WO2020052546A1 - 抗cd38抗体、其抗原结合片段及医药用途 - Google Patents

抗cd38抗体、其抗原结合片段及医药用途 Download PDF

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WO2020052546A1
WO2020052546A1 PCT/CN2019/105119 CN2019105119W WO2020052546A1 WO 2020052546 A1 WO2020052546 A1 WO 2020052546A1 CN 2019105119 W CN2019105119 W CN 2019105119W WO 2020052546 A1 WO2020052546 A1 WO 2020052546A1
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seq
amino acid
sequence
antibody
heavy chain
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English (en)
French (fr)
Inventor
叶鑫
孙乐
宋明娟
傅蓓蓓
王小华
张蕾
陶维康
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Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
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Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
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Priority to CA3111651A priority Critical patent/CA3111651A1/en
Priority to AU2019338999A priority patent/AU2019338999A1/en
Priority to EP19859106.7A priority patent/EP3851456A4/en
Priority to US17/274,082 priority patent/US20220275100A1/en
Priority to MX2021002531A priority patent/MX2021002531A/es
Priority to BR112021004130-3A priority patent/BR112021004130A2/pt
Priority to KR1020217009542A priority patent/KR20210057752A/ko
Priority to JP2021512532A priority patent/JP2021536254A/ja
Priority to CN201980049843.1A priority patent/CN112513087B/zh
Publication of WO2020052546A1 publication Critical patent/WO2020052546A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IG], 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/5759—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds localised on the membrane of tumour or cancer cells
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/6857—Antibody fragments
    • 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
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    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91091—Glycosyltransferases (2.4)
    • G01N2333/91148—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)

Definitions

  • the present disclosure belongs to the field of biotechnology, and more particularly, the present disclosure relates to therapeutic uses of anti-CD38 antibodies and compositions thereof, and methods for producing the antibody molecules.
  • Multiple myeloma (Multiple myeloma, MM) is a malignant plasma cell disease. Its tumor cells originate from plasma cells in the bone marrow, and plasma cells are cells that develop B lymphocytes to the final functional stage. Therefore, multiple myeloma can also be classified as B lymphocytic lymphoma.
  • M protein monoclonal immunoglobulin or light chain
  • China's domestic incidence is estimated to be 2-3 persons per 100,000, with a male to female ratio of 1.6: 1.
  • Most patients are older than 40 years old and mostly older than 60 years old.
  • Multiple myeloma accounts for about 10% of various hematomas. As the aging trend intensifies, the number of patients will further intensify.
  • multiple myeloma is an incurable malignant tumor of the blood system.
  • the main treatments available include: bone marrow transplantation, multiple small molecule chemotherapy, especially protease inhibitors represented by carfilzomib.
  • the immune modulator represented by lenalidomide significantly prolongs the survival of patients with MM.
  • the disease almost always recurs in the end, and the average survival period after relapse is only about 9 months.
  • CD38 is a type II transmembrane multifunctional protein with an extracellular domain of 256 amino acids. On the one hand, it has been proved indirectly that CD38 exerts the role of lateral signal transmission through its ligand CD31, which can cause cell adhesion and play a role in lymphocyte activation and B cell differentiation, but there is no direct evidence for this function in biochemistry.
  • CD38 has the functions of cyclase and hydrolase, and can promote the conversion of NAD (nicotinamide adenine dinucleotide) to cADPR (cycloadenosine diphosphate ribose) through the action of cyclase, and also can pass cADPR Hydrolytic enzymes promote the conversion of cADPR to ADPR (adenosine diphosphate ribose).
  • CD38 can regulate Ca2 + flow, and also can promote cell production of adenosine to suppress immunity. Changes in Ca2 + flow may affect insulin secretion.
  • CD38 knockout mice have shown normal survival, but with different symptoms such as decreased humoral immune response, decreased insulin, and heart / pulmonary muscle defects.
  • RNA levels show higher expression in prostate and thymus, and especially in a variety of hematoma cells are significantly overexpressed.
  • the clinical data of darlimumab also verified the efficacy and safety of CD38 antibody in the field of multiple myeloma, suggesting the potential value of CD38 target development.
  • Various literature studies show that the mechanism of action of anti-CD38 antibodies in the treatment of MM includes:
  • ADCC mainly related to epitopes and IgG1-Fc;
  • CD38 target has become a hot spot in the treatment of multiple myeloma.
  • Another important CD38-targeting antibody is Sanofi's Isatuximab. It has also conducted multiple clinical trials of single-use and combination with small-molecule drugs. From the available data, it is seen Xiximab has the same efficacy and safety as darlimumab.
  • antibodies such as MOR202 (Morphasys) and TAK-079 (Takeda) have already entered the clinic, and there are also CD38 / CD3 bispecific antibodies and CD38-CAR-T in the preclinical research stage.
  • WO2006099875 WO2007042309, WO2008047242, WO2012092612, WO2016164656, WO2017149122, etc.
  • the present disclosure provides a series of CD38 antibodies with higher affinity, better tumor suppressive activity in vivo, and good metabolic activity in vivo. Specifically, the present disclosure provides a monoclonal antibody or an antigen-binding fragment that specifically binds to human CD38.
  • the disclosure provides an anti-CD38 antibody or antigen-binding fragment thereof that specifically binds human CD38, the antibody or antigen-binding fragment thereof comprising a CDR as shown below:
  • the CDR variants of the anti-CD38 antibody or antigen-binding fragment including 3 heavy chain CDRs and 3 light chain CDRs) having 3, 2 or 1 amino acid differences are screened by affinity maturation methods CDR variants obtained with 3, 2 or 1 amino acid differences.
  • the affinity (KD) of the anti-CD38 antibody or antigen-binding fragment to human CD38 is less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, or less than 10 -11 M.
  • the anti-CD38 antibody is a murine antibody, a chimeric antibody, or a humanized antibody, preferably a humanized antibody.
  • the antibody is a murine antibody or a chimeric antibody, and the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 3, 5, 7 or the same as SEQ ID NO: 3 , 5, 7 have at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and / or the light chain variable region of the antibody, the amino acid sequence of which is as SEQ ID NO: 4 , 6, 8, or have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 4, 6, and 8.
  • the anti-CD38 antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region as shown below:
  • a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 3 or has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 3,
  • Light chain variable region the amino acid sequence of which is shown in SEQ ID NO: 4 or has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 4;
  • variable region of the heavy chain the amino acid sequence of which is shown in SEQ ID NO: 5 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
  • Light chain variable region the amino acid sequence of which is shown in SEQ ID NO: 6 or has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 6;
  • a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 7 or has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7, and
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 8 or has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8.
  • the antibody is a humanized antibody comprising a framework (FR) region or a framework region variant derived from a human germline, said framework region variant Chain framework regions and / or heavy chain framework regions each having up to 10 (e.g., 10, 9, 8, 7, 6, 6, 5, 4, 3, 2 or 1) amino acids Back mutation.
  • the humanized antibody comprises any one selected from the following (d) to (f):
  • a heavy chain variable region comprising: a heavy chain HCDR1, HCDR2, HCDR3, and a heavy chain framework region, wherein:
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 9 or has 3, 2 or 1 amino acid difference from SEQ ID NO: 9,
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 10 or has 3, 2 or 1 amino acid difference from the SEQ ID NO: 10 sequence,
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 11 or has 3, 2 or 1 amino acid difference from the SEQ ID NO: 11 sequence,
  • the heavy chain framework region has one or more back mutations selected from the group consisting of 2F, 38K, 44S, 48I, 67A, 66K, 69L, 71V, and 73Q; and / or
  • Light chain variable region the light chain variable region includes: a light chain LCDR1, LCDR2, LCDR3 and a light chain framework region, wherein:
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 12 or has 3, 2 or 1 amino acid difference from SEQ ID ID NO: 12,
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 13 or has 3, 2 or 1 amino acid difference from the SEQ ID NO: 13 sequence,
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 14 or has 3, 2 or 1 amino acid difference from the SEQ ID NO: 14 sequence,
  • the light chain framework region has one or more back mutations selected from 2F, 43S, 49K, and 87F;
  • a heavy chain variable region comprising: a heavy chain HCDR1, HCDR2, HCDR3, and a heavy chain framework region, wherein:
  • the sequence of the HCDR1 amino acid is shown in SEQ ID NO: 15 or has 3, 2 or 1 amino acid difference from SEQ ID NO: 15;
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 16 or has 3, 2 or 1 amino acid difference from the SEQ ID NO: 16 sequence,
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 17 or has 3, 2 or 1 amino acid difference from the SEQ ID NO: 17 sequence,
  • the heavy chain framework region has one or more back mutations selected from the group consisting of 79F, 82A T, 91S, and 76S; and / or;
  • Light chain variable region the light chain variable region includes: a light chain LCDR1, LCDR2, LCDR3 and a light chain framework region, wherein:
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 18 or has 3, 2 or 1 amino acid difference from SEQ ID NO: 18,
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 19 or has 3, 2 or 1 amino acid difference from the SEQ ID NO: 19 sequence,
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 20 or has 3, 2 or 1 amino acid difference from the SEQ ID NO: 20 sequence,
  • the light chain framework region has one or more back mutations selected from 58I, 68R, and 85T;
  • a heavy chain variable region comprising: a heavy chain HCDR1, HCDR2, HCDR3, and a heavy chain framework region, wherein:
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 15 or has 3, 2 or 1 amino acid difference from SEQ ID NO: 15;
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 21 or has 3, 2 or 1 amino acid difference from the SEQ ID NO: 21 sequence,
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 17 or has 3, 2 or 1 amino acid difference from the SEQ ID NO: 17 sequence,
  • the heavy chain framework region has one or more back mutations selected from 48I, 77T, and 82A T; and / or
  • Light chain variable region the light chain variable region includes: a light chain LCDR1, LCDR2, LCDR3 and a light chain framework region, wherein:
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 22 or has 3, 2 or 1 amino acid difference from SEQ ID ID NO: 22,
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 19 or has 3, 2 or 1 amino acid difference from the SEQ ID NO: 19 sequence,
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 23 or has 3, 2 or 1 amino acid difference from the SEQ ID ID NO: 23 sequence,
  • the light chain framework region has one or more back mutations selected from 4L, 9A, 22S, 58I, 60A, and 68R;
  • 82A T 82A refers to the first bit position 82A (numbering according to Kabat numbering convention) the amino acid backmutated threonine (Thr or abbreviated T).
  • the antibody heavy chain FR region sequence is selected from or has at least 95%, 96%, 97%, 98%, 99%, or a combination of human germline IGHV1-3 * 01 and hJH4.1 or 100% sequence identity.
  • the light chain FR region is selected from the combination of human germline IGKV3-11 * 01 and hJK4.1 and has at least 95%, 96%, 97%, 98%, 99% or 100% sequence Identity.
  • sequence of the heavy chain FR region is selected from the combination of human germline IGHV3-7 * 01 and FR4 of hJH6.1 or has at least 95%, 96%, 97%, 98%, 99 % Or 100% sequence identity.
  • the light chain FR region sequence is selected from or has at least 95%, 96%, 97%, 98%, 99%, or a combination of human germline IGKV4-1 * 01 and hJK4.1 or 100% sequence identity.
  • the humanized antibody comprises a heavy chain variable region or a variant thereof having the sequence shown in SEQ ID NO: 24, 32, or 37; wherein the variant is a heavy chain variable region
  • the framework region in the variable region has 1-10 (eg, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1) amino acid mutations.
  • the amino acid mutation is selected from any of the following:
  • the humanized antibody comprises:
  • a heavy chain variable region having a sequence such as SEQ ID NO: 26, 27, 28, 29, 34, or 39, or a sequence having at least 95%, 96% with SEQ ID NO: 26, 27, 28, 29, 34, or 39 Heavy chain variable regions with%, 97%, 98%, 99%, or 100% sequence identity.
  • the humanized antibody comprises a light chain variable region or a variant thereof having the sequence shown in SEQ ID NO: 25, 33, or 38; the variant is in the sequence SEQ ID NO: 25, 33, or 38 in the light chain variable region has 1 to 10 framework regions (e.g., 10, 9, 8, 7, 6, 6, 5, 4, 3 , 2 or 1) amino acid mutations.
  • amino acid mutation is selected from any one of (j) to (l):
  • the mutation sites are numbered according to Kabat numbering rules.
  • the humanized antibody comprises: a light chain variable region having a sequence such as SEQ ID NO: 30, 31, 35, 36, 40, 41, or 42, or a sequence identical to SEQ ID NO: 30, 31, 35, 36, 40, 41 or 42 a light chain variable region represented by an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the humanized antibody comprises:
  • the humanized antibody comprises:
  • the antibody further comprises a constant region; preferably, wherein the antibody is a chimeric or humanized antibody whose heavy chain constant region is derived from a human antibody IgG1, IgG2, IgG3, or IgG4, or Conventional variants of IgG1, IgG2, IgG3, or IgG4; the light chain constant region is derived from a human antibody kappa, lambda chain, or a conventional variant thereof.
  • the amino acid sequence of the constant region of the heavy chain is as shown in SEQ ID NO: 43 or 44 or has the same sequence as at least 95%, 96%, 97%, 98%, 99%, or 100%
  • the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 45 or has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it.
  • the heavy chain is as shown in SEQ ID NO: 46, 48, 49, 51, 52, or 54 or has at least 85% with SEQ ID NO: 46, 48, 49, 51, 52, or 54 , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; and /or
  • the light chain is as shown in SEQ ID NO: 47, 50, or 53 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91% of SEQ ID NO: 47, 50, or 53 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.
  • the anti-CD38 antibody comprises:
  • the anti-CD38 antibody comprises:
  • the anti-CD38 antibody or antigen-binding fragment thereof has enhanced ADCC activity, and the improvement of the activity is achieved by changing the affinity of the Fc region of the antibody or antigen-binding fragment thereof with FcyIIIa, for example, refer to MacroGenics The F243L, R292P, Y300L mutations and combinations thereof mentioned in patent WO2008140603, such as the S239D, I332E mutation or combination thereof in the IgG1 Fc region mentioned in Xencor patent US20080260731, and other mutations that can enhance ADCC function have been disclosed in the art.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab ', F (ab') 2, scFv, diabody, and dsFv.
  • the present disclosure also provides an anti-CD38 antibody that competitively binds human CD38 with the antibody or antigen-binding fragment thereof according to any one of the foregoing, or binds the same as the antibody or antigen-binding fragment with any one of the foregoing.
  • CD38 epitope CD38 epitope
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of an anti-CD38 antibody or an antigen-binding fragment thereof as described above, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.
  • Agent preferably, the unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of an anti-CD38 antibody or an antigen-binding fragment thereof; or the amount of the antibody or antigen-binding fragment thereof in a unit dose of the pharmaceutical composition is preferably 0.1-2000 mg, More preferably, it is 1-1000 mg.
  • the present disclosure also provides an isolated nucleic acid molecule encoding an anti-CD38 antibody or an antigen-binding fragment thereof according to any of the foregoing.
  • the present disclosure also provides a vector comprising the above-mentioned nucleic acid molecule.
  • the present disclosure also provides a host cell transformed (or transduced, transfected) with a vector as described above.
  • a host cell comprising a vector as described above.
  • the host cell is selected from prokaryotic cells and eukaryotic cells.
  • the host cell does not include any human cells capable of developing into a complete individual, such as human embryonic stem cells, fertilized eggs, germ cells; preferably, all
  • the host cell is a eukaryotic cell, more preferably a mammalian cell, wherein the mammalian cell includes, but is not limited to, CHO, 293, NSO, and sugar editing of the mammalian cell can change the sugar of the antibody or its antigen-binding fragment.
  • Cells that modify the ADCC function of the antibody or its antigen-binding fragment such as by knocking out genes such as FUT8 or GnT-III; in some embodiments, the mammalian cells do not include human cells.
  • the present disclosure also provides a method of preparing an anti-CD38 antibody or an antigen-binding fragment thereof as described above, the method comprising the steps of culturing the above-mentioned host cell and then recovering the anti-CD38 antibody or an antigen-binding fragment thereof; optionally including an anti-CD38 The antibody or antigen-binding fragment thereof is subjected to a purification step.
  • the present disclosure also provides a method for detecting or measuring human CD38, the method comprising contacting the above-mentioned anti-CD38 antibody or an antigen-binding fragment thereof to a test sample.
  • the present disclosure also provides a reagent for detecting or measuring human CD38, the reagent comprising the anti-CD38 antibody or an antigen-binding fragment thereof according to any one of the above.
  • the present disclosure also provides a diagnostic agent for a human CD38-related disease, the diagnostic agent comprising an anti-CD38 antibody or an antigen-binding fragment thereof according to the above.
  • the present disclosure also provides a method for diagnosing a human CD38-related disease, the method comprising detecting or determining human CD38 or CD38-positive cells using the above-mentioned anti-CD38 antibody or an antigen-binding fragment thereof.
  • the present disclosure also provides use of the above-mentioned anti-CD38 antibody or antigen-binding fragment thereof in the preparation of a diagnostic agent for a disease related to human CD38.
  • the present disclosure also provides a method for treating or preventing a disease, the method comprising administering to a subject a therapeutically effective or preventatively effective amount of the above-mentioned anti-CD38 antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the above-mentioned, or a nucleic acid as described above molecule.
  • the disease or disorder is a tumor or immune disease.
  • the disease or disorder is a CD38-positive disease or disorder.
  • the aforementioned disease or disorder is a tumor.
  • the aforementioned tumor is selected from the group consisting of leukemia, B-cell lymphoma, plasma cell malignant tumor, T / NK cell lymphoma, and myeloma.
  • the leukemia is selected from the group consisting of: acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute promyelocytic leukemia, chronic lymphocytic leukemia, acute and chronic myeloid leukemia.
  • the myeloma is selected from the group consisting of multiple myeloma, promyelocytic tumors, light amyloidosis, and the like.
  • the lymphoma is non-Hodgkin's lymphoma or Hodgkin's lymphoma.
  • the aforementioned tumor is a B-cell lymphoma / leukemia, for example, selected from a mature B-cell tumor or a precursor B-cell lymphoblastic leukemia / lymphoma, or a B-cell non-Hodgkin's lymphoma or B-cell Hodgkin's lymphoma.
  • the tumor is selected from the group consisting of: B-cell chronic lymphocytic leukemia (CLL) or small lymphocytic leukemia (SLL), B-cell acute lymphocytic leukemia, B-cell prelymphocytic leukemia, and lymphoplasmic cells Lymphoma, mantle cell lymphoma (MCL), low / intermediate / advanced follicular lymphoma (FL), skin follicular central lymphoma, marginal zone B cell lymphoma (including MALT type, lymph node MZBL type, spleen MZBL Type), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma (Burkitt lymphoma), plasmacytoma / plasma cell myeloma, plasma cell leukemia, lymphoproliferative disease after transplantation, Waldenstrom macroglobulin Anemia, plasma cell leukemia, anaplastic large cell lymphoma (ALCL), hairy cell lympho
  • CLL
  • the tumor is a B-cell lymphoma or multiple myeloma.
  • the tumor is multiple myeloma.
  • the aforementioned disease or disorder is an immune disease, such as an immune disease involving CD38-expressing B cells, plasma cells, monocytes, and T cells.
  • the immune disease includes, but is not limited to, rheumatoid arthritis, psoriasis, ankylosing spondylitis, joint psoriasis, dermatitis, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD ), Crohn's disease (Crohn's disease), ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, gastritis, uveitis, glomerulonephritis, eczema, asthma, arteriosclerosis, leukocyte adhesion defects Disease, Raynaud syndrome, Sjogren syndrome, juvenile diabetes, Reiter disease, Behcet disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia symptoms (such as acute idiopathic thrombocytopenic purpura, chronic idiopathic Thrombocytopenic purpura), hemolytic
  • IBD
  • the immune disease is selected from: rheumatoid arthritis, systemic lupus erythematosus, asthma, inflammatory bowel disease, multiple sclerosis, Crohn's disease, gastritis, Hashimoto's thyroid Inflammation, ankylosing spondylitis, and grafts against host disease.
  • the disease or disorder is rheumatoid arthritis.
  • the present disclosure further provides the use of an anti-CD38 antibody or an antigen-binding fragment thereof as described above or a pharmaceutical composition or a nucleic acid molecule as described above in the manufacture of a medicament for treating or preventing a disease or disorder.
  • the disease or disorder is a tumor or immune disease.
  • the disease or disorder is a CD38-positive disease or disorder.
  • the aforementioned disease or condition may be a tumor, for example, the disease is characterized by the presence of tumor cells expressing CD38.
  • the aforementioned tumor is selected from the group consisting of leukemia, B-cell lymphoma, plasma cell malignant tumor, T / NK cell lymphoma, and myeloma.
  • the leukemia is selected from the group consisting of: acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute promyelocytic leukemia, chronic lymphocytic leukemia, acute and chronic myeloid leukemia.
  • the myeloma is selected from the group consisting of: multiple myeloma, promyelocytic tumor, and light amylinosis.
  • the lymphoma is non-Hodgkin's lymphoma or Hodgkin's lymphoma.
  • the aforementioned tumor is a B-cell lymphoma / leukemia, for example, selected from a mature B-cell tumor or a precursor B-cell lymphoblastic leukemia / lymphoma, or a B-cell non-Hodgkin's lymphoma or B-cell Hodgkin's lymphoma.
  • the aforementioned tumor is selected from the group consisting of: B-cell chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), B-cell acute lymphocytic leukemia, B-cell prelymphocytic leukemia, and lymphoplasmacytoid Lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (including low, intermediate, or advanced FL), skin follicular central lymphoma, marginal zone B-cell lymphoma (including MALT type, lymph node MZBL type, spleen MZBL Type), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma (Burkitt lymphoma), plasma cell tumor, plasma cell myeloma, plasma cell leukemia, lymphoproliferative disease after transplantation, Waldenstrom macroglobulin Anemia, plasma cell leukemia and anaplastic large cell lymphoma (ALCL), hairy
  • CLL
  • the tumor is a B-cell lymphoma or multiple myeloma.
  • the tumor is multiple myeloma.
  • the disease or disorder is an immune disease, such as an immune disease involving CD38-expressing B cells, plasma cells, monocytes, and T cells.
  • the immune disease may be selected from: rheumatoid arthritis, psoriasis, ankylosing spondylitis, joint psoriasis, dermatitis, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD ), Crohn's disease (Crohn's disease), ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, gastritis, uveitis, glomerulonephritis, eczema, asthma, arteriosclerosis, leukocyte adhesion defects Disease, Raynaud syndrome, Sjogren syndrome, juvenile diabetes, Reiter disease, Behcet disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia symptoms (such as acute idiopathic thrombocytopenic purpura, chronic idiopathic Thrombocytopenic purpura), hemolytic anemia,
  • IBD
  • the immune disease is selected from rheumatoid arthritis, systemic lupus erythematosus, asthma, inflammatory bowel disease, multiple sclerosis, Crohn's disease, gastritis, Hashimoto's thyroiditis, Ankylosing spondylitis and grafts fight host disease. In some embodiments, wherein the immune disease or disorder is rheumatoid arthritis.
  • the present disclosure further provides an anti-CD38 antibody or an antigen-binding fragment thereof, or a pharmaceutical composition, or a nucleic acid molecule for use in treating or preventing the aforementioned disease or condition.
  • the disease or disorder is a tumor or immune disease; in some embodiments, the disease or disorder is a CD38-positive disease or disorder.
  • the aforementioned disease or condition is a tumor, for example, the disease is characterized by the presence of tumor cells expressing CD38.
