Nothing Special   »   [go: up one dir, main page]

WO2021104052A1 - 药物组合物及其制备方法和应用 - Google Patents

药物组合物及其制备方法和应用 Download PDF

Info

Publication number
WO2021104052A1
WO2021104052A1 PCT/CN2020/128820 CN2020128820W WO2021104052A1 WO 2021104052 A1 WO2021104052 A1 WO 2021104052A1 CN 2020128820 W CN2020128820 W CN 2020128820W WO 2021104052 A1 WO2021104052 A1 WO 2021104052A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
acid sequence
variant
amino acid
cdr2
Prior art date
Application number
PCT/CN2020/128820
Other languages
English (en)
French (fr)
Inventor
于俊涛
徐刚
陈博
Original Assignee
康诺亚生物医药科技（成都）有限公司
上海苓樾生物医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 康诺亚生物医药科技（成都）有限公司, 上海苓樾生物医药科技有限公司 filed Critical 康诺亚生物医药科技（成都）有限公司
Priority to EP20892076.9A priority Critical patent/EP4071170A4/en
Priority to US17/780,852 priority patent/US20230132604A1/en
Priority to KR1020227022100A priority patent/KR20220119393A/ko
Priority to AU2020390028A priority patent/AU2020390028A1/en
Priority to JP2022532596A priority patent/JP2023505123A/ja
Publication of WO2021104052A1 publication Critical patent/WO2021104052A1/zh
Priority to JP2024005374A priority patent/JP2024026820A/ja

Links

Images

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to a CD38 antibody and its preparation method and application.
  • CD38 is a 46kDa type II transmembrane glycoprotein.
  • the N-terminal contains 21 amino acid residues and is located in the cell.
  • the transmembrane region contains 21 amino acids and the C-terminal is an extracellular region composed of 258 amino acid residues.
  • CD38 plays a role in regulating migration and receptor-mediated adhesion through the interaction with CD31 or hyaluronic acid.
  • CD38 also has extracellular enzyme activity and participates in the production of nucleotide metabolites. Play a role in storage control.
  • CD38 is expressed at a relatively low level; on the contrary, in plasma cells and some malignant cells, CD38 is usually expressed at a high level, including multiple For myeloma (multiple myeloma, MM) and chronic lymphocytic leukemia (chronic lymphocytic leukemia, CLL), the difference in expression abundance makes CD38 a target molecule for targeted multiple myeloma cell therapy.
  • multiple myeloma multiple myeloma
  • CLL chronic lymphocytic leukemia
  • Multiple myeloma is a type of plasma cell carcinoma characterized by the accumulation of malignant cells in the bone marrow and the production of monoclonal immunoglobulin (M protein).
  • M protein monoclonal immunoglobulin
  • Daratumumab is a CD38 targeting antibody that has been approved for marketing in relapsed/refractory multiple myeloma.
  • monoclonal antibodies that are undergoing clinical trials include isatuximab, MOR202 and TAK-079, etc., except for Relapsed/refractory multiple myeloma also includes newly diagnosed multiple myeloma.
  • CD38 antibody also shows good therapeutic effects in other malignant tumors, such as: NK/T cell lymphoma, T cell acute lymphoblastic leukemia and immunoglobulin light chain amyloidosis.
  • Rheumatoid arthritis (rheumatoidarthritis, RA) is an autoimmune disease in which a large number of B lymphocytes proliferate in the diseased synovium.
  • Flow cytometry results showed that the expression rate of CD38+, CD3+CD38+ and CD56+CD38+ subpopulation cells in the peripheral blood of RA patients was significantly increased, and the expression rate of CD38+ cells was significantly correlated with the level of rheumatoid factor (RF) in RA patients; immunohistochemical results It shows that CD38 is specifically and highly expressed in the synovium of RA patients; ELISA experiments have confirmed that after siRNA interference with CD38 gene, the expression levels of IL-1 ⁇ and IL- ⁇ in RASF medium are significantly reduced, and the increase of CD38 gene expression may be involved in RA Immune activation of patients (CD38 expression in peripheral blood and synovial tissues of rheumatoid arthritis.
