WO2022095698A1 - 抗人cd38抗体及其制备方法和用途 - Google Patents

抗人cd38抗体及其制备方法和用途 Download PDF

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WO2022095698A1
WO2022095698A1 PCT/CN2021/124806 CN2021124806W WO2022095698A1 WO 2022095698 A1 WO2022095698 A1 WO 2022095698A1 CN 2021124806 W CN2021124806 W CN 2021124806W WO 2022095698 A1 WO2022095698 A1 WO 2022095698A1
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antibody
sequence
seq
amino acid
antigen
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PCT/CN2021/124806
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French (fr)
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朱玲巧
党尉
张成海
袁玉菁
郭锦林
吴易潘
邹秋玲
李致科
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上海麦济生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of antibodies, and more particularly, the present invention relates to an antibody against human CD38 and a preparation method and application thereof.
  • CD38 is a 46kDa type II transmembrane glycoprotein with an extracellular segment consisting of 258 amino acids.
  • the functions of CD38 include receptor-mediated adhesion and signaling and bifunctional in vitro enzymatic activity to promote intracellular calcium mobilization. Under normal conditions, CD38 is expressed at low levels on lymphoid and myeloid cells, but in multiple myeloma, malignant tumor cells express CD38 specifically, making CD38 an ideal myeloma target.
  • Multiple myeloma is a type of malignant plasma cell disease that occurs in B lymphocytes. Usually, myeloma cells proliferate in the bone marrow and in the spongy soft tissue of the bone, causing osteolytic bone destruction.
  • the incidence of multiple myeloma in China has surpassed that of acute leukemia, and it is the second most common hematological malignancy after non-Hodgkin's lymphoma.
  • the number of patients in China is about 80,000 to 100,000. With the arrival of an aging society and the deterioration of the environment and other factors, it is found that the incidence rate of multiple myeloma is increasing.
  • CD38 monoclonal antibody kills tumor cells mainly through Fc-dependent immune effector mechanisms, including complement-mediated cytotoxicity (CDC), antibody-mediated cytotoxicity (ADCC), antibody-mediated phagocytosis ( ADCP), and through apoptosis.
  • CD38 antibodies have immunomodulatory effects: they regulate myeloma cells by reducing immunomodulatory effects such as CD38+ immunosuppression, regulating cells, and promoting T cell expansion and activity.
  • antibody drugs targeting CD38 such as Johnson & Johnson's Daratumumab and Sanofi's Isatuximab, are already on the market, and Tianjing Bio's MOR202 is in clinical trials.
  • Antibodies targeting CD38 have very significant efficacy advantages in the treatment of multiple myeloma compared with current drugs and macromolecular and small molecule drugs under development. good competitive advantage.
  • the main treatment methods for myeloma in my country are stem cell transplantation and combination regimens such as Janssen's Velcade (bortezomib) or Celgene's REVLIMID (lenalidomide).
  • the FDA has approved a number of new drug molecules for the treatment of multiple myeloma. With the continuous emergence of new therapeutic drugs, the efficacy has been improved and the prognosis has been improved.
  • the median survival time is 7-8 years, and multiple The transformation of myeloma into a chronic disease has objectively driven the growth of the multiple myeloma treatment market.
  • the arrival of CD38 monoclonal antibody provides a new option for patients with multiple myeloma, especially relapsed and refractory patients. It is expected that more breakthroughs will be made in the field of CD38 antibody research to benefit more patients.
  • the inventors of the present invention have conducted a large number of experiments and obtained a group of monoclonal antibodies that can specifically bind to CD38 on the cell surface, and these antibodies can also bind to CD38 of cynomolgus monkeys.
  • a series of anti-human CD38 antibodies with different affinities of high, medium and low were obtained, which were able to kill tumor cells through an Fc-dependent immune effector mechanism.
  • the application provides an antibody or antigen-binding portion thereof that specifically binds to human CD38, comprising a heavy chain variable region comprising a HCDR3 sequence, optionally further comprising HCDR1 and/or or HCDR2 sequence.
  • the HCDR3 sequence described above comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45 and 48 .
  • the HCDR2 sequence described above comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44 and 47 .
  • the HCDR1 sequence described above comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43 and 46 .
  • the above-described heavy chain variable region comprises an amino acid sequence having at least 80% homology to an amino acid sequence selected from the group consisting of SEQ ID Nos: 97, 99, 101, 103, 105, 107, 109 and 111, or The heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 97, 99, 101, 103, 105, 107, 109 and 111.
  • the antibody or antigen-binding portion thereof that specifically binds human CD38 further comprises a light chain variable region, wherein the light chain variable region comprises an LCDR1, LCDR2 and/or LCDR3 sequence.
  • the LCDR1 sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91 and 94 amino acid sequence.
  • the LCDR1 sequence further comprises an amino acid sequence selected from the group consisting of SEQ ID NO:115.
  • the LCDR2 sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92 and 95 amino acid sequence.
  • the LCDR2 sequence further comprises an amino acid sequence selected from the group consisting of SEQ ID NO:116.
  • the LCDR3 sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93 and 96 amino acid sequence.
  • the LCDR3 sequence further comprises an amino acid sequence selected from the group consisting of SEQ ID NO:117.
  • the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 98, 100, 102, 104, 106, 108, 110 and 112.
  • the antigen binding moiety described above is selected from the group consisting of Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments, Fd fragments and single domain antibodies.
  • the antibody that specifically binds to human CD38 according to the first aspect is a murine monoclonal antibody.
  • the antibody that specifically binds human CD38 of the first aspect is a humanized antibody.
  • the present application provides an expression vector, the expression vector contains a nucleotide molecule that can encode the amino acid sequence as described above.
  • the expression vector is pTT5, pUC57, pDR1, pcDNA3.1(+), pDHFF, or pCHO 1.0, and the like.
  • the present application provides a host cell containing the expression vector as described above.
  • the host cell is HEK293, COS, CHO, NSO, sf9, sf21, DH5 ⁇ , BL21(DE3), or TG1, and the like.
  • the present application provides a method for preparing the antibody or antigen-binding portion thereof that specifically binds to human CD38 according to the first aspect, the method comprising the following steps:
  • the present application provides a pharmaceutical composition, the composition comprising the anti-human CD38 antibody or its antigen-binding portion according to the first aspect and a pharmaceutically acceptable carrier.
  • the composition is used to treat human CD38-related diseases.
  • the CD38-related disease includes multiple myeloma, non-Hodgkin's lymphoma, AL amyloidosis, T-cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, acute lymphoblastic Cell leukemia, plasma cell leukemia, acute myeloid leukemia, non-small cell lung cancer, pancreatic cancer, colorectal cancer and triple negative breast cancer, etc.
  • the present application provides methods of preventing or treating human CD38-related diseases, comprising administering to an individual in need thereof the antibody or antigen-binding portion thereof of the first aspect, or the pharmaceutical composition of the fifth aspect.
  • the anti-human CD38 antibody or its antigen-binding portion of the present invention can specifically bind to human CD38, and has high, medium and low affinity anti-human CD38 antibodies, which can kill tumor cells through an Fc-dependent immune effector mechanism.
  • the anti-human CD38 antibody or antigen-binding portion thereof of the present invention can be used to prevent or treat CD38-related diseases, such as multiple myeloma, non-Hodgkin's lymphoma, AL amyloidosis, T-cell lymphoma and diffuse lymphoma B cell lymphoma, etc.
  • FIG. 1 shows the experimental results of humanized CD38 monoclonal antibody binding to Daudi cells
  • Figure 2 shows the experimental results of humanized CD38 antibody-induced apoptosis of Daudi lymphoma cells
  • Figure 3 shows the experimental results of anti-Fc cross-linking of humanized CD38 antibody to induce apoptosis of Daudi lymphoma cells
  • the present application provides novel anti-CD38 antibodies or antigen-binding portions thereof that specifically bind to human CD38.
  • the antibody or antigen-binding portion thereof of the present application has high, medium, and low affinity binding activity to CD38, while binding to cynomolgus monkey CD38.
  • the present application also provides amino acids of the antibody or antigen-binding fragment thereof, a carrier comprising the amino acid, a host cell comprising the amino acid or the carrier, a method for preparing and purifying the antibody, and the medical treatment of the antibody or antigen-binding fragment thereof and biological applications, such as the prevention or treatment of CD38-related diseases or disorders.
  • antibody refers to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (eg, IgM ).
  • Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH).
  • the heavy chain constant region comprises three domains, namely CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated as CL).
  • the light chain constant region contains one domain (CL1).
  • the VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • antigen-binding portion of an antibody refers to a portion or segment of an intact antibody molecule responsible for binding an antigen.
  • the antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both.
  • Antigen-binding fragments of antibodies can be prepared from whole antibody molecules using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques, among others.
  • Non-limiting examples of antigen-binding moieties include: Fab fragments; F(ab')2 fragments; Fd fragments; Fv fragments; single-chain Fv (scFv) molecules; The smallest recognition unit composed of residues (eg, isolated CDRs).
  • the term "antigen binding portion” also includes other engineered molecules such as diabodies, tribodies, tetrabodies, minibodies, and the like.
  • VH heavy chain variable region
  • VL light chain variable region
  • CDRs complementarity determining regions
  • the CDR region sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition.
  • the CDR sequences are defined using Kabat.
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2 and HCDR3, respectively;
  • CDR1, CDR2 and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2 and LCDR3, respectively.
  • variable region sequence of a given antibody the CDR region sequences in the variable region sequence can be analyzed in various ways, for example, can be determined using the online software Abysis (http://www.abysis.org/).
  • binding refers to a non-random binding reaction between two molecules, eg, binding of an antibody to an epitope, eg, an antibody with an affinity that is at least two-fold greater than its affinity for a non-specific antigen. Affinity The ability to bind to a specific antigen. It will be appreciated, however, that antibodies are capable of specifically binding to two or more antigens whose sequences are related. For example, the antibodies of the invention can specifically bind to CD38 in humans and non-humans (eg, mice or non-human primates).
  • Monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in a small number of individuals.
  • Monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while The remainder of the chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, provided they exhibit the desired (See, U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
  • homology is defined as the percentage of identical residues in a variant of an amino acid or nucleotide sequence after sequence alignment and introduction of gaps, if desired, to a maximum percent homology. Methods and computer programs for alignment are well known in the art.