  • the tumor is selected from the group consisting of leukemia, B-cell lymphoma, plasma cell malignant tumor, T / NK cell lymphoma and myeloma.
  • the leukemia is selected from the group consisting of: acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute promyelocytic leukemia, chronic lymphocytic leukemia, acute and chronic myeloid leukemia.
  • the myeloma is selected from the group consisting of: multiple myeloma, promyelocytic tumor, and light amylinosis.
  • the lymphoma is non-Hodgkin's lymphoma or Hodgkin's lymphoma.
  • the tumor is a B-cell lymphoma / leukemia, such as selected from a mature B-cell tumor or a precursor B-cell lymphoblastic leukemia / lymphoma, or a B-cell non-Hodgkin's lymphoma or B Cell Hodgkin's lymphoma.
  • the tumor is selected from the group consisting of: B-cell chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), B-cell acute lymphocytic leukemia, B-cell prelymphocytic leukemia, and lymphoplasmic cells Lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (including low, intermediate, or advanced FL), skin follicular central lymphoma, marginal zone B cell lymphoma (including MALT type, lymph node MZBL type, spleen MZBL type), hairy cell leukemia, diffuse large B cell lymphoma, Burkitt lymphoma (Burkitt lymphoma), plasmacytoma, plasma cell myeloma, plasma cell leukemia, post-transplant lymphoproliferative disease, Waldenstrom giant ball Proteinemia, plasma cell leukemia, and anaplastic large cell lymphoma (ALCL), hairy cell lymphoma.
  • CLL
  • the aforementioned disease or disorder is an immune disease, such as an immune disease involving CD38-expressing B cells, plasma cells, monocytes, and T cells.
  • the immune disease may be selected from: rheumatoid arthritis, psoriasis, joint psoriasis, dermatitis, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD), Crohn disease, Ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, arteriosclerosis, leukocyte adhesion deficiency disease, multiple sclerosis, Raynaud syndrome, Sjogren syndrome, adolescents Diabetes, Reiter disease, Behcet disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, symptoms of immune-mediated thrombocytopenia (such as acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura), hemolysis Anemia, myasthenia gravis, lupus nep
  • IBD
  • the immune disease is selected from rheumatoid arthritis, systemic lupus erythematosus, asthma, inflammatory bowel disease, multiple sclerosis, Crohn's disease, gastritis, Hashimoto's thyroiditis , Ankylosing spondylitis and grafts against host disease. In some embodiments, wherein the immune disease is rheumatoid arthritis.
  • the anti-CD38 antibody or antigen-binding fragment thereof disclosed in the present disclosure shows good results in biochemical tests or in vitro and in vivo drug efficacy tests.
  • the KD value of the antibody hu9E and human CD38 in the present disclosure is 1.31 nM
  • the KD value of hu11E and human CD38 is 0.568 nM
  • the KD value of hu160E and human CD38 is 0.0585 nM
  • the KD of the control antibody was 2.35 nM
  • the antibodies in this disclosure have high affinity (Table 18).
  • the antibodies of hu11E and hu160E in the present disclosure can effectively inhibit tumor growth in mice.
  • the tumor inhibition rate of hu11E is as high as 93.14%.
  • the tumor inhibition rate was as high as 70.02%, which was significantly higher than the control antibody Dara (tumor inhibition rate was 56.83%) (Table 20).
  • the CD38 monoclonal antibody or antigen-binding fragment of the present disclosure has good metabolic kinetic characteristics in rats, showing a long half-life and high bioavailability.
  • FIG. 1A In vitro ADCP test results of CD38 antibody on Molp-8 cells.
  • FIG. 1B In vitro ADCP test results of CD38 antibody on Daudi cells.
  • FIG. 1 Antitumor effect of CD38 antibody in mice.
  • the “antibody” in the present disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure formed by connecting two identical heavy chains and two identical light chains through interchain disulfide bonds.
  • the amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes called immunoglobulins, that is, IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , Alpha, and epsilon chains.
  • Igs of the same class can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by different constant regions.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of the heavy and light chains of the antibody varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). Three hypervariable regions determine the specificity of an antibody, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (VL or LCVR) and heavy chain variable region (VH or HCVR) are composed of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminal to the carboxy terminal is: FR1, CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.
  • the antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, and preferably humanized antibodies.
  • mouse antibody in the present disclosure is a monoclonal antibody against human CD38 prepared according to the knowledge and skill in the art. Test subjects are injected with CD38 antigen during preparation, and then hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated.
  • the mouse-derived CD38 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse-derived kappa, lambda chain or a variant thereof, or further include a mouse-derived IgG1, IgG2 , IgG3 or a variant of the heavy chain constant region thereof.
  • chimeric antibody refers to an antibody that fuses the variable region of an antibody of one species (such as a mouse) with the constant region of an antibody of another species (such as a human), and can reduce the induction of murine antibodies. Immune response. To establish a chimeric antibody, first establish a hybridoma that secretes a mouse-specific monoclonal antibody, and then clone the variable region gene from the mouse hybridoma cell; then, clone the constant region gene of the human antibody as needed; The gene is linked to the human constant region gene to form a chimeric gene and inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.
  • the antibody light chain of the CD38 chimeric antibody further comprises a light chain constant region of a human-derived ⁇ , ⁇ chain, or a variant thereof.
  • the antibody heavy chain of the CD38 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a conventional variant thereof, preferably a human IgG1 heavy chain constant region, and more preferably a function of enhancing CDC Amino acid mutations (such as the E333A mutation) of the IgG1 heavy chain constant region.
  • humanized antibody refers to an antibody produced by transplanting a mouse CDR sequence into a human antibody variable region framework, that is, a different type of human germline antibody framework sequence. It can overcome the heterogeneous response induced by the chimeric antibody because it carries a large amount of murine protein components.
  • framework sequences can be obtained from a public DNA database including germline antibody gene sequences or published references.
  • Germline DNA sequences such as human heavy and light chain variable region genes can be obtained in the "VBase" human germline sequence database (Internet www.mrccpe.com.ac.uk/vbase), and in Kabat, EA et al. , 1991 Sequences of Proteins of Immunological Interest, 5th edition.
  • the human antibody variable region framework sequence may be subjected to a minimum of reverse mutations (or back mutations) to maintain the activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies further obtained by affinity maturation of CDRs from phage display or yeast display.
  • CDRs may result in a decrease in the affinity of the produced antibody or antigen-binding fragment thereof for the antigen due to changes in the framework residues in contact with the antigen. Such interactions can be the result of highly mutated somatic cells. Therefore, it may still be necessary to transplant such donor framework amino acids to the framework of a humanized antibody.
  • Amino acid residues involved in antigen binding from non-human antibodies or antigen-binding fragments thereof can be identified by examining the sequence and structure of the variable regions of animal monoclonal antibodies. Residues in the CDR donor framework that differ from the germline can be considered to be related.
  • the sequence can be compared to the consensus of a subtype consensus sequence or a murine sequence with a high percentage of similarity. Rare framework residues are thought to be the result of high somatic mutations and thus play an important role in binding.
  • the antibody light chain of the CD38 humanized antibody further comprises a light chain constant region of a human-derived ⁇ , ⁇ chain, or a conventional variant thereof.
  • the antibody heavy chain of the CD38 humanized antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a conventional variant thereof, preferably a human IgG1 heavy chain constant region, and more preferably a CDC-enhancing region.
  • the IgG1 heavy chain constant region of a functional amino acid mutation (such as the E333A mutation).
  • the "conventional variant" of the human antibody heavy chain constant region and the human antibody light chain constant region described in the present disclosure refers to the heavy chain constant region of human origin that does not change the structure and function of the variable region of the antibody, as disclosed in the prior art. Or light chain constant region variants.
  • Exemplary variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants that perform site-directed modification and amino acid substitution of the heavy chain constant region, specifically replacing YTE as known in the art.
  • back mutation refers to mutating the amino acid residues of the FR region derived from the human antibody to the corresponding amino acid residues of the original source antibody, usually to avoid the decrease in immunogenicity and activity caused by the humanized antibody.
  • the humanized antibody variable region can be subjected to minimal reverse mutations to maintain the activity of the antibody.
  • Human antibodies (HuMAb)", “human antibodies”, “fully human antibodies”, “fully human antibodies” can be used interchangeably, and can be antibodies derived from humans or antibodies obtained from a transgenic organism, The transgenic organism is "engineered” to produce specific human antibodies in response to antigenic stimulation, and can be produced by any method known in the art. In some technologies, elements of human heavy and light chain loci are introduced into cell lines of organisms derived from embryonic stem cell lines, where endogenous heavy and light chain loci are targeted damage. Transgenic organisms can synthesize human antibodies specific for human antigens, and the organisms can be used to produce hybridomas that secrete human antibodies.
  • a human antibody can also be an antibody in which the heavy and light chains are encoded by a nucleotide sequence derived from one or more personal DNA sources.
  • Fully human antibodies can also be constructed by gene or chromosome transfection methods and phage display technology, or by activated B cells in vitro, all of which are known in the art.
  • full-length antibody “whole antibody”, “whole antibody” and “whole antibody” are used interchangeably herein and refer to a substantially complete form of the antibody, distinguished from the antigen-binding fragments as defined below.
  • the term refers specifically to antibodies that include constant regions in the light and heavy chains.
  • the full-length antibodies of the present disclosure include a light chain variable region and a light chain constant region shown in Table 1 below, and a heavy chain variable region and a heavy chain constant region are linked to form a full-length antibody.
  • a person skilled in the art can select different light chain constant regions and heavy chain constant regions from different antibodies, for example, light chain constant regions (or conventional variants) and heavy chain constant regions (or conventional variants thereof) derived from human antibodies. body). Meanwhile, the combination of the light chain variable region and the heavy chain variable region in Table 1 may form a single chain antibody (scFv), a Fab, or other scFv or Fab-containing antigen-binding fragment forms.
  • Table 1 Combination of light chain variable region and heavy chain variable region of anti-CD38 humanized antibody
  • h009-01V means the light / heavy chain variable region pair numbered h009-01V, the heavy chain variable region is h009VH1 (SEQ ID NO: 24), and the light chain variable region is h009VL1 ( SEQ ID NO: 25), and so on.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, in addition to possible variant antibodies (e.g., containing naturally occurring mutations or mutations produced during the manufacture of monoclonal antibody preparations, these variants Except usually present in small amounts), the individual antibodies constituting the population are the same and / or bind the same epitope.
  • polyclonal antibody preparations which typically contain different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation (formulation) is directed against a single determinant on an antigen.
  • monoclonal indicates the properties of an antibody as obtained from a substantially homogeneous antibody population and should not be construed as requiring the antibody to be made by any particular method.
  • monoclonal antibodies used in accordance with the present disclosure can be prepared by various techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and the use of transgenic animals containing all or part of a human immunoglobulin locus Methods, such methods, and other exemplary methods for making monoclonal antibodies are described herein.
  • antigen-binding fragment or “functional fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, CD38). It has been shown that fragments of a full-length antibody can be used to achieve the antigen-binding function of the antibody.
  • binding fragments included in the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) a F (ab ') 2 fragment, through a hinge A bivalent fragment formed by two Fab fragments connected to a disulfide bridge on the region, (iii) an Fv fragment consisting of the VH and VL domains of the one arm of the antibody; (iv) dsFv, consisting of VH and VL Stable antigen-binding fragments formed by disulfide bonds; and (v) diabody, bispecific antibody, and multispecific antibody comprising fragments such as scFv, dsFv, Fab, and the like.
  • VL and VH are encoded by separate genes
  • recombinant methods can be used to link them through a synthetic linker, resulting in a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called a monovalent molecule Chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883).
  • scFv monovalent molecule Chain Fv
  • Such single chain antibodies are also intended to be included in the term "antigen-binding fragment”.
  • Antigen-binding moieties can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the antigen-binding fragments of the present disclosure include Fab, F (ab ') 2, Fab', single chain antibody (scFv), dimerized V region (diabody), disulfide-stabilized V (DsFv) and so on.
  • Fab is an antibody fragment obtained by treating an IgG antibody molecule with a protease papain (amino acid residue at position 224 of the H chain), which has a molecular weight of about 50,000 and has antigen binding activity in the obtained fragment, in which the H chain N Approximately half of the ends and the entire L chain are held together by disulfide bonds.
  • protease papain amino acid residue at position 224 of the H chain
  • the Fab of the present disclosure can be produced by treating the monoclonal antibody of the present disclosure with papain.
  • the Fab can be produced by inserting DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab.
  • F (ab ') 2 refers to an antibody fragment having a molecular weight of approximately 100,000 and having antigen-binding activity, obtained by digesting the two disulfide bonds downstream of the IgG hinge region with pepsin, which contains two Fabs The zones are connected at hinge positions.
  • F (ab ') 2 of the present disclosure can be produced by treating a monoclonal antibody of the present disclosure with pepsin.
  • the F (ab ') 2 can be produced by connecting a Fab' described below with a thioether bond or a disulfide bond.
  • Fab ' is an antibody fragment having a molecular weight of about 50,000 and having an antigen-binding activity obtained by cutting the disulfide bond of the hinge region of F (ab') 2 described above.
  • the Fab 'of the present disclosure can be produced by treating the F (ab') 2 of the present disclosure with a reducing agent such as dithiothreitol.
  • the Fab ' can be produced by inserting DNA encoding a Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab '.
  • single chain antibody single chain Fv or “scFv” is meant to include an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) linked by a linker Molecule.
  • Such scFv molecules may have a general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA90: 6444-6448) .
  • linkers useful in the present disclosure are by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immuno l. 31: 94-106, Hu et al. (1996) , Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the scFv of the present disclosure can be produced by obtaining the cDNA encoding VH and VL of the monoclonal antibody of the present disclosure, constructing a DNA encoding the scFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then The expression vector is introduced into a prokaryote or a eukaryote to express scFv.
  • a “diabody” is an antibody fragment in which scFv is dimerized, and is an antibody fragment having bivalent antigen-binding activity. In a bivalent antigen-binding activity, the two antigens may be the same or different.
  • Bispecific antibodies and multispecific antibodies refer to antibodies that can simultaneously bind two or more antigens or epitopes, and include scFv or Fab fragments that can bind CD38.
  • the diabody of the present disclosure can be produced by obtaining the cDNA encoding VH and VL of the monoclonal antibody of the present disclosure, constructing a DNA encoding scFv such that the amino acid sequence of the peptide linker is 8 residues or less, and The DNA is inserted into a prokaryotic or eukaryotic expression vector, and the expression vector is then introduced into a prokaryotic or eukaryotic organism to express a diabody.
  • DsFv is obtained by linking a polypeptide in which one amino acid residue in each of VH and VL is replaced by a cysteine residue via a disulfide bond between the cysteine residues.
  • the amino acid residues substituted with cysteine residues can be selected according to a known method (Protein Engineering, 7,697 (1994)) based on the three-dimensional structure prediction of the antibody.
  • the dsFv of the present disclosure can be produced by obtaining the cDNA encoding VH and VL of the monoclonal antibody of the present disclosure, constructing a DNA encoding dsFv, and inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, The expression vector is then introduced into a prokaryote or eukaryote to express dsFv.
  • framework (FR) refers to a part of an antibody's variable domain VL or VH, which serves as a scaffold for the antigen-binding loop (CDR) of the variable domain.
  • framework regions are variable domains that do not have CDRs.
  • amino acid difference or “amino acid mutation” refers to the difference between a polypeptide and its variant, and refers to the presence of amino acid changes or mutations in the variant protein or polypeptide compared to the original protein or polypeptide, including the occurrence of the original protein or polypeptide1 , 2, 3, or more amino acids are substituted, inserted or deleted.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • LCDR1, LCDR2, LCDR3 three CDRs in each light chain variable region.
  • the amino acid sequence boundaries of the CDRs can be determined using any of a variety of well-known schemes, including the "Kabat” numbering convention (see Kabat et al.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the CDRs are Amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89 in human VL -97 (LCDR3).
  • the number of CDR amino acid residues in VH is approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the number of CDR amino acid residues in VL Approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3).
  • the CDR regions of antibodies can be determined using the program IMGT / DomainGap Align.
  • epitope refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on a CD38 molecule).
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols Methods Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).
  • antibodies bind with an affinity (KD) of less than about 10 -8 M, such as about less than 10 -9 M, 10 -10 M, 10 -11 M or less.
  • KD refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.
  • the antibodies of the present disclosure bind to CD38 with a dissociation equilibrium constant (KD) of less than about 10 -7 M, such as less than about 10 -8 M, 10 -9 M, or 10 -10 M or less, for example, using a surface Plasmon resonance (SPR) technology was measured in a BIACORE instrument.
  • SPR surface Plasmon resonance
  • the term “competition” is used to compete for antigen-binding proteins (such as neutralizing antigen-binding proteins or neutralizing antibodies) for the same epitope, it means competition between antigen-binding proteins, which is determined by the following assay Assay:
  • the antigen-binding protein e.g., an antibody or functional fragment thereof
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, eg, Stahli et al., 1983, Methodsin Enzymology 9: 242-253
  • solid phase direct biotin-avidin EIA see, eg, Kirkland et al., 1986, J. Immunol.
  • the assay involves the use of purified antigens that are capable of binding to unlabeled detection antigen binding proteins and labeled reference antigen binding proteins (the antigens are on a solid surface or a cell surface).
  • the antigens are on a solid surface or a cell surface.
  • the amount of marker bound to a solid surface or a cell surface is measured to measure competitive inhibition.
  • the test antigen-binding protein is present in excess.
  • Antigen-binding proteins identified by competitive assays include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and tables that are sufficiently close to the epitope bound by the reference antigen-binding protein Binding antigen binding protein, the two epitopes spatially prevent each other from binding. Additional details on methods for determining competitive binding are provided in the examples of this disclosure. Usually when a competitive antigen binding protein is present in excess, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70 -75% or more specific binding of a reference antigen binding protein to a common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97% or more.
  • nucleic acid molecule refers to DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operatively linked to the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
  • the vector is a "plasmid”, which refers to a circular double-stranded DNA loop into which an additional DNA segment can be ligated.
  • the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in host cells into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial origins of replication) or can be integrated into the host cell's genome after introduction into the host cell, thereby accompanying The host genome is replicated together (eg, non-episomal mammalian vectors).
  • mice can be immunized with human CD38 or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibody or antigen-binding fragment according to the invention is genetically engineered to add one or more human-derived FR regions to a CDR region of non-human origin.
  • the human FR germline sequence can be obtained by aligning the IMGT human antibody variable region germline gene database with MOE software from the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001ISBN012441351 obtain.
  • IMGT ImMunoGeneTics
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include microorganisms (e.g., bacteria), plant or animal cells. Easily transformed bacteria include members of the enterobacteriaceae, such as strains of Escherichiacoli or Salmonella; Bacilillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomycescerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary Cell Line) and NSO cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains are cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems cause glycosylation of antibodies, especially the highly conserved N-terminal site in the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human CD38. Positive clones were expanded in serum-free medium in the bioreactor to produce antibodies.
  • the culture medium in which the antibody is secreted can be purified by conventional techniques.
  • a or G Sepharose FF columns with adjusted buffer for purification. Non-specifically bound components are washed away. Then bound antibody was eluted by pH gradient method, and antibody fragments were detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product sometimes needs to be immediately frozen, such as -70 ° C, or lyophilized.
  • administering when applied to an animal, human, subject, cell, tissue, organ, or biological fluid refer to an exogenous drug, therapeutic agent, diagnostic agent, or composition and animal, Human, subject, cell, tissue, organ or biological fluid contact.
  • administering may refer to, for example, treatment, pharmacokinetics, diagnostics, research, and experimental methods.
  • Treatment of a cell includes contact of a reagent with a cell, and contact of a reagent with a fluid, wherein the fluid is in contact with the cell.
  • administering “administering,” and “treating” also mean treating cells in vitro and ex vivo by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to a human, veterinary or research subject refers to therapeutic treatment, preventive or prophylactic measures, research and diagnostic applications.
  • Treatment means the administration to a patient of an internal or external therapeutic agent, such as a composition comprising any one of the antibodies or antigen-binding fragments thereof or a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, said patient
  • an internal or external therapeutic agent such as a composition comprising any one of the antibodies or antigen-binding fragments thereof or a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof
  • a therapeutic agent is administered in a treated patient or population in an amount effective to alleviate one or more diseases or symptoms to induce the deterioration of such symptoms or to inhibit the development of such symptoms to any clinically measurable degree.
  • the amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease or condition can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Evaluate whether the symptoms of the disease have been alleviated by any clinical test method that a doctor or other health care professional usually uses to assess the severity or progression of the symptoms.
  • any statistical test method known in the art such as Student's test, Chi-square test, Mann and Whitney's The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce symptoms of the target disease in a statistically significant number of subjects.
  • Constant modification or “conservative substitution or substitution” refers to the replacement of amino acids in proteins by other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.) Changes are made without altering the biological activity of the protein.
  • Those skilled in the art know that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub.Co. ,, P. 224, (4th edition)).
  • structural or functionally similar amino acid substitutions are unlikely to disrupt biological activity. Exemplary conservative substitutions are set out in Table 2 below, “Exemplary conservative amino acid substitutions.”
  • an “effective amount” or “effective dose” refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired results.
  • beneficial or desirable results include elimination or reduction of risk, reduction of severity or delayed onset of the disorder, including the biochemistry of the disorder, its complications, and intermediate pathological phenotypes presented during the development of the disorder, Academic and / or behavioral symptoms.
  • beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-associated disorders of the present disclosure or improving one or more symptoms of the disorder, reducing other agents required to treat the disorder Dosage, enhances the efficacy of another agent, and / or delays the progression of a target antigen-related disorder of the present disclosure in a patient.
  • Exogenous refers to a substance that is produced outside the organism, cell, or human as appropriate.
  • Endogenous refers to a substance that is produced in an organism, cell, or human body as appropriate.
  • the “mutated sequence” described in the present disclosure refers to a nucleotide sequence and an amino acid sequence of the present disclosure that are different from the nucleotide sequence and the amino acid sequence of the present disclosure in the case of appropriate modification such as substitution, insertion, or deletion. Percent sequence identity nucleotide and amino acid sequences.
  • the sequence identity described in this disclosure may be at least 85%, 90%, or 95%, non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.
  • homology refers to sequence similarity between two polynucleotide sequences or between two polypeptides.
  • a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by the same base, then the molecules are identical at that position Source.
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the total number of positions compared x 100.
  • sequences are optimally aligned, if 6 of the 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous; if the 100 positions in the two sequences are 95 Matches or is homologous, then the two sequences are 95% homologous.
  • comparisons are made when the two sequences are aligned to give the maximum percent homology.
  • the comparison can be performed by the BLAST algorithm, where the parameters of the algorithm are selected to give the largest match between the individual sequences over the entire length of the individual reference sequences.
  • the following references relate to BLAST algorithms often used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS): Altschul, SF et al. (1990) J. Mol.
  • PCR polymerase chain reaction
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from the total genome, and cDNA, phage, or plasmid sequences transcribed from total cellular RNA. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51: 263; edited by Erlich, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).
  • the PCR used herein is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample.
  • the method includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or Produce specific parts of nucleic acids.
  • isolated refers to a purified state, and in this case means that the specified molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media.
  • isolated is not intended to mean the absence of these materials at all or the absence of water, buffers, or salts, unless they are present in an amount that significantly interferes with the experimental or therapeutic use of a compound as described herein.
