照抗体Daratumumab(Dara)针对稳定表达人CD38的CHO细胞株1-T-14诱导抗体依赖性细胞毒性(ADCC)。
使用工程改造的Jurkat细胞作为效应细胞，该细胞稳定表达FcγRIIIa-FcεRIaγ杂合受体，由NFAT应答元件驱动表达萤火虫萤光素酶。抗体在ADCC作用机制中的生物活性通过NFAT通路活化产生的萤光素酶定量。
将1.5E5个效应细胞分别与终浓度33μg/ml至85pg/ml的本发明的抗体和对照抗体Daratumumab混匀，然后将2.5E4个靶细胞即稳定表达人CD38的CHO细胞株1-T-14(效应细胞与靶细胞E:T比例为6:1)加入其中，在37℃下孵育16小时。其后，通过Promega公司试剂盒Bio-Glo TM Luciferase Assay System进行检测，最终通过MD酶标仪检测LUM值，并使用下式进行诱导倍数和EC50计算。
诱导倍数＝(受检孔读值–背景值)/(阴性对照孔读值–背景值)
诱导倍数采用prism7软件进行4参数拟合曲线，计算样品的EC50。
结果见图4和表8。
表8 抗体的抗体依赖性细胞毒性
板号 抗体 EC50(ng/mL) 相对活性(％)
P1 Dara 31.76 100.0
P1 8B7-H1L1 60.59 52.4
P1 8B7-H3L3 37.94 83.7
P2 Dara 39.01 100.0
P2 20M19-H1L0 31.55 123.6
P2 20M19-H1L1 41.63 93.7
P3 Dara 32.59 100.0
P3 32A7-H1L1 60.23 54.1
P3 32A7-H2L1 56.42 57.8
P4 Dara 31.61 100.0
P4 5E19-H3L1 51.46 61.4
P4 无关抗体 ～ ～
实施例9 抗人CD38人源化抗体的体外结合亲和力和动力学实验
采用GE公司BIAcore仪器S200测定抗体抗原相互作用力。
参考GE公司Biotin CAPture Kit(货号28-9202-34，Lot.10265137)操作说明，首先在传感芯片CAP分析通道和对照样品通道都偶联His标签的人CD38蛋白抗原，然后在分析通道和对照样品通道一起流过梯度稀释的抗体样品(起始浓度20nM，1:3稀释8个浓度点，并且设定0.741nm浓度点重复)，测定抗体抗原结合后发生的光反应值。经仪器软件拟合分析，最终得到抗体的结合常数Kon和解离常数Koff，以及亲和力常数KD。
结果见表9。
表9 人源化改造抗体的结合动力学
  ka(1/Ms) kd(1/s) KD(M)
20M19-H1L1 1.049E+6 7.136E-4 6.804E-10
8B7-H3L3 6.623E+5 0.001197 1.808E-9
8B7-H4L3 1.277E+6 0.002919 2.287E-9
实施例10 抗人CD38人源化抗体的细胞凋亡活性测定
材料：Daudi-Luc(ATCC，Cat No.CCL-213)细胞
试剂盒：Alex FluorTM 488 Annexin V/Dead细胞凋亡试剂盒(invitrogen,Cat:V13245,Lot:1923636)
工具抗体：AffinipureF(ab')2 Fragment Goat-anti-human IgG Fcr Fragment Specific(Immo Research,Cat:109-006-008,Lot:129913,Conc.1.3mg/ml)
实验步骤：
用预冷的PBS洗1遍Daudi细胞，然后以1E5/孔将细胞种入96孔板内，加入0.7nM抗CD38抗体孵育30分钟。加入5μg/ml羊抗人的工具抗体至96孔板内，4℃孵育24小时。用预冷的PBS洗2遍细胞后，弃掉上清，用100μl 1x Annexin溶液重悬细胞。加入5μl Alexa
Figure PCTCN2020116191-appb-000073
488 annexin V和1μL 100μg/mL PI工作溶液至96孔板内，室温避光孵育15分钟。用400μl 1X annexin结合溶液终止反应后，将96孔板放置于冰上，最后用流式细胞分析仪测量荧光激发530nM和发射488nM的读值。
结果见表10。
表10 本发明抗体的细胞凋亡活性
Figure PCTCN2020116191-appb-000074
表中的“交联”和“非交联”分别为桥接Fab和不桥接Fab的形式。
实施例11 抗人CD38人源化抗体的成药性表征
1.抗体表位分析
采用Fortebio公司的In tandem方法进行本发明抗体的抗原表位分析。简言之，先用本发明的含鼠源抗体的单克隆上清液结合抗原细胞即人CD38高 表达CHO细胞株1-T-14，然后加入对照抗体与鼠源抗体竞争与抗原表位的结合。检测所标记的对照抗体的结合信号，对照抗体的结合信号越低，越能证明鼠源抗体占据了对照抗体识别的抗原表位，本发明的抗体与对照抗体的抗原结合表位靠近或一致。