  • CD38 is significantly elevated in peripheral blood of other autoimmune diseases such as systemic lupus erythematosus (SLE), and plays an important role in the occurrence and development of SLE.
  • SLE systemic lupus erythematosus
  • a variety of CD38 antibodies have shown very significant anti-tumor effects, clinical studies have found that there is significant heterogeneity in the therapeutic effect and response duration of different antibodies, and this This difference may be related to the mechanism of action of antibodies. For example, after CD38 antibody treatment, the NK cells in the patient’s blood are quickly depleted.
  • Antibodies that mainly use antibody-dependent cytotoxicity (ADCC) may have limited effect due to the lack of effector cells; at the same time they have another mechanism of action.
  • Antibodies (such as daratumumab with complement-dependent cytotoxicity, CDC), show that the total effective rate and survival time of treatment are higher than other CD38 antibodies in the clinical research stage.
  • Some studies have introduced mutations on the Fc of IgG1, or replaced the hinge region of IgG1 with the hinge region of IgG3 to improve the CDC efficiency of the antibody. Recently, there have also been studies that the Fc mutation of IgG1 makes the monoclonal antibody form a hexamer, which effectively enhances the killing efficiency of the CD38 antibody, but brings some difficulties in production and development.
  • the present inventors immunized mice with CD38 recombinant protein and obtained a variety of new high-affinity CD38 antibodies, which have the strongest CDC, ADCC and antibody-dependent cellular phagocytosis (ADCP) activities of their kind, and they also have the ability to inhibit CD38 extracellular enzymes. Activity, and the function of causing apoptosis under Fc cross-linking conditions.
  • the inventors further introduced new mutations in the Fc segment of the antibody, and the tumor-killing efficiency of the new CD38 antibody obtained was much higher than that of similar antibodies currently on the market.
  • the present disclosure provides antibodies or antigen binding portions thereof that bind to CD38 protein.
  • the present disclosure provides a nucleic acid molecule encoding the antibody or antigen binding portion thereof according to the aforementioned aspect.
  • the present disclosure provides a vector containing the nucleic acid of the aforementioned aspect.
  • the present disclosure provides a cell containing the vector of the aforementioned aspect.
  • the antibody or antigen-binding portion thereof according to any one of the preceding aspects, wherein the antibody or antigen-binding portion thereof is humanized.
  • the present disclosure provides a pharmaceutical composition or kit comprising the antibody or antigen-binding portion or nucleic acid encoding thereof according to any one of the foregoing aspects and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method for treating CD38-related disorders, which includes the steps of: administering to the mammal a therapeutically effective amount of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, cell, and / Or pharmaceutical composition.
  • the antibody can bind to human CD38 with high affinity, has an inhibitory effect on the CD38 enzyme, has CDC, ADCC and/or ADCP killing activity on different tumor cells, and has an anti-tumor function. Moreover, the antibody does not cause red blood cell lysis.
  • FIG. 11-1 Antibody-dependent cytotoxicity (ADCC) effect of anti-CD38 humanized antibody on Ramos.2G6.4C10
  • FIG. 12-1 Anti-CD38 humanized antibody's antibody-dependent cellular phagocytosis (ADCP) effect on Daudi
  • FIG. 12-4 Anti-CD38 humanized antibody's antibody-dependent cellular phagocytosis (ADCP) effect on Raji
  • antibodies that specifically bind CD38 and antigen-binding fragments thereof.
  • a monoclonal anti-CD38 antibody that specifically binds CD38 wherein the anti-CD38 antibody includes a variant of the parent antibody.
  • antibodies that specifically bind to CD38 e.g., human CD38.
  • an anti-CD38 antibody comprising one or more modifications in amino acid residues (for example, 5-13 amino acid substitutions in the framework region of the heavy chain variable region), and without such modifications Compared with the parent antibody, it maintains the affinity to the antigen.
  • the term “about” or “approximately” means within plus or minus 10% of a given value or range. Where an integer is required, the term refers to within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.
  • the phrase "substantially identical" can be understood as showing at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, Antibody chains with 97%, 98%, 99% or more sequence identity.