  • at least 80% homology refers to any value between 80% and 100% homology, such as 85%, 90%, 95%, 99%, and the like.
  • CD38-related disease includes multiple myeloma, non-Hodgkin's lymphoma, AL amyloidosis, T-cell lymphoma, and diffuse large B-cell lymphoma, among others.
  • the application provides an antibody or antigen-binding portion thereof that specifically binds CD38, comprising a heavy chain variable region and/or a light chain variable region.
  • the CDR, heavy and light chain variable region amino acid sequences suitable for use with the antibodies disclosed herein are exemplified in Tables 1-3.
  • the HCDR3 is selected from the amino acid sequences set forth in SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45 and 48 . In another specific embodiment, the HCDR3 is selected from the amino acid sequences set forth in SEQ ID NOs: 3, 9, 18, 21, 27, 33, 36 and 45. In a preferred embodiment, the HCDR3 is selected from the amino acid sequences shown in 9, 18, 21, 27, 33, 36 and 45.
  • the HCDR2 is selected from the amino acid sequences set forth in SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44 and 47 . In another specific embodiment, the HCDR2 is selected from the amino acid sequences set forth in SEQ ID NOs: 2, 8, 17, 20, 26, 32, 35 and 44. In a preferred embodiment, the HCDR2 is selected from the amino acid sequences shown in 8, 17, 20, 26, 32, 35 and 44.
  • HCDR1 is selected from the amino acid sequences set forth in SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43 and 46 . In another specific embodiment, HCDR1 is selected from the amino acid sequences set forth in SEQ ID NOs: 1, 7, 16, 19, 25, 31, 34 and 43. In a preferred embodiment, HCDR1 is selected from the amino acid sequences shown in 7, 16, 19, 25, 31, 34 and 43.
  • the antibody heavy chain variable regions disclosed herein comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 97, 99, 101, 103, 105, 107, 109 and 111.
  • the heavy chain variable region consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 97, 99, 101, 103, 105, 107, 109 and 111.
  • the antibody or antigen-binding portion thereof disclosed herein may further comprise a light chain variable region in addition to the heavy chain variable region.
  • the CDR3 (LCDR3) of the light chain variable region is selected from SEQ ID NOs: 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, The amino acid sequences shown at 90, 93, 96 and 117.
  • LCDR3 is selected from the amino acid sequences set forth in SEQ ID NOs: 51, 57, 66, 69, 75, 81, 84 and 117.
  • LCDR3 is selected from the amino acid sequences set forth in SEQ ID NOs: 57, 66, 69, 75, 81, 84 and 117.
  • LCDR2 is selected from the amino acids set forth in SEQ ID NOs: 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95 and 116 sequence, or selected from the amino acid sequences shown in SEQ ID NOs: 50, 65, 68, 74, 80, 83, 92 and 116. In a preferred embodiment, LCDR2 is selected from the amino acid sequences set forth in SEQ ID NOs: 65, 68, 74, 80, 83, 92 and 116.
  • LCDR1 is selected from the amino acid sequences of SEQ ID NOs: 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94 and 115, Alternatively selected from the amino acid sequences of SEQ ID NOs: 55, 64, 67, 73, 79, 82, 91 and 115. In a preferred embodiment, LCDR1 is selected from the amino acid sequences set forth in SEQ ID NOs: 55, 64, 67, 73, 79, 82 and 91.
  • the antibody light chain variable regions disclosed herein comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 98, 100, 102, 104, 106, 108, 110 and 112.
  • the light chain variable region consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 98, 100, 102, 104, 106, 108, 110 and 112.
  • the heavy chain or heavy chain variable region, light chain or light chain variable region of the antibodies disclosed herein can be substituted, deleted or added at least one of the specific amino acid sequences listed above for each corresponding to amino acid, and the resulting variant still retains human CD38 binding activity.
  • the number of amino acid substitutions, deletions or additions described above is 1-30, preferably 1-20, more preferably 1-10.
  • the sequence variant differs from the original amino acid sequence by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and/or additions.
  • the sequence variant differs from the original amino acid sequence by about 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions.
  • the amino acid substitutions are conservative substitutions.
  • the antibody disclosed herein is antibody 57H, 105H, 116H, or 145H, wherein the heavy chain variable region sequence of antibody 57H is shown in SEQ ID NO: 99, and the light chain variable region sequence is shown in SEQ ID NO: 99 NO: 100, wherein LCDR2 is preferably shown in SEQ ID NO: 116, and the remaining CDR sequences are the same as those of murine antibody 57; the heavy chain variable region sequence of antibody 105H is shown in SEQ ID NO: 107, and the light chain can be The variable region sequence is shown in SEQ ID NO: 108, wherein the CDR sequence is the same as that of the murine antibody 105; the heavy chain variable region sequence of antibody 116H is shown in SEQ ID NO: 109, and the light chain variable region sequence is shown in SEQ ID NO.
  • the antibodies disclosed herein are monoclonal antibodies. In specific embodiments, the antibodies disclosed herein are humanized antibodies.
  • the antibodies disclosed herein, or antigen-binding portions thereof are capable of specifically binding human CD38.
  • the antibody or antigen-binding portion thereof specifically binds human CD38 or cynomolgus CD38.
  • the antibody or antigen-binding portion thereof specifically binds human CD38.
  • any suitable expression vector can be used in the present application.
  • the expression vector can be one of pTT5, pUC57, pDR1, pcDNA3.1(+), pDHFF and pCHO 1.0. Fusion DNA sequences ligated with appropriate transcriptional and translational regulatory sequences may be included in the expression vector.
  • useful host cells are cells containing the above-mentioned expression vectors, which can be eukaryotic cells, such as mammalian or insect host cell culture systems, which can be used for the expression of the antibodies or antigen-binding portions thereof of the present application.
  • eukaryotic cells such as mammalian or insect host cell culture systems
  • the host cell may also be a prokaryotic cell containing the above-mentioned expression vector, such as DH5 ⁇ , BL21(DE3) or TG1, etc.
  • the method for preparing an anti-human CD38 monoclonal antibody disclosed herein comprises: culturing a host cell under suitable expression conditions, thereby expressing an anti-human CD38 monoclonal antibody; isolating and purifying the expressed anti-human CD38 monoclonal antibody Antibody.
  • the recombinant protein can be purified to a substantially homogeneous material, eg, as a single band on SDS-PAGE.
  • the anti-human CD38 antibody disclosed herein can be separated and purified by the method of affinity chromatography. According to the characteristics of the used affinity column, conventional methods such as high salt buffer, changing pH, etc. can be used. Methods The anti-human CD38 antibody bound to the affinity column was eluted.
  • the humanized anti-human CD38 monoclonal antibodies disclosed herein are obtained by immunizing Balb/c mice with laboratory-prepared human CD38 antigen, and comparing the titers of multiple immunized mice The mouse spleen cells were fused with hybridoma cells, and the hybridoma cell lines with different affinities were screened out. More specifically, through a large number of experiments, the inventors of the present application first expressed human CD38 antigen respectively, and on this basis, mixed immunized mice with different adjuvants and human CD38 antigen, and then further immunized the spleen cells of the above-mentioned mice with hybridization.
  • the tumor cell line sp2/0 was fused, and the fused hybridoma was screened with human CD38 antigen to select a positive cell line, and the target cell line was obtained after verifying its binding to human CD38 and Daudi cells.
  • the target molecule was humanized
  • the light chain and heavy chain genes were simultaneously cloned into the eukaryotic expression vector pCHO1.0.
  • the above-mentioned expression vector was transfected into CHO cells by liposome method, and then positive cell clones were screened with puromycin and methotrexate, and the high-expression clones obtained by screening were expanded and cultured with serum-free medium, and protein A affinity column was used to expand the culture. Isolation or purification of humanized anti-human CD38 antigen monoclonal antibodies.
  • the parental antibody of murine origin can be further altered using routine techniques in the art, such as PCR mutagenesis, to generate chimeric or humanized or other variant forms of the antibody.
  • the parent antibody of the present application can be mutated, for example, within the complementarity determining region (CDR) domains to generate variant antibodies, which can be screened for the presence of properties of interest, such as binding affinity (lower KD), IC50, specificity, preferential binding, etc. Wait.
  • properties of interest in the variant antibody is an improvement over the property in the parent antibody.
  • Amino acid substitution variant antibodies are preferred, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues of the parent antibody molecule are removed and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis are one or more CDR regions, but framework region (FR) changes are also contemplated. Conservative amino acid substitutions are preferred, but non-conservative amino acid changes can also be introduced and the resulting variant antibodies screened for properties of interest.
  • compositions comprising an antibody or antigen-binding portion thereof disclosed herein and a pharmaceutically acceptable carrier.
  • the above-mentioned anti-human CD38 monoclonal antibody disclosed herein can be formulated into a pharmaceutical preparation together with a pharmaceutically acceptable carrier, so that the therapeutic effect can be more stably exerted.
  • these formulations can preserve the conformational integrity of the amino acid core sequence of the anti-human CD38 monoclonal antibodies disclosed herein, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamidation, or oxidation) .
  • liquid formulations are generally stable at 2°C-8°C for at least one year.
  • lyophilized formulations are stable at 30°C for at least six months.
  • the anti-human CD38 antibody monoclonal antibody preparation can be a suspension, water injection, freeze-dried preparation commonly used in the pharmaceutical field, preferably a water injection or freeze-dried preparation, for the anti-human CD38 monoclonal antibody disclosed herein
  • pharmaceutically acceptable excipients include, but are not limited to, surfactants, solution stabilizers, isotonicity regulators and buffers or combinations thereof.
  • surfactants include, but are not limited to: non-ionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80), poloxamers (such as poloxamer 188), Triton, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, tetradecyl, linoleyl or octadecyl sarcosine, Pluronics, MONAQUATTM, etc., the amount of which should be added should make the anti-human CD38 monoclonal antibody The tendency to granulate is minimal.
  • non-ionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80), poloxamers (such as poloxamer 188), Triton, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, tetradecyl, linoleyl or octadec
  • solution stabilizers include, but are not limited to, one or a combination of the following: sugars, such as reducing sugars and non-reducing sugars; amino acids, such as monosodium glutamate or histidine; alcohols such as trihydric alcohols, higher sugar alcohols, propylene glycol, polyethylene glycol, and the like.