本发明的抗体与Daratumumab、Mor202、Isatuximab的竞争结果分别见图5。
实验证明，本发明的抗体与抗原的结合位点与对照抗体的表位是一致的。
2.抗体单体率分析
实验仪器：UPLC CLASS ACQUITYH(WATERS)
分析柱：TSKgel G3000SWXL 7.8*300(TOSHI，Cat No 003C03326C
分析溶液：DPBS(GIBCO，Cat No.14190-136，Lot#1967705)
分析方法：将50μl浓度为1mg/ml的抗体进样至预平衡好的层析柱内，室温，0.7ml/min的流速流动35min，并同时检测机器A280和A214的吸收值，进入根据出峰时间和出峰体积来判断抗体的单体含量和比例。
结果见表11。
表11 本发明抗体的主峰保留时间和单体率
Figure PCTCN2020116191-appb-000075
3.抗体疏水性质分析
实验仪器：ARC(Waters)
实验用分析柱：TSKgel Butyl-NPR(4.6mmX3.5cm，Cat No 14947)
分析溶液：a.20mM Histidine，pH6.0；b.20mM Histidine，1.6M(NH 4) 2SO 4
分析方法：根据疏水层析柱使用说明，来分析抗体的疏水性质。
结果见表12。
表12 本发明抗体的疏水性质
Figure PCTCN2020116191-appb-000076
4.抗体体外猴(cyno)血清稳定性研究
实验材料：待测抗体，FBS，抗原人CD38重组蛋白，anti-huIgG Fab单克隆抗体(Sigma,I5260-1ML)，HRP标记山羊抗人IgG二抗(Jackson,code:109-035-098)。
实验仪器：37℃培养箱，酶标仪。
实验步骤：
样品制备：
1)待测抗体调浓度至20μg/ml，过滤除菌，分装为250μl/管备用；
2)FBS等体积加入分装好的待测抗体，即终浓度为50％血清浓度，10μg/ml抗体浓度；
3)共制备7个样品，封口膜封口，放置于37℃，全程需保持无菌；
4)分别于第0天、3天、7天、10天、14天、21天取样放置于4℃保存待测，其中第21天取样应至少在4℃放置一天。
检测方法：
1)分别PBS包被结合抗原、anti-IgG Fab单克隆抗体于96孔酶联板，0.2μg/ml，100μl/孔，4℃过夜；
2)配置所需试剂：
封闭液 5％BSA+PBS
抗体稀释液 5％BSA+PBS+50％FBS
酶联板洗涤液 0.1％Tween+PBS
3)包被好的酶联板PBS清洗三遍，300μl/孔，洗去游离的未包被抗原；
4)加入封闭液，200μl/孔，37℃封闭1h；
5)待测抗体使用抗体稀释液稀释至2μg/ml，并3倍稀释共8个梯度；
6)倒掉封闭液，两种包被方法的酶联板分别加入稀释好的抗体，100μl/孔，于37℃孵育1h；
7)PBST洗板3遍；
8)1:5000稀释二抗，加入洗涤好的酶联板中，100μl/孔，37℃孵育40min；
9)PBST洗板3遍；
10)TMB显色，100μl/孔，避光10min；
11)加入50μl 2M HCL终止，450nm读数。
结果处理：
根据ELISA显色值制定结合曲线，观察放置不同时间的结合曲线变化，评估放置后抗体结合活性稳定性。
结果见图6。
5.抗体小鼠体内药物代谢分析
实验材料：待测抗体，小鼠不同时间点采集的血清，待测抗体的结合抗原人CD38，抗体标准品Dara，anti-huIgG Fab单克隆抗体(Sigma,I5260-1ML)，HRP标记山羊抗人IgG二抗(Jackson,code:109-035-098)。
实验方法：
血清收集：
1)雌性Balb/C小鼠，3只/组，分别于尾静脉或者腹腔给药，200μg待测抗体/只；
2)按照实验设计时间点尾静脉采血，收集血样，室温放置30min以上，4000rpm，15min收集血清，-20℃保存，为防止血清蒸发，最后血清收集体积尽量大于20μl；
3)最后一次收集血清至少于-20℃冻存24h。
检测方法：
1)分别PBS包被结合抗原、anti-IgG Fab单克隆抗体于96孔酶联板，0.2μg/ml，100μl/孔，4℃过夜；
2)配置所需试剂：
封闭液 5％BSA+PBS
抗体稀释液 5％BSA+PBS+20％空白小鼠血清
酶联板洗涤液 0.1％Tween+PBS
3)包被好的酶联板PBS清洗三遍，300μl/孔；
4)加入封闭液，200μl/孔，37℃封闭1h；
5)起始血清使用封闭液稀释至合适浓度，用含有相同血清浓度的抗体稀释液稀释至合适的浓度范围，具体浓度的稀释倍数需根据预实验来调整，原则上使检测的血清最终显色值在标曲显色值范围内；
6)抗体稀释液稀释抗体标准品，标准品的稀释仍然根据预实验来调整，使标准品拟合出线性曲线(如有合适软件，也可拟合出S形曲线)。
7)倒掉封闭液，两种包被方法的酶联板分别加入稀释好的抗体标准品与待测血清，100μl/孔，于37℃孵育1h；
8)PBST洗板3遍；