  • nucleic acid sequence the term can be understood as showing that the reference nucleic acid sequence is at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. %, 99% or higher sequence identity nucleotide sequence.
  • Sequence "identity” or “identity” has the art-recognized meaning, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of the polynucleotide or polypeptide or along the region of the molecule (see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Pres, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
  • substitution type variant is a variant in which at least one amino acid residue in the natural sequence is removed and a different amino acid is inserted into the same position.
  • the substitution may be single, in which only one amino acid in the molecule is substituted; or may be multiple, in which two or more amino acids in the same molecule are substituted. Multiple substitutions can be located at consecutive positions.
  • an amino acid may be substituted by multiple residues, where such variants include both substitutions and insertions.
  • An “insertion type” variant is a variant in which one or more amino acids are inserted at a specific position in the immediate vicinity of a natural sequence. Immediately adjacent to an amino acid means to be attached to the ⁇ -carboxy or ⁇ -amino functional group of the amino acid.
  • a “deletion type” variant is a variant in which one or more amino acids in the natural amino acid sequence have been removed. Normally, deletion variants have one or two amino acids deleted in a specific region of the molecule.
  • variable domains of antibodies refers to certain parts of related molecules that have broad sequence differences between antibodies and are used for specific recognition and binding of specific antibodies against their specific targets.
  • variability is not evenly distributed throughout the variable domains of antibodies.
  • the variability is concentrated in three segments called complementarity determining regions (CDRs; namely CDR1, CDR2 and CDR3) or hypervariable regions, all of which are located in the variable domains of the light and heavy chains.
  • CDRs complementarity determining regions
  • FR framework regions or framework sequences.
  • Each variable domain of the natural heavy chain and light chain includes four FR regions, which mainly adopt a ⁇ -sheet configuration, which are connected by three CDRs, and the CDRs form a loop that connects the ⁇ -sheet structure and In some cases, a partial ⁇ -sheet structure is formed.
  • the CDRs of each chain are usually connected by FR regions adjacent to each other, and with the help of CDRs from other chains, help the formation of antibody target binding sites (epitopes or determinants) (see Kabat et al. Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)).
  • the numbering of immunoglobulin amino acid residues is based on the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise specified.
  • a CDR may have the ability to specifically bind to cognate epitopes.
  • an "antibody fragment” or “antigen-binding fragment” of an antibody refers to any part of a full-length antibody that is less than full-length but contains at least a portion of the variable region (e.g., one or more CDR and/or one or more antibody binding sites), and thus retain the binding specificity and at least part of the specific binding capacity of the full-length antibody. Therefore, an antigen-binding fragment refers to an antibody fragment that includes an antigen-binding portion that binds the same antigen as the antibody from which the antibody fragment is derived.
  • Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, For example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov).
  • the fragment may include multiple chains linked together, for example by disulfide bonds and/or by peptide linkers.
  • Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids.
  • Antigen-binding fragments include any antibody fragment that, when inserted into the antibody framework (for example, by replacing the corresponding region), obtains an antibody that immunospecifically binds (ie, exhibits a Ka of at least or at least about 10 7 -10 8 M-1) antigen .
  • “Functional fragments” or “anti-CD38 antibody analogs” are fragments or analogs that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction.
  • a functional fragment generally has the same meaning as an "antibody fragment", and in the case of an antibody, it can refer to a fragment that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction, such as Fv, Fab , F(ab') 2 and so on.
  • the "Fv" fragment is composed of a dimer (V H- VL dimer) formed by a non-covalent combination of the variable domain of a heavy chain and the variable domain of a light chain.
  • V H- VL dimer dimer
  • the three CDRs of each variable domain interact to determine the target binding site on the surface of the VH- VL dimer, as in the case of intact antibodies.
  • the six CDRs confer the target binding specificity of a complete antibody.
  • even a single variable domain or half of the Fv that includes only 3 target-specific CDRs) can still have the ability to recognize and bind to the target.
  • Bispecific antibody refers to the ability of antibodies and/or antigen-binding molecules to specifically bind to two different antigenic determinants, usually, bispecific antibodies and/or antigen-binding molecules Contains two antigen binding sites, each of which is specific for a different antigenic determinant.