  • sugars such as reducing sugars and non-reducing sugars
  • amino acids such as monosodium glutamate or histidine
  • alcohols such as trihydric alcohols, higher sugar alcohols, propylene glycol, polyethylene glycol, and the like.
  • the amount of solution stabilizer added should be such that the resulting formulation remains stable for a period of time considered to be stable by those skilled in the art.
  • Isotonicity modifiers include, but are not limited to, sodium chloride, mannitol, or combinations thereof.
  • Buffers include, but are not limited to, Tris, histidine buffer, phosphate buffer, or a combination thereof.
  • the present application also provides methods of preventing or treating CD38-related diseases, comprising administering to an individual an anti-human CD38 antibody, or a composition comprising an anti-human CD38 antibody.
  • the anti-human CD38 antibodies disclosed herein can effectively prevent and/or treat CD38-related diseases.
  • the CD38-related disease includes multiple myeloma, non-Hodgkin's lymphoma, AL amyloidosis, T-cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, acute lymphoblastic Cell leukemia, plasma cell leukemia, acute myeloid leukemia, non-small cell lung cancer, pancreatic cancer, colorectal cancer and triple negative breast cancer, etc.
  • the dosage varies depending on the age and weight of the patient, the nature and severity of the disease, and the route of administration. Reference can be made to animals. According to the results of the experiment and the comprehensive situation, the total dosage cannot exceed a certain range.
  • the dose and frequency of administration of the antibody or composition thereof may vary depending on the prevention or treatment of the disease.
  • a composition containing an antibody of the present application or a mixture thereof is administered to a patient who is not already in a disease state to increase patient resistance, and this amount is defined as a "prophylactically effective dose.”
  • the specific dosage again depends on the patient's health and systemic immunity. Relatively lower doses are usually administered at relatively infrequent intervals for longer periods of time.
  • Those of ordinary skill in the art can easily grasp the specific dosage and frequency according to actual needs.
  • the human CD38 antigen sequence was purchased from Yiqiao Shenzhou (Cat. No.: HG10818-M).
  • the 43-300th amino acid fragment of the N-terminal of human CD38 was spliced with hFc fragment and Flag tag (DYKDDDDK) by PCR method.
  • the hFc fragment and Flag tag were located at the C-terminus, and constructed into pTT5 expression vector (stored in the laboratory) to obtain For pTT5 (hCD38-ECD-hFc) and pTT5 (hCD38-ECD-Flag), clones with completely correct sequencing results were selected for plasmid extraction and transfection.
  • the cynomolgus monkey CD38 sequence was purchased from Yiqiao Shenzhou (Cat. No.: CG90050-G).
  • the N-terminal 44-301 amino acid fragment was spliced with the Flag tag (DYKDDDDK) by PCR method, and the Flag tag was located at the C-terminal, and constructed into the pTT5 expression vector (stored in the laboratory) to obtain pTT5 (cyno-CD38-ECD -Flag), select clones with completely correct sequencing results for plasmid extraction and transfection.
  • the amino acid sequence of Daratumumab comes from IMGT, and after codon optimization, the nucleotide sequence of the variable region of the heavy chain and light chain is synthesized in the whole gene.
  • the variable region of Daratumumab heavy chain was linked to the constant region of IgG1 by PCR method; the variable region of Daratumumab light chain was linked to the constant region of Kappa chain.
  • Plasmids were transfected into HEK293E cell line (laboratory preservation) by PEI method. After 5 days of culture in Freestyle 293 medium (purchased from Gibco) containing 3 mM valproic acid, protein A affinity chromatography (purchased from Pharmacia) or Flag affinity chromatography (purchased from Genscript) was used from the cell culture. The target protein was purified from the serum. The quantification of the protein was carried out by the Bicinchoninic acid (BCA) method, and the purified protein was used for the following further analysis and research. The purified protein was used for the following mouse immunization and further analysis and research.
  • BCA Bicinchoninic acid
  • mice 100 ⁇ g/mouse of hCD38-ECD-hFc antigen was diluted with normal saline to 75 ⁇ l, mixed with an equal volume of Freund’s complete adjuvant, and subjected to phacoemulsification to 4-5 weeks old Balb/c mice (purchased).
  • Subcutaneous multi-point injection was performed from Shanghai Lingchang Biotechnology Co., Ltd., animal production license number: SCXK (Shanghai) 2013-0018).
  • 50 ⁇ g/mouse protein was also diluted to 75 ⁇ l and mixed with an equal volume of incomplete Freund's adjuvant. After phacoemulsification was complete, the mice were injected subcutaneously at multiple points, and the immunization was repeated two weeks later.
  • mice with serum titer >10000 pulse immunization was performed one week after blood collection: 10 ⁇ g antigen/100 ⁇ l normal saline/mouse was injected into the tail vein.
  • ELISA plate was coated with hCD38-ECD-hFc antigen at a concentration of 1 ⁇ g/ml, 100 ⁇ l per well, and coated overnight at 4°C. Plates were washed twice with PBST (PBS containing 0.5% Tween-20) and patted dry. 200 ⁇ l of coating solution containing 1% BSA was added to each well for blocking, and the cells were blocked at room temperature for 4 hours, patted dry, and stored in a -20° C. refrigerator for later use. During the detection, 100 ⁇ l of mouse serum of different concentrations was added to each well of the ELISA plate, two duplicate wells were set, and the cells were incubated at room temperature for 1.5 hours.
  • Hybridoma sp2/0 cells (from the cell bank of the Type Culture Collection, Chinese Academy of Sciences, with accession number TCM-18) were cultured in a 37° C., 5% CO 2 incubator, and the medium was changed one day before fusion. Three days after the shock immunization of mice, mouse splenocytes were harvested for fusion. The fusion and screening methods are as follows: the mouse spleen is taken, and the spleen cells are counted after grinding and washing. Splenocytes and sp2/0 cells were mixed at a ratio of 10:1 and centrifuged at 1500 rpm for 7 minutes. Wash off the supernatant.
  • the supernatant was taken from an enzyme-labeled plate coated with hCD38-ECD-hFc antigen for ELISA detection.
  • the ELISA detection method was the same as that described in Example 2.
  • the positive hybridoma clones were selected and expanded in a 24-well plate, subcloned by limiting dilution method, and the hybridoma strains stably expressing the target antibody were obtained for seed conservation and library construction.
  • antibodies 1, 57, 71, 73, 77, 78, 105, 116 and 145 were selected to construct chimeric and humanized antibodies.
  • the chimeric antibody was constructed by intercepting the variable region of the heavy chain and the variable region of the light chain of the murine antibody, and using overlapping PCR to connect the light and heavy chain constant regions of human IgG1 respectively.
  • the amino acid sequences of the light chain variable region and the heavy chain variable region of the murine anti-human CD38 monoclonal antibody were analyzed according to Kabat's rule, and 3 CDRs and 4 FRs were determined. Taking antibody No. 105 as an example, by comparing the homology between NCBI IgBlast and human IgG germline sequence (Germline), IGHV1-46*01 was selected as the heavy chain CDR transplantation template, and the mouse-derived anti-human CD38 monoclonal antibody 105 was selected. The heavy chain CDR region of No. 1 was grafted into the IGHV1-46*01 framework region to construct a CDR-grafted antibody of the heavy chain.
  • IGKV1-16*01 was selected as the light chain CDR transplantation template, and the light chain CDR region of mouse anti-human CD38 monoclonal antibody No. 105 was transplanted into IGKV1-16
  • the framework region of *01 was constructed as a light chain CDR-grafted antibody.
  • some amino acid positions in the framework region were backmutated.
  • the amino acid sequence was Kabat coded, and the position of the site was indicated by the Kabat code.
  • the F at position 36 encoded by Kabat is reverted to L of murine origin
  • the A at position 43 is mutated to T
  • the P at position 44 is reverted to I of murine origin
  • the The S at position 46 is reverted to the R of mouse origin
  • the G at the 66th position is reverted to the R of mouse origin
  • the T of the 69th position is reverted to the S of mouse origin.
  • the M at position 48 encoded by Kabat is reverted to murine I
  • the V at position 67 is reverted to murine A
  • the M at position 69 is reverted to murine L
  • the 71st position is reverted to murine L.
  • variable region gene sequences were codon-optimized and synthesized by Cricetulus griseus according to the codon usage preference of Cricetulus griseus.
  • the synthetic humanized variable region sequence was linked to the human IgG1 constant region, and this antibody was defined as the humanized antibody of Antibody No. 105 (105-Humanization, 105H).
  • the remaining antibodies were similarly humanized using the same principles described above.
  • the amino acid sequence numbering of the variable region of the humanized antibody is shown in Table 3, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 113, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 114.
  • Transient expression vectors of humanized heavy chain and light chain were respectively constructed using pTT5 vector, and the combination of light and heavy chain was transiently transfected and expressed antibody using HEK293E system.
  • HEK293E cells were cultured in Free Style 293Expression Medium (purchased from Gibco), and the plasmid was transferred into cells by PEI transfection method for 5 days, and the cell supernatant was collected. After purification by Protein A, each humanized monoclonal antibody was obtained.
  • the humanized heavy chain variable region amino acid sequence of antibody No. 1 is shown in SEQ ID NO: 97; the humanized light chain variable region amino acid sequence is shown in SEQ ID NO: 98. After ligation with the human IgG1 constant region, the 1H antibody sequence was finally obtained.
  • the humanized heavy chain variable region amino acid sequence of antibody 57 is shown in SEQ ID NO: 99; the humanized light chain variable region amino acid sequence is shown in SEQ ID NO: 100. After ligation with the human IgG1 constant region, the antibody sequence of 57H was finally obtained.
  • the humanized heavy chain variable region amino acid sequence of antibody No. 71 is shown in SEQ ID NO: 101; the humanized light chain variable region amino acid sequence is shown in SEQ ID NO: 102. After ligation with the human IgG1 constant region, the antibody sequence of 71H was finally obtained.
  • the humanized heavy chain variable region amino acid sequence of antibody No. 73 is shown in SEQ ID NO: 103; the humanized light chain variable region amino acid sequence is shown in SEQ ID NO: 104. After ligation with the human IgG1 constant region, the antibody sequence of 73H was finally obtained.