9)1:5000稀释二抗，加入洗涤好的酶联板中，100μl/孔，37℃孵育40min；
10)PBST洗板3遍；
11)TMB显色，100μl/孔，避光10min；
12)加入50μl 2M HCL终止，450nm读数。
结果见表13。
表13-1 本发明抗体的小鼠体内药物代谢性质
Figure PCTCN2020116191-appb-000077
Figure PCTCN2020116191-appb-000078
表13-2 本发明抗体的小鼠体内药物代谢性质
Figure PCTCN2020116191-appb-000079
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。

Claims (19)

  1. 一种抗体分子或其片段，其特征在于，具有以下重链CDRs区：
    VH-CDR1：具有SEQ ID NO:16所示氨基酸序列或在SEQ ID NO:16所示氨基酸序列的基础上进行一个或几个氨基酸突变；
    VH-CDR2：具有SEQ ID NO:17所示氨基酸序列或在SEQ ID NO:17所示氨基酸序列的基础上进行一个或几个氨基酸突变；
    VH-CDR3：具有SEQ ID NO:18所示氨基酸序列或在SEQ ID NO:18所示氨基酸序列的基础上进行一个或几个氨基酸突变；
    以及，以下轻链CDRs区：
    VL-CDR1：具有SEQ ID NO:4所示氨基酸序列或在SEQ ID NO:4所示氨基酸序列的基础上进行一个或几个氨基酸突变；
    VL-CDR2：具有SEQ ID NO:5所示氨基酸序列或在SEQ ID NO:5所示氨基酸序列的基础上进行一个或几个氨基酸突变；
    VL-CDR3：具有SEQ ID NO:19所示氨基酸序列或在SEQ ID NO:19所示氨基酸序列的基础上进行一个或几个氨基酸突变；
    其中，所述抗体CDRs区的氨基酸突变使突变后抗体6个CDRs组合能够形成抗原结合位点，且至少部分的保留了CDRs区突变前抗体分子或其片段的生物活性。
  2. 根据权利要求1所述抗体分子或其片段，其特征在于，所述CDRs区突变前抗体分子或其片段包括SEQ ID NO:16所示的VH-CDR1、SEQ ID NO:17所示的VH-CDR2、SEQ ID NO:18所示的VH-CDR3、SEQ ID NO:4所示的VL-CDR1、SEQ ID NO:5所示的VL-CDR2、SEQ ID NO:19所示的VL-CDR3。
  3. 根据权利要求1或2所述的抗体或其片段，其特征在于，所述抗体分子或其片段特异性结合细胞膜表面CD38，且能够通过结合细胞膜表面CD38诱导CD38+细胞凋亡。
  4. 根据权利要求1或2所述的抗体分子或其片段，其特征在于，所述抗体分子或其片段对于CD38+细胞具有抗体依赖性细胞介导的细胞毒作用(ADCC)、补体依赖性细胞毒作用(CDC)。
  5. 根据权利要求1或2所述的抗体分子或其片段，其特征在于所述抗体 分子或其片段能够与至少一种选自下组的抗体分子竞争结合CD38：Daratumumab、Isatuximab、Mor202。
  6. 一种抗体分子或其片段，所述抗体分子或其片段包含重链可变区(VH)，其中所述重链可变区包含选自如SEQ ID NO:1、SEQ ID NO:7、SEQ ID NO:11、SEQ ID NO:16、SEQ ID NO:20、SEQ ID NO:26、SEQ ID NO:31、SEQ ID NO:41和SEQ ID NO:147所示的CDR1(VH-CDR1)，选自如SEQ ID NO:2、SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:17、SEQ ID NO:21、SEQ ID NO:27、SEQ ID NO:32、SEQ ID NO:37和SEQ ID NO:42所示的CDR2(VH-CDR2)，和选自如SEQ ID NO:3、SEQ ID NO:9、SEQ ID NO:13、SEQ ID NO:18、SEQ ID NO:22、SEQ ID NO:28、SEQ ID NO:33、SEQ ID NO:38、SEQ ID NO:43、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48和SEQ ID NO:137所示的CDR3(VH-CDR3)；和
    所述抗体分子或其片段包含轻链可变区(VL)，其中所述轻链可变区包含选自如SEQ ID NO:4、SEQ ID NO:14、SEQ ID NO:23、SEQ ID NO:29、SEQ ID NO:34、SEQ ID NO:39、SEQ ID NO:44、SEQ ID NO:49、SEQ ID NO:136和SEQ ID NO:148所示的CDR1(VL-CDR1)，选自如SEQ ID NO:5、SEQ ID NO:24和SEQ ID NO:35所示的CDR2(VL-CDR2)，选自如SEQ ID NO:6、SEQ ID NO:10、SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:25、SEQ ID NO:30、SEQ ID NO:36、SEQ ID NO:40、SEQ ID NO:45、SEQ ID NO:138和SEQ ID NO:139所示的CDR3(VL-CDR3)。