  • “Pharmaceutical composition” means a mixture containing one or more of the antibodies or antigen-binding fragments thereof described herein with other chemical components, such as a physiological / pharmaceutically acceptable carrier or excipient.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and then exerts the biological activity.
  • pharmaceutically acceptable carrier refers to any inactive substance suitable for use in a formulation for the delivery of antibodies or antigen-binding fragments.
  • the carrier can be an anti-adhesive, adhesive, coating, disintegrant, filler or diluent, preservative (such as antioxidant, antibacterial or antifungal agent), sweetener, absorption delaying agent, wetting Agents, emulsifiers, buffers, etc.
  • suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and the like
  • Osmotic agents are, for example, sugars, polyols, sorbitol and sodium chloride.
  • a "CD38-positive disease or disorder” is a disease or disorder in which cells expressing CD38 are present.
  • immune diseases involving CD38-expressing B cells, plasma cells, monocytes, and T cells one of the characteristics of the disease is a tumor disease in which tumor cells expressing CD38 are present, such as leukemia expressing CD38, B-cell lymphoma, plasma cell malignant tumor, T / NK cell lymphoma, and myeloma.
  • the leukemia is selected from the group consisting of: acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute promyelocytic leukemia, chronic lymphocytic leukemia, acute and chronic myeloid leukemia.
  • the myeloma is selected from the group consisting of: multiple myeloma, promyelocytic tumor, and light amylinosis.
  • the lymphoma is non-Hodgkin's lymphoma or Hodgkin's lymphoma.
  • the tumor may be selected from B-cell lymphoma / leukemia, including but not limited to: precursor B-cell lymphoblastic leukemia / lymphoma, B-cell non-Hodgkin lymphoma, or B-cell Hodgkin Lymphoma, mature B-cell tumor.
  • the tumor is selected from the group consisting of: B-cell chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), B-cell acute lymphocytic leukemia, B-cell prelymphocytic leukemia, and lymphoplasmic cells Lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (including low, intermediate, or advanced FL), skin follicular central lymphoma, marginal zone B cell lymphoma (including MALT type, lymph node MZBL type, spleen MZBL type), hairy cell leukemia, diffuse large B cell lymphoma, Burkitt lymphoma (Burkitt lymphoma), plasmacytoma, plasma cell myeloma, plasma cell leukemia, post-transplant lymphoproliferative disease, Waldenstrom giant ball Proteinemia, plasma cell leukemia, and anaplastic large cell lymphoma (ALCL), hairy cell lymphoma.
  • CLL
  • the tumor is multiple myeloma.
  • the immune disease is selected from: rheumatoid arthritis, psoriasis, ankylosing spondylitis, joint psoriasis, dermatitis, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD) , Crohn's disease (Crohn's disease), ulcerative colitis, respiratory distress syndrome, meningitis, gastritis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, arteriosclerosis, leukocyte adhesion deficiency disease , Raynaud syndrome, Sjogren syndrome, juvenile diabetes, Reiter disease, Behcet disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia symptoms (such as acute idiopathic thrombocytopenic purpura, chronic thrombocytopenia symptoms (
  • the immune disease is selected from rheumatoid arthritis, systemic lupus erythematosus, asthma, inflammatory bowel disease, multiple sclerosis, Crohn's disease, gastritis, Hashimoto's thyroiditis , Ankylosing spondylitis and grafts against host disease. In some embodiments, wherein the immune disease is rheumatoid arthritis.
  • the present disclosure provides a medicament for treating or preventing a disease associated with a target antigen (eg, CD38) -positive cells, the medicament comprising an anti-CD38 antibody of the present disclosure or an antigen-binding fragment thereof as an active ingredient, which can be administered to a subject in need
  • a therapeutically effective or prophylactically effective amount of the agent to treat or prevent a CD38-positive disease can inhibit CD38-induced disease-related activities or eliminate or reduce the number of CD38-expressing cells.
  • the therapeutically or prophylactically effective amount is a unit dose composition containing 0.1-3000 mg (preferably 0.1-2000 mg, more preferably 1-1000 mg) of the anti-CD38 antibody or antigen-binding fragment thereof as described above.
  • the present disclosure relates to a method for immunodetection or measurement of a target antigen (e.g., CD38), a reagent for immunodetection or measurement of a target antigen (e.g., CD38), and a cell for immunodetection or measurement of a target antigen (e.g., CD38).
  • a method for detecting or determining the amount of a target antigen may be any known method.
  • it includes immunological detection or assay methods.
  • the immunodetection or measurement method is a method for detecting or measuring the amount of the antibody or the amount of the labeled antigen or antibody.
  • Examples of the immunodetection or measurement method include radioactive substance-labeled immune antibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, western blotting, physicochemical method Wait.
  • the above-mentioned diseases associated with CD38-positive cells can be diagnosed by detecting or measuring cells expressing CD38 with the antibodies or antibody fragments of the present disclosure.
  • a known immunodetection method can be used, and an immunoprecipitation method, a fluorescent cell staining method, an immunotissue staining method, or the like is preferably used.
  • a fluorescent antibody staining method using the FMAT8100HTS system can be used.
  • a sample for detecting or measuring a target antigen for example, CD38
  • a target antigen for example, CD38
  • cells expressing the target antigen such as tissue cells, blood, plasma, Serum, pancreatic fluid, urine, feces, tissue fluid or culture fluid.
  • the diagnostic agent containing the monoclonal antibody or the antibody fragment thereof of the present disclosure may further contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction according to a required diagnostic method.
  • Reagents for performing an antigen-antibody reaction include buffers, salts, and the like.
  • the reagent for detection includes reagents commonly used in immunodetection or measurement methods, such as a labeled second antibody that recognizes the monoclonal antibody, an antibody fragment thereof, or a conjugate thereof, a substrate corresponding to the label, and the like.
  • UniProt ADP-ribose cyclase / Cyclic ADP-ribose hydrolase 1 (human CD38 protein, Uniprot number: P28907) was used as a template for CD38 to design the amino acid sequence of the antigen and detection protein used in the present disclosure.
  • CD38 protein is fused with different tags such as His tag or Fc. They were cloned into pTT5 vector (Biovector, Cat #: 102762) or pTargeT vector (Promega, A1410), and were transiently expressed in 293 cells or stably expressed in CHO-S cells, purified, and the antigens and detection proteins of the present disclosure were obtained.
  • CD38 extracellular domain and mouse IgG2a-Fc fusion protein CD38-ECD-mFc, as an immunogen
  • the underlined part is the mouse IgG2a-Fc part
  • CD38 extracellular domain and human IgG1Fc fusion protein CD38-ECD-Fc, as an immunogen
  • the underlined part is the human IgG1-Fc part
  • CD38 extracellular domain fusion protein with His tag CD38-ECD-His, as an immunogen or detection reagent:
  • the underlined part is a 6 ⁇ His label
  • the cell expression supernatant sample was centrifuged at high speed to remove impurities, the buffer was replaced with PBS, and imidazole was added to a final concentration of 5 mM. Equilibrate the nickel column with 5 mM imidazole in PBS and rinse 2-5 times the column volume. The replaced cell supernatant sample was applied to a Ni Sepharose excel column (GE, 17-3712-02). The column was rinsed with 5 mM imidazole in PBS until the A280 reading dropped to baseline. The column was then washed with PBS + 10 mM imidazole to remove non-specifically bound heteroproteins, and the effluent was collected.
  • Ni Sepharose excel column GE, 17-3712-02
  • the target protein was eluted with 300 mM imidazole in PBS, and the elution peak was collected. The collected eluate was concentrated and further purified by gel chromatography Superdex200 (GE, 28-9893-35). The mobile phase was PBS. The polymer peak was removed and the eluted peak was collected. The obtained protein was identified as correct after electrophoresis, peptide mapping, and LC-MS.
  • the His-labeled CD38-ECD-His SEQ ID NO: 57
  • CD38-ECD-His can also be used as an immunogen to stimulate mouse immunity after coupling reaction with KLH by in vitro chemistry.
  • the cell expression supernatant sample was centrifuged at high speed to remove impurities, and the supernatant was subjected to MabSelectSure (GE, 17-5438-01) affinity chromatography.
  • the MabSelect Sure column was first regenerated with 0.1M NaOH, washed with pure water, and equilibrated with PBS. After the supernatant was combined, the column was washed with PBS until the A280 reading dropped to baseline.
  • the target protein was eluted with 0.1M acetic acid buffer at pH 3.5, and neutralized with 1MTris-HCl.
  • the eluted samples were appropriately concentrated and further purified by Superdex200 (GE, 28-9893-35), which was well-equipped with PBS gel chromatography.
  • the receiver tubes where the target proteins were collected were collected and concentrated to the appropriate concentration.
  • This method is used to purify a CD38-ECD-Fc (SEQ ID NO: 2) fusion protein, and this method can also be used to purify humanized antibody proteins involved in this disclosure.
  • Example 3 Obtaining and preparing anti-human CD38 hybridoma monoclonal antibody
  • Anti-human CD38 monoclonal antibodies are produced by immunizing mice. SJL white mice for experiments, female, 4-6 weeks of age (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001).
  • mice SPF level. After the mice were purchased, they were raised in a laboratory environment for 1 week, a 12/12 hour light / dark cycle, a temperature of 20-25 ° C, and a humidity of 40-60%. The adapted mice were immunized according to different protocols, 3-5 mice per group.
  • Immune antigens can be CD38-ECD-His, CD38-ECD-Fc, CD38-FL-CHOS (transfected with human CD38 full-length CHOS cells), etc., a single agent can be used with different immune adjuvants or different types of immunity Proto-cross immunity.
  • the immune site can be subcutaneously in the abdominal cavity or the back, or alternately in both locations.
  • Exemplary immunization methods are, for example, immunization with Titermax (sigma Lot Num: T2684) or alum (Thremo Lot Num: 77161).
  • the ratio of antigen to adjuvant is 1: 1, and the ratio of antigen to adjuvant (alum) is 4: 1, 25-50 ⁇ g or 1 ⁇ 10 7 cells / head (first immunization), 25-50 ⁇ g or 1 ⁇ 10 7 cells / only (boost immunity).
  • the antigen was injected intraperitoneally (IP) or subcutaneously (SC) on day 0, and the immunization was repeated every two weeks after the first immunization. Blood was collected every three weeks, and antibody titers in mouse serum were determined by ELISA. After the 8-12th immunization, mice with high antibody titers and plateaus in the serum were selected for splenocyte fusion. Immunization was strengthened 3 days before splenocyte fusion, and an intraperitoneal (IP) injection of an antigen solution prepared with 25-50 ⁇ g or 1 ⁇ 10 7 cells / physiological saline was performed.
  • IP intraperitoneal
  • mice Sera with high antibody titers were selected (see Test Example 1, ELISA method combined with CD38), and the mice whose titers tended to plateau were fused with spleen cells. The selected mice were immunized 3 days before the fusion. The spleen lymphocytes and myeloma cells Sp2 / 0 cells ( CRL-8287 TM ) to obtain hybridoma cells. The fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI), and aliquoted into 96-well cell culture plates (1 ⁇ 10 5 cells / 150 ⁇ l / well), 37 ° C, 5% CO 2 incubation.
  • HAT complete medium RPMI-1640 medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI
  • HAT complete medium On the fifth day after fusion, HAT complete medium was added, 50 ⁇ l / well, and incubated at 37 ° C. and 5% CO 2 . From 7 to 8 days after fusion, according to the cell growth density, the medium was completely changed.
  • the medium was HT complete medium (RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI), 200 ⁇ l / well, 37 ° C, 5% CO 2 incubation.
  • CD38-binding ELISA was used to detect the cell growth density (see Test Example 1).
  • the cell supernatants of positive wells tested by ELISA were detected by FACS method combined with CD38-FL-CHO-S (see Test Example 2), the positive wells were exchanged, and the cells were expanded into a 24-well plate according to the cell density.
  • the cell lines transferred to the 24-well plate were re-tested for seed conservation and first subcloning.
  • the first subclone screening (see Test Examples 1, 2) was positive for seed conservation, and the second subcloning was performed.
  • the second subclone was positive (see Test Examples 1, 2) for seed conservation and protein expression. Multiple fusions yielded hybridoma cells with high CD38 affinity.
  • Hybridoma clones m009, m011, and m160 were obtained through screening experiments and binding experiments, and antibodies were further prepared by serum-free cell culture.
  • the antibodies were purified according to the purification examples for use in detection examples.
  • variable region of the mouse antibody of hybridoma clone 009 was determined as follows:
  • the underline in the above sequence is the CDR sequence determined according to the Kabat numbering rule, the FR sequence in italics, and the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • variable region of the mouse antibody of hybridoma clone m011 is as follows:
  • the underline in the above sequence is the CDR sequence determined according to the Kabat numbering rule, the FR sequence in italics, and the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • variable region of the mouse antibody of hybridoma clone m160 is as follows:
  • the underline in the above sequence is the CDR sequence determined according to the Kabat numbering rule, the FR sequence in italics, and the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • Candidate molecules obtained by hybridoma screening can be amplified and sequenced to obtain variable region coding gene sequences.
  • Primer and tail primers were designed based on the sequence obtained.
  • heavy / light chain variable region germline genes with high homology to m009, m011 and m160 were selected as templates, respectively.
  • the CDRs of the three mouse-derived antibodies were transplanted into the corresponding human-derived templates to form humanized antibody variable region sequences in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the humanized light chain template of the mouse antibody m009 is IGKV3-11 * 01 and hJK4.1, and the humanized heavy chain template is IGHV1-3 * 01 and hJH4.1.
  • the CDRs of m009 are transplanted onto the humanized template.
  • the humanized variable region sequence was obtained as follows:
  • the underline in the sequence is the CDR sequence determined according to the Kabat numbering rule, the FR sequence in italics, and the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the transplantation means that the mouse-derived antibody CDR is implanted into the human germline FR region; "A43S” means that the 43rd (according to the Kabat numbering system) A is mutated back to S on the basis of transplantation, and so on.
  • the humanized antibody variable region sequence of hybridoma clone m009 is as follows:
  • the aforementioned humanized light chain variable region and heavy chain variable region are respectively linked to the human germline light chain constant region (e.g., human kappa, lambda chain light chain constant region) and heavy constant region (e.g., human IgG1, IgG2, IgG3, or The heavy chain constant region of IgG4 or a variant thereof) is combined to form the heavy and light chains of a humanized antibody, thereby obtaining a complete humanized antibody of m009 (h009).
  • the heavy chain variable region and light chain variable region of the aforementioned h009 antibody are respectively lighter than the human IgG1 heavy chain constant region and the sequence shown in SEQ ID NO: 45 as shown in SEQ ID NO: 43. Combination of constant chain regions to obtain full-length humanized antibodies (h009-01 to h009-15).
  • the sequence of the variable region is shown in Table 5:
  • h009-07 in the table means h009-07, the heavy chain variable region of the humanized antibody is h009VH2, the light chain variable region is h009VL2, and so on for other antibodies.
  • the humanized light chain template of the mouse antibody m011 is IGKV4-1 * 01 and hJK4.1, and the humanized heavy chain template is IGHV3-7 * 01 and hJH6.1.
  • Human germline IGHV3-7 * 01 and hJH6.1 introduced N 82A T in the FR region (according to the Kabat numbering system, the 82A amino acid was mutated from asparagine (abbreviation: N or Asn) to threonine (abbreviation: T Or Thr)) mutation; and N76S (mutation of the amino acid at position 76 from asparagine (abbreviation: N or Asn) to threonine (abbreviation: S or Ser)) according to Kabat numbering system, CDR of m011 Transplanted into the human source template, the humanized variable region sequence was obtained as follows:
  • the underline in the sequence is the CDR sequence determined according to the Kabat numbering rule, the FR sequence in italics, and the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • Hybridoma clone m011 reverse mutation design see Table 6 below:
  • the transplantation indicates that the mouse-derived antibody CDR is implanted into the human germline FR region sequence.
  • G68R means that the 68th (according to the Kabat numbering system) G is back-mutated to R on the basis of transplantation, and so on.
  • variable region of h011 humanized antibody is as follows:
  • the aforementioned humanized light chain variable region and heavy chain variable region are respectively linked to the human germline light chain constant region (e.g., human kappa, lambda chain light chain constant region) and heavy constant region (e.g., human IgG1, IgG2, IgG3 or The heavy chain constant region of IgG4 or a variant thereof) is combined to form the heavy and light chains of a humanized antibody, thereby obtaining a complete humanized antibody (h011) of m011.
  • the heavy chain variable region and light chain variable region of the above h011 antibody are respectively related to the human IgG1 heavy chain constant region and the sequence shown in SEQ ID NO: 43 and human kappa of the sequence shown in SEQ ID NO: 45.
  • the light chain constant regions were combined to obtain full-length humanized antibodies (h011-01 to h011-06).
  • the sequences of the variable regions are shown in Table 7:
  • h011-04" in the table indicates that the humanized antibody numbered h011-04 is h011VH2 for the heavy chain variable region, h011VL2 for the light chain variable region, and so on for other antibodies.
  • the humanized light chain template of the mouse antibody m160 is IGKV4-1 * 01 and hJK4.1 and the humanized heavy chain template is IGHV3-7 * 01 and hJH6.1.
  • the CDRs of m160 were transplanted to the human template, and the sequence of the humanized variable region obtained was as follows:
  • the underline in the sequence is the CDR sequence determined according to the Kabat numbering rule, the FR sequence in italics, and the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • transplantation represents the insertion of the mouse antibody CDR into the human germline FR region sequence.
  • M4L means that the 4th position (according to the Kabat numbering system) is back-mutated to L on the basis of transplantation, and so on.
  • variable region of h160 humanized antibody is as follows:
  • the aforementioned humanized light chain variable region and heavy chain variable region are respectively linked to the human germline light chain constant region (e.g., human kappa, lambda chain light chain constant region) and heavy constant region (e.g., human IgG1, IgG2, IgG3, or The heavy chain constant region of IgG4 or a variant thereof) is combined to form the heavy and light chains of a humanized antibody, thereby obtaining a complete humanized antibody of m160 (h160).
  • the heavy chain variable region and light chain variable region of the above h160 antibody are respectively linked to the human IgG1 heavy chain constant region and the sequence shown in SEQ ID NO: 43 and human kappa shown in SEQ ID NO: 45.
  • the light chain constant regions were combined to obtain full-length humanized antibodies (h160-01 to h160-08).
  • the sequences of the variable regions are shown in Table 9:
  • h160-07 in the table means h160-07 of the humanized antibody with the heavy chain variable region of h160VH2, the light chain variable region of h160 and VL3, and other antibodies and so on.
  • Example 5 Construction and expression of an anti-human CD38 humanized antibody IgG1 or IgG1-E333A
  • Primer PCR was designed to construct each humanized antibody VH / VK gene fragment, and then homologous recombination with the expression vector pHr (with signal peptide and constant region gene (CH1-FC / CL) fragment, laboratory construction) to construct the full length of the antibody Expression vector VH-CH1-FC-pHr / VK-CL-pHr.
  • the light chain constant region of a humanized antibody may be selected from the human kappa and lambda chain light chain constant regions, and the heavy chain constant region may be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4, or variants thereof, without limitation.
  • Implementation includes optimizing the design of the constant regions of human IgG1, IgG2, and IgG4 to improve antibody functions.
  • the IgG1-E333A constant region can be obtained by E333A point mutation of IgG1. This mutation can enhance the binding capacity of IgG1-Fc to C1q and further enhance the antibody Function of CDC (see US6528624).
  • the following specific light / heavy chain constant regions are not limited to the constant regions of antibodies of the present disclosure, and other light / heavy chain constant regions and mutants thereof known in the art can also be selected.
  • Exemplary heavy and light chain constant regions are as follows:
  • IgG1 heavy chain constant region 1 + IgG1 heavy chain constant region:
  • IgG1-E333A heavy chain constant region IgG1-E333A heavy chain constant region
  • the humanized light chain variable region and heavy chain variable region of the aforementioned hybridoma clones m009, m011, and m160 are respectively linked to the human IgG1 heavy chain constant region and the sequence shown in SEQ ID NO: 43, such as SEQ ID: NO: 45, the combination of the human kappa light chain constant regions, the full-length humanized antibodies obtained are shown in Table 5, Table 7, and Table 9; another exemplary, the aforementioned hybridoma clones m009, m011
  • the light chain constant regions are combined to obtain a full-length humanized antibody.
  • the sequence of the light and heavy chain variable regions of the obtained humanized antibody is shown in Table 10:
  • the anti-CD38 antibody darlimumab (referred to as Dara, the sequence source refers to WHO Drug Information, Vol. 24, No. 1, 2010) as a control antibody
  • the heavy chain and light chain sequences are as follows:
  • Test example 1 ELISA experiment of CD38 antibody binding to CD38 protein
  • the affinity of the anti-CD38 antibody was detected by the amount of binding of the antibody to CD38 immobilized on the ELISA plate.
  • 2 ⁇ g / ml streptavidin (Abcam, CAT # ab123480) was coated on a 96-well ELISA plate (Costar, CAT # 3590). After the plate was washed and blocked, 2 ⁇ g / ml biotin-labeled CD38-ECD-His was added. After incubation, add anti-CD38 antibody samples diluted at different concentrations, and then wash the plate and add horseradish peroxidase-sheep anti-human F (ab ') 2 antibody (Jackson, CAT # 109-036-097), and then wash the plate. Tetramethylbenzidine solution was added for color development. Finally, a stop solution was added. OD450 was measured on a microplate reader and the EC50 value was calculated. The results are shown in Table 11.
  • CHO-S FreeStyle TM CHO-S cells, Invitrogen, R80007) cells (CD38-FL-CHOS) stably transfected with full-length human CD38 (Uniprot number: P28907) in CD CHO medium (Gibco, REF # 10743- 029), 1 ⁇ 10 6 cells / ml CD38-FL-CHOS cells were blocked with 1% BSA, anti-CD38 antibody samples diluted at different concentrations were added, washed twice, and then Alexa Fluor 488-goat anti-human ( H + L) antibody (Invitrogen, CAT # A11013). After washing twice, the fluorescence signal value was read using a flow cytometer. The results are shown in Table 12.
  • FACS detection results show that the humanized antibodies of the present disclosure have strong binding ability to the natural CD38 on the cell surface.
  • CD38-ECD-His was prepared with a 20 mM Tris-HCl (pH 6.5) buffer solution to a concentration of 4 ⁇ g / ml.
  • anti-CD38 antibody samples were prepared in different concentrations with buffer. Take 25 ⁇ l of CD38-ECD-His and anti-CD38 antibody samples to the bottom-through black wall 96-well plate (Corning, CAT # 3603). After 15 minutes of incubation at room temperature, 50 ⁇ l of a substrate NGD (Sigma, CAT # N5131-25MG) at a concentration of 200 ⁇ M was added.
  • a substrate NGD Sigma, CAT # N5131-25MG
  • PBMC Peripheral blood mononuclear cells
  • the experimental results show that the humanized antibodies in the present disclosure have a strong effect on ADCC of Molp-8 and Daudi cells in vitro, and can cause target cell lysis significantly.
  • Test example 5 CD38 antibody on CDC of Molp-8 and Daudi cells
  • the experimental results show that the humanized antibodies in the present disclosure have a strong effect on CDC of Molp-8 and Daudi cells in vitro, and can cause target cell lysis significantly.
  • the cells were plated at 2.5 ⁇ 10 4 cells / well (25 ⁇ l / well) in a 96-well plate (Corning, CAT # 3903), 25 ⁇ l of anti-CD38 antibody and a negative control at different concentrations were added, and incubated for 30 minutes (37 ° C., 5% CO 2 ).
  • Jurkat (Jurkat-Lucia TM NFAT Cells, Invivogene) cells stably transfecting full-length human Fc ⁇ IIa (Uniprot: P12318) were collected as effector cells, and the effector cells were added to target cells at 7.5 ⁇ 10 4 cells / well (50 ⁇ l / well) and Antibodies were incubated in 96-well plates.
  • a Biacore instrument was used to test the affinity of the disclosed chimeric antibodies (ch-009, -01, -160, see the preparation method of Example 3), humanized antibodies, and Dara for the human CD38-ECD-His antigen.
  • the human Fc capture molecule was covalently coupled to the CM5 biosensor chip (CAT # BR-1005-30, GE) according to the method described in the instruction manual of the human Fc capture kit (CAT # BR-1008-39, GE). To capture the antibody to be tested. Then the human CD38-ECD-His antigen was passed on the surface of the chip, and the reaction signal was detected in real time using a Biacore instrument to obtain the binding and dissociation curves, and the affinity value was obtained by fitting. After the dissociation of each cycle was completed in the experiment, the biochip was washed and regenerated with the regeneration solution provided in the human Fc capture kit (GE). The results are shown in Table 17 and Table 18.
  • Test example 8 CD38 antibody in vivo pharmacokinetic experiment
  • the animals were provided by Xippel-Bikai Experimental Animal Co., Ltd .; they were administered intravenously and subcutaneously at a dose of 3 mg / kg.
  • 8 h, 1 d, 2 d, 4 d, 7 d, 10 d, 14 d, 21 d, and 28 d collect 0.2 ml of whole blood without anticoagulation; place the blood at 4 ° C for 30 min, and centrifuge at 1000 g for 15 min.
  • the serum (serum) was placed in an EP tube and stored at -80 ° C.
  • the subcutaneous injection administration group was collected before and after 1h, 8h, 1d, 2d, 4d, 7d, 10d, 14d, 21, 28d. Blood, separated serum was placed in EP tube and stored at -80 ° C.
  • T1 / 2 in the table indicates half-life
  • IV indicates intravenous injection
  • SC indicates subcutaneous injection.
  • PK pharmacokinetics, pharmacokinetics
  • PK pharmacokinetics, pharmacokinetics
  • hu9E, hu11E, and hu160E at 3mpk doses were measured in rats, and the results showed that all three had good PK performance in rats: subcutaneous injections had higher PK performance Bioavailability, the average T1 / 2 of intravenous injection was 11.4 days, 13.3 days, and 13.6 days; the average T1 / 2 of subcutaneous injection was 11.1 days, 12.1 days, and 10.9 days, suggesting that the antibody stability in rats is good and has development Possibility of subcutaneous preparation.
  • Test example 9 CD38 antibody tumor tumor in vivo efficacy test
  • Balb / c nude mice SPF, 14-16g, female, purchased from Shanghai Slark Laboratory Animal Co., Ltd.
  • Balb / c nude mice were adaptively bred in a laboratory environment for 6 days (days), and AMO-1 cells were inoculated subcutaneously in the right rib (Nanjing Kebai, CBP60242, 5 ⁇ 10 6 + 50% matrigel Matrigel / only, Matrigel basement membrane Matrigel, BD company, article number # 356237), grouped after 9 days, 8 animals / group, average tumor volume is about 197.21 ⁇ 9.25mm 3 (d0), a total of 7 groups, respectively:
  • Intraperitoneal injection twice a week for 3 weeks. Tumor volume and weight were measured twice a week, and data were recorded. Observed to the required time after all the administration was completed, the mice were sacrificed and tumors were taken.
  • Relative volume (RTV) V T / V 0
  • Tumor inhibition rate (%) (C RTV -T RTV ) / C RTV (%)
  • V 0 and V T are the tumor volume at the beginning and end of the experiment, respectively.
  • C RTV and T RTV are the relative tumor volumes of the control group and the experimental group at the end of the experiment. The results are shown in Table 20 and Figure 2.
  • the results of in vivo pharmacological experiments of mouse tumors show that the humanized antibodies hu11E and hu160E of the present disclosure can significantly inhibit tumor growth.
  • the tumor inhibition rates of the hu11E (1mg / kg) group and the hu160E (1mg / kg) group were 93.14% and 70.02%, respectively; the hu11E (3mg / kg) group and hu160E (3mg / kg) group kg) group tumor inhibition rate reached 99.52% and 89.89%, respectively.
  • the weight of the animals in each group was normal, suggesting that the antibodies of the present disclosure have no significant toxic and side effects.

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Abstract

本公开提供了抗CD38抗体、其抗原结合片段及医药用途。具体的，本公开提供了包含所述抗CD38抗体CDR区的鼠源抗体、嵌合抗体或人源化抗体，以及包含抗CD38抗体或其抗原结合片段的药物组合物，以及其作为药物的用途。特别地，本公开提供了一种人源化的抗CD38抗体在制备用于治疗CD38阳性疾病或病症的药物中的用途。

Description

抗CD38抗体、其抗原结合片段及医药用途 技术领域
本公开属于生物技术领域，更具体地，本公开涉及抗CD38抗体及其组合物的治疗性用途和用于生产所述抗体分子的方法。
背景技术
这里的陈述仅提供与本公开有关的背景信息，而不必然地构成现有技术。
多发性骨髓瘤(Multiple myeloma，MM)是一种恶性浆细胞病，其肿瘤细胞起源于骨髓中的浆细胞，而浆细胞是B淋巴细胞发育到最终功能阶段的细胞。因此，多发性骨髓瘤也可以归到B淋巴细胞淋巴瘤的范围。
多发性骨髓瘤特征为骨髓浆细胞异常增生伴有单克隆免疫球蛋白或轻链(M蛋白)的过度生成，极少数患者是不产生M蛋白的未分泌型MM。多发性骨髓瘤常伴有多发性溶骨性损害、高钙血症、贫血、肾脏损害。由于正常免疫球蛋白的生成受到抑制，因此容易出现各种细菌性感染。
中国国内发病率估计为2至3人/10万，男女比例为1.6：1，大多患者年龄>40岁，多为>60岁的老人。目前，中国国内预计有7万左右的患者，全球大概每年新增8万多患者。多发性骨髓瘤在各种血液瘤中占比约10％左右。随着老龄化趋势的加重，患者的数量有进一步加剧的趋势。
目前，多发性骨髓瘤是一种无法治愈的血液系统恶性肿瘤，现有主要的治疗手段包括：骨髓移植、多重小分子药物化疗，尤其是卡非佐米(carfilzomib)为代表的蛋白酶抑制剂，和来那度胺(lenalidomide)为代表的免疫调节剂，大大延长了MM患者的生存期。但是，该病最后几乎都会复发，复发后平均生存周期仅9个月左右。在复发性或难治性MM患者(Refractory multiple myeloma，RRMM)身上，单用Janssen公司的达雷木单抗(Daratumumab，Dara)表现出良好效果，因而被FDA视为突破性药物，并于2015年11月获批上市，用于RRMM患者的治疗。目前在开展多个Dara和小分子联用的临床试验，Dara和小分子药物的联用可以大大提高临床疗效。
CD38是具有256氨基酸胞外域的II型跨膜多功能蛋白。一方面，通过间接方式证明CD38通过配体CD31发挥横向信号传递的作用，可引起细胞粘附，在淋巴细胞活化和B细胞分化等方面起作用，但是该功能未有生化方面的直接证据。另一方面，CD38具有环化酶及水解酶的功能，可以通过环化酶的作用促进NAD(烟酰胺腺嘌呤二核苷酸)转化成cADPR(环腺苷二磷酸核糖)，也可以通过cADPR水解酶作用，促进cADPR转化成ADPR(二磷酸腺苷核糖)。CD38可以调节Ca2+流，也可以促进细胞产生腺苷从而抑制免疫，Ca2 +流的变化可能会影响胰岛素的分泌。敲除CD38基因的小鼠显示能够正常存活，但存在体液免疫应答下降、胰岛 素下降、心/肺肌肉缺陷等不同症状。人体内CD38在造血细胞、免疫细胞及多种正常组织如脑、胰脏、肾脏、肌肉等中表达，RNA水平检测显示在前列腺及胸腺中有较高表达，而在多种血液瘤细胞尤其是多发性骨髓瘤细胞中明显高表达，达雷木单抗的临床数据也验证了CD38抗体在多发性骨髓瘤领域的药效及安全性，提示CD38靶点开发的潜在价值。各类文献研究显示抗CD38的抗体在治疗MM中的作用机制主要包括：
1)ADCC、CDC、ADCP，主要和表位及IgG1-Fc相关的作用机制；
2)凋亡引起的直接杀伤作用；
3)通过抑制CD38酶活性对于肿瘤细胞的生存产生的影响；
4)潜在的对于表达CD38的Treg细胞的清除作用。
CD38靶点成为多发性骨髓瘤治疗的一大热点。另一个重要的靶向CD38的抗体是Sanofi的伊沙妥昔单抗(Isatuximab)，同样开展了多个单用及与小分子药物联用的临床实验，从现有的数据来看伊沙妥昔单抗具有和达雷木单抗相当的疗效及安全性。目前还有MOR202(Morphasys)及TAK-079(Takeda)等抗体已进入临床，也有CD38/CD3双特异性抗体及CD38-CAR-T等在临床前研究阶段。其他针对CD38靶点的抗体相关专利可参见如：WO2006099875、WO2007042309、WO2008047242、WO2012092612、WO2016164656、WO2017149122等。
继续开发具有高选择性、高亲和力和良好药效的抗体仍是目前急需的。
发明内容
本公开提供一系列亲和力(affinity)更高，体内抑瘤活性更好，且具有良好体内代谢活性的CD38抗体。具体地，本公开提供一种与人CD38特异性结合的单克隆抗体或抗原结合片段。
在一些实施方案中，本公开提供一种抗CD38抗体或其抗原结合片段，所述抗体与人CD38特异性结合，所述抗体或其抗原结合片段包含，如下所示的CDR：
(i)分别如SEQ ID NO：9、10、11所示的重链HCDR1、HCDR2、HCDR3或与SEQ ID NO：9、10、11所示的HCDR1、HCDR2和HCDR3分别具有3、2或1个氨基酸差异的HCDR变体；和分别如SEQ ID NO：12、13、14氨基酸序列所示的轻链LCDR1、LCDR2、LCDR3或与SEQ ID NO：12、13、14所示的LCDR1、LCDR2和LCDR3分别具有3、2或1个氨基酸差异的LCDR变体；或
(ii)分别如SEQ ID NO：15、16、17所示的重链HCDR1、HCDR2、HCDR3或与SEQ ID NO：15、16、17所示的HCDR1、HCDR2和HCDR3分别具有3、2或1个氨基酸差异的HCDR变体；和分别如SEQ ID NO：18、19、20所示的轻链LCDR1、LCDR2、LCDR3或与SEQ ID NO：18、19、20所示的LCDR1、LCDR2和LCDR3分别具有3、2或1个氨基酸差异的LCDR变体；或
(iii)分别如SEQ ID NO：15、21、17所示的重链HCDR1、HCDR2、HCDR3 或与SEQ ID NO：15、21、17所示的HCDR1、HCDR2和HCDR3分别具有3、2或1个氨基酸差异的HCDR变体；和分别如SEQ ID NO：22、19、23所示的轻链LCDR1、LCDR2、LCDR3或与SEQ ID NO：22、19、23所示的LCDR1、LCDR2和LCDR3分别具有3、2或1个氨基酸差异的LCDR变体。
在一些实施方式中，所述抗CD38抗体或抗原结合片段的CDR(包括3个重链CDR和3个轻链CDR)具有3、2或1个氨基酸差异的CDR变体是经亲和力成熟方法筛选获得的具有3、2或1个氨基酸差异的CDR变体。
在一些实施方案中，所述抗CD38抗体或抗原结合片段与人CD38的亲和力(KD)小于10 -8M、小于10 -9M、小于10 -10M或小于10 -11M。
在一些实施方案中，所述抗CD38抗体为鼠源抗体、嵌合抗体或人源化抗体，优选人源化抗体。
在一些实施方案中，所述抗体为鼠源抗体或嵌合抗体，所述抗体的重链可变区，其氨基酸序列如SEQ ID NO：3、5、7所示或与SEQ ID NO：3、5、7具有至少95％、96％、97％、98％、99％或100％序列同一性；和/或所述的抗体的轻链可变区，其氨基酸序列如SEQ ID NO：4、6、8所示或与SEQ ID NO：4、6、8具有至少95％、96％、97％、98％、99％或100％序列同一性。
在一些实施方案中，所述的抗CD38抗体或抗原结合片段包含如下所示的重链可变区和轻链可变区：
(a)：
重链可变区，其氨基酸序列如SEQ ID NO：3所示或与SEQ ID NO：3具有至少95％、96％、97％、98％、99％或100％序列同一性，且
轻链可变区，其氨基酸序列如SEQ ID NO：4所示或与SEQ ID NO：4具有至少95％、96％、97％、98％、99％或100％序列同一性；
(b)：
重链可变区，其氨基酸序列如SEQ ID NO：5所示或与SEQ ID NO：5具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性，且
轻链可变区，其氨基酸序列如SEQ ID NO：6所示或与SEQ ID NO：6具有至少95％、96％、97％、98％、99％或100％序列同一性；
(c)：
重链可变区，其氨基酸序列如SEQ ID NO：7所示或与SEQ ID NO：7具有至少95％、96％、97％、98％、99％或100％序列同一性，且
轻链可变区，其氨基酸序列如SEQ ID NO：8所示或与SEQ ID NO：8具有至少95％、96％、97％、98％、99％或100％序列同一性。
在一些实施方案中，所述抗体为人源化抗体，所述人源化抗体包含来源于人种系的框架(FR)区或框架区变体，所述框架区变体为在人抗体的轻链框架区和/ 或重链框架区上分别具有至多10个(例如10个、9个、8个、7个、6个、5个、4个、3个、2个或1个)氨基酸的回复突变。在一些实施方案中，所述人源化抗体包含选自以下(d)至(f)中的任一项：
(d)重链可变区，所述重链可变区包含：重链HCDR1、HCDR2、HCDR3以及重链框架区，其中：
所述HCDR1的氨基酸序列如SEQ ID NO：9所示或与SEQ ID NO：9具有3、2或1个氨基酸差异，
所述HCDR2的氨基酸序列如SEQ ID NO：10所示或与SEQ ID NO：10序列具有3、2或1个氨基酸差异，
所述HCDR3的氨基酸序列如SEQ ID NO：11所示或与SEQ ID NO：11序列具有3、2或1个氨基酸差异，
所述重链框架区具有选自2F、38K、44S、48I、67A、66K、69L、71V和73Q中的一个或更多个回复突变；和/或
轻链可变区，所述轻链可变区包含：轻链LCDR1、LCDR2、LCDR3以及轻链框架区，其中：
所述LCDR1的氨基酸序列如SEQ ID NO：12所示或与SEQ ID NO：12具有3、2或1个氨基酸差异，
所述LCDR2的氨基酸序列如SEQ ID NO：13所示或与SEQ ID NO：13序列具有3、2或1个氨基酸差异，
所述LCDR3的氨基酸序列如SEQ ID NO：14所示或与SEQ ID NO：14序列具有3、2或1个氨基酸差异，
所述轻链框架区具有选自2F、43S、49K和87F中的一个或更多个回复突变；
(e)重链可变区，所述重链可变区包含：重链HCDR1、HCDR2、HCDR3以及重链框架区，其中：
所述HCDR1氨基酸的序列如SEQ ID NO：15所示或与SEQ ID NO：15具有3、2或1个氨基酸差异，
所述HCDR2的氨基酸序列如SEQ ID NO：16所示或与SEQ ID NO：16序列具有3、2或1个氨基酸差异，
所述HCDR3的氨基酸序列如SEQ ID NO：17所示或与SEQ ID NO：17序列具有3、2或1个氨基酸差异，
所述重链框架区具有选自79F、 82A T、91S和76S中的一个或更多个回复突变；和/或；
轻链可变区，所述轻链可变区包含：轻链LCDR1、LCDR2、LCDR3以及轻链框架区，其中：
所述LCDR1的氨基酸序列如SEQ ID NO：18所示或与SEQ ID NO：18具有3、2或1个氨基酸差异，
所述LCDR2的氨基酸序列如SEQ ID NO：19所示或与SEQ ID NO：19序列具有3、2或1个氨基酸差异，
所述LCDR3的氨基酸序列如SEQ ID NO：20所示或与SEQ ID NO：20序列具有3、2或1个氨基酸差异，
所述轻链框架区具有选自58I、68R和85T中的一个或更多个回复突变；和
(f)重链可变区，所述重链可变区包含：重链HCDR1、HCDR2、HCDR3以及重链框架区，其中：
所述HCDR1的氨基酸序列如SEQ ID NO：15所示或与SEQ ID NO：15具有3、2或1个氨基酸差异，
所述HCDR2的氨基酸序列如SEQ ID NO：21所示或与SEQ ID NO：21序列具有3、2或1个氨基酸差异，
所述HCDR3的氨基酸序列如SEQ ID NO：17所示或与SEQ ID NO：17序列具有3、2或1个氨基酸差异，
所述重链框架区具有选自48I、77T和 82A T中的一个或更多个回复突变；和/或
轻链可变区，所述轻链可变区包含：轻链LCDR1、LCDR2、LCDR3以及轻链框架区，其中：
所述LCDR1的氨基酸序列如SEQ ID NO：22所示或与SEQ ID NO：22具有3、2或1个氨基酸差异，
所述LCDR2的氨基酸序列如SEQ ID NO：19所示或与SEQ ID NO：19序列具有3、2或1个氨基酸差异，
所述LCDR3的氨基酸序列如SEQ ID NO：23所示或与SEQ ID NO：23序列具有3、2或1个氨基酸差异，
所述轻链框架区具有选自4L、9A、22S、58I、60A和68R中的一个或更多个回复突变；
其中，前述回复突变的位点根据Kabat编号规则编号，前述突变例如“2F”是指将第2位(根据Kabat编号规则编号的第2位)氨基酸回复突变为苯丙氨酸(缩写Phe或F)，“ 82A T”是指将第 82A位(根据Kabat编号规则编号的第 82A位)氨基酸回复突变为苏氨酸(缩写Thr或T)。
在一些实施方案中，所述的抗体重链FR区序列选自人种系IGHV1-3*01和hJH4.1的组合或与其具有至少95％、96％、97％、98％、99％或100％序列同一性。
在一些实施方案中，所述的轻链FR区选自人种系IGKV3-11*01和hJK4.1的组合与其具有至少95％、96％、97％、98％、99％或100％序列同一性。
在另一些实施方案中，所述的重链FR区序列选自人种系IGHV3-7*01和hJH6.1的FR4的组合或与其具有至少95％、96％、97％、98％、99％或100％序列同一性。
在另一些实施方案中，所述的轻链FR区序列选自人种系IGKV4-1*01和hJK4.1的组合或与其具有至少95％、96％、97％、98％、99％或100％序列同一性。
在一些实施方案中，所述的人源化抗体包含序列如SEQ ID NO：24、32或37所示的重链可变区或其变体；其中所述的变体是在上述重链可变区中的框架区上具有1-10个(例如10个、9个、8个、7个、6个、5个、4个、3个、2个或1个)氨基酸突变。在一些实施方案中，其中所述的氨基酸突变选自以下任一项所述：
(g)在序列如SEQ ID NO：24所示的重链可变区中的框架区上具有选自2F、38K、44S、48I、67A、66K、69L、71V和73Q中的一个或更多个氨基酸的回复突变；
(h)在序列如SEQ ID NO：32所示的重链可变区中的框架区上具有选自79F和91S中的一个或更多个氨基酸回复突变；
(i)在序列如SEQ ID NO：37所示的重链可变区中的框架区上具有48I氨基酸回复突变。
在一些实施方案中，所述的人源化抗体包含：
序列如SEQ ID NO：26、27、28、29、34或39所示的重链可变区，或序列与SEQ ID NO：26、27、28、29、34或39具有至少95％、96％、97％、98％、99％或100％序列同一性的重链可变区。
在一些实施方案中，所述的人源化抗体包含序列如SEQ ID NO：25、33或38所示的轻链可变区或其变体；所述的变体是在序列SEQ ID NO：25、33或38任一项所示的轻链可变区中的框架区上具有1-10个(例如10个、9个、8个、7个、6个、5个、4个、3个、2个或1个)氨基酸突变。
在一些实施方案中，其中所述的氨基酸突变选自以下(j)至(l)任一项：
(j)在序列如SEQ ID NO：25所示的轻链可变区中的框架区上具有选自2F、43S、49K和87F中的一个或更多个氨基酸回复突变；
(k)在序列如SEQ ID NO：33所示的轻链可变区中的框架区上具有选自58I、68R和85T中的一个或更多个回复突变；
(l)在序列如SEQ ID NO：38所示的轻链可变区中的框架区上具有选自4L、9A、22S、58I、60A和68R中的一个或更多个回复突变；