  • the bispecific antibody and/or antigen binding molecule can simultaneously bind to two antigenic determinants, especially two antigenic determinants expressed on two different cells.
  • monoclonal antibody refers to a population of the same antibody, meaning that each individual antibody molecule in the monoclonal antibody population is the same as other antibody molecules. This characteristic is in contrast to the characteristic of the polyclonal population of antibodies, which contains antibodies with multiple different sequences.
  • Monoclonal antibodies can be prepared by many well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117).
  • monoclonal antibodies can be prepared by immortalizing B cells, for example by fusion with myeloma cells to generate hybridoma cell lines or by infecting B cells with viruses such as EBV.
  • Recombinant technology can also be used to prepare antibodies from a clonal population of host cells in vitro by transforming host cells with plasmids carrying artificial sequences of nucleotides encoding the antibodies.
  • hybridomas refers to a cell or cell line (usually a myeloma or lymphoma cell) produced by fusing antibody-producing lymphocytes and antibody-non-producing cancer cells.
  • hybridomas can proliferate and continue to be supplied to produce specific monoclonal antibodies. Methods for generating hybridomas are known in the art (see, for example, Harlow & Lane, 1988).
  • hybridodoma or “hybridoma cell” is referred to, it also includes subclones and progeny cells of the hybridoma.
  • a full-length antibody has two full-length heavy chains (for example, VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and hinge regions
  • VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4 and VL-CL full-length light chains
  • VL-CL full-length light chains
  • chimeric antibody refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as where the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody Of antibodies.
  • Humanized antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or Other antigen-binding subsequences of antibodies) contain minimal sequences derived from non-human immunoglobulins.
  • the humanized antibody is a human immunoglobulin (recipient antibody), in which the complementarity determining region (CDR) residues of the recipient antibody are derived from a non-human species with the desired specificity, affinity and ability ( Donor antibody) such as mouse, rat or rabbit CDR residue replacement.
  • CDR complementarity determining region
  • PCR-mediated mutations can be introduced to introduce mutations, and their impact on antibody binding or other functional properties can be assessed using the in vitro or in vivo tests described herein. Usually, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions. In addition, there are usually no more than one or two mutations in the CDR. Therefore, the humanized antibody of the present invention also encompasses antibodies containing 1 or 2 amino acid mutations in the CDR.
  • CDR refers to a complementarity-determining region, and it is known that each heavy chain and light chain of an antibody molecule has 3 CDRs. CDR is also called hypervariable region, and exists in the variable region of each heavy chain and light chain of an antibody, and has a very high variability site in the primary structure of the CDR.
  • the CDR of the heavy chain is represented by CDR1, CDR2, and CDR3 derived from the amino terminal of the amino terminal sequence of the heavy chain
  • CDR of the light chain is represented by CDR1, CDR2, CDR3 derived from the amino terminal of the amino terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.
  • epitope refers to any epitope on the antigen to which the paratope of an antibody binds.
  • Epitope determinants usually include chemically active surface typing of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics.
  • an antibody that immunospecifically binds (or specifically binds) an antigen has an affinity constant Ka ( or 1 ⁇ 10 -7 M or 1 ⁇ 10 7 M -1 or 1 ⁇ 10 8 M -1 or greater).
  • Ka affinity constant
  • Kd dissociation constant
  • the affinity constant can be determined by standard kinetic methods of antibody reaction, for example, immunoassay, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123: 1599), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art (see, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Patent No. 7,229,619 describing exemplary SPR and ITC methods for calculating the binding affinity of antibodies).
  • SPR surface plasmon resonance
  • ITC isothermal titration calorimetry
  • nucleic acid molecule refers to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including those usually linked together by phosphodiester bonds.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleic acid molecule is intended to include DNA molecules and RNA molecules.
  • the nucleic acid molecule can be single-stranded or double-stranded, and can be cDNA.
  • an isolated nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule.
  • isolated nucleic acid molecules such as cDNA molecules may be substantially free of other cellular materials or culture media when prepared by recombinant technology, or be substantially free of chemical precursors or other chemical components when chemically synthesized.