  7. 根据权利要求6所述的抗体分子或其片段，其特征在于，所述重链可变区包含选自以下的CDR组合：
    (1)如SEQ ID NO:1所示的VH-CDR1、如SEQ ID NO:2所示的VH-CDR2、如SEQ ID NO:3所示的VH-CDR3；
    (2)如SEQ ID NO:7所示的VH-CDR1、如SEQ ID NO:8所示的VH-CDR2、如SEQ ID NO:9所示的VH-CDR3；
    (3)如SEQ ID NO:11所示的VH-CDR1、如SEQ ID NO:12所示的VH-CDR2、如SEQ ID NO:13所示的VH-CDR3；
    (4)如SEQ ID NO:16所示的VH-CDR1、如SEQ ID NO:17所示的VH-CDR2、如SEQ ID NO:18所示的VH-CDR3；
    (5)如SEQ ID NO:20所示的VH-CDR1、如SEQ ID NO:21所示的 VH-CDR2、如SEQ ID NO:22所示的VH-CDR3；
    (6)如SEQ ID NO:26所示的VH-CDR1、如SEQ ID NO:27所示的VH-CDR2、如SEQ ID NO:28所示的VH-CDR3；
    (7)如SEQ ID NO:31所示的VH-CDR1、如SEQ ID NO:32所示的VH-CDR2、如SEQ ID NO:33所示的VH-CDR3；
    (8)如SEQ ID NO:26所示的VH-CDR1、如SEQ ID NO:37所示的VH-CDR2、如SEQ ID NO:38所示的VH-CDR3；
    (9)如SEQ ID NO:41所示的VH-CDR1、如SEQ ID NO:42所示的VH-CDR2、如SEQ ID NO:43所示的VH-CDR3；
    (10)如SEQ ID NO:11所示的VH-CDR1、如SEQ ID NO:12所示的VH-CDR2、如SEQ ID NO:46所示的VH-CDR3；
    (11)如SEQ ID NO:11所示的VH-CDR1、如SEQ ID NO:12所示的VH-CDR2、如SEQ ID NO:47所示的VH-CDR3；
    (12)如SEQ ID NO:11所示的VH-CDR1、如SEQ ID NO:12所示的VH-CDR2、如SEQ ID NO:48所示的VH-CDR3；
    (13)如SEQ ID NO:16所示的VH-CDR1、如SEQ ID NO:17所示的VH-CDR2、如SEQ ID NO:137所示的VH-CDR3；
    (14)如SEQ ID NO:147所示的VH-CDR1、如SEQ ID NO:42所示的VH-CDR2、如SEQ ID NO:43所示的VH-CDR3；
    和/或，所述抗体分子或其片段包含轻链可变区(VL)，其中所述轻链可变区包含选自以下的CDR组合：
    (1)如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:6所示的VL-CDR3；
    (2)如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:10所示的VL-CDR3；
    (3)如SEQ ID NO:14所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:15所示的VL-CDR3；
    (4)如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:19所示的VL-CDR3；
    (5)如SEQ ID NO:23所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:25所示的VL-CDR3；
    (6)如SEQ ID NO:29所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:30所示的VL-CDR3；
    (7)如SEQ ID NO:34所示的VL-CDR1、如SEQ ID NO:35所示的VL-CDR2、如SEQ ID NO:36所示的VL-CDR3；
    (8)如SEQ ID NO:39所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:40所示的VL-CDR3；