  • the humanized heavy chain variable region amino acid sequence of antibody No. 78 is shown in SEQ ID NO: 105; the humanized light chain variable region amino acid sequence is shown in SEQ ID NO: 106. After ligation with the human IgG1 constant region, the antibody sequence of 78H was finally obtained.
  • the humanized heavy chain variable region amino acid sequence of antibody No. 105 is shown in SEQ ID NO: 107; the humanized light chain variable region amino acid sequence is shown in SEQ ID NO: 108. After ligation with the human IgG1 constant region, the antibody sequence of 105H was finally obtained.
  • the humanized heavy chain variable region amino acid sequence of antibody No. 116 is shown in SEQ ID NO: 109; the humanized light chain variable region amino acid sequence is shown in SEQ ID NO: 110. After ligation with the human IgG1 constant region, the antibody sequence of 116H was finally obtained.
  • the humanized heavy chain variable region amino acid sequence of antibody No. 145 is shown in SEQ ID NO: 111; the humanized light chain variable region amino acid sequence is shown in SEQ ID NO: 112. After ligation with the human IgG1 constant region, the antibody sequence of 145H was finally obtained.
  • Table 3 Amino acid sequences of exemplary humanized anti-human CD38 antibody heavy and light chain variable regions
  • Daudi cells (preserved in the laboratory) in good growth state were sampled and counted, and after centrifugation, they were resuspended in PBS supplemented with 1% FBS, and the density was adjusted to 2 ⁇ 10 7 .
  • the highest concentration of antibody samples was 10 ⁇ g/ml, and the samples were added after three-fold serial dilution (8 gradients in total).
  • 20ul/well (4 ⁇ 10 5 /well) of Daudi cells was added, and 30 ⁇ l of serially diluted antibody drug was added, and incubated at 4° C. for 30 minutes. Add 180 ⁇ l of 1% FBS PBS to wash, and centrifuge at 1200 rpm for 6 min.
  • Example 7 Humanized CD38 antibody induces apoptosis of Daudi lymphoma cells
  • Example 8 Anti-Fc cross-linking of humanized CD38 antibody induces apoptosis of Daudi lymphoma cells
  • the affinity of the expressed purified humanized antibody was tested by Biacore T200 (GE healthcare).
  • the specific experimental method is as follows: using the Protein-A CM5 sensor chip (GE healthcare), with FC1 (Flow cell 1) as the reference channel and FC2 (Flow cell 2) as the sample channel. Human antibody or control antibody were captured in the FC2 channel, respectively, and then different concentrations of hCD38-Flag or cyno-CD38-Flag were injected.
  • the cycling conditions were as follows: analytes were injected at 50 ⁇ l/min for 4 min in all channels of the FCs, the dissociation time was 20 min, 6M guanidine hydrochloride (Sinopharm Chemical Reagent Co., Ltd.) was injected at a rate of 10 ⁇ l/min for 30 s for surface regeneration, and then Biacore T200 was used for surface regeneration.
  • Evaluation Software Ver 1.0 calculates the difference between the signal of the captured antibody and the signal of the non-captured antibody and the affinity of the interaction. As shown in Table 5 and Table 6. The experimental results show that the preferred antibodies can bind to CD38 of human and cynomolgus monkeys with high, medium and low affinities.
  • the inventors of the present invention have conducted a large number of experiments and obtained a set of monoclonal antibodies that can specifically bind to CD38 on the cell surface. These antibodies can also bind to CD38 of cynomolgus monkeys. A series of anti-human CD38 antibodies with different affinities of high, medium and low were obtained, which were able to kill tumor cells through an Fc-dependent immune effector mechanism.

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Abstract

本发明公开了能够结合人CD38的抗体以及其制备方法和应用。本发明的抗体具有高、中和低不同亲和力的抗人CD38抗体，同时可以和食蟹猴的CD38结合，其能够通过Fc依赖性免疫效应机制杀死肿瘤细胞。可应用于治疗CD38相关疾病，例如多发性骨髓瘤、非霍奇金淋巴瘤、AL型淀粉样变性、T细胞淋巴瘤和弥漫性大B细胞淋巴癌。

Description

抗人CD38抗体及其制备方法和用途 技术领域
本发明涉及抗体领域，更具体地，本发明涉及抗人CD38的抗体及其制备方法和应用。
背景技术
CD38是一个46kDa的二型跨膜糖蛋白，胞外段由258个氨基酸组成，CD38的功能包括受体介导的粘附和信号传导及双功能体外酶活性促进胞内钙动员。在正常状态下，CD38低水平表达在淋系和髓系细胞上，但在多发性骨髓瘤中，恶性肿瘤细胞特异性高表达CD38，使CD38成为理想的骨髓瘤靶点。
多发性骨髓瘤是一类发生于B淋巴细胞的恶性浆细胞病。通常骨髓瘤细胞在骨髓内及骨骼海绵软组织内克隆性增生，引起溶骨性骨骼破坏，愈后不良多伴有贫血、肾衰竭和骨髓瘤细胞髓外浸润所导致的多种损害。中国多发性骨髓瘤的发病率已经超过急性白血病，是仅次于非霍奇金淋巴瘤之后居于第二位的血液系统恶性肿瘤。MM在中国每年新发病例约1.5～2万例，患者的中位生存期约4～5年，中国患病人数大约8-10万例。随着老龄化社会的到来和环境的恶化等多种因素的影响，发现多发性骨髓瘤呈发病比率上升。
研究发现CD38单克隆抗体主要通过Fc依赖性免疫效应机制杀死肿瘤细胞，包括补体介导的细胞毒作用(CDC)、抗体介导的细胞毒作用(ADCC)、抗体介导的细胞吞噬作用(ADCP)，以及通过细胞凋亡(apoptosis)。此外，CD38抗体具有免疫调节作用：通过减少CD38+免疫抑制、调节细胞及促进T细胞扩增和活性等免疫调节作用，对骨髓瘤细胞进行调控。