其中，所述突变的位点根据Kabat编号规则编号。
在一些实施方案中，所述的人源化抗体包含：序列如SEQ ID NO：30、31、35、36、40、41或42所示的轻链可变区，或序列与SEQ ID NO：30、31、35、36、40、41或42具有至少95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列所示的轻链可变区。
在一些实施方案中，所述的人源化抗体包含：
(m)序列如SEQ ID NO：24、26、27、28或29所示的重链可变区，和序列 如SEQ ID NO:25、30或31所示的轻链可变区；或
(n)序列如SEQ ID NO：32或34所示的重链可变区，和序列如SEQ ID NO:33、35或36所示的轻链可变区；或
(o)序列如SEQ ID NO：37或39所示的重链可变区，和序列如SEQ ID NO:38、40、41或42所示的轻链可变区；
在一些实施方案中，所述人源化抗体包含：
(p)序列如SEQ ID NO：26所示的重链可变区，和序列如SEQ ID NO:30所示的轻链可变区；或
(q)序列如SEQ ID NO：32所示的重链可变区，和序列如SEQ ID NO:33所示的轻链可变区；或
(r)序列如SEQ ID NO：37所示的重链可变区，和序列如SEQ ID NO:38序列所示的轻链可变区。
在一些实施方案中，所述的抗体进一步包含恒定区；优选地，其中所述抗体为嵌合抗体或人源化抗体，其重链恒定区来源于人抗体IgG1、IgG2、IgG3或IgG4，或IgG1、IgG2、IgG3或IgG4的常规变体；轻链恒定区来源于人抗体κ、λ链或其常规变体。在一些具体的实施方案中，所述重链恒定区的氨基酸序列如SEQ ID NO：43或44所示或与其具有至少95％、96％、97％、98％、99％或100％序列同一性，所述轻链恒定区的氨基酸序列如SEQ ID NO：45所示或与其具有至少95％、96％、97％、98％、99％或100％序列同一性。
在一些实施方案中，所述重链如SEQ ID NO：46、48、49、51、52或54所示，或与SEQ ID NO：46、48、49、51、52或54具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性；和/或
所述轻链如SEQ ID NO：47、50或53所示，或与SEQ ID NO：47、50或53具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性。
在一些实施方案中，所述抗CD38抗体包含：
(i)序列如SEQ ID NO：46或48所示的重链，和序列如SEQ ID NO：47所示的轻链；或
(ii)序列如SEQ ID NO：49或51所示的重链，和序列如SEQ ID NO：50所示的轻链；或
(iii)序列如SEQ ID NO：52或54所示的重链，和序列如SEQ ID NO：53所示的轻链。
在一些具体的实施方案中，所述抗CD38抗体包含：
(iv)序列如SEQ ID NO：48所示的重链和序列如SEQ ID NO：47所示的轻链；或
(v)序列如SEQ ID NO：51所示的重链和序列如SEQ ID NO：50所示的轻链；或
(vi)序列如SEQ ID NO：54所示的重链和序列如SEQ ID NO：53所示的轻链。
在一些实施方案中，所述的抗CD38抗体或其抗原结合片段具有增强的ADCC活性，该活性的提高通过在改变抗体或其抗原结合片段的Fc区与FcyIIIa的亲和力实现，比如，可参照MacroGenics专利WO2008140603提到的F243L、R292P、Y300L突变及其组合，如Xencor专利US20080260731提到的在IgG1Fc区进行S239D、I332E突变或其组合，以及其他本领域中已经公开的其他可增强ADCC功能的突变。
在一些实施方案中，所述抗原结合片段选自Fab、Fab'、F(ab')2、scFv、双抗体和dsFv。
本公开还提供一种抗CD38抗体，所述抗体与前述任一项所述的抗体或其抗原结合片段竞争性结合人CD38，或与前述任一项所述的抗体或其抗原结合片段结合相同的CD38抗原表位。
本公开还提供一种药物组合物，其含有治疗有效量的如上所述的抗CD38抗体或其抗原结合片段，以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。优选地，所述药物组合物单位剂量中可含有0.01至99重量％的抗CD38抗体或其抗原结合片段；或药物组合物单位剂量中含抗体或其抗原结合片段的量优选为0.1-2000mg，更优选为1-1000mg。
本公开还提供一种分离的核酸分子，其编码如前面任一所述的抗CD38抗体或其抗原结合片段。
本公开还提供一种载体，其包含上述的核酸分子。
本公开还提供一种用如上所述的载体转化(或转导、转染)的宿主细胞。还公开了一种宿主细胞，其包含如上所述的载体。所述宿主细胞选自原核细胞和真核细胞，在一些实施方案中，所述宿主细胞不包括任何能够发育成完整个体的人细胞，如人胚胎干细胞、受精卵、生殖细胞；优选地，所述宿主细胞为真核细胞，更优选哺乳动物细胞，其中所述的哺乳动物细胞包括但不限于CHO、293、NSO，以及在哺乳动物细胞中进行基因编辑可改变抗体或其抗原结合片段的糖基化修饰，进而改变抗体或其抗原结合片段的ADCC功能的细胞，例如，敲除FUT8或GnT-III等基因；在一些实施方案中，所述哺乳动物细胞不包括人细胞。
本公开还提供一种制备如上所述的抗CD38抗体或其抗原结合片段的方法，所述方法包括培养上述宿主细胞，然后回收抗CD38抗体或其抗原结合片段的步骤；任选地包括对抗CD38抗体或其抗原结合片段进行纯化步骤。
本公开还提供用于检测或测定人CD38的方法，所述方法包括使上述抗CD38抗体或其抗原结合片段接触待测样本。
本公开还提供用于检测或测定人CD38的试剂，所述试剂包含上述任一项所述的抗CD38抗体或其抗原结合片段。
本公开还提供用于与人CD38相关的疾病的诊断剂，所述诊断剂包含根据上述的抗CD38抗体或其抗原结合片段。
本公开还提供用于诊断与人CD38相关的疾病的方法，所述方法包括使用上述抗CD38抗体或其抗原结合片段检测或测定人CD38或CD38阳性细胞。
本公开还提供上述抗CD38抗体或其抗原结合片段在制备与人CD38相关的疾病的诊断剂中的应用。
本公开还提供治疗或预防疾病的方法，所述方法包括向受试者施用治疗有效量或预防有效量的上述抗CD38抗体或其抗原结合片段，或包含上述的药物组合物，或上述的核酸分子。
在一些实施方案中，所述疾病或病症为肿瘤或免疫性疾病。
在一些实施方案中，所述疾病或病症为CD38阳性疾病或病症。
在一些实施方案中，前述疾病或病症为肿瘤。
在一些实施方案中，前述肿瘤选自白血病、B细胞淋巴瘤、浆细胞恶性瘤、T/NK细胞淋巴瘤和骨髓瘤。在一些实施方案中，所述白血病选自：急性淋巴细胞白血病、急性成淋巴细胞白血病、急性早幼粒细胞白血病、慢性淋巴细胞白血病、急性和慢性骨髓型白血病。在一些实施方案中，所述骨髓瘤选自：多发性骨髓瘤、前髓细胞肿瘤和轻链淀粉样变性等。在一些实施方案中，所述淋巴瘤为非何杰金淋巴瘤或何杰金淋巴瘤。在一些实施方案中，前述肿瘤为B细胞淋巴瘤/白血病，例如选自成熟B细胞肿瘤或前体B细胞成淋巴细胞白血病/淋巴瘤，或选自B细胞非霍奇金淋巴瘤或B细胞霍奇金淋巴瘤。在一些实施方案中，所述肿瘤选自：B细胞慢性淋巴细胞性白血病(CLL)或小淋巴细胞性白血病(SLL)、B细胞急性淋巴细胞性白血病、B细胞前淋巴细胞白血病、淋巴浆细胞样淋巴瘤、套细胞淋巴瘤(MCL)、低级/中级/高级滤泡性淋巴瘤(FL)、皮肤滤泡中心淋巴瘤、边缘区B细胞淋巴瘤(包括MALT型、淋巴结MZBL型、脾MZBL型)、毛细胞白血病、弥漫性大B细胞淋巴瘤、伯基特淋巴瘤(Burkitt淋巴瘤)、浆细胞瘤/浆细胞骨髓瘤、浆细胞白血病、移植后淋巴增生性疾病、Waldenstrom巨球蛋白血症、浆细胞白血病、间变性大细胞淋巴瘤(ALCL)、多毛细胞淋巴瘤。
在一些实施方式实施方案中，所述肿瘤为B-细胞淋巴瘤或多发性骨髓瘤。
在一些实施方案中，所述肿瘤为多发性骨髓瘤。
在一些实施方案中，前述疾病或病症为免疫性疾病，例如由表达CD38的B细胞、浆细胞、单核细胞和T细胞参与的免疫性疾病。
在一些实施方案中，所述免疫性疾病包括但不限于类风湿性关节炎、牛皮癣、强直性脊柱炎、关节银屑病、皮炎、系统性硬皮病及硬化症、炎症性肠病(IBD)、Crohn病(克罗恩氏病)、溃疡性结肠炎、呼吸窘迫综合征、脑膜炎、脑炎、胃炎、 葡萄膜炎、肾小球肾炎、湿疹、哮喘、动脉硬化、白细胞粘附缺陷病、Raynaud症候群、Sjogren症候群、青少年糖尿病、Reiter病、Behcet病、免疫复合物性肾炎、IgA肾病、IgM多发性神经病、免疫介导的血小板减少症状(如急性特发性血小板减少性紫癜、慢性特发性血小板减少性紫癜)、溶血性贫血、重症肌无力、狼疮性肾炎、系统性红斑狼疮、风湿关节炎(RA)、异位性皮炎、天疱疮、Graves病、桥本氏甲状腺炎、Wegener肉芽肿、Omenn症候群、慢性肾功能衰竭、急性传染性单核细胞增多征、多发性硬化症、HIV和疱疹病毒相关的疾病、严重急性呼吸综合征和脉络视网膜炎(choreoretinitis)、移植物对抗宿主疾病、以及病毒感染引起的免疫性疾病(如伊波病毒(EBV)感染B细胞引起或介导的疾病。
在一些实施方式中，所述免疫性疾病选自：类风湿性关节炎、系统性红斑狼疮、哮喘、炎症性肠道疾病、多发性硬化症、克罗恩氏病、胃炎、桥本氏甲状腺炎、强直性脊柱炎和移植物对抗宿主疾病。在一些实施方案中，所述疾病或病症为类风湿性关节炎。
本公开进一步提供如上所述的抗CD38抗体或其抗原结合片段或如上所述的药物组合物或核酸分子在制备用于治疗或预防疾病或病症的药物中的应用。
在一些实施方案中，所述疾病或病症为肿瘤或免疫性疾病。
在一些实施方案中，所述疾病或病症为CD38阳性疾病或病症。
在一些实施方案中，前述疾病或病症可以是肿瘤，例如疾病的特征在于存在表达CD38的肿瘤细胞。
在一些实施方案中，前述述肿瘤选自白血病、B细胞淋巴瘤、浆细胞恶性瘤、T/NK细胞淋巴瘤和骨髓瘤。
在一些实施方案中，所述白血病选自：急性淋巴细胞白血病、急性成淋巴细胞白血病、急性早幼粒细胞白血病、慢性淋巴细胞白血病、急性和慢性骨髓型白血病。
在一些实施方案中，所述骨髓瘤选自：多发性骨髓瘤、前髓细胞肿瘤和轻链淀粉样变性。
在一些实施方案中，所述淋巴瘤为非何杰金淋巴瘤或何杰金淋巴瘤。
在一些实施方案中，前述肿瘤为B细胞淋巴瘤/白血病，例如选自成熟B细胞肿瘤或前体B细胞成淋巴细胞白血病/淋巴瘤，或选自B细胞非霍奇金淋巴瘤或B细胞霍奇金淋巴瘤。
在一些实施方案中，前述肿瘤选自：B细胞慢性淋巴细胞性白血病(CLL)、小淋巴细胞性白血病(SLL)、B细胞急性淋巴细胞性白血病、B细胞前淋巴细胞白血病、淋巴浆细胞样淋巴瘤、套细胞淋巴瘤(MCL)、滤泡性淋巴瘤(包括低级、中级或高级FL)、皮肤滤泡中心淋巴瘤、边缘区B细胞淋巴瘤(包括MALT型、淋巴结MZBL型、脾MZBL型)、毛细胞白血病、弥漫性大B细胞淋巴瘤、伯基特淋巴瘤(Burkitt淋巴瘤)、浆细胞瘤、浆细胞骨髓瘤、浆细胞白血病、移 植后淋巴增生性疾病、Waldenstrom巨球蛋白血症、浆细胞白血病和间变性大细胞淋巴瘤(ALCL)、多毛细胞淋巴瘤。
在一些实施方案中，所述肿瘤为B-细胞淋巴瘤或多发性骨髓瘤。
在一些实施方案中，所述肿瘤为多发性骨髓瘤。
在一些实施方案中，所述疾病或病症为免疫性疾病，例如是由表达CD38的B细胞、浆细胞、单核细胞和T细胞参与的免疫性疾病。
在一些实施方案中，所述免疫性疾病可选自：类风湿性关节炎、牛皮癣、强直性脊柱炎、关节银屑病、皮炎、系统性硬皮病及硬化症、炎症性肠病(IBD)、Crohn病(克罗恩氏病)、溃疡性结肠炎、呼吸窘迫综合征、脑膜炎、脑炎、胃炎、葡萄膜炎、肾小球肾炎、湿疹、哮喘、动脉硬化、白细胞粘附缺陷病、Raynaud症候群、Sjogren症候群、青少年糖尿病、Reiter病、Behcet病、免疫复合物性肾炎、IgA肾病、IgM多发性神经病、免疫介导的血小板减少症状(如急性特发性血小板减少性紫癜、慢性特发性血小板减少性紫癜)、溶血性贫血、重症肌无力、狼疮性肾炎、系统性红斑狼疮、风湿关节炎(RA)、异位性皮炎、天疱疮、Graves病、桥本氏甲状腺炎、Wegener肉芽肿、Omenn症候群、慢性肾功能衰竭、急性传染性单核细胞增多征、多发性硬化症、HIV和疱疹病毒相关的疾病、严重急性呼吸综合征、脉络视网膜炎(choreoretinitis)、移植物对抗宿主疾病、以及病毒感染引起的免疫性疾病(如伊波病毒(EBV)感染B细胞引起或介导的疾病)。在一些实施方式中，所述免疫性疾病选自类风湿性关节炎、系统性红斑狼疮、哮喘、炎症性肠道疾病、多发性硬化、克罗恩氏病、胃炎、桥本氏甲状腺炎、强直性脊柱炎和移植物对抗宿主疾病。在一些实施方案中，其中所述免疫疾病或病症是类风湿性关节炎。
本公开进一步提供一种抗CD38抗体或其抗原结合片段、或药物组合物、或核酸分子，其用于治疗或预防前述疾病或病症。
在一些实施方案中，所述疾病或病症为肿瘤或免疫性疾病；在一些实施方案中，所述疾病或病症为CD38阳性疾病或病症。
在一些实施方案中，前述疾病或病症是肿瘤，例如疾病的特征在于存在表达CD38的肿瘤细胞，在一些实施方案中，所述肿瘤选自白血病、B细胞淋巴瘤、浆细胞恶性瘤、T/NK细胞淋巴瘤和骨髓瘤。在一些实施方案中，所述白血病选自：急性淋巴细胞白血病、急性成淋巴细胞白血病、急性早幼粒细胞白血病、慢性淋巴细胞白血病、急性和慢性骨髓型白血病。在一些实施方案中，所述骨髓瘤选自：多发性骨髓瘤、前髓细胞肿瘤和轻链淀粉样变性。在一些实施方案中，所述淋巴瘤为非何杰金淋巴瘤或何杰金淋巴瘤。在一些实施方案中，所述肿瘤为B细胞淋巴瘤/白血病，例如选自成熟B细胞肿瘤或前体B细胞成淋巴细胞白血病/淋巴瘤，或选自B细胞非霍奇金淋巴瘤或B细胞霍奇金淋巴瘤。在一些实施方案中，所述肿瘤选自：B细胞慢性淋巴细胞性白血病(CLL)、小淋巴细胞性白血病(SLL)、 B细胞急性淋巴细胞性白血病、B细胞前淋巴细胞白血病、淋巴浆细胞样淋巴瘤、套细胞淋巴瘤(MCL)、滤泡性淋巴瘤(包括低级、中级或高级FL)、皮肤滤泡中心淋巴瘤、边缘区B细胞淋巴瘤(包括MALT型、淋巴结MZBL型、脾MZBL型)、毛细胞白血病、弥漫性大B细胞淋巴瘤、伯基特淋巴瘤(Burkitt淋巴瘤)、浆细胞瘤、浆细胞骨髓瘤、浆细胞白血病、移植后淋巴增生性疾病、Waldenstrom巨球蛋白血症、浆细胞白血病和间变性大细胞淋巴瘤(ALCL)、多毛细胞淋巴瘤。在一些实施方案中，所述肿瘤为B-细胞淋巴瘤或多发性骨髓瘤。在一些实施方案中，所述肿瘤为多发性骨髓瘤。
在一些实施方案中，前述疾病或病症为免疫性疾病，例如是由表达CD38的B细胞、浆细胞、单核细胞和T细胞参与的免疫性疾病。
在一些实施方案中，所述免疫性疾病可选自：类风湿性关节炎、牛皮癣、关节银屑病、皮炎、系统性硬皮病及硬化症、炎症性肠病(IBD)、Crohn病、溃疡性结肠炎、呼吸窘迫综合征、脑膜炎、脑炎、葡萄膜炎、肾小球肾炎、湿疹、哮喘、动脉硬化、白细胞粘附缺陷病、多发性硬化症、Raynaud症候群、Sjogren症候群、青少年糖尿病、Reiter病、Behcet病、免疫复合物性肾炎、IgA肾病、IgM多发性神经病、免疫介导的血小板减少症状(如急性特发性血小板减少性紫癜、慢性特发性血小板减少性紫癜)、溶血性贫血、重症肌无力、狼疮性肾炎、系统性红斑狼疮、风湿关节炎(RA)、异位性皮炎、天疱疮、Graves病、桥本甲状腺炎、Wegener肉芽肿、Omenn症候群、慢性肾功能衰竭、急性传染性单核细胞增多征、HIV和疱疹病毒相关的疾病、严重急性呼吸综合征、脉络视网膜炎(choreoretinitis)、以及病毒感染引起的免疫性疾病(如伊波病毒(EBV)感染B细胞引起或介导的疾病)。在一些实施方案中，所述免疫性疾病选自类风湿性关节炎、系统性红斑性狼疮、哮喘、炎症性肠道疾病、多发性硬化、克罗恩氏病、胃炎、桥本氏甲状腺炎、强直性脊柱炎和移植物对抗宿主疾病。在一些实施方案中，其中所述免疫性疾病是类风湿性关节炎。
本公开的抗CD38抗体或其抗原结合片段在生化测试或体内外药效试验中，均表现出良好的效果。其中，在抗体与抗原的亲和力检测中，本公开中的抗体hu9E与人CD38的KD值为1.31nM，hu11E与人CD38的KD值为0.568nM，hu160E与人CD38的KD值为0.0585nM，而对照抗体的KD为2.35nM，本公开中的抗体具有高亲和力(表18)。
在体内抑瘤活性的实验中发现，本公开中的hu11E和hu160E抗体均能有效的抑制小鼠体内的肿瘤生长，其中在1mpk/kg的剂量时，hu11E的抑瘤率高达93.14％，hu160E的抑瘤率高达70.02％，均明显高于对照抗体Dara(抑瘤率为56.83％)(表20)。
此外，本公开CD38单克隆抗体或抗原结合片段在大鼠体内具有良好的的代谢动力学特性，显示出很长的半衰期和很高的生物利用度。
附图说明
图1A：CD38抗体对Molp-8细胞的体外ADCP测试结果。
图1B：CD38抗体对Daudi细胞的体外ADCP测试结果。
图2：CD38抗体在小鼠体内抑瘤效果。
具体实施方式
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
本公开所述的“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL或LCVR)和重链可变区(VH或HCVR)由3个CDR区和4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的抗人CD38的单克隆抗体。制备时用CD38抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本公开一个优选的实施方案中，所述的鼠源CD38抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将某物种(如鼠)的抗体的可变区与另一物种(如人)抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后 从鼠杂交瘤细胞中克隆可变区基因；再根据需要，克隆人抗体的恒定区基因；将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。在本公开一个具体的实施方案中，所述的CD38嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的CD38嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其常规变体的重链恒定区，优选包含人源IgG1重链恒定区，更优选包含增强CDC的功能氨基酸突变(如E333A突变)的IgG1重链恒定区。
术语“人源化抗体(humanized antibody)”，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服由于嵌合抗体携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(因特网www.mrccpe.com.ac.uk/vbase)中获得，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变(或回复突变)，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示或酵母菌展示对CDR进行亲和力成熟后获得的人源化抗体。
CDR的移植可由于与抗原接触的构架残基的变化而导致产生的抗体或其抗原结合片段对抗原的亲和力减弱。此类相互作用可以是体细胞高度突变的结果。因此，可能仍然需要将此类供体构架氨基酸移植至人源化抗体的构架。来自非人抗体或其抗原结合片段的参与抗原结合的氨基酸残基可通过检查动物单克隆抗体可变区序列和结构来鉴定。CDR供体构架中与种系不同的的各残基可被认为是相关的。如果不能确定最接近的种系，那么可将序列与亚型共有序列或具有高相似性百分数的鼠序列的共有序列相比较。稀有构架残基被认为可能是体细胞高度突变的结果，从而在结合中起着重要作用。
在本公开一些具体的实施方案中，所述的CD38人源化抗体的抗体轻链进一步包含人源κ、λ链或其常规变体的轻链恒定区。所述的CD38人源化抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其常规变体的重链恒定区，优选包含人源IgG1重链恒定区，更优选包含增强CDC的功能氨基酸突变(如E333A突变)的IgG1重链恒定区。
本公开中所述人抗体重链恒定区和人抗体轻链恒定区的“常规变体”是指现有技术已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体，示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3或IgG4重链恒定区变体，具体替换如现有技术已知的YTE突变，L234A和/或L235A突变、S228P突变、E333A突变、和/或获得knob-into-hole结构的突变(使得抗体重链具有knob-Fc和hole-Fc组合)等，这些突变已被证实使得抗体具 有新的性能，但不改变抗体可变区的功能。
术语“回复突变”是指将人抗体来源的FR区氨基酸残基突变成原始来源抗体对应位置的氨基酸残基，通常是为了避人源化抗体引起的免疫原性下降和活性下降，对所述的人源化抗体可变区可进行最少反向突变，以保持抗体的活性。
“人抗体(HuMAb)”、“人源抗体”、“全人抗体”、“完全人抗体”可以互换使用，可以是源于人的抗体或者是从一种转基因生物体中获得的抗体，该转基因生物体经“改造”以响应于抗原刺激而产生特异性人抗体，并且可以通过本领域已知的任何方法产生。在某些技术中，将人重链和轻链基因座的元件引入到源于胚胎干细胞系的生物体的细胞株中，这些细胞系中的内源性重链和轻链基因座被靶向破坏。转基因生物可以合成对人抗原具有特异性的人抗体，并且该生物可以用于产生分泌人抗体的杂交瘤。人抗体还可以是一种抗体，其中重链和轻链是由源于一个或更多个人DNA来源的核苷酸序列编码的。完全人抗体还可以通过基因或染色体转染方法以及噬菌体展示技术来构建，或者由体外活化的B细胞构建，所有的这些都是本领域已知的。
术语“全长抗体”、“完整抗体”、“完全抗体”和“全抗体”在本文中可互换使用，指基本上完整形式的抗体，与下文定义的抗原结合片段相区分。该术语特别指在轻链和重链中包含恒定区的抗体。
在一些实施方案中，本公开的全长抗体包括由下表1所示的轻链可变区与轻链恒定区，重链可变区与重链恒定区连接后形成全长抗体。本领域技术人员可以根据实际需要选择不同的抗体来源的轻链恒定区、重链恒定区，例如人抗体来源的轻链恒定区(或其常规变体)和重链恒定区(或其常规变体)。同时，表1中轻链可变区和重链可变区的组合可以形成单链抗体(scFv)、Fab或其他包含scFv或Fab的抗原结合片段形式。
表1：抗CD38人源化抗体轻链可变区和重链可变区的组合
可变区的组合 重链可变区VH 轻链可变区VL
h009-01V h009 VH1 h009 VL1
h009-02V h009 VH2 h009 VL1
h009-03V h009 VH3 h009 VL1
h009-04V h009 VH4 h009 VL1
h009-05V h009 VH5 h009 VL1
h009-06V h009 VH1 h009 VL2
h009-07V h009 VH2 h009 VL2
h009-08V h009 VH3 h009 VL2
h009-09 V h009 VH4 h009 VL2
h009-10V h009 VH5 h009 VL2
h009-11V h009 VH1 h009 VL3
h009-12V h009 VH2 h009 VL3
h009-13V h009 VH3 h009 VL3
h009-14V h009 VH4 h009 VL3
h009-15V h009 VH5 h009 VL3
h011-01V h011 VH1 h011VL1
h011-02V h011 VH2 h011VL2
h011-03V h011 VH1 h011VL3
h011-04V h011 VH2 h011VL1
h011-05V h011 VH1 h011VL2
h011-06V h011 VH2 h011VL3
h160-01V h160 VH1 h160 VL1
h160-02V h160 VH1 h160 VL2
h160-03V h160 VH1 h160 VL3
h160-04V h160 VH1 h160 VL4
h160-05V h160 VH2 h160 VL1
h160-06V h160 VH2 h160 VL2
h160-07V h160 VH2 h160 VL3
h160-08V h160 VH2 h160 VL4
注：例如“h009-01V”，其表示编号为h009-01V的轻/重链可变区对，其重链可变区为h009VH1(SEQ ID NO：24)，轻链可变区为h009VL1(SEQ ID NO：25)，其它依此类推。
术语“单克隆抗体”是指从基本上均质抗体的群体获得的抗体，即除可能的变体抗体(例如含有天然存在的突变或在制造单克隆抗体制剂的期间产生的突变，这些变体通常以少量存在)之外，构成所述群体的个别抗体相同和/或结合相同表位。与通常包含针对不同决定簇(表位)的不同抗体的多克隆抗体制备物不同，单克隆抗体制备物(制剂)的每个单克隆抗体是针对抗原上的单一决定簇的。因此，修饰语“单克隆”指示如从基本上均质抗体群体获得的抗体的特性，且不应解释为需要通过任何特定方法来制造抗体。例如，根据本公开使用的单克隆抗体可通过各种技术制备，所述技术包括但不限于杂交瘤方法、重组DNA方法、噬菌体展示方法以及利用含有全部或部分人免疫球蛋白基因座的转基因动物的方法，此类方法以及用于制备单克隆抗体的其他示例性方法在本文中进行描述。