    (9)如SEQ ID NO:44所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:45所示的VL-CDR3；
    (10)如SEQ ID NO:49所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:40所示的VL-CDR3；
    (11)如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:138所示的VL-CDR3；
    (12)如SEQ ID NO:136所示的VL-CDR1、如SEQ ID NO:35所示的VL-CDR2、如SEQ ID NO:36所示的VL-CDR3；
    (13)如SEQ ID NO:136所示的VL-CDR1、如SEQ ID NO:35所示的VL-CDR2、如SEQ ID NO:139所示的VL-CDR3；
    (14)如SEQ ID NO:148所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:45所示的VL-CDR3。
  8. 根据权利要求6或7所述的抗体分子或其片段，其特征在于，所述重链可变区包含选自SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68至SEQ ID NO:88、SEQ ID NO:140至SEQ ID NO:142中任一个所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的氨基酸序列；和/或
    所述轻链可变区包含选自SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:89至SEQ ID NO:105、SEQ ID NO:134和SEQ ID NO:135、SEQ ID NO:143至SEQ ID NO:146中任一个所示的氨基酸序列或与所示的氨基酸序列具有至少75％同一性的氨基酸序列。
  9. 根据权利要求6至8中任一项所述的抗体分子或其片段，其特征在于，所述抗体分子或其片段包含选自以下的CDR组合：
    (1)如SEQ ID NO:1所示的VH-CDR1、如SEQ ID NO:2所示的VH-CDR2、如SEQ ID NO:3所示的VH-CDR3；如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:6所示的VL-CDR3；
    (2)如SEQ ID NO:7所示的VH-CDR1、如SEQ ID NO:8所示的VH-CDR2、如SEQ ID NO:9所示的VH-CDR3；如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:10所示的VL-CDR3；
    (3)如SEQ ID NO:11所示的VH-CDR1、如SEQ ID NO:12所示的VH-CDR2、如SEQ ID NO:13所示的VH-CDR3；如SEQ ID NO:14所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:15所示的VL-CDR3；
    (4)如SEQ ID NO:16所示的VH-CDR1、如SEQ ID NO:17所示的VH-CDR2、如SEQ ID NO:18所示的VH-CDR3；如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:19所示的VL-CDR3；
    (5)如SEQ ID NO:20所示的VH-CDR1、如SEQ ID NO:21所示的VH-CDR2、如SEQ ID NO:22所示的VH-CDR3；如SEQ ID NO:23所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:25所示的VL-CDR3；
    (6)如SEQ ID NO:26所示的VH-CDR1、如SEQ ID NO:27所示的VH-CDR2、如SEQ ID NO:28所示的VH-CDR3；如SEQ ID NO:29所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:30所示的VL-CDR3；
    (7)如SEQ ID NO:31所示的VH-CDR1、如SEQ ID NO:32所示的VH-CDR2、如SEQ ID NO:33所示的VH-CDR3；如SEQ ID NO:34所示的VL-CDR1、如SEQ ID NO:35所示的VL-CDR2、如SEQ ID NO:36所示的VL-CDR3；
    (8)如SEQ ID NO:26所示的VH-CDR1、如SEQ ID NO:37所示的VH-CDR2、如SEQ ID NO:38所示的VH-CDR3；如SEQ ID NO:39所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:40所示的 VL-CDR3；