国际上针对CD38的抗体药物如强生的Daratumumab和赛诺菲的Isatuximab已上市，天境生物的MOR202在临床试验中。靶向CD38的抗体在多发性骨髓瘤治疗中相比于现行药物及在研的大分子、小分子药物均有非常显著的疗效优势，单药及联合用药均显示疗效优势，因此将能保持较好的竞争优势。
我国目前骨髓瘤的主要治疗方法是干细胞移植及杨森的万珂(硼替佐米)或Celgene的瑞复美(来那度胺)等联合用药方案治疗。近两年FDA批准了多个全新药物分子用于多发性骨髓瘤治疗，随着治疗新药的不断涌现，疗效得到提高、改善了预后，目前中位生存期为7-8年，正逐步将多发性骨髓瘤转为慢性病，客 观上也带动了多发性骨髓瘤治疗市场的增长。CD38单抗的到来，为多发性骨髓瘤患者，特别是复发和难治性患者提供了新的选择。期待CD38抗体领域的研究能取得更多突破，造福更多患者。
发明内容
本发明的发明人进行了大量试验，得到了一组可以特异性结合细胞表面CD38的单克隆抗体，这些抗体同时可以和食蟹猴的CD38结合。获得了一系列具有高、中和低不同亲和力的抗人CD38抗体，其能够通过Fc依赖性免疫效应机制杀死肿瘤细胞。
第一方面，本申请提供了一种特异性结合人CD38的抗体或其抗原结合部分，其包含重链可变区，所述重链可变区包含HCDR3序列，任选地还包含HCDR1和/或HCDR2序列。在一些实施方案中，上述HCDR3序列包含选自SEQ ID NOs:3，6，9，12，15，18，21，24，27，30，33，36，39，42，45和48的氨基酸序列。在一些实施方案中，上述HCDR2序列包含选自SEQ ID NOs:2，5，8，11，14，17，20，23，26，29，32，35，38，41，44和47的氨基酸序列。在一些实施方案中，上述HCDR1序列包含选自SEQ ID NOs:1，4，7，10，13，16，19，22，25，28，31，34，37，40，43和46的氨基酸序列。
在一些实施方案中，上述重链可变区包含与选自SEQ ID Nos：97，99，101，103，105，107，109和111的氨基酸序列具有至少80％同源性的氨基酸序列，或者所述重链可变区包含选自SEQ ID NOs:97，99，101，103，105，107，109和111的氨基酸序列。
在一些实施方案中，特异性结合人CD38的抗体或其抗原结合部分还包含轻链可变区，其中所述轻链可变区包含LCDR1、LCDR2和/或LCDR3序列。在某些实施方案中，所述LCDR1序列包含选自SEQ ID NOs:49，52，55，58，61，64，67，70，73，76，79，82，85，88，91和94的氨基酸序列。在一些实施方案中，LCDR1序列还包含选自SEQ ID NO:115的氨基酸序列。在某些实施方案中，所述LCDR2序列包含选自SEQ ID NOs:50，53，56，59，62，65，68，71，74，77，80，83，86，89，92和95的氨基酸序列。在一些实施方案中，LCDR2序列还包含选自SEQ ID NO:116的氨基酸序列。在某些实施方案中，所述LCDR3序列包含选自SEQ ID NOs:51，54，57，60，63，66，69，72，75，78，81，84， 87，90，93和96的氨基酸序列。在一些实施方案中，LCDR3序列还包含选自SEQ ID NO:117的氨基酸序列。
在一些实施方案中，上述轻链可变区包含与选自SEQ ID NOs:98，100，102，104，106，108，110和112的氨基酸序列具有至少80％同源性的氨基酸序列；或者所述轻链可变区包含选自SEQ ID NOs:98，100，102，104，106，108，110和112的氨基酸序列。
在可选的实施方案中，上述抗原结合部分选自Fab片段、Fab’片段、F(ab’)2片段、Fv片段、scFv片段、Fd片段和单域抗体。
在一些实施方案中，第一方面所述的特异性结合人CD38的抗体为鼠源单克隆抗体。
在一些实施方案中，第一方面所述的特异性结合人CD38的抗体为人源化抗体。
第二方面，本申请提供了一种表达载体，所述表达载体含有的核苷酸分子可编码如上所述的氨基酸序列。
在一些实施方案中，所述表达载体为pTT5、pUC57、pDR1、pcDNA3.1(+)、pDHFF或pCHO 1.0等。
第三方面，本申请提供了一种宿主细胞，所述宿主细胞含有如上所述的表达载体。在一些实施方案中，所述宿主细胞为HEK293、COS、CHO、NS0、sf9、sf21、DH5α、BL21(DE3)或TG1等。
第四方面，本申请提供了一种制备第一方面所述的特异性结合人CD38的抗体或其抗原结合部分的方法，所述方法包括以下步骤：
a)在使得第三方面所述的宿主细胞能够产生所述抗体或其抗原结合部分的表达条件下，培养所述的宿主细胞，从而表达所述抗体或其抗原结合部分；以及b)分离并纯化a)表达的所述抗体或其抗原结合部分。
第五方面，本申请提供了一种药物组合物，所述组合物包含第一方面所述的抗人CD38抗体或其抗原结合部分以及药学上可接受的载体。
在一些实施方案中，所述组合物用于治疗人CD38相关的疾病。
在一些实施方案中，所述CD38相关疾病包括多发性骨髓瘤、非霍奇金淋巴瘤、AL型淀粉样变性、T细胞淋巴瘤、弥漫性大B细胞淋巴癌、慢性淋巴细胞白血病、急性淋巴细胞白血病、浆细胞性白血病、急性髓性白血病、非小细胞肺癌、 胰腺癌、结直肠癌和三阴乳腺癌等。
在其他方面，本申请提供了预防或治疗人CD38相关疾病的方法，包括给予有需要的个体第一方面所述的抗体或其抗原结合部分、或第五方面所述的药物组合物。
本发明的抗人CD38抗体或其抗原结合部分能够特异性与人CD38结合，具有高、中和低不同亲和力的抗人CD38抗体，其能够通过Fc依赖性免疫效应机制杀死肿瘤细胞。本发明的抗人CD38抗体或其抗原结合部分可以用于预防或治疗CD38相关的疾病，例如多发性骨髓瘤、非霍奇金淋巴瘤、AL型淀粉样变性、T细胞淋巴瘤和弥漫性大B细胞淋巴癌等。
附图说明
图1为人源化CD38单克隆抗体与Daudi细胞结合的实验结果
图2为人源化CD38抗体诱导Daudi淋巴瘤细胞凋亡的实验结果
图3为人源化CD38抗体抗Fc交联诱导Daudi淋巴瘤细胞凋亡的实验结果
具体实施方式
本申请提供了特异性结合于人CD38的新的抗CD38抗体或其抗原结合部分。在优选实施方案中，本申请的抗体或其抗原结合部分具有高、中和低不同亲和力结合CD38的活性，同时可以结合食蟹猴CD38。本申请还提供了该抗体或其抗原结合片段的氨基酸、包含所述氨基酸的载体、包含所述氨基酸或载体的宿主细胞、制备和纯化该抗体的方法以及所述抗体或其抗原结合片段的医学和生物学应用，例如预防或治疗CD38相关疾病或病症。
为容易地理解本申请，首先定义本文中使用的某些术语。
本文所用术语“抗体”指包含四条多肽链，即通过双硫键互连的两条重链(H)链及两条轻链(L)的免疫球蛋白分子，以及其多聚体(例如IgM)。各重链包含重链可变区(缩写为VH)及重链恒定区(缩写为CH)。重链恒定区包含三个域，即CH1、CH2及CH3。各轻链包含轻链可变区(缩写为VL)及轻链恒定区(缩写为CL)。轻链恒定区包含一个域(CL1)。VH及VL区可进一步细分称为互补决定区(CDR)的高变区，其中穿插有称为构架区(FR)的保守区。
如本文所用，术语抗体的“抗原结合部分”是指负责结合抗原的完整抗体分子的一部分或区段。抗原结合域可以包含重链可变区(VH)、轻链可变区(VL)或上述两者。抗体的抗原结合片段可使用任何适合的标准技术从完整抗体分子制备， 所述标准技术包括蛋白水解消化或重组遗传工程化技术等。抗原结合部分的非限制性实例包括：Fab片段；F(ab')2片段；Fd片段；Fv片段；单链Fv(scFv)分子；单域抗体；dAb片段及由模拟抗体高变区的氨基酸残基组成的最小识别单元(例如分离的CDR)。术语“抗原结合部分”也包括其它工程化的分子，如双抗体、三抗体、四抗体及微型抗体等。
如本文所用，术语“重链可变区(VH)”及“轻链可变区(VL)”分别指单一抗体可变重链及轻链区，其包含FR1、2、3及4及CDR 1、2及3。
本领域技术人员公知，互补决定区(CDR，通常有CDR1、CDR2及CDR3)是可变区中对抗体的亲和力和特异性影响最大的区域。VH或VL的CDR序列有两种常见的定义方式，即kabat定义和Chothia定义，例如参见Kabat et al,“Sequences of Proteins of Immunological Interest”，National Institutes of Health,Bethesda，Md.(1991)；A1-Lazikani et al.，J.Mol.Biol.273:927-948(1997)；以及Martin et al.，Proc.Natl.Acad.Sci.USA86:9268-9272(1989)。对于给定抗体的可变区序列，可以根据Kabat定义或者Chothia定义来确定VH和VL序列中CDR区序列。在本申请的实施方案中，利用Kabat定义CDR序列。在本文中，重链可变区的CDR1、CDR2及CDR3分别简称为HCDR1、HCDR2及HCDR3；轻链可变区的CDR1、CDR2及CDR3分别简称为LCDR1、LCDR2及LCDR3。
对于给定抗体的可变区序列，可以通过多种方式分析可变区序列中CDR区序列，例如可以利用在线软件Abysis确定(http://www.abysis.org/)。
如本文所用术语“特异性结合”，是指两个分子之间的非随机结合反应，例如抗体至抗原表位的结合，例如抗体以比其对非特异性抗原的亲和性大至少两倍的亲和性结合于特异性抗原的能力。然而应了解，抗体能够特异性结合于两种或更多其序列相关的抗原。例如，本发明的抗体可特异性结合于人类与非人类(例如小鼠或非人类灵长动物)的CD38。
本文所用术语“单克隆抗体”指由基本同质的抗体群体获得的抗体，即，除了可能在少量个体中存在自然发生的突变以外，组成群体的各个抗体是相同的。本文所述单克隆抗体特别包括“嵌合”抗体，其中重链和/或轻链的一部分与来源于具体物种或属于具体抗体类或亚类的抗体中的对应序列相同或同源，而重链和/或轻链的余下部分与来源于另一物种或属于另一抗体类或亚类的抗体中的对 应序列相同或同源，并且还包括这样的抗体的片段，只要它们能表现出所期望的生物学活性(参见，美国专利号4,816,567；和Morrison et al,Proc.Natl.Acad.Sci.USA 81:6851-6855(1984))。
如本文所用，术语“同源性”被定义为经过序列比对和引入空位后，氨基酸或核苷酸序列变体中相同的残基的百分比，如果需要，达到最大百分比的同源性。用于比对的方法和计算机程序在本领域内是公知的。本文所述的“至少80％同源性”是指同源性为80％至100％中的任一值，例如85％、90％、95％、99％等。
如本文所用，术语“CD38相关疾病”包括多发性骨髓瘤、非霍奇金淋巴瘤、AL型淀粉样变性、T细胞淋巴瘤和弥漫性大B细胞淋巴癌等。