术语抗体的“抗原结合片段”或“功能片段”是指抗体的保持特异性结合抗原(例如，CD38)的能力的一个或多个片段。已显示可利用全长抗体的片段来实现抗体的抗原结合功能。术语抗体的“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，通过铰链区上的二硫桥所连接的两个Fab片段形成的二价片段，(iii)由抗体的单臂的VH和VL结构域组成的Fv片段；(iv)dsFv，由VH和VL经链间二硫键形成的稳定的抗原结合片段；和(v)包含scFv、dsFv、Fab等片段的双抗体、双特异性抗体和多特异性抗体。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法通过合成接头连接它们，从而产生单个蛋白质链，在其中VL和VH区配对形成单价分子(称为单链Fv(scFv)；参见，例如， Bird等人(1988)Science 242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性能筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。在一些实施方案中，本公开的抗原结合片段包括Fab、F(ab')2、Fab'、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)等。
“Fab”是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子，在所获得的片段中具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
本公开的Fab可以通过用木瓜蛋白酶处理本公开的单克隆抗体来生产。此外，可以通过将编码所述抗体的Fab的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab来生产所述Fab。
“F(ab')2”是指，通过胃蛋白酶消化IgG铰链区中两个二硫键的下游部分，而获得的分子量约100,000并具有抗原结合活性的抗体片段，其所包含的两个Fab区在铰链位置相连。
本公开的F(ab')2可以通过用胃蛋白酶处理本公开的单克隆抗体来生产。此外，可以通过用硫醚键或二硫键连接下面描述的Fab'来生产所述F(ab')2。
“Fab'”是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。本公开的Fab'可以通过用还原剂例如二硫苏糖醇处理本公开的F(ab')2来生产。此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
本公开的scFv可以通过以下步骤来生产：获得编码本公开单克隆抗体的VH和VL的cDNA，构建编码scFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达scFv。
“双抗体”是其中scFv被二聚体化的抗体片段，是具有二价抗原结合活性的抗体片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。
双特异性抗体和多特异性抗体是指能同时结合两个或多个抗原或抗原决定簇的抗体，其中包含能结合CD38的scFv或Fab片段。
本公开的双抗体可以通过以下步骤来生产：获得本公开的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA以使肽接头的氨基酸序列长度为8个残基或更少，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达双抗体。
“dsFv”是通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得的。可以按照已知方法(Protein Engineering,7,697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。
本公开的dsFv可以通过以下步骤来生产：获得本公开的单克隆抗体的VH和VL的编码cDNA，构建编码dsFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达dsFv。
本文中使用的术语“框架(FR)”，是指抗体可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，框架区是不具有CDR的可变结构域。
术语“氨基酸差异”或“氨基酸突变”是指多肽与其变体之间，指相较于原蛋白质或多肽，变体蛋白质或多肽存在氨基酸的改变或突变，包括在原蛋白质或多肽的基础上发生1个、2个、3个或更多个氨基酸获替换、插入或缺失。
术语“互补决定区”、“CDR”或“高变区”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2) 和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位(例如，CD38分子上的特定部位)。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -8M，例如大约小于10 -9M、10 -10M、10 -11M或更小的亲和力(KD)结合。
术语“KD”是指特定抗体-抗原相互作用的解离平衡常数。通常，本公开的抗体以小于大约10 -7M，例如小于大约10 -8M、10 -9M或10 -10M或更小的解离平衡常数(KD)结合CD38，例如，如使用表面等离子体共振(SPR)技术在BIACORE仪中测定的。
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和性抗原结合蛋白或中和性抗体)的情况中，意指在抗原结合蛋白之间的竞争，其通过以下测定法来测定：待检测的抗原结合蛋白(例如抗体或其功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如CD38抗原或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619；Cheung，等，1990，Virology176：546-552)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用纯化抗原，其能与带有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白结合(所述抗原在固态表面或细胞表面上)。在待测抗原结合蛋白存在下，测量结合于固态表面或细胞表面的标记的量，来测量竞争性抑制。通常，待测抗原结合蛋白是过量存在的。由竞争性测定(竞争性抗原结合蛋 白)鉴定的抗原结合蛋白包括：与参考抗原结合蛋白相同的表位发生结合的抗原结合蛋白；以及，与参考抗原结合蛋白结合的表位所充分接近的表位发生结合的抗原结合蛋白，所述两个表位在空间上互相妨碍结合的发生。在本公开实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争性抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或更多。
本文中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”或“表达载体”是指能够运输与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人CD38或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。同样可以用常规方法制备抗原结合片段。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括微生物(例如细菌)、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，将编码重链和轻链的cDNA序列克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统 会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人CD38特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物有时需立即冷冻，如-70℃，或者冻干。
“给予”、“施用”和“处理”当应用于动物、人、受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”、“施用”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本公开的任一种抗体或其抗原结合片段的组合物或编码抗体或其抗原结合片段的核酸分子，所述患者具有一种或多种疾病或症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病或症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床可测量的程度。有效缓解任何具体疾病或症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。示例性保守取代于下表2“示例性氨基酸保守取代”中陈述。
表2.示例性氨基酸保守取代
原始残基 保守取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His；Asp
Asp(D) Glu；Asn
Cys(C) Ser；Ala；Val
Gln(Q) Asn；Glu
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr；Phe
Tyr(Y) Trp；Phe
Val(V) Ile；Leu
“有效量”或“有效剂量”是指得任一种或多种有益的或所需的结果所必需的药物、化合物或药物组合物的量。对于预防用途，有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作，包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用，有益的或所需的结果包括临床结果，诸如减少各种本公开靶抗原相关病症的发病率或改善所述病症的一个或更多个症状，减少治疗病症所需的其它药剂的剂量，增强另一种药剂的疗效，和/或延缓患者的本公开靶抗原相关病症的进展。
“外源性”指根据情况在生物、细胞或人体外产生的物质。
“内源性”指根据情况在生物、细胞或人体内产生的物质。
本公开所述的“突变序列”是指对本公开的核苷酸序列和氨基酸序列进行适当的替换、插入或缺失等突变修饰情况下，得到的与本公开的核苷酸序列和氨基酸序列具有不同百分比序列同一性程度的核苷酸序列和氨基酸序列。本公开中所述的序列同一性可以至少为85％、90％或95％，非限制性实施例包括85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％。
本文中“同源性”、“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被相同的碱基占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除 以比较的全部位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个是匹配或同源的，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。通常，当比对两个序列时进行比较以给出最大百分比同源性。例如，可以通过BLAST算法执行比较，其中选择算法的参数以在各个参考序列的整个长度上给出各个序列之间的最大匹配。以下参考文献涉及经常用于序列分析的BLAST算法：BLAST算法(BLAST ALGORITHMS):Altschul，S.F.等人，(1990)J.Mol.Biol.215:403-410；Gish，W.等人，(1993)Nature Genet.3:266-272；Madden，T.L.等人，(1996)Meth.Enzymol.266:131-141；Altschul，S.F.等人，(1997)Nucleic Acids Res.25:3389-3402；Zhang，J.等人，(1997)Genome Res.7:649-656。其他如NCBI BLAST提供的常规BLAST算法也为本领域技术人员所熟知。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本文使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4,683,195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press,N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“分离的”指纯化状态，并且在这种情况下意味着在指定的分子基本上不含其他生物分子，例如核酸、蛋白质、脂质、碳水化合物或其他材料，例如细胞碎片和生长培养基。通常，术语“分离的”并不意图指完全不存在这些材料或不存在水、缓冲液或盐，除非它们以显著干扰如本文所述的化合物的实验或治疗用途的量存在。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述抗体或其抗原结合片段与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体或赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学上可接受的载体”指适合用于制剂中用于递送抗体或抗原结合片段的任何无活性物质。载体可以是抗粘附剂、粘合剂、包衣、崩解剂、充填剂或稀释剂、防腐剂(如抗氧化剂、抗菌剂或抗真菌剂)、增甜剂、吸收延迟剂、润湿剂、乳化剂、缓冲剂等。合适的药学上可接受的载体的示例包括水、乙醇、多元醇(例如甘油、丙二醇、聚乙二醇等)右旋糖、植物油(例如橄榄油)、盐水、缓冲液、缓冲的盐水和等渗剂例如糖、多元醇、山梨糖醇和氯化钠。
“CD38阳性疾病或病症”是存在表达CD38的细胞的疾病或病症。非限制性地，例如由表达CD38的B细胞、浆细胞、单核细胞和T细胞参与的免疫性疾病，疾病的特征之一为存在表达CD38的肿瘤细胞的肿瘤疾病，例如表达CD38的白血病、B细胞淋巴瘤、浆细胞恶性瘤、T/NK细胞淋巴瘤和骨髓瘤。本公开在一些实施方案中，所述白血病选自：急性淋巴细胞白血病、急性成淋巴细胞白血病、急性早幼粒细胞白血病、慢性淋巴细胞白血病、急性和慢性骨髓型白血病。在一些实施方案中，所述骨髓瘤选自：多发性骨髓瘤、前髓细胞肿瘤和轻链淀粉样变性。在一些实施方案中，所述淋巴瘤为非何杰金淋巴瘤或何杰金淋巴瘤。在一些实施方案中，所述肿瘤可选自B细胞淋巴瘤/白血病，包括但不限于：前体B细胞成淋巴细胞白血病/淋巴瘤、B细胞非霍奇金淋巴瘤或B细胞霍奇金淋巴瘤、成熟B细胞肿瘤。在一些实施方案中，所述肿瘤选自：B细胞慢性淋巴细胞性白血病(CLL)、小淋巴细胞性白血病(SLL)、B细胞急性淋巴细胞性白血病、B细胞前淋巴细胞白血病、淋巴浆细胞样淋巴瘤、套细胞淋巴瘤(MCL)、滤泡性淋巴瘤(包括低级、中级或高级FL)、皮肤滤泡中心淋巴瘤、边缘区B细胞淋巴瘤(包括MALT型、淋巴结MZBL型、脾MZBL型)、毛细胞白血病、弥漫性大B细胞淋巴瘤、伯基特淋巴瘤(Burkitt淋巴瘤)、浆细胞瘤、浆细胞骨髓瘤、浆细胞白血病、移植后淋巴增生性疾病、Waldenstrom巨球蛋白血症、浆细胞白血病和间变性大细胞淋巴瘤(ALCL)、多毛细胞淋巴瘤。在一些实施方案中，所述肿瘤为多发性骨髓瘤。在一些实施方案中，所述免疫性疾病选自：类风湿性关节炎、牛皮癣、强直性脊柱炎、关节银屑病、皮炎、系统性硬皮病及硬化症、炎症性肠病(IBD)、Crohn病(克罗恩氏病)、溃疡性结肠炎、呼吸窘迫综合征、脑膜炎、胃炎、脑炎、葡萄膜炎、肾小球肾炎、湿疹、哮喘、动脉硬化、白细胞粘附缺陷病、Raynaud症候群、Sjogren症候群、青少年糖尿病、Reiter病、Behcet病、免疫复合物性肾炎、IgA肾病、IgM多发性神经病、免疫介导的血小板减少症状(如急性特发性血小板减少性紫癜、慢性特发性血小板减少性紫癜)、溶血性贫血、重症肌无力、狼疮性肾炎、系统性红斑狼疮、风湿关节炎(RA)、异位性皮炎、天疱疮、Graves病、桥本氏甲状腺炎、Wegener肉芽肿、Omenn症候群、慢性肾功能衰竭、急性传染性单核细胞增多征、多发性硬化症、HIV和疱疹病毒相关的疾病、严重急性呼吸综合征、脉络视网膜炎(choreoretinitis)、移植物对抗宿主疾病、以及病毒感染引起的免疫性疾病(如伊波病毒(EBV)感染B细胞引起或介导的疾病)。在一些 实施方案中，所述免疫性疾病选自类风湿性关节炎、系统性红斑性狼疮、哮喘、炎症性肠道疾病、多发性硬化、克罗恩氏病、胃炎、桥本氏甲状腺炎、强直性脊柱炎和移植物对抗宿主疾病。在一些实施方案中，其中所述免疫疾病是类风湿性关节炎。
此外，本公开提供用于治疗或预防与目标抗原(例如CD38)阳性细胞相关疾病的药剂，所述药剂包含本公开的抗CD38抗体或其抗原结合片段作为活性成分，可以对有需要的对象给予治疗有效量或预防有效量的该药剂，以治疗或预防CD38阳性疾病。抗CD38抗体或其抗原结合片段可抑制CD38诱导的与疾病相关的活性或消除或降低CD38表达细胞的数目。所述治疗或预防有效量为单位剂量的组合物中含有0.1-3000mg(优选为0.1-2000mg，更优选为1-1000mg)的如前所述的抗CD38抗体或其抗原结合片段。
另外，本公开涉及用于免疫检测或测定目标抗原(例如CD38)的方法、用于免疫检测或测定目标抗原(例如CD38)的试剂、用于免疫检测或测定表达目标抗原(例如CD38)的细胞的方法和用于诊断与目标抗原(例如CD38)阳性细胞相关的疾病的诊断剂，其包含本公开的特异性识别目标抗原(例如人CD38)结合的抗体或抗体片段作为活性成分。
在本公开中，用于检测或测定目标抗原(例如CD38)的量的方法可以是任何已知方法。例如，它包括免疫检测或测定方法。
免疫检测或测定方法是使标记的抗原或抗体检测或测定抗体量或抗原量的方法。免疫检测或测定方法的实例包括放射性物质标记的免疫抗体方法(RIA)、酶免疫测定法(EIA或ELISA)、荧光免疫测定法(FIA)、发光免疫测定法、蛋白质免疫印迹法、物理化学方法等。
上述与CD38阳性细胞相关的疾病可以通过用本公开的抗体或抗体片段检测或测定表达CD38的细胞来诊断。
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS系统(Applied Biosystem)的荧光抗体染色法等。
在本公开中，对用于检测或测定目标抗原(例如CD38)的样品没有特别限制，只要它具有包含表达目标抗原(例如CD38)的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
根据所需的诊断方法，含有本公开的单克隆抗体或其抗体片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方法的试剂，例如识别所述单克隆抗体、其抗体片段或其结合物的标记的第二抗体和与所述标记对应的底物等。
在以上说明书中提出了本公开一种或多种实施方案的细节。虽然可使用与本 文所述类似或相同的任何方法和材料来实施或测试本公开，但是以下描述优选的方法和材料。通过说明书和权利要求书，本公开的其他特点、目的和优点将是显而易见的。
在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形式包括复数指代物的情况。除非另有定义，本文使用的所有技术和科学术语都具有本公开所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。提出以下实施例是为了更全面地说明本公开的优选实施方案。这些实施例不应以任何方式理解为限制本公开的范围，本公开的范围由权利要求书限定。
实施例与测试例
以下结合实施例与测试例进一步描述本公开，但这些实施例并非限制着本公开的范围。本公开实施例与测试例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.CD38抗原的制备
以UniProt ADP-核糖环化酶/环ADP-核糖水解酶1(人CD38蛋白，Uniprot号：P28907)作为CD38的模板，设计本公开使用的抗原及检测用蛋白的氨基酸序列，可选地，在CD38蛋白基础上融合不同的标签如His标签或Fc等。分别克隆到pTT5载体上(Biovector，Cat#:102762)或pTargeT载体上(Promega,A1410)，在293细胞瞬时表达或在CHO-S细胞中稳定表达，纯化，获得本公开抗原及检测用蛋白。
CD38胞外域与小鼠IgG2a-Fc融合蛋白：CD38-ECD-mFc，作为免疫原；
Figure PCTCN2019105119-appb-000001
注释：划线部分为小鼠IgG2a-Fc部分；
CD38胞外域与人IgG1Fc融合蛋白：CD38-ECD-Fc，作为免疫原；
Figure PCTCN2019105119-appb-000002
注释：划线部分为人IgG1-Fc部分；
带His标签的CD38胞外域融合蛋白：CD38-ECD-His，作为免疫原或检测试剂：
Figure PCTCN2019105119-appb-000003
注释：划线部分为6×His标签；
实施例2.CD38相关重组蛋白的纯化
1.带His标签的重组蛋白的纯化步骤：
将细胞表达上清样品高速离心去除杂质，并将缓冲液置换为PBS，加入咪唑至终浓度为5mM。用含有5mM咪唑的PBS溶液平衡镍柱，冲洗2-5倍柱体积。将置换后的细胞上清样品上Ni Sepharose excel柱(GE，17-3712-02)。用含有5mM咪唑的PBS溶液冲洗柱子，至A280读数降至基线。后用PBS+10mM咪唑冲洗层析柱，除去非特异结合的杂蛋白，并收集流出液。再用含有300mM咪唑的PBS溶液洗脱目的蛋白，并收集洗脱峰。收集的洗脱液浓缩后用凝胶层析Superdex200(GE，28-9893-35)进一步纯化，流动相为PBS。去除聚体峰，收集洗脱峰。所得到的蛋白经电泳、肽图、LC-MS鉴定为正确后分装备用。得到带His标签的CD38-ECD-His(SEQ ID NO：57)，用作制备本公开抗体的免疫原或检测试剂。CD38-ECD-His也可以通过体外化学法与KLH进行偶联反应后作为免疫原刺激小鼠免疫。
2.CD38-ECD-Fc融合蛋白的纯化步骤：
将细胞表达上清样品高速离心去除杂质，上清进行MabSelect Sure(GE， 17-5438-01)亲和层析。MabSelect Sure层析柱先用0.1M NaOH再生，利用纯水冲洗后用PBS平衡柱子，将上清结合后，利用PBS进行洗涤至A280读数降至基线。用0.1M醋酸缓冲液在pH3.5条件下洗脱目的蛋白，用1MTris-HCl中和。洗脱样品适当浓缩后利用PBS平衡好的凝胶层析Superdex200(GE，28-9893-35)进一步纯化，合并收集目的蛋白所在接收管浓缩至适当浓度。此方法用来纯化CD38-ECD-Fc(SEQ ID NO：2)融合蛋白，该方法也可以用来纯化本公开中涉及的人源化抗体蛋白。
实施例3.抗人CD38杂交瘤单克隆抗体的获得和制备
1.免疫
抗人CD38单克隆抗体通过免疫小鼠产生。实验用SJL白小鼠，雌性，4-6周龄(北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2012-0001)。
饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期，温度20-25℃；湿度40-60％。将已适应环境的小鼠按不同方案免疫，每组3-5只。
免疫抗原可以是CD38-ECD-His、CD38-ECD-Fc、CD38-FL-CHOS(转染人CD38全长的CHOS细胞)等，可以使用单独一种试剂配合不同的免疫佐剂或者不同类型免疫原交叉免疫。免疫部位可以是腹腔或者背部皮下，或者两种位置交替免疫。示例性免疫方法如用Titermax(sigma Lot Num：T2684)或明矾(Thremo Lot Num：77161)免疫。抗原与佐剂(titermax)比例为1:1，抗原与佐剂(明矾)比例为4:1，25-50μg或1×10 7cells/只(首次免疫)，25-50μg或1×10 7cells/只(加强免疫)。第0天腹膜内(IP)注射或皮下(SC)注射抗原，首免后每两周重复免疫一次。每三周取血，用ELISA方法确定小鼠血清中的抗体滴度。在第8-12次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射25-50μg或1×10 7cells/只的生理盐水配制的抗原溶液。
2.细胞融合
选择血清中的抗体滴度高(见测试例1，结合CD38的ELISA方法)并且滴度趋于平台的小鼠进行脾细胞融合，融合前3天冲刺免疫所选小鼠。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2019105119-appb-000004
CRL-8287 TM)进行融合得到杂交瘤细胞。融合好的杂交瘤细胞用HAT完全培养基(含20％FBS、1×HAT和1×OPI的RPMI-1640培养基)重悬，分装于96孔细胞培养板中(1×10 5细胞/150μl/孔)，37℃，5％CO 2孵育。融合后的第5天加入HAT完全培养基，50μl/孔，37℃，5％CO 2孵育。融合后第7天至8天，根据细胞生长密度，全换液，培养基为HT完全培养基(含20％FBS、1×HT和1×OPI的RPMI-1640培养基)，200μl/孔，37℃，5％CO 2孵育。
3.杂交瘤细胞筛选
融合后第10-11天，根据细胞生长密度，进行结合CD38的ELISA方法检测(见测试例1)。并将ELISA检测阳性孔的细胞上清进行结合CD38-FL-CHO-S的FACS方法检测(见测试例2)，阳性孔换液，并根据细胞密度及时扩大至24孔板中。移入24孔板的细胞株经过复测后进行保种和第一次亚克隆。第一次亚克隆筛选(见测试例1、2)为阳性的进行保种，并进行第二次亚克隆。第二次亚克隆为阳性(见测试例1、2)的进行保种和蛋白表达。多次融合获得具有高CD38亲和力的杂交瘤细胞。