    (9)如SEQ ID NO:41所示的VH-CDR1、如SEQ ID NO:42所示的VH-CDR2、如SEQ ID NO:43所示的VH-CDR3；如SEQ ID NO:44所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:45所示的VL-CDR3；
    (10)如SEQ ID NO:11所示的VH-CDR1、如SEQ ID NO:12所示的VH-CDR2、如SEQ ID NO:47所示的VH-CDR3；如SEQ ID NO:14所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:15所示的VL-CDR3；
    (11)如SEQ ID NO:16所示的VH-CDR1、如SEQ ID NO:17所示的VH-CDR2、如SEQ ID NO:137所示的VH-CDR3；如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:19所示的VL-CDR3；
    (12)如SEQ ID NO:16所示的VH-CDR1、如SEQ ID NO:17所示的VH-CDR2、如SEQ ID NO:137所示的VH-CDR3；如SEQ ID NO:4所示的VL-CDR1、如SEQ ID NO:5所示的VL-CDR2、如SEQ ID NO:138所示的VL-CDR3；
    (13)如SEQ ID NO:31所示的VH-CDR1、如SEQ ID NO:32所示的VH-CDR2、如SEQ ID NO:33所示的VH-CDR3；如SEQ ID NO:136所示的VL-CDR1、如SEQ ID NO:35所示的VL-CDR2、如SEQ ID NO:36所示的VL-CDR3；
    (14)如SEQ ID NO:147所示的VH-CDR1、如SEQ ID NO:42所示的VH-CDR2、如SEQ ID NO:43所示的VH-CDR3；如SEQ ID NO:44所示的VL-CDR1、如SEQ ID NO:24所示的VL-CDR2、如SEQ ID NO:45所示的VL-CDR3；
    优选地，所述抗体分子或其片段包含的重链可变区和轻链可变区选自以下组合：
    (1)如SEQ ID NO:50所示的氨基酸序列或与如SEQ ID NO:50所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:51所示的氨基酸序列或与如SEQ ID NO:51所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (2)如SEQ ID NO:52所示的氨基酸序列或与如SEQ ID NO:52所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:53所示的氨基酸序列或与如SEQ ID NO:53所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (3)如SEQ ID NO:54所示的氨基酸序列或与如SEQ ID NO:54所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:55所示的氨基酸序列或与如SEQ ID NO:55所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (4)如SEQ ID NO:56所示的氨基酸序列或与如SEQ ID NO:56所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:57所示的氨基酸序列或与如SEQ ID NO:57所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (5)如SEQ ID NO:58所示的氨基酸序列或与如SEQ ID NO:58所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:59所示的氨基酸序列或与如SEQ ID NO:59所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (6)如SEQ ID NO:60所示的氨基酸序列或与如SEQ ID NO:60所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:61所示的氨基酸序列或与如SEQ ID NO:61所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (7)如SEQ ID NO:62所示的氨基酸序列或与如SEQ ID NO:62所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:63所示的氨基酸序列或与如SEQ ID NO:63所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (8)如SEQ ID NO:64所示的氨基酸序列或与如SEQ ID NO:64所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:65所示的氨基酸序列或与如SEQ ID NO:65所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (9)如SEQ ID NO:66所示的氨基酸序列或与如SEQ ID NO:66所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:67所示的氨基酸序列或与如SEQ ID NO:67所示的氨基酸序列具有至少75％同一性 的氨基酸序列；
    (10)如SEQ ID NO:69所示的氨基酸序列或与如SEQ ID NO:69所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:90所示的氨基酸序列或与如SEQ ID NO:90所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (11)如SEQ ID NO:71所示的氨基酸序列或与如SEQ ID NO:71所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:90所示的氨基酸序列或与如SEQ ID NO:90所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (12)如SEQ ID NO:74所示的氨基酸序列或与如SEQ ID NO:74所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:92所示的氨基酸序列或与如SEQ ID NO:92所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (13)如SEQ ID NO:75所示的氨基酸序列或与如SEQ ID NO:75所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:94所示的氨基酸序列或与如SEQ ID NO:94所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (14)如SEQ ID NO:76所示的氨基酸序列或与如SEQ ID NO:76所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:94所示的氨基酸序列或与如SEQ ID NO:94所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (15)如SEQ ID NO:77所示的氨基酸序列或与如SEQ ID NO:77所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:95所示的氨基酸序列或与如SEQ ID NO:95所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (16)如SEQ ID NO:83所示的氨基酸序列或与如SEQ ID NO:83所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:102所示的氨基酸序列或与如SEQ ID NO:102所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (17)如SEQ ID NO:84所示的氨基酸序列或与如SEQ ID NO:84所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:102所 示的氨基酸序列或与如SEQ ID NO:102所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (18)如SEQ ID NO:85所示的氨基酸序列或与如SEQ ID NO:85所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:134所示的氨基酸序列或与如SEQ ID NO:134所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (19)如SEQ ID NO:88所示的氨基酸序列或与如SEQ ID NO:88所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:103所示的氨基酸序列或与如SEQ ID