一方面，本申请提供了特异性结合CD38的抗体或其抗原结合部分，其包含重链可变区和/或轻链可变区。表1-3中示例性列出了适用于本申请公开的抗体的CDR、重链和轻链可变区氨基酸序列。
在具体的实施方案中，HCDR3选自SEQ ID NOs:3，6，9，12，15，18，21，24，27，30，33，36，39，42，45和48所示的氨基酸序列。在另一具体的实施方案中，HCDR3选自SEQ ID NOs:3，9，18，21，27，33，36和45所示的氨基酸序列。在优选的实施方案中，HCDR3选自9，18，21，27，33，36和45所示的氨基酸序列。
在具体的实施方案中，HCDR2选自SEQ ID NOs:2，5，8，11，14，17，20，23，26，29，32，35，38，41，44和47所示的氨基酸序列。在另一具体的实施方案中，HCDR2选自SEQ ID NOs:2，8，17，20，26，32，35和44所示的氨基酸序列。在优选的实施方案中，HCDR2选自8，17，20，26，32，35和44所示的氨基酸序列。
在具体的实施方案中，HCDR1选自SEQ ID NOs:1，4，7，10，13，16，19，22，25，28，31，34，37，40，43和46所示的氨基酸序列。在另一具体的实施方案中，HCDR1选自SEQ ID NOs:1，7，16，19，25，31，34和43所示的氨基酸序列。在优选的实施方案中，HCDR1选自7，16，19，25，31，34和43所示的氨基酸序列。
在一些实施方案中，本文公开的抗体重链可变区包含选自SEQ ID NOs:97，99，101，103，105，107，109和111的氨基酸序列。在具体的实施方案中，所述重链可变区由选自SEQ ID NOs:97，99，101，103，105，107，109和111 的氨基酸序列组成。
本文公开的抗体或其抗原结合部分在包含重链可变区的基础上还可以进一步包含轻链可变区。
在一些实施方案中，所述轻链可变区的CDR3(LCDR3)选自SEQ ID NOs:51，54，57，60，63，66，69，72，75，78，81，84，87，90，93，96和117所示的氨基酸序列。在另一具体的实施方案中LCDR3选自SEQ ID NOs:51，57，66，69，75，81，84和117所示的氨基酸序列。在优选的实施方案中，LCDR3选自SEQ ID NOs:57，66，69，75，81，84和117所示的氨基酸序列。
在一些实施方案中，LCDR2选自SEQ ID NOs:50，53，56，59，62，65，68，71，74，77，80，83，86，89，92，95和116所示的氨基酸序列，或者选自SEQ ID NOs:50，65，68，74，80，83，92和116所示的氨基酸序列。在优选的实施方案中，LCDR2选自SEQ ID NOs:65，68，74，80，83，92和116所示的氨基酸序列。
在一些实施方案中，LCDR1选自SEQ ID NOs:49，52，55，58，61，64，67，70，73，76，79，82，85，88，91，94和115的氨基酸序列，或者选自SEQ ID NOs:55，64，67，73，79，82，91和115的氨基酸序列。在优选的实施方案中，LCDR1选自SEQ ID NOs:55，64，67，73，79，82和91所示的氨基酸序列。
在一些实施方案中，本文公开的抗体轻链可变区包含选自SEQ ID NOs:98，100，102，104，106，108，110和112的氨基酸序列。在具体的实施方案中，所述轻链可变区由选自SEQ ID NOs:98，100，102，104，106，108，110和112的氨基酸序列组成。
在一些实施方案中，本文公开的抗体的重链或重链可变区、轻链或轻链可变区可以在上述所列举的各自对应的具体氨基酸序列的基础上取代、缺失或添加至少一个氨基酸，且得到的变体仍然保持结合人CD38的活性。
在某些实施方案中，上述氨基酸取代、缺失或添加的数目为1-30个，优选为1-20个，更优选为1-10个。在优选的实施方案中，序列变体与原氨基酸序列相差约1、2、3、4、5、6、7、8、9、或10个氨基酸的取代、缺失和/或添加。在更优选的实施方案中，序列变体与原氨基酸序列相差约1、2、3、4或5个氨基酸的取代、缺失或添加。在具体的实施方案中，所述氨基酸取代为保守性取代。
在优选的实施方案中，本文公开的抗体为抗体57H、105H、116H、或145H， 其中抗体57H的重链可变区序列如SEQ ID NO：99所示，轻链可变区序列如SEQ ID NO：100所示，其中LCDR2优选为SEQ ID NO：116所示，其余CDR序列与鼠源抗体57的相同；抗体105H的重链可变区序列如SEQ ID NO：107所示，轻链可变区序列如SEQ ID NO：108所示，其中CDR序列与鼠源抗体105相同；抗体116H的重链可变区序列如SEQ ID NO：109所示，轻链可变区序列如SEQ ID NO：110所示，其中CDR序列与鼠源抗体116相同；抗体145H的重链可变区序列如SEQ ID NO：111所示，轻链可变区序列如SEQ ID NO：112所示，其中LCDR3优选为SEQ ID NO：117所示，其余CDR序列与鼠源抗体145的相同。
在一些的实施方案中，本文公开的抗体为单克隆抗体。在具体的实施方案中，本文公开的抗体为人源化的抗体。
本文公开的抗体或其抗原结合部分能够特异性结合人CD38。在具体的实施方案中，所述抗体或其抗原结合部分特异性结合人CD38或食蟹猴CD38。在优选的实施方案中，所述抗体或其抗原结合部分特异性结合人CD38。
在一些实施方案中，任何合适的表达载体都可以用于本申请。例如，所述表达载体可以为pTT5、pUC57、pDR1、pcDNA3.1(+)、pDHFF及pCHO 1.0中的一种。表达载体中可以包括连接有合适的转录和翻译调节序列的融合DNA序列。
在一些实施方案中，可用的宿主细胞为含有上述表达载体的细胞，可以是真核细胞，如哺乳动物或昆虫宿主细胞培养系统均可用于本申请的抗体或其抗原结合部分的表达。例如，HEK293细胞、COS、CHO、NS0、sf9及sf21等均可适用于本发明。所述宿主细胞也可以为含有上述表达载体的原核细胞，例如可以为DH5α、BL21(DE3)或TG1等。
在一些实施方案中，本文公开的抗人CD38单克隆抗体的制备方法包括：在适合的表达条件下，培养宿主细胞，从而表达抗人CD38单克隆抗体；分离和纯化表达的抗人CD38单克隆抗体。利用上述方法，可以将重组蛋白纯化为基本均一的物质，例如在SDS-PAGE电泳上为单一条带。
在一些实施方案中，可以利用亲和层析的方法对本文公开的抗人CD38抗体进行分离纯化，根据所利用的亲和柱的特性，可以使用常规的方法例如高盐缓冲液、改变PH等方法洗脱结合在亲和柱上的抗人CD38抗体。
在一些实施方案中，本文公开的人源化的抗人CD38单克隆抗体是通过以下方法得到的：利用实验室制备的人CD38抗原免疫Balb/c小鼠，在多次免疫小鼠 滴度较高后取小鼠脾细胞与杂交瘤细胞融合并筛选出具有不同亲和力的杂交瘤细胞株。更具体地，本申请的发明人通过大量实验，首先分别表达了人CD38抗原，在此基础上利用不同的佐剂与人CD38抗原混合免疫小鼠，然后进一步将上述小鼠的脾细胞与杂交瘤细胞株sp2/0融合，融合后的杂交瘤利用人CD38抗原筛选出阳性细胞株，在验证其对人CD38结合和与Daudi细胞的结合后获得目标细胞株。将目标分子进行人源化改造后，将轻链和重链基因同时克隆到真核表达载体pCHO1.0中。将上述表达载体通过脂质体法转染CHO细胞，然后用嘌呤霉素和甲胺蝶呤筛选阳性细胞克隆，将筛选得到的高表达克隆用无血清培养基扩大培养，用Protein A亲和柱分离或纯化人源化的抗人CD38抗原单克隆抗体。
在另外一些实施方案中，可以使用本领域的常规技术，例如PCR诱变进一步改变鼠源的亲本抗体来产生抗体的嵌合或人源化形式或其他变异形式。本申请的亲本抗体可以在例如抗原互补决定区(CDR)结构域内被突变来产生变异抗体，可筛选其目的性质的存在，例如结合亲和力(更低的KD)、IC50、特异性、优先结合等等。优选地，变异抗体中目的性质是相对于亲本抗体中性质的改善。优选氨基酸替代变异抗体，并且亲本抗体分子的至少1、2、3、4、5、6、7、8、9或10个氨基酸残基被去除且在它的位置上插入不同的残基。用于替代诱变的最感兴趣的位点是一个或更多个CDR区，但是也考虑框架区(FR)改变。优选保守的氨基酸替代，也可引入非保守氨基酸改变并用获得的变异抗体筛选目的性质。
本申请提供了药物组合物，其包含本文公开的抗体或其抗原结合部分以及药学上可接受的载体。本文公开的上述抗人CD38单克隆抗体，可以和药学上可接受的载体一起配制成药物制剂，从而更稳定地发挥疗效。在一些实施方案中，这些制剂可以保证本文公开的抗人CD38单克隆抗体的氨基酸核心序列构象的完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱酰胺或氧化)。在一些实施方案中，对于液体制剂，通常可以在2℃-8℃条件下保存至少稳定一年。在一些实施方案中，对于冻干制剂，在30℃下至少六个月保持稳定。
在一些实施方案中，所述抗人CD38抗体单克隆抗体制剂可为制药领域常用的混悬、水针、冻干等制剂，优选水针或冻干制剂，对于本文公开的抗人CD38单克隆抗体的水针或冻干制剂，药学上可以接受的辅料包括但不限于：表面活性剂、溶液稳定剂、等渗调节剂和缓冲液或其组合。在一些实施方案中，表面活性 剂包括但不限于：非离子型表面活性剂如聚氧乙烯山梨醇脂肪酸酯(吐温20或80)，泊洛沙姆(如泊洛沙姆188)，Triton，十二烷基硫酸钠(SDS)，月桂硫酸钠，十四烷基、亚油基或十八烷基肌氨酸，Pluronics，MONAQUATTM等，其加入量应使抗人CD38单克隆抗体的颗粒化趋势最小。在一些实施方案中，溶液稳定剂包括但不限于以下列举的一种或其组合：糖类，例如还原性糖和非还原性糖；氨基酸类，例如谷氨酸单钠或组氨酸；醇类，例如三元醇、高级糖醇、丙二醇、聚乙二醇等。溶液稳定剂的加入量应该使最后形成的制剂在本领域的技术人员认为达到稳定的时间内保持稳定状态。等渗调节剂包括但不限于氯化钠、甘露醇或其组合。缓冲液包括但不限于：Tris、组氨酸缓冲液、磷酸盐缓冲液或其组合。
本申请还提供了预防或治疗CD38相关疾病的方法，其包括给予个体抗人CD38抗体、或者包含抗人CD38抗体的组合物。具体地说，本文公开的抗人CD38抗体能够有效地预防和/或治疗CD38相关疾病。
在一些实施方案中，所述CD38相关疾病包括多发性骨髓瘤、非霍奇金淋巴瘤、AL型淀粉样变性、T细胞淋巴瘤、弥漫性大B细胞淋巴癌、慢性淋巴细胞白血病、急性淋巴细胞白血病、浆细胞性白血病、急性髓性白血病、非小细胞肺癌、胰腺癌、结直肠癌和三阴乳腺癌等。