通过阻断实验和结合实验筛选得到杂交瘤克隆m009、m011和m160，用无血清细胞培养法进一步制备抗体，按纯化实例纯化抗体，供在检测例中使用。
其中测得杂交瘤克隆009的鼠源抗体可变区序列如下：
>m009 VH：m009重链可变区序列
Figure PCTCN2019105119-appb-000005
>m009 mVL：m009轻链可变区序列
Figure PCTCN2019105119-appb-000006
注：上述序列中下划线为根据Kabat编号规则确定的CDR序列，斜体为FR序列，顺序依次为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
杂交瘤克隆m011的鼠源抗体可变区序列如下：
>m011 VH：m011重链可变区序列
Figure PCTCN2019105119-appb-000007
>m011 VL：m011轻链可变区序列
Figure PCTCN2019105119-appb-000008
注：上述序列中下划线为根据Kabat编号规则确定的CDR序列，斜体为FR序列，顺序依次为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
杂交瘤克隆m160的鼠源抗体可变区序列如下：
>m160 VH：m160重链可变区序列
Figure PCTCN2019105119-appb-000009
>m160 VL：m160轻链可变区序列
Figure PCTCN2019105119-appb-000010
注：上述序列中下划线为根据Kabat编号规则确定的CDR序列，斜体为FR序列，顺序依次为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
各重链及轻链CDR区序列如表3所示：
表3.重链、轻链CDR区序列
Figure PCTCN2019105119-appb-000011
4.人IgG1嵌合抗体制备
杂交瘤筛选所获得的候选分子经过扩增测序可得到可变区编码基因序列。以测序所得序列设计首尾引物，以测序基因为模板，经过PCR搭建各抗体的VH/VK基因片段，再与表达载体pHr(带信号肽及hIgG1/hkappa恒定区基因(CH1-Fc/CL)片段)进行同源重组，构建重组嵌合抗体全长表达质粒VH-CH1-Fc-pHr/VL-CL-pHr，形成杂交瘤克隆m009、m011和m160的嵌合抗体ch-009、ch-011和ch-160。
实施例4.抗CD38杂交瘤单克隆抗体的人源化
通过比对IMGT人类抗体重和轻链可变区种系基因数据库和MOE软件分析，分别挑选与m009、m011和m160同源性高的重/轻链可变区种系基因作为模板。将这三个鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的人源化抗体可变区序列。
人源FR区的选择和FR区氨基酸的回复突变：在所获得的鼠源抗体VH/VL CDR典型结构的基础上，从人源种系数据库中搜索轻链可变区(VL)和重链可变区(VH)的同源序列，按FR的同源性由高到低排序，选取FR同源性最高的种系作为主要模板；将鼠源抗体的CDR区移植到人源模板上；进一步地，以鼠源抗体的三维结构为基础，通过软件对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变；并对化学不稳定氨基酸残基优化，得到最终的人源化分子。
1.杂交瘤克隆m009人源化构架选择
鼠源抗体m009的人源化轻链模板为IGKV3-11*01和hJK4.1，人源化重链模板为IGHV1-3*01和hJH4.1，将m009的CDR移植到人源模板上，获得人源化可变区序列如下：
>h009VH-CDR移植
Figure PCTCN2019105119-appb-000012
>h009VL-CDR移植
Figure PCTCN2019105119-appb-000013
注：序列中下划线为根据Kabat编号规则确定的CDR序列，斜体为FR序列，顺序依次为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
2.杂交瘤克隆m009人源化回复突变设计，见表4：
表4.杂交瘤克隆m009的人源化回复突变
Figure PCTCN2019105119-appb-000014
Figure PCTCN2019105119-appb-000015
注：移植代表鼠源抗体CDR植入人种系FR区；“A43S”表示在移植基础上，将第43位(依照Kabat编号系统编号)的A回复突变为S，其它以此类推。
杂交瘤克隆m009的人源化抗体可变区序列如下：
>h009 VH1(SEQ ID NO：24)
Figure PCTCN2019105119-appb-000016
>h009 VH2(SEQ ID NO：26)
Figure PCTCN2019105119-appb-000017
>h009 VH3(SEQ ID NO：27)
Figure PCTCN2019105119-appb-000018
>h009 VH4(SEQ ID NO：28)
Figure PCTCN2019105119-appb-000019
>h009 VH5(SEQ ID NO：29)
Figure PCTCN2019105119-appb-000020
>h009 VL1(SEQ ID NO：25)
Figure PCTCN2019105119-appb-000021
>h009 VL2(SEQ ID NO：30)
Figure PCTCN2019105119-appb-000022
>h009 VL3(SEQ ID NO：31)
Figure PCTCN2019105119-appb-000023
将上述人源化轻链可变区和重链可变区分别与人种系轻链恒定区(例如人κ、λ链轻链恒定区)和重恒定区(例如人IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区)组合形成人源化抗体的重链和轻链，进而获得完整的m009的人源化抗体(h009)。示例性的，上述h009抗体的重链可变区和轻链可变区分别与如SEQ ID NO：43所示的人IgG1重链恒定区和序列如SEQ ID NO：45所示的人kappa轻链恒定区组合，获得全长人源化抗体(h009-01至h009-15)，可变区序列如表5所示：
表5.h009人源化抗体的重链可变区和轻链可变序列
可变区 h009 VL1 h009 VL2 h009 VL3
h009 VH1 h009-01 h009-06 h009-11
h009 VH2 h009-02 h009-07 h009-12
h009 VH3 h009-03 h009-08 h009-13
h009 VH4 h009-04 h009-09 h009-14
h009 VH5 h009-05 h009-10 h009-15
注：表中例如“h009-07”，其表示编号为h009-07的人源化抗体的重链可变区为的h009 VH2、轻链可变区为的h009VL2，其它抗体以此类推。
3.杂交瘤克隆m011人源化构架选择
鼠源抗体m011的人源化轻链模板为IGKV4-1*01和hJK4.1，人源化重链模板为IGHV3-7*01和hJH6.1，为了消除潜在的热点(hot spot)，在人种系IGHV3-7*01和hJH6.1的FR区引入N  82A T(依照Kabat编号系统，将第82A位氨基酸由天冬酰胺(缩写：N或Asn)突变为苏氨酸(缩写：T或Thr))突变；和N76S(依照Kabat编号系统编号，将第76位氨基酸由天冬酰胺(缩写：N或Asn)突变为苏氨酸(缩写：S或Ser))突变，将m011的CDR移植到人源模板上，获得人源化可变区序列如下：
>h011VH-CDR移植
Figure PCTCN2019105119-appb-000024
>h011VL-CDR移植
Figure PCTCN2019105119-appb-000025
Figure PCTCN2019105119-appb-000026
注：序列中下划线为根据Kabat编号规则确定的CDR序列，斜体为FR序列，顺序依次为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
4.杂交瘤克隆m011回复突变设计，见下表6：
表6.杂交瘤克隆m011人源化回复突变
Figure PCTCN2019105119-appb-000027
注：移植代表鼠源抗体CDR植入人种系FR区序列，G68R表示在移植基础上将第68位(依照Kabat编号系统编号)的G回复突变为R，其它以此类推。
h011人源化抗体可变区具体序列如下：
>h011 VH1(SEQ ID NO：32)
Figure PCTCN2019105119-appb-000028
>h011 VH2(SEQ ID NO：34)
Figure PCTCN2019105119-appb-000029
>h011 VL1(SEQ ID NO：33)
Figure PCTCN2019105119-appb-000030
>h011 VL2(SEQ ID NO：35)
Figure PCTCN2019105119-appb-000031
>h011VL3(SEQ ID NO：36)
Figure PCTCN2019105119-appb-000032
将上述人源化轻链可变区和重链可变区分别与人种系轻链恒定区(例如人κ、 λ链轻链恒定区)和重恒定区(例如人IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区)组合形成人源化抗体的重链和轻链，进而获得完整的m011的人源化抗体(h011)。示例性的，上述h011抗体的重链可变区和轻链可变区分别与序列如SEQ ID NO：43所示的人IgG1重链恒定区和序列如SEQ ID NO：45所示的人kappa轻链恒定区组合，获得全长人源化抗体(h011-01至h011-06)，其可变区序列如表7所示：
表7.h0011人源化抗体的重轻链可变区相互组合表
h011VL1 h011VL2 h011 VL3
h011 VH1 h011-01 h011-03 h011-05
h011 VH2 h011-02 h011-04 h011-06
注：表中例如“h011-04”，其表示编号为h011-04的人源化抗体的重链可变区为的h011VH2、轻链可变区为的h011VL2，其它抗体以此类推。
5.杂交瘤克隆m160人源化构架选择
鼠源抗体m160的人源化轻链模板为IGKV4-1*01和hJK4.1人源化重链模板为IGHV3-7*01和hJH6.1，为了消除人种系FR区潜在的热点(hot spot)，在人种系IGHV3-7*01和hJH6.1的FR区引入S77T突变(依照Kabat编号系统编号，将第77位 氨基酸由苏氨酸(缩写：S或Ser)突变为苏氨酸(缩写：T或Thr))、N  82A T突变(依照Kabat编号系统编号，将第 82A位氨基酸由天冬酰胺(缩写：N或 Asn)突变为苏氨酸(缩写：T或Thr))，将m160的CDR移植到人源模板上，获得的人源化可变区序列如下：
>h160VH-CDR移植
Figure PCTCN2019105119-appb-000033
>h160VL-CDR移植
Figure PCTCN2019105119-appb-000034
注：序列中下划线为根据Kabat编号规则确定的CDR序列，斜体为FR序列，顺序依次为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
6.杂交瘤克隆m160的人源化回复突变设计，见下表8：
表8.杂交瘤克隆m160人源化回复突变
Figure PCTCN2019105119-appb-000035
Figure PCTCN2019105119-appb-000036
注：移植代表鼠抗体CDR植入人种系FR区序列，“M4L”表示在移植基础上将第4位(依照Kabat编号系统编号)的M回复突变为L，其它以此类推。
h160人源化抗体可变区具体序列如下：
>h160 VH1(SEQ ID NO：37)
Figure PCTCN2019105119-appb-000037
>h160 VH2(SEQ ID NO：39)
Figure PCTCN2019105119-appb-000038
>h160 VL1(SEQ ID NO：38)
Figure PCTCN2019105119-appb-000039
>h160 VL2(SEQ ID NO：40)
Figure PCTCN2019105119-appb-000040
>h160 VL3(SEQ ID NO：41)
Figure PCTCN2019105119-appb-000041
>h160 VL4(SEQ ID NO：42)
Figure PCTCN2019105119-appb-000042
将上述人源化轻链可变区和重链可变区分别与人种系轻链恒定区(例如人κ、λ链轻链恒定区)和重恒定区(例如人IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区)组合形成人源化抗体的重链和轻链，进而获得完整的m160的人源化抗体(h160)。示例性的，上述h160抗体的重链可变区和轻链可变区分别与序列如 SEQ ID NO：43所示的人IgG1重链恒定区和序列如SEQ ID NO：45所示的人kappa轻链恒定区组合，获得全长人源化抗体(h160-01至h160-08)，其可变区序列如表9所示：
表9.h016人源化抗体的重链可变区和轻链可变区
h160 VL1 h160 VL2 h160 VL3 h160 VL4
h160 VH1 h160-01 h160-02 h160-03 h160-04
h160 VH2 h160-05 h160-06 h160-07 h160-08
注：表中例如“h160-07”，其表示编号为h160-07的人源化抗体的重链可变区为的h160 VH2、轻链可变区为的h160 VL3，其它抗体以此类推。
实施例5.构建和表达抗人CD38人源化抗体IgG1或IgG1-E333A的形式
设计引物PCR搭建各人源化抗体VH/VK基因片段，再与表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段，实验室构建)进行同源重组，构建抗体全长表达载体VH-CH1-FC-pHr/VK-CL-pHr。人源化抗体的轻链恒定区可选自人κ、λ链轻链恒定区，重链恒定区可选自人IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，非限制性实施包括对人IgG1、IgG2、IgG4的恒定区进行优化设计，改善抗体功能，例如通过IgG1进行E333A点突变获得IgG1-E333A恒定区，该突变可以增强IgG1-Fc与C1q的结合能力，进一步增强抗体CDC的功能(参见US6528624)。以下具体轻/重链恒定区并非作为本公开抗体恒定区的限制，也可选用本领域其它已知的抗体轻/重链恒定区及其突变体。
示例性的重轻链恒定区如下所示：
IgG1重链恒定区：
Figure PCTCN2019105119-appb-000043
IgG1-E333A重链恒定区：
Figure PCTCN2019105119-appb-000044
Figure PCTCN2019105119-appb-000045
kappa轻链恒定区：
Figure PCTCN2019105119-appb-000046
示例性地，将前述杂交瘤克隆m009、m011、m160的人源化轻链可变区和重链可变区分别与序列如SEQ ID NO：43所示的人IgG1重链恒定区和序列如SEQ ID NO：45所示的人kappa轻链恒定区组合，获得的全长人源化抗体参见表5、表7和表9所示；另一示例性地，将前述杂交瘤克隆m009、m011、m160的人源化轻链可变区和重链可变区分别与序列如SEQ ID NO：44所示的人IgG1-E333A重链恒定区和序列如SEQ ID NO：45所示的人kappa轻链恒定区组合，获得全长人源化抗体，所获得的人源化抗体的轻重链可变区序列见表10：
表10.人源化抗体(重链恒定区为人IgG1-E333A，轻链恒定区kappa)的可变区序列
Figure PCTCN2019105119-appb-000047
Figure PCTCN2019105119-appb-000048
示例性的，人源化抗体h009-07、hu9E、h011-01、hu11E、h160-01和hu160E的全长氨基酸序列如下：
h009-07抗体的重链序列:
Figure PCTCN2019105119-appb-000049
h009-07抗体的轻链序列:
Figure PCTCN2019105119-appb-000050
hu9E抗体的重链序列:
Figure PCTCN2019105119-appb-000051
hu9E抗体的轻链序列:
Figure PCTCN2019105119-appb-000052
Figure PCTCN2019105119-appb-000053
h011-01抗体的重链序列：
Figure PCTCN2019105119-appb-000054
h011-01抗体的轻链序列:
Figure PCTCN2019105119-appb-000055
hu11E抗体的重链序列：
Figure PCTCN2019105119-appb-000056
hu11E抗体的轻链序列:
Figure PCTCN2019105119-appb-000057
h160-01抗体的重链序列：
Figure PCTCN2019105119-appb-000058
Figure PCTCN2019105119-appb-000059
h160-01抗体的轻链序列:
Figure PCTCN2019105119-appb-000060
hu160E抗体的重链序列：
Figure PCTCN2019105119-appb-000061
hu160E抗体的轻链序列:
Figure PCTCN2019105119-appb-000062
本公开将抗CD38的抗体达雷木单抗(简称Dara，序列来源参照WHO Drug Information,Vol.24,No.1,2010)作为对照抗体，其重链和轻链序列如下所示：
Dara的重链序列：
Figure PCTCN2019105119-appb-000063
Dara的轻链序列：
Figure PCTCN2019105119-appb-000064
用以下测试方法验证本公开抗体性能及有益效果。
测试例1.CD38抗体结合CD38蛋白的ELISA实验
抗CD38抗体的亲和力通过抗体与固定在ELISA板上CD38的结合的量来检测。包被2μg/ml链霉亲和素(Abcam，CAT#ab123480)于96孔ELISA板(Costar，CAT#3590)上，洗板封闭后，加入2μg/ml生物素标记的CD38-ECD-His。孵育后，加入不同浓度稀释的抗CD38抗体样品，再洗板后加入辣根过氧化物酶-羊抗人F(ab’) 2抗体(Jackson，CAT#109-036-097)，再洗板加入四甲基联苯胺溶液显色，最后加入终止液，在酶标仪上测量OD450并计算其EC50值，结果如表11所示。
表11.CD38人源化抗体的亲和力
抗体 EC50(μg/ml)
hu9E 0.02594
hu11E 0.02762
hu160E 0.02801
结果显示本公开的人源化抗体可与CD38蛋白特异性结合，并且结合能力较强。
测试例2.CD38抗体结合CD38-FL-CHO-S细胞的实验
稳定转染有全长人CD38(Uniprot号：P28907)的CHO-S(FreeStyle TM CHO-S细胞,Invitrogen,R80007)细胞(CD38-FL-CHOS)于CD CHO培养基(Gibco，REF#10743-029)中培养，1×10 6细胞/ml CD38-FL-CHOS细胞用1％BSA封闭后，加入不同浓度稀释的抗CD38抗体样品，洗两次后，再加入Alexa Fluor 488-羊抗人(H+L)抗体(Invitrogen，CAT#A11013)，洗两次后，使用流式细胞仪读取荧光信号值，结果如表12所示。
表12.CD38人源化抗体的亲和力
抗体 EC50(μg/ml)
hu9E 0.4020
hu11E 0.4813
hu160E 0.4740
FACS检测结果显示本公开的人源化抗体对细胞表面的天然CD38具有很强的结合能力。
测试例3.CD38抗体对CD38酶活抑制的实验
将CD38-ECD-His用20mM Tris-HCl(pH 6.5)缓冲液配成浓度为4μg/ml的溶液。同样，将抗CD38抗体样品用缓冲液配成不同的浓度。各取25μl CD38-ECD-His及抗CD38抗体样品加入底透黑壁96孔板(Corning，CAT#3603)。在常温孵育15分钟后，加入50μl浓度为200μM的底物NGD(Sigma，CAT#N5131-25MG)。常温孵育2小时后，用FlexStation 3(Molecular Devices)使用发射波长410nm(激发光300nm)检测环GDP核糖(cGDPR)的产生，结果如表13所示。
表13.CD38人源化抗体对CD38酶活抑制结果
抗体 IC50(μg/ml) Imax(最大抑制率，％)
Dara 3.147 44.54
hu9E 1.559 89.63
hu11E 1.827 44.31
hu160E 1.199 47.67
实验结果显示hu9E的最大酶活抑制率达89.63％，明显优于对照抗体Dara，而hu11E和hu160E的最大酶活抑制率分别达44.31％和47.67％，与对照抗体相当。
测试例4.CD38抗体对Molp-8、Daudi细胞体外ADCC的实验
收集人多发性骨髓瘤细胞系Molp-8(南京科佰，CBP60562)或人Burkitt's淋巴瘤细胞系Daudi细胞(ATCC，CCL-123)，1000rpm离心5分钟，用含10％超低IgG胎牛血清(Gibco，CAT# 1921005PJ)的无酚红RPMI 1640培养基(Gibco，CAT#11835-030)重悬，用细胞计数仪(Countstar，IC1000)计数，稀释细胞至1×10 5细胞/ml。
使用Ficoll(GE，CAT# 17-5442-02)从新鲜人血液中分离外周血单核细胞(PBMC)，于无酚红RPMI 1640培养基中重悬，用细胞计数仪计数，稀释细胞至3×10 6细胞/ml。
将Molp-8或Daudi与不同浓度的CD38抗体或阴性对照IgG(C25-hIgG1(WT)，实验室制备)按1:1的比例，在96孔板中各加入50μl，孵育30分钟(37℃，5％CO 2)后，加入50μl效应细胞PBMC，效应细胞比目标细胞比率为30:1。孵育4小时(37℃，5％CO 2)后，使用CytoTox 96非放射细胞毒性测试试剂盒(Promega，CAT#G1780)检测LDH(乳糖脱氢酶，lactate dehydrogenase)释放。取50μl细胞上清，再加入50μl CytoTox
Figure PCTCN2019105119-appb-000065
Reagent，室温孵育30分钟后加入终止液，用FlexStation 3(Molecular Devices)检测吸收光(490nm)，结果如表14所示。
表14.CD38人源化抗体对靶细胞ADCC结果
Figure PCTCN2019105119-appb-000066
Figure PCTCN2019105119-appb-000067
实验结果显示本公开中的人源化抗体对Molp-8、Daudi细胞体外ADCC的作用较强，可显著造成靶细胞裂解。
测试例5.CD38抗体对Molp-8、Daudi细胞体外CDC的实验
收集Molp-8或Daudi细胞后，1000rpm离心5分钟，重悬。用细胞计数仪(Countstar，IC1000)计数，将细胞以1×10 6细胞/ml重悬在含10％超低IgG胎牛血清(Gibco，CAT# 1921005PJ)的无酚红RPMI 1640培养基(Gibco，CAT# 11835-030)中。随后，将细胞以5×10 4细胞/孔(50μl/孔)铺于96孔板(Corning，CAT#3903)中。然后，加入50μl不同浓度的抗CD38抗体和阴性对照。孵育30分钟(37℃，5％CO 2)后，向每个孔中加入50μl人血清(实验室制备)。孵育2小时(37℃，5％CO 2)后，向每个孔中加入16.6μl Alamar Blue试剂(Thermo，CAT#DAL1025)，孵育20小时(37℃，5％CO 2)。最后，用FlexStation 3(Molecμlar Devices)检测发射波长585nm(激发波长570nm)，结果如表15所示。
表15.CD38人源化抗体对靶细胞CDC结果
Figure PCTCN2019105119-appb-000068
实验结果显示本公开中的人源化抗体对Molp-8、Daudi细胞体外CDC的作用较强，可显著造成靶细胞细胞裂解。
测试例6.CD38抗体对Molp-8、Daudi细胞的体外ADCP报告系统测试
收集Molp-8或Daudi细胞后，1000rpm离心5分钟，重悬。用细胞计数仪(Countstar，IC1000)计数，将细胞以1×10 6细胞/ml重悬在含10％超低IgG胎牛血清(Gibco，CAT# 1921005PJ)的无酚红RPMI 1640培养基(Gibco，CAT# 11835-030)中。随后，将细胞以2.5×10 4细胞/孔(25μl/孔)铺于96孔板(Corning，CAT#3903)中，加入25μl不同浓度的抗CD38抗体和阴性对照，孵育30分钟(37℃，5％CO 2)。收集稳转全长人FcγIIa(Uniprot号：P12318)的Jurkat(Jurkat-Lucia TM  NFAT Cells，Invivogene)细胞作为效应细胞，将效应细胞以7.5×10 4细胞/孔(50μl/孔)加入靶细胞和抗体孵育的96孔板中。孵育6小时(37℃，5％CO 2)后吸出10μl上清到新的96孔板(Corning，CAT#3903)，加入90μl/孔的QUANTI-Luc(Invivogene，rep-qlc1)，用VITOR(VITOR3，PerkinElmer)检测化学发光Luminescence，结果如表16和图1所示。