NO:103所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (20)如SEQ ID NO:88所示的氨基酸序列或与如SEQ ID NO:88所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:104所示的氨基酸序列或与如SEQ ID NO:104所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (21)如SEQ ID NO:140所示的氨基酸序列或与如SEQ ID NO:140所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:143所示的氨基酸序列或与如SEQ ID NO:143所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (22)如SEQ ID NO:141所示的氨基酸序列或与如SEQ ID NO:141所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:144所示的氨基酸序列或与如SEQ ID NO:144所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (23)如SEQ ID NO:142所示的氨基酸序列或与如SEQ ID NO:142所示的氨基酸序列具有至少75％同一性的氨基酸序列；和，如SEQ ID NO:144所示的氨基酸序列或与如SEQ ID NO:144所示的氨基酸序列具有至少75％同一性的氨基酸序列。
  10. 根据权利要求6至9中任一项所述的抗体分子或其片段，其特征在于，所述抗体分子或其片段为单克隆抗体、单链抗体、双功能抗体、单域抗体、纳米抗体、完全或部分人源化的抗体或者嵌合抗体等任意形式，或者，所述抗体分子或其片段为半抗体或半抗体的抗原结合片段，例scFv、BsFv、dsFv、(dsFv) 2、Fab、Fab'、F(ab') 2或Fv；
    优选地，所述抗体分子或其片段还包含人或鼠的恒定区，优选包含人或鼠的轻链恒定区(CL)和/或重链恒定区(CH)；
    更优选地，所述抗体分子或其片段包含选自IgG、IgA、IgM、IgD或IgE的重链恒定区和/或κ或λ型轻链恒定区。
  11. 根据权利要求6至10中任一项所述的抗体分子或其片段，其特征在于，所述抗体分子为单克隆抗体，优选为鼠源、嵌合或人源化的单克隆抗体；优选地，所述单克隆抗体的重链恒定区为IgG1或IgG4亚型，轻链恒定区为κ型；
    优选地，所述单克隆抗体的重链恒定区包含如SEQ ID NO:106所示的氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列；
    优选地，所述单克隆抗体的轻链恒定区包含如SEQ ID NO:107所示氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列。
  12. 一种缀合物或融合蛋白，所述缀合物或融合蛋白包含权利要求1至11中任一项所述的抗体分子或其片段。
  13. 一种核酸分子，其编码权利要求1至11中任一项所述的抗体分子或其片段或者编码所述抗体分子或其片段中包含的重链CDR、轻链CDR、轻链可变区、重链可变区、重链或轻链。
  14. 一种载体，其包含权利要求13所述的核酸分子。
  15. 一种宿主细胞，所述宿主细胞包含权利要求13所述的核酸分子和/或权利要求14所述的载体，或者所述宿主细胞被权利要求13所述的核酸分子和/或权利要求14所述的载体转化或转染。
  16. 一种药物组合物，其包含权利要求1至11中任一项所述的抗体分子或其片段、权利要求12所述的缀合物或融合蛋白、权利要求13所述的核酸分子、权利要求14所述的载体或权利要求15所述的宿主细胞，以及任选地药学上可接受的辅料。
  17. 权利要求1至11中任一项所述的抗体分子或其片段、权利要求12所述的缀合物或融合蛋白、权利要求13所述的核酸分子、权利要求14所述的载体或权利要求15所述的宿主细胞在制备药物中的用途，所述药物用于治疗与CD38高表达相关的疾病；
    优选地，所述疾病为恶性血液肿瘤，优选为多发性骨髓瘤或非霍奇金淋巴瘤。
  18. 一种试剂盒，所述试剂盒包括权利要求1至11中任一项所述的抗体分子或其片段、权利要求12所述的缀合物或融合蛋白、权利要求13所述的核酸分子、权利要求14所述的载体、权利要求15所述的宿主细胞或权利要求16所述的药物组合物。
  19. 一种预防或治疗与CD38表达相关或由CD38介导的疾病的方法，所述方法包括给有此需要的受试者施用本发明的抗体分子或其片段、缀合物或融合蛋白、核酸分子、载体、宿主细胞和/或药物组合物，以及任选的其他药物或手段。
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