本文公开的抗人CD38单克隆抗体及其组合物在对包括人在内的动物给药时，给药剂量因病人的年龄和体重、疾病特性和严重性以及给药途径而异，可以参考动物实验的结果和综合情况，总给药量不能超过一定范围。
抗体或其组合物的施用剂量和频率可根据对疾病进行预防或治疗而变化。在预防性应用中，向尚未处于疾病状态的患者施用含有本申请的抗体或其混合物的组合物以增强患者抵抗力，此量定义为“预防性有效剂量”。在此用途中，具体的剂量又视患者健康状况及全身免疫性而定。通常以相对不频繁的间隔施用相对较低剂量较长时间。在治疗性应用中，有时需要以相对较短间隔施用相对较高剂量直至疾病进展减缓或终止为止，且优选直至患者显示疾病症状部分或完全改善为止。此后，可向患者施用预防性方案。本领域普通技术人员可以容易地根据实际需要掌握具体的剂量和频率。
本说明书和权利要求书中，词语“包括”、“包含”和“含有”意指“包括但不限于”，且并非意图排除其他部分、添加物、组分、或步骤。
应该理解，在本申请的特定方面、实施方案或实施例中描述的特征、特性、 组分或步骤，可适用于本文所描述的任何其他的方面、实施方案或实施例，除非与之矛盾。
上述公开内容总体上描述了本发明。以下具体的实施例是对本发明作进一步的说明，不应理解为对本发明的限制。实施例不包括对传统常规方法的详细描述，这样的方法对于本领域具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，例如分子克隆手册、冷泉港的抗体技术实验手册。未注明试剂来源的为常规试剂。
具体实施例
实施例1 人CD38胞外段的Fc标签和Flag标签抗原、食蟹猴CD38胞外段Flag 标签蛋白、参比抗体Daratumumab的制备
人CD38抗原序列购买于义翘神州(货号：HG10818-M)。通过PCR法将人CD38N端第43-300位氨基酸片段分别与hFc片段和Flag标签(DYKDDDDK)进行拼接，hFc片段和Flag标签位于C端，并构建至pTT5表达载体(实验室保存)上，获得pTT5(hCD38-ECD-hFc)和pTT5(hCD38-ECD-Flag)，选取测序结果完全正确的克隆进行质粒抽提后转染。
食蟹猴CD38序列购买于义翘神州(货号：CG90050-G)。通过PCR法将其N端第44-301位氨基酸片段与Flag标签(DYKDDDDK)进行拼接，Flag标签位于C端，并构建至pTT5表达载体(实验室保存)上，获得pTT5(cyno-CD38-ECD-Flag)，选取测序结果完全正确的克隆进行质粒抽提后转染。
Daratumumab的氨基酸序列来自IMGT，经过密码子优化后全基因合成重链、轻链的可变区核苷酸序列。通过PCR法将Daratumumab重链的可变区与IgG1的恒定区连接；Daratumumab轻链可变区与Kappa链的恒定区连接。然后将这些片段克隆至pTT5表达载体，测序验证确认后进行质粒抽提以备转染。
通过PEI法将质粒转染至HEK293E细胞系(实验室保存)。利用含3mM的丙戊酸的Freestyle293培养基(购自Gibco公司)培养5天后，利用Protein A亲和层析(购自Pharmacia公司)或Flag亲和层析(购自Genscript公司)从细胞培养上清中纯化目的蛋白。蛋白的定量通过二喹啉甲酸(Bicinchoninic acid，BCA)方法进行，纯化得到的蛋白用于以下的进一步分析与研究。纯化得到的蛋白用于以下的小鼠 免疫及进一步分析与研究。
实施例2  hCD38-ECD-hFc的免疫
将100μg/鼠的hCD38-ECD-hFc抗原用生理盐水稀释成75μl后，与等体积的弗氏完全佐剂混合，并经超声乳化完全后对4-5周龄的Balb/c小鼠(购自上海灵畅生物科技有限公司，动物生产许可证号：SCXK(沪)2013-0018)进行皮下多点注射。三周后，将50μg/鼠的蛋白同样稀释成75μl后与等体积弗氏不完全佐剂混合，超声乳化完全后对小鼠进行皮下多点注射，两周后再次重复此免疫。所有小鼠在第三次免疫后一周剪尾取血分离血清，利用包被hCD38-ECD-hFc抗原的ELISA进行血清滴度的检测。对于血清抗体效价>10000的小鼠，在取血后一周进行冲击免疫：尾静脉注射10μg抗原/100μl生理盐水/鼠。
滴度的检测通过ELISA方法进行：利用hCD38-ECD-hFc抗原包被ELISA板，包被浓度为1μg/ml，每孔100μl，4℃包被过夜。PBST(含0.5％Tween-20的PBS)洗板2次后拍干。每孔加入含1％BSA的包被液封闭200μl，常温下封闭4小时后拍干，至-20℃冰箱中保存待用。检测时在ELISA板中每孔加入不同浓度的小鼠血清100μl，设2个复孔，室温孵育1.5小时。PBST洗涤3次后拍干。加入用PBST1:10000倍稀释的HRP标记的兔抗鼠Ig抗体(购自Sigma公司)100μl，室温孵育1小时。PBST洗涤3次后拍干。每孔加100μl显色液(临用前将ELISA显色A液与显色B液按照1：1的体积比混匀)显色，随后每孔加入100μl 2M H2SO4终止液终止反应。立即用酶标仪(Molecular Device)在450nm波长处测量各孔OD值。
实施例3 杂交瘤融合和筛选
杂交瘤sp2/0细胞(来自中国科学院典型培养物保藏委员会细胞库，保藏号为TCM-18)在37℃、5％CO 2培养箱中培养，融合前一天换液。小鼠冲击免疫三天后取小鼠脾细胞进行融合。融合与筛选方法如下：取小鼠脾脏，研磨洗涤后进行脾细胞计数。将脾细胞和sp2/0细胞以10:1的比例混合，1500rpm离心7分钟。洗去上清液。1分钟内加入1ml PEG(1450)，轻摇90秒，在2.5分钟内加入无血清DMEM培养液(购自Gibco公司)5ml，再一次性加5ml无血清培养液终止反应，静置5分钟，1280rpm离心8分钟。按照一块96孔板两百万个sp2/0细胞的数量，将细胞均匀接种入96孔板，每孔200μl。先用含次黄嘌呤(hypoxanthine， H)、甲胺蝶呤(aminopterin，A)和胸腺嘧啶核苷(thymidine，T)的HAT培养基筛选，每3～4天半量换液，第10天改用HT培养基。10天后，待杂交瘤细胞铺满96孔板底部大于10％时，取上清用hCD38-ECD-hFc抗原包被的酶标板进行ELISA检测。ELISA检测方法如实施例2中所述方法相同。挑选出阳性杂交瘤克隆于24孔板中扩大培养，有限稀释法进行亚克隆，获得稳定表达目的抗体的杂交瘤株后进行保种建库。
实施例4 鼠源的抗人CD38单克隆抗体序列的测定
亲和力较高的阳性孔经过2～3轮的亚克隆最后得到了150株杂交瘤株。对这些杂交瘤细胞株进行抗体序列调取。使用Trizol提取各杂交瘤细胞株的总RNA，用逆转录试剂盒(购自ABI公司)将mRNA逆转录成cDNA。以文献报道的引物通过PCR扩增鼠源的抗人CD38单克隆抗体的轻链可变区和重链可变区基因，然后将PCR产物克隆入pGEM-T载体，测序并分析可变区基因序列。在GenBank中对获得的序列进行比对分析，所有序列均符合小鼠IgG可变区基因的特征。表1和表2列举了优选抗体的CDR区域的氨基酸序列。
表1：示例性鼠源抗人CD38抗体的重链CDR氨基酸序列
Figure PCTCN2021124806-appb-000001
实施例5 抗人CD38单克隆抗体的人源化
根据序列分析结果，挑取1，57，71，73，77，78，105，116和145号抗体进行了嵌合抗体和人源化抗体的构建。嵌合抗体的构建通过截取鼠源抗体的重链可变区和轻链可变区，利用overlapping PCR分别与人IgG1的轻重链恒定区连接而成。
表2：示例性鼠源抗人CD38抗体的轻链CDR氨基酸序列
Figure PCTCN2021124806-appb-000002
根据Kabat法则对鼠源的抗人CD38单克隆抗体的轻链可变区和重链可变区的氨基酸序列进行分析并确定了3个CDR和4个FR。以105号抗体为例，通过在NCBI IgBlast与人IgG胚系序列(Germline)进行同源性比较，选择IGHV1-46*01为重链CDR移植模板，将鼠源的抗人CD38单克隆抗体105号的重链CDR区移植入IGHV1-46*01骨架区，构建成重链的CDR移植抗体。同样地，经过与人IgG胚系序列同源性比较，选择IGKV1-16*01为轻链CDR移植模板，将鼠源的抗人CD38单克隆抗体105号的轻链CDR区移植入IGKV1-16*01的骨架区，构建成轻链的CDR移植抗体。同时，在此基础上，对一些框架区的氨基酸位点进行了回复突变。在进行回复突变时，将氨基酸序列进行了Kabat编码，位点的位置由Kabat码指示。优选地，对于轻链可变区序列，将Kabat编码第36位的F回复为鼠源的L，将第43位的A突变成T，第44位的P回复为鼠源的I，第46位的S回复为鼠源的R，第66位的G回复为鼠源的R，第69位的T回复为鼠源 的S。对于重链可变区序列，将Kabat编码第48位的M回复为鼠源的I，第67位的V回复为鼠源的A，第69位的M回复为鼠源的L，第71位的R回复为鼠源的A，第73位的T回复为鼠源的K，第78位的V回复为鼠源的A。上述可变区基因序列由生工生物按照Cricetulus griseus的密码子使用偏好进行密码子优化并合成。将合成的人源化可变区序列与人IgG1恒定区相连，此抗体定义为105号抗体的人源化抗体105-Humanization，105H)。
利用上述相同原理，对其余抗体同样进行了人源化。人源化抗体的可变区氨基酸序列编号见表3，重链恒定区的氨基酸序列为SEQ ID NO：113所示，轻链恒定区的氨基酸序列为SEQ ID NO：114所示。利用pTT5载体分别构建人源化重链、轻链的瞬时表达载体，将上述轻重链组合利用HEK293E系统进行瞬时转染并表达抗体。HEK293E细胞在Free Style 293Expression Medium(购自Gibco公司)培养基中培养，利用PEI转染法将质粒转入细胞5天后收取细胞上清，利用Protein A纯化后获得各个人源化单克隆抗体。
最终，1号抗体人源化后的重链可变区氨基酸序列如SEQ ID NO：97所示；人源化后的轻链可变区氨基酸序列如SEQ ID NO：98所示。与人IgG1恒定区相连后，最终获得1H的抗体序列。
57号抗体人源化后的重链可变区氨基酸序列如SEQ ID NO：99所示；人源化后的轻链可变区氨基酸序列如SEQ ID NO：100所示。与人IgG1恒定区相连后，最终获得57H的抗体序列。
71号抗体人源化后的重链可变区氨基酸序列如SEQ ID NO：101所示；人源化后的轻链可变区氨基酸序列如SEQ ID NO：102所示。与人IgG1恒定区相连后，最终获得71H的抗体序列。
73号抗体人源化后的重链可变区氨基酸序列如SEQ ID NO：103所示；人源化后的轻链可变区氨基酸序列如SEQ ID NO：104所示。与人IgG1恒定区相连后，最终获得73H的抗体序列。