表16.CD38人源化抗体对靶细胞的体外ADCP报告系统测试结果
Figure PCTCN2019105119-appb-000069
实验结果显示本公开中的人源化抗体对Molp-8、Daudi细胞的体外ADCP报告系统测试效果均明显优于对照抗体Dara。
测试例7.BIAcore检测CD38抗体亲和力实验
用Biacore仪器测试本公开嵌合抗体(ch-009、-011、-160参见实施例3的制备方法)、人源化抗体及Dara对人CD38-ECD-His抗原的亲和力。
按照人Fc捕获试剂盒(CAT#BR-1008-39，GE)说明书中所述的方法，将人Fc捕获分子共价偶联于CM5生物传感芯片(CAT#BR-1005-30，GE)上，从而亲和捕获待测抗体。然后于芯片表面流经人CD38-ECD-His抗原，利用Biacore仪器实时检测反应信号，从而获得结合和解离曲线，通过拟合得到亲和力数值。在实验中每个循环解离完成后，用人Fc捕获试剂盒(GE)里配置的再生溶液将生物芯片洗净再生，结果见表17和表18。
表17.抗CD38抗体与人CD38亲和力的BIAcore检测结果
抗体 ka(1/Ms) kd(1/s) KD(M)
ch-009 2.04E+06 4.77E-04 2.34E-10
h009-07 2.11E+06 2.10E-03 9.95E-10
h009-08 1.89E+06 2.23E-03 1.18E-09
h009-09 2.29E+06 4.89E-03 2.14E-09
h009-10 2.04E+06 4.33E-03 2.12E-09
h009-11 2.33E+06 1.21E-02 5.17E-09
h009-12 2.24E+06 9.17E-04 4.09E-10
h009-13 2.28E+06 1.06E-03 4.65E-10
h009-14 2.27E+06 1.85E-03 8.15E-10
h009-15 2.28E+06 1.70E-03 7.45E-10
ch-011 7.46E+06 1.48E-03 1.99E-10
h011-01 6.68E+06 2.08E-03 3.11E-10
h011-02 6.59E+06 1.95E-03 2.97E-10
h011-03 6.97E+06 2.13E-03 3.06E-10
h011-04 6.76E+06 2.02E-03 2.99E-10
h011-05 6.89E+06 2.13E-03 3.09E-10
h011-06 6.71E+06 2.01E-03 3.00E-10
ch-160 2.74E+06 1.70E-04 6.20E-11
h160-01 1.99E+06 1.36E-04 6.87E-11
h160-02 1.93E+06 1.63E-04 8.46E-11
h160-03 2.40E+06 1.87E-04 7.79E-11
h160-04 2.12E+06 1.84E-04 8.67E-11
h160-05 2.05E+06 1.41E-04 6.91E-11
h160-06 2.09E+06 1.64E-04 7.82E-11
h160-07 2.43E+06 1.82E-04 7.48E-11
h160-08 2.29E+06 1.79E-04 7.82E-11
结果显示，本公开所得的人源化抗体对人CD38均有很高的亲和力。
表18.抗CD38抗体与人CD38亲和力的BIAcore检测结果
Figure PCTCN2019105119-appb-000070
结果显示本公开所得的hu9E、hu11E、hu160E抗体对人CD38均有很高的亲和力，其KD值小于对照抗体Dara的KD值，优于对照抗体。
体内活性生物学评价
测试例8.CD38抗体的体内药代动力学实验
SD大鼠18只，雄性，平均分成6组，动物由西普尔-必凯实验动物有限公司提供；分别静脉注射和皮下注射给药，给药剂量3mg/kg，静脉注射给药组于给药 前及给药后5min、8h、1d、2d、4d、7d、10d、14d、21d、28d采集全血0.2ml，不加抗凝；取血后在4℃放置30min，1000g离心15min；取上清(血清)置于EP管中，于-80℃保存；皮下注射给药组于给药前及给药后1h、8h、1d、2d、4d、7d、10d、14d、21、28d采集全血，分离血清置于EP管中，于-80℃保存。
根据测试例1抗CD38抗体结合CD38蛋白的ELISA实验中的方法作不同样品的标准曲线，血清样品1：1000代替抗CD38抗体加入反应体系后根据OD450换算不同时间点抗CD38抗体于血清中的浓度，所得数据由Phoenix WinNonlin软件分析计算药代动力学相关参数。hu9E、hu11E和hu160E抗体的体内药代动力学结果分别如表19所示。
表19.抗体大鼠药代动力学评价表
Figure PCTCN2019105119-appb-000071
备注：表中T1/2表示半衰期，IV表示静脉注射，SC表示皮下注射。
在大鼠中测定了hu9E、hu11E和hu160E 3mpk剂量下静脉注射和皮下注射的PK(pharmacokinetics，药物代谢动力学)，结果显示三者均具有良好的大鼠体内PK表现：皮下注射均具有较高的生物利用度，静脉注射平均T1/2分别为11.4天，13.3天和13.6天；皮下注射平均T1/2分别为11.1天，12.1天和10.9天，提示大鼠体内抗体稳定性良好，具有开发皮下制剂的可能性。
测试例9.CD38抗体小鼠肿瘤体内药效实验
Balb/c裸鼠，SPF，14-16g，雌性，购自上海斯莱克实验动物有限责任公司。Balb/c裸鼠于实验室环境适应性饲养6d(天)，在右肋部皮下接种AMO-1细胞(南京科佰，CBP60242，5×10 6+50％matrigel基质胶/只，Matrigel基底膜基质胶，BD公司，货号#356237)，9天后分组，8只/组，平均瘤体积约197.21±9.25mm 3(d0)，共7组，分别为：
空白对照IgG(3mg/kg)(C25-hIgG1(WT)，实验室制备)组、
Dara(1mg/kg)组、
Dara(3mg/kg)组、
hu11E(1mg/kg)组、
hu11E(3mg/kg)组、
hu160E(1mg/kg)组、
hu160E(3mg/kg)组；
腹腔注射，一周二次，连续给药3周。每周测量2次瘤体积和体重，记录数据。给药全部结束观察至要求的时间，处死小鼠，取瘤。
使用Excel 2003统计软件：平均值以avg计算；SD值以STDEV计算；SEM值以STDEV/SQRT计算；组间差异P值以TTEST计算。
肿瘤体积(V)计算公式为：V＝1/2×L 长×L 短 2
相对体积(RTV)＝V T/V 0
抑瘤率(％)＝(C RTV-T RTV)/C RTV(％)
其中V 0、V T分别为实验开始时及实验结束时的肿瘤体积。C RTV、T RTV分别为实验结束时的对照组及实验组的相对肿瘤体积，结果如表20和图2所示。
表20.抗CD38抗体体内抑瘤结果
Figure PCTCN2019105119-appb-000072
备注：mpk表示mg/kg
小鼠肿瘤体内药效实验结果显示，本公开的人源化抗体hu11E和hu160E均能够明显的抑制肿瘤生长。相比空白对照IgG(3mg/kg)组，hu11E(1mg/kg)组和hu160E(1mg/kg)组抑瘤率分别达93.14％和70.02％；hu11E(3mg/kg)组和hu160E(3mg/kg)组抑瘤率分别达99.52％和89.89％。给药过程中各组动物体重正常，提示本公开的抗体无明显毒副作用。

Claims (23)

  1. 一种抗CD38抗体或其抗原结合片段，所述抗CD38抗体或抗原结合片段与人CD38特异性结合，所述抗体或其抗原结合片段包含：
    (i)重链HCDR1，所述HCDR1的氨基酸序列如SEQ ID NO：9所示或与SEQ ID NO：9具有3、2或1个氨基酸差异，
    重链HCDR2，所述HCDR2的氨基酸序列如SEQ ID NO：10所示或与SEQ ID NO：10序列具有3、2或1个氨基酸差异，
    重链HCDR3，所述HCDR3的氨基酸序列如SEQ ID NO：11所示或与SEQ ID NO：11序列具有3、2或1个氨基酸差异，
    轻链LCDR1，所述LCDR1的氨基酸序列如SEQ ID NO：12所示或与SEQ ID NO：12具有3、2或1个氨基酸差异，
    轻链LCDR2，所述LCDR2的氨基酸序列如SEQ ID NO：13所示或与SEQ ID NO：13序列具有3、2或1个氨基酸差异，和
    轻链LCDR3，所述LCDR3的氨基酸序列如SEQ ID NO：14所示或与SEQ ID NO：14序列具有3、2或1个氨基酸差异；或者
    (ii)重链HCDR1，所述HCDR1的氨基酸序列如SEQ ID NO：15所示或与SEQ ID NO：15具有3、2或1个氨基酸差异，
    重链HCDR2，所述HCDR2的氨基酸序列如SEQ ID NO：16所示或与SEQ ID NO：16序列具有3、2或1个氨基酸差异，
    重链HCDR3，所述HCDR3的氨基酸序列如SEQ ID NO：17所示或与SEQ ID NO：17序列具有3、2或1个氨基酸差异，
    轻链LCDR1，所述LCDR1的氨基酸序列如SEQ ID NO：18所示或与SEQ ID NO：18具有3、2或1个氨基酸差异，
    轻链LCDR2，所述LCDR2的氨基酸序列如SEQ ID NO：19所示或与SEQ ID NO：19序列具有3、2或1个氨基酸差异，和
    轻链LCDR3，所述LCDR3的氨基酸序列如SEQ ID NO：20所示或与SEQ ID NO：20序列具有3、2或1个氨基酸差异；或者
    (iii)重链HCDR1，所述HCDR1的氨基酸序列如SEQ ID NO：15所示或与SEQ ID NO：15具有3、2或1个氨基酸差异，
    重链HCDR2，所述HCDR2的氨基酸序列如SEQ ID NO：21所示或与SEQ ID NO：21序列具有3、2或1个氨基酸差异，
    重链HCDR3，所述HCDR3的氨基酸序列如SEQ ID NO：17所示或与SEQ ID NO：17序列具有3、2或1个氨基酸差异，
    轻链LCDR1，所述LCDR1的氨基酸序列如SEQ ID NO：22所示或与SEQ ID NO：22具有3、2或1个氨基酸差异，
    轻链LCDR2，所述LCDR2的氨基酸序列如SEQ ID NO：19所示或与SEQ ID NO：19序列具有3、2或1个氨基酸差异，和
    轻链LCDR3，所述LCDR3的氨基酸序列如SEQ ID NO：23所示或与SEQ ID NO：23序列具有3、2或1个氨基酸差异。
  2. 根据权利要求1所述的抗CD38抗体或其抗原结合片段，其中所述抗体为鼠源抗体、嵌合抗体或人源化抗体。
  3. 根据权利要求2所述的抗CD38抗体或其抗原结合片段，其中所述的鼠源抗体或嵌合抗体包含重链可变区和轻链可变区，其中：
    (a)所述重链可变区的氨基酸序列如SEQ ID NO：3所示或与SEQ ID NO：3具有至少95％序列同一性，且所述轻链可变区的氨基酸序列如SEQ ID NO：4所示或与SEQ ID NO：4具有至少95％序列同一性；
    (b)所述重链可变区的氨基酸序列如SEQ ID NO：5所示或与SEQ ID NO：5具有至少95％序列同一性，且所述轻链可变区的氨基酸序列如SEQ ID NO：6所示或与SEQ ID NO：6具有至少95％序列同一性；或
    (c)所述重链可变区的氨基酸序列如SEQ ID NO：7所示或与SEQ ID NO：7具有至少95％序列同一性，且所述轻链可变区的氨基酸序列如SEQ ID NO：8所示或与SEQ ID NO：8具有至少95％序列同一性。
  4. 根据权利要求2所述的抗CD38抗体或其抗原结合片段，其中所述抗体为人源化抗体，所述人源化抗体包含来源于人抗体的框架区或框架区变体，所述框架区变体在人抗体的轻链框架区和/或重链框架区上分别具有至多10个氨基酸的回复突变；
    优选地，所述人源化抗体包含选自以下(d)至(f)中的任一项：
    (d)重链可变区，所述重链可变区包含：重链HCDR1、HCDR2、HCDR3以及重链框架区，其中，所述HCDR1的氨基酸序列如SEQ ID NO：9所示或与SEQ ID NO：9具有3、2或1个氨基酸差异，所述HCDR2的氨基酸序列如SEQ ID NO：10所示或与SEQ ID NO：10具有3、2或1个氨基酸差异，所述HCDR3的氨基酸序列如SEQ ID NO：11所示或与SEQ ID NO：11具有3、2或1个氨基酸差异，所述重链框架区包含选自以下一个或更多个回复突变：2F、38K、44S、48I、67A、66K、69L、71V和73Q；和/或
    轻链可变区，所述轻链可变区包含：轻链LCDR1、LCDR2、LCDR3以及轻链框架区，其中，所述LCDR1的氨基酸序列如SEQ ID NO：12所示或与SEQ ID NO：12具有3、2或1个氨基酸差异，所述LCDR2的氨基酸序列如SEQ ID NO：13所示或与SEQ ID NO：13具有3、2或1个氨基酸差异，所述LCDR3的氨基酸 序列如SEQ ID NO：14所示或与SEQ ID NO：14具有3、2或1个氨基酸差异，所述轻链框架区包含选自以下一个或更多个回复突变：2F、43S、49K和87F；
    (e)重链可变区，所述重链可变区包含：重链HCDR1、HCDR2、HCDR3以及重链框架区，其中，所述HCDR1的氨基酸序列如SEQ ID NO：15所示或与SEQ ID NO：15具有3、2或1个氨基酸差异，所述HCDR2的氨基酸序列如SEQ ID NO：16所示或与SEQ ID NO：16具有3、2或1个氨基酸差异，所述HCDR3的氨基酸序列如SEQ ID NO：17所示或与SEQ ID NO：17具有3、2或1个氨基酸差异，所述重链框架区包含选自以下一个或更多个回复突变：79F、 82A T、91S和76S；和/或；
    轻链可变区，所述轻链可变区包含：轻链LCDR1、LCDR2、LCDR3以及轻链框架区，其中，所述LCDR1的氨基酸序列如SEQ ID NO：18所示或与SEQ ID NO：18具有3、2或1个氨基酸差异，所述LCDR2的氨基酸序列如SEQ ID NO：19所示或与SEQ ID NO：19具有3、2或1个氨基酸差异，所述LCDR3的氨基酸序列如SEQ ID NO：20或与SEQ ID NO：20序列具有3、2或1个氨基酸差异，所述轻链框架区包含选自以下一个或更多个回复突变：58I、68R和85T；和
    (f)重链可变区，所述重链可变区包含：重链HCDR1、HCDR2、HCDR3以及重链框架区，其中，所述HCDR1的氨基酸序列如SEQ ID NO：15所示或与SEQ ID NO：15具有3、2或1个氨基酸差异，所述HCDR2的氨基酸序列如SEQ ID NO：21所示或与SEQ ID NO：21具有3、2或1个氨基酸差异，所述HCDR3的氨基酸序列如SEQ ID NO：17所示或与SEQ ID NO：17具有3、2或1个氨基酸差异，所述重链框架区包含选自以下一个或更多个回复突变：48I、77T和 82A T；和/或
    轻链可变区，所述轻链可变区包含：轻链LCDR1、LCDR2、LCDR3以及轻链框架区，其中，所述LCDR1的氨基酸序列如SEQ ID NO：22所示或与SEQ ID NO：22具有3、2或1个氨基酸差异，所述LCDR2的氨基酸序列如SEQ ID NO：19所示或与SEQ ID NO：19具有3、2或1个氨基酸差异，所述LCDR3的氨基酸序列如SEQ ID NO：23所示或与SEQ ID NO：23具有3、2或1个氨基酸差异，所述轻链框架区包含选自以下一个或更多个回复突变：4L、9A、22S、58I、60A和68R；
    其中，所述回复突变的位点根据Kabat编号规则编号。
  5. 根据权利要求4所述的抗CD38抗体或其抗原结合片段，其中所述的人源化抗体包含：序列如SEQ ID NO：24、32或37所示的重链可变区或其变体，所述变体在序列如SEQ ID NO：24、32或37所示的重链可变区中的框架区上具有1-10个氨基酸突变。
  6. 根据权利要求5所述的抗CD38抗体或其抗原结合片段，其中所述的变体 选自以下(g)至(i)中的任一项：
    (g)在序列如SEQ ID NO：24所示的重链可变区中的框架区上具有选自以下一个或更多个回复突变：2F、38K、44S、48I、67A、66K、69L、71V和73Q；
    (h)在序列如SEQ ID NO：32所示的重链可变区中的框架区上具有选自以下一个或更多个回复突变：79F和91S；和
    (i)在序列如SEQ ID NO：37所示的重链可变区中的框架区上具有48I回复突变；
    优选地，其中所述的人源化抗体包含：
    序列如SEQ ID NO：26、27、28、29、34或39所示的重链可变区、或
    序列与SEQ ID NO：26、27、28、29、34或39具有至少95％序列同一性的重链可变区。
  7. 根据权利要求4所述的抗CD38抗体或其抗原结合片段，其中所述的人源化抗体包含序列如SEQ ID NO：25、33或38所示的轻链可变区或其变体，所述的变体在序列如SEQ ID NO：25、33或38所示的轻链可变区中的框架区上具有1-10个氨基酸突变。
  8. 根据权利要求7所述的抗CD38抗体或其抗原结合片段，其中所述的变体选自以下(j)至(l)任一项：
    (j)在序列如SEQ ID NO：25所示的轻链可变区中的框架区上具有选自以下一个或更多个回复突变：2F、43S、49K和87F；
    (k)在序列如SEQ ID NO：33所示的轻链可变区中的框架区上具有选自以下一个或更多个回复突变：58I、68R和85T；
    (l)在序列如SEQ ID NO：38所示的轻链可变区中的框架区上具有选自以下一个或更多个回复突变：4L、9A、22S、58I、60A和68R；
    优选地，其中所述的人源化抗体包含：
    序列如SEQ ID NO：30、31、35、36、40、41或42所示的轻链可变区、或序列与SEQ ID NO：30、31、35、36、40、41或42具有至少95％序列同一性的轻链可变区。
  9. 根据权利要求1所述的抗CD38抗体或其抗原结合片段，其包含：
    (m)序列如SEQ ID NO：24、26、27、28或29所示的重链可变区，和
    序列如SEQ ID NO：25、30或31所示的轻链可变区；
    (n)序列如SEQ ID NO：32或34所示的重链可变区，和序列如SEQ ID NO：33、35或36所示的轻链可变区；或
    (o)序列如SEQ ID NO：37或39所示的重链可变区，和序列如SEQ ID NO： 38、40、41或42所示的轻链可变区；
    优选地，所述人源化抗体包含：
    (p)序列如SEQ ID NO：26所示的重链可变区和序列如SEQ ID NO：30所示的轻链可变区；或
    (q)序列如SEQ ID NO：32所示的重链可变区和序列如SEQ ID NO：33所示的轻链可变区；或
    (r)序列如SEQ ID NO：37所示的重链可变区和序列如SEQ ID NO：38所示的轻链可变区。
  10. 根据权利要求1至9中任一项所述的抗CD38抗体或其抗原结合片段，其中，所述的抗体包含恒定区；
    优选地，所述抗体为嵌合抗体或人源化抗体，其重链恒定区来源于人抗体IgG1、IgG2、IgG3或IgG4，或IgG1、IgG2、IgG3或IgG4的常规变体，轻链恒定区来源于人抗体κ、λ链，或κ、λ链的常规变体；
    更优选地，所述重链恒定区的氨基酸序列如SEQ ID NO：43或44所示或与SEQ ID NO：43或44具有至少95％序列同一性，
    更优选地，所述轻链恒定区的氨基酸序列如SEQ ID NO：45所示或与SEQ ID NO：45具有至少95％序列同一性。
  11. 根据权利要求10所述的抗CD38抗体或其抗原结合片段，其包含：
    氨基酸序列如SEQ ID NO：46、48、49、51、52或54所示或与SEQ ID NO：46、48、49、51、52或54具有至少85％序列同一性的重链；和/或
    氨基酸序列如SEQ ID NO：47、50或53所示或与SEQ ID NO：47、50或53具有至少85％序列同一性的轻链。
  12. 根据权利要求11所述的抗CD38抗体或其抗原结合片段，其中所述抗体包含：
    序列如SEQ ID NO：46所示的重链和序列如SEQ ID NO：47所示的轻链；
    序列如SEQ ID NO：48所示的重链和序列如SEQ ID NO：47所示的轻链；
    序列如SEQ ID NO：49所示的重链和序列如SEQ ID NO：50所示的轻链；
    序列如SEQ ID NO：51所示的重链和序列如SEQ ID NO：50所示的轻链；
    序列如SEQ ID NO：52所示的重链和序列如SEQ ID NO：53所示的轻链；或
    序列如SEQ ID NO：54所示的重链和序列如SEQ ID NO：53所示的轻链。
  13. 一种分离的单克隆抗体或其抗原结合片段，其与权利要求1至12中任一项所述的抗CD38抗体或其抗原结合片段竞争性结合人CD38。
  14. 一种药物组合物，其含有：
    治疗有效量的权利要求1至13中任一项所述的抗CD38抗体或其抗原结合片段，以及
    一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
  15. 一种分离的核酸分子，其编码权利要求1至13中任一项所述的抗CD38抗体或其抗原结合片段。
  16. 一种载体，其包含权利要求15所述的核酸分子。
  17. 一种宿主细胞，其包含权利要求16所述的载体；优选地，所述宿主细胞选自原核细胞和真核细胞，优选为真核细胞，更优选哺乳动物细胞。
  18. 一种制备权利要求1至13中任一项所述抗CD38抗体或其抗原结合片段的方法，所述方法包括步骤：
    培养权利要求17所述的宿主细胞，
    回收所述抗CD38抗体或其抗原结合片段；
    任选地，对所述抗CD38抗体或其抗原结合片段进行纯化。
  19. 一种用于检测或测定人CD38的方法，所述方法包括：
    使权利要求1至13中任一项所述的抗CD38抗体或其抗原结合片段接触待测样本；
    确定所述待测样本中人CD38的存在或水平。
  20. 一种用于检测或测定人CD38的试剂，所述试剂包含：
    权利要求1至13中任一项所述的抗CD38抗体或其抗原结合片段；或
    权利要求16所述的药物组合物。
  21. 选自以下(m)至(o)任一项的物质在制备治疗或预防疾病或病症的药物中的用途：
    (m)权利要求1至13中任一项所述的抗CD38抗体或其抗原结合片段，
    (n)权利要求14所述的药物组合物，和
    (o)权利要求15所述的核酸分子；
    优选地，所述疾病或病症为肿瘤或免疫性疾病。
  22. 根据权利要求21所述的用途，其中所述疾病或病症为CD38阳性疾病或病症。
  23. 根据权利要求21或22所述的用途，其中：
    所述疾病或病症为肿瘤或免疫性疾病，
    所述免疫性疾病选自：类风湿性关节炎、牛皮癣、强直性脊柱炎、关节银屑病、皮炎、系统性硬皮病及硬化症、炎症性肠病、克罗恩氏病、溃疡性结肠炎、呼吸窘迫综合征、脑膜炎、脑炎、胃炎、葡萄膜炎、肾小球肾炎、湿疹、哮喘、动脉硬化、白细胞粘附缺陷病、Raynaud症候群、Sjogren症候群、青少年糖尿病、Reiter病、Behcet病、免疫复合物性肾炎、IgA肾病、IgM多发性神经病、免疫介导的血小板减少症状、溶血性贫血、重症肌无力、狼疮性肾炎、系统性红斑狼疮、风湿关节炎、异位性皮炎、天疱疮、Graves病、桥本氏甲状腺炎、Wegener肉芽肿、Omenn症候群、慢性肾功能衰竭、急性传染性单核细胞增多征、多发性硬化症、HIV和疱疹病毒相关的疾病、严重急性呼吸综合征和脉络视网膜炎、移植物对抗宿主疾病、以及病毒感染引起的免疫性疾病；
    优选地，所述免疫性疾病选自：类风湿性关节炎、系统性红斑狼疮、哮喘、炎症性肠病、多发性硬化症、克罗恩氏病、胃炎、桥本氏甲状腺炎、强直性脊柱炎、移植物对抗宿主疾病、免疫介导的血小板减少症状；
    优选地，所述免疫介导的血小板减少症状是急性特发性血小板减少性紫癜或慢性特发性血小板减少性紫癜；
    更优选地，所述免疫性疾病为类风湿性关节炎；
    所述肿瘤选自：白血病、B细胞淋巴瘤、T细胞淋巴瘤、NK细胞淋巴瘤、浆细胞恶性瘤和骨髓瘤；
    优选地，所述B细胞淋巴瘤选自：成熟B细胞肿瘤、前体B细胞成淋巴细胞白血病/淋巴瘤、B细胞非霍奇金淋巴瘤、B细胞霍奇金淋巴瘤；
    更优选地，所述肿瘤选自：急性淋巴细胞白血病、急性成淋巴细胞白血病、急性早幼粒细胞白血病、慢性淋巴细胞白血病、急性或慢性骨髓型白血病、多发性骨髓瘤、前髓细胞肿瘤、轻链淀粉样变性、B细胞慢性淋巴细胞性白血病、小淋巴细胞性白血病、B细胞急性淋巴细胞性白血病、B细胞前淋巴细胞白血病、淋巴浆细胞样淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、皮肤滤泡中心淋巴瘤、边缘区B细胞淋巴瘤、毛细胞白血病、弥漫性大B细胞淋巴瘤、伯基特淋巴瘤、浆细胞瘤、浆细胞骨髓瘤、浆细胞白血病、移植后淋巴增生性疾病、Waldenstrom巨球蛋白血症、浆细胞白血病和间变性大细胞淋巴瘤、多毛细胞淋巴瘤；
    更优选地，所述肿瘤是多发性骨髓瘤。
PCT/CN2019/105119 2018-09-11 2019-09-10 抗cd38抗体、其抗原结合片段及医药用途 Ceased WO2020052546A1 (zh)

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CN113912727A (zh) * 2021-11-16 2022-01-11 福州迈新生物技术开发有限公司 抗cd38蛋白单克隆抗体、细胞系及其制备方法和应用
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