78号抗体人源化后的重链可变区氨基酸序列如SEQ ID NO：105所示；人源化后的轻链可变区氨基酸序列如SEQ ID NO：106所示。与人IgG1恒定区相连后，最终获得78H的抗体序列。
105号抗体人源化后的重链可变区氨基酸序列如SEQ ID NO：107所示；人源化后的轻链可变区氨基酸序列如SEQ ID NO：108所示。与人IgG1恒定区相连 后，最终获得105H的抗体序列。
116号抗体人源化后的重链可变区氨基酸序列如SEQ ID NO：109所示；人源化后的轻链可变区氨基酸序列如SEQ ID NO：110所示。与人IgG1恒定区相连后，最终获得116H的抗体序列。
145号抗体人源化后的重链可变区氨基酸序列如SEQ ID NO：111所示；人源化后的轻链可变区氨基酸序列如SEQ ID NO：112所示。与人IgG1恒定区相连后，最终获得145H的抗体序列。
表3：示例性人源化抗人CD38抗体重链和轻链可变区的氨基酸序列
Figure PCTCN2021124806-appb-000003
实施例6 人源化CD38单克隆抗体与Daudi细胞的结合
将生长状态良好的Daudi细胞(实验室保存)取样计数，离心后用添加1％FBS的PBS重悬，密度调节为2 X 10 7。抗体样品最高浓度10μg/ml，三倍梯度稀释(共8个梯度)后加样。在96孔培养板中，加入20ul/孔(4 X 10 5/孔)Daudi细胞，并加入30μl梯度稀释的抗体药物，4℃孵育30分钟。加入180μl 1％FBS PBS清洗，1200rpm离心6min。用真空泵吸去上清后，加入羊抗人IgG-FITC(用1％FBS PBS 1：1000稀释)50μl，在4℃孵育30分钟。加入180μl 1％FBS PBS后1200rpm离心6分钟后用160μl PBS重悬。最后用流式细胞仪检测细胞表面荧光强度。利用GraphPad Prism6软件进行数据分析。实验结果见图1和表4。
表4：人源化CD38抗体对Daudi细胞的结合
Figure PCTCN2021124806-appb-000004
Figure PCTCN2021124806-appb-000005
实施例7 人源化CD38抗体诱导Daudi淋巴瘤细胞凋亡
在96孔培养板200μl/孔的实验体系中，加入1X 10 5Daudi细胞/孔，并加入1.85和1.03μg/孔的抗体药物，培养24小时结束后，在培养孔中直接加入1：100的PI(Propidium Iodide)溶液(2μl)，继续培养30分钟，离心去除上清，用PBS洗涤，离心去除上清，然后加入160μl PBS重悬，用流式细胞仪完成检测。设置未染色孔作为空白对照，参比抗体为Daratumumab(图2中简写为Dara)。图2的结果表明73H相较参比抗体显示了更强的诱导凋亡活性，其余抗体和参比抗体一致，并未见明显的诱导凋亡活性。
实施例8 人源化CD38抗体抗Fc交联诱导Daudi淋巴瘤细胞凋亡
在96孔培养板200μl/孔的实验体系中，加入1X 105Daudi细胞/孔，并加入1.85和1.03μg/孔的抗体药物，在细胞培养箱中孵育30分钟。然后加入1μg/孔抗人Fc抗体，继续培养24小时。培养24小时。培养结束后在培养孔中直接加入1：100的PI溶液(2μl)，继续培养30分钟，离心去除上清，用PBS洗涤，离心去除上清，然后加入160μl PBS重悬，用流式细胞仪完成检测。设置未染色孔作为空白对照，参比抗体为Daratumumab(图3中简写为Dara)。图3的结果表明5个优选抗体相较参比抗体具有一致的较强的抗Fc交联诱导细胞凋亡活性。
实施例9 人源化CD38单克隆抗体的亲和力测定
表达纯化的人源化抗体的亲和力通过Biacore T200(GE healthcare)检测。具体实验方法为：利用Protein-A CM5传感芯片(GE healthcare)，以FC1(Flow cell 1)为参照通道，FC2(Flow cell 2)为样品通道。在FC2通道分别捕获人源抗体或对照抗体，随后注射不同浓度的hCD38-Flag或者cyno-CD38-Flag。循环条件为：在FCs所有通道中以50μl/min注射4min分析物，解离时间为20min，以10μl/min速率注射6M盐酸胍(国药集团化学试剂有限公司)30s进行表面再生，然后利用Biacore T200 Evaluation Software Ver 1.0计算捕获抗体的信 号和无捕获抗体的信号差值及相互作用的亲和力。如表5和表6所显示。实验结果表明优选抗体均能够与人和食蟹猴的CD38结合，具有高、中和低不同亲和力。
表5：人源化CD38抗体结合人CD38的亲和力
抗体编号 Ka(1/Ms) Kd(1/s) KD(nM)
57H 2.641E+05 1.009E-04 0.382
105H 2.976E+04 9.748E-04 32.755
116H 1.472E+06 9.530E-05 0.065
145H 6.125E+05 1.975E-03 3.224
表6：人源化CD38抗体结合猴CD38的亲和力
抗体编号 Ka(1/Ms) Kd(1/s) KD(nM)
57H 5.492E+04 9.201E-05 1.675
105H 6.113E+04 2.080E-04 3.402
116H 2.513E+05 1.019E-04 0.405
145H 9.782E+04 3.930E-03 40.176
结论：本发明的发明人进行了大量试验，得到了一组可以特异性结合细胞表面CD38的单克隆抗体，这些抗体同时可以和食蟹猴的CD38结合。获得了一系列具有高、中和低不同亲和力的抗人CD38抗体，其能够通过Fc依赖性免疫效应机制杀死肿瘤细胞。
可以理解，尽管本申请以某种形式被说明，但本申请并不局限于本说明书中所显示和描述的内容。对本领域的技术人员显而易见的是，在不偏离本申请的范围的前提下还可对所述实施方式和/或某一特征或参数做出各种变化。这些变化都在本申请要求保护的范围内。

Claims (14)

  1. 特异性结合人CD38的抗体或其抗原结合部分，其包含至少1个选自以下序列，其突变序列或者与其具有至少95％序列同一性的氨基酸序列。其包含重链可变区，所述重链可变区包含HCDR3序列，任选地还包含HCDR1和/或HCDR2序列，其中所述HCDR3序列包含选自SEQ ID NOs:3，6，9，12，15，18，21，24，27，30，33，36，39，42，45和48的氨基酸序列；和/或所述HCDR2序列包含选自SEQ ID NOs:2，5，8，11，14，17，20，23，26，29，32，35，38，41，44和47的氨基酸序列；和/或所述HCDR1序列包含选自SEQ ID NOs:1，4，7，10，13，16，19，22，25，28，31，34，37，40，43和46的氨基酸序列。
  2. 如权利要求1所述的抗体或其抗原结合部分，其中所述重链可变区包含选自SEQ ID NOs:97，99，101，103，105，107，109和111的氨基酸序列或者具有至少80％同源性的氨基酸序列。
  3. 如权利要求1或2所述的抗体或其抗原结合部分，其还包含轻链可变区。其包含以下序列，其突变序列或者与其具有至少95％序列同一性的氨基酸序列。所述轻链可变区包含LCDR1、LCDR2和/或LCDR3序列，其中所述LCDR1序列包含选自SEQ ID NOs:49，52，55，58，61，64，67，70，73，76，79，82，85，88，91和94的氨基酸序列；和/或所述LCDR2序列包含选自SEQ ID NOs:50，53，56，59，62，65，68，71，74，77，80，83，86，89，92和95的氨基酸序列；和/或所述LCDR3序列包含选自SEQ ID NOs:51，54，57，60，63，66，69，72，75，78，81，84，87，90，93和96的氨基酸序列。
  4. 如权利要求3所述的抗体或其抗原结合部分，其中所述CDR区突变序列为CDR区发生1-3个优化抗体活性的氨基酸突变。其中所述的LCDR1序列突变序列优选为SEQ ID NO：115；其中所述的LCDR2序列突变序列优选为SEQ ID NO：116；其中所述的LCDR3序列突变序列优选为SEQ ID NO：117。
  5. 如权利要求3和4所述的抗体或其抗原结合部分，其中所述轻链可变区包含选自SEQ ID NOs:98，100，102，104，106，108，110和112的氨基酸序列或者具有至少80％同源性的氨基酸序列。
  6. 如前述权利要求任一项所述的抗体或其抗原结合部分，优选的所述抗体57H、105H、116H、或145H。其中抗体57H的重链可变区序列如SEQ ID NO：99所示，轻链可变区序列如SEQ ID NO：100所示，其中LCDR2优选为SEQ ID NO：116所示，其余CDR序列与鼠源抗体57的相同；抗体105H的重链可变区序列如 SEQ ID NO：107所示，轻链可变区序列如SEQ ID NO：108所示，其中CDR序列与鼠源抗体105相同；抗体116H的重链可变区序列如SEQ ID NO：109所示，轻链可变区序列如SEQ ID NO：110所示，其中CDR序列与鼠源抗体116相同；抗体145H的重链可变区序列如SEQ ID NO：111所示，轻链可变区序列如SEQ ID NO：112所示，其中LCDR3优选为SEQ ID NO：117所示，其余CDR序列与鼠源抗体145的相同。
  7. 优选地，权利要求1-6所述抗原结合部分选自Fab片段、Fab’片段、F(ab’)2片段、Fv片段、scFv片段、Fd片段和单域抗体。
  8. 如前述权利要求任一项所述的抗体或其抗原结合部分，其中所述抗体或其抗原结合部分特异性结合于人CD38或食蟹猴CD38，其中优选地，所述抗体具有高、中和低不同亲和力。
  9. 药物组合物，其包含权利要求1-7中任一项所述的抗体或其抗原结合部分以及药学上可接受的载体。
  10. 表达载体，其包含权利要求9所述的抗体氨基酸序列。
  11. 宿主细胞，其包含权利要求9所述的抗体氨基酸序列或权利要求10所述的表达载体。
  12. 产生权利要求1-7中任一项所述的抗体或其抗原结合部分的方法，其包括：
    a)在使得权利要求11所述的宿主细胞能够产生所述抗体或其抗原结合部分的表达条件下，培养所述宿主细胞，从而表达抗体或其抗原结合部分；以及b)分离并纯化步骤a)中表达的所述抗体或其抗原结合部分。
  13. 权利要求1-7中任一项所述的抗体或其抗原结合部分、或者权利要求9所述的组合物在制备用于预防或治疗CD38相关疾病的药物中的用途。
  14. 权利要求13所述的用途，其中所述CD38相关疾病选自多发性骨髓瘤、非霍奇金淋巴瘤、AL型淀粉样变性、T细胞淋巴瘤和弥漫性大B细胞淋巴癌。
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