  • the exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.
  • operably linked with respect to nucleic acid sequences, regions, elements, or domains means that the nucleic acid regions are functionally related to each other.
  • a promoter can be operably linked to a nucleic acid encoding a polypeptide, so that the promoter regulates or mediates transcription of the nucleic acid.
  • the "conservative sequence modification" of the sequence described in the sequence listing described herein is also provided, that is, the nucleotide and amino acid sequence modifications that do not eliminate the binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence to the antigen.
  • conservative sequence modifications include conservative nucleotide and amino acid substitutions, and nucleotide and amino acid additions and deletions.
  • modifications can be introduced into the sequence listing described herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative sequence modifications include conservative amino acid substitutions in which amino acid residues are replaced with amino acid residues with similar side chains. Families of amino acid residues with similar side chains are already defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • amino acids with acidic side chains e.g., aspartic acid, glutamic acid
  • side chains with no electrical polarity e.g., Amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan)
  • amino acids with non-polar side chains e.g., alanine, valine
  • Acid, leucine, isoleucine, proline, phenylalanine, methionine amino acids with ⁇ -branched side chains (e.g.
  • the predicted non-essential amino acid residue in the anti-CD38 antibody is preferably replaced by another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (for example, see Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12 ( 10): 879-884 (1999); Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).
  • mutations can be randomly introduced along all or part of the anti-CD38 antibody coding sequence by, for example, saturation mutagenesis, and the resulting modified anti-CD38 antibody can be screened for improved binding activity.
  • expression refers to the process of producing a polypeptide through the transcription and translation of polynucleotides.
  • the expression level of the polypeptide can be evaluated by any method known in the art, including, for example, a method of measuring the amount of the polypeptide produced from the host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.
  • a "host cell” is a cell used to receive, maintain, replicate, and amplify a vector.
  • the host cell can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid.
  • the host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include but are not limited to CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.
  • a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell.
  • the vector includes those vectors into which a nucleic acid encoding a polypeptide or a fragment thereof can be introduced by restriction digestion and ligation.
  • Concerning vectors also include those containing nucleic acid encoding a polypeptide.
  • the vector is used to introduce the nucleic acid encoding the polypeptide into the host cell, to amplify the nucleic acid or to express/display the polypeptide encoded by the nucleic acid.
  • the vector usually remains free, but can be designed to integrate the gene or part of it into the chromosome of the genome.
  • Vectors for artificial chromosomes are also considered, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art.
  • vectors also include “viral vectors” or “viral vectors”.
  • the viral vector is an engineered virus that is operably linked to a foreign gene to transfer the foreign gene (as a vehicle or shuttle) into the cell.
  • expression vector includes a vector capable of expressing DNA that is operably linked to a regulatory sequence capable of affecting the expression of such DNA fragments, such as a promoter region. Such additional fragments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which when introduced into an appropriate host cell, results in the expression of the cloned DNA. Appropriate expression vectors are well known to those skilled in the art, and include expression vectors that are replicable in eukaryotic cells and/or prokaryotic cells, and expression vectors that remain free or that are integrated into the host cell genome.
  • treating an individual suffering from a disease or disease condition means that the individual's symptoms are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment and/or cure. Prevention refers to preventing underlying diseases and/or preventing symptoms from worsening or disease progression. Treatment also includes any pharmaceutical use of any of the provided antibodies or antigen-binding fragments thereof and the compositions provided herein.
  • therapeutic effect refers to the effect resulting from the treatment of an individual, which changes, generally improves or ameliorates the symptoms of a disease or disease condition, or cures the disease or disease condition.
  • terapéuticaally effective amount or “therapeutically effective dose” refers to the amount of a substance, compound, material, or composition containing the compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Therefore, it is the amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of the disease or disorder.
  • prophylactically effective amount or “prophylactically effective dose” refers to the amount of a substance, compound, material, or composition containing the compound that will have the expected preventive effect when administered to a subject, for example, to prevent or delay a disease or symptom The occurrence or recurrence of the disease, reducing the possibility of the occurrence or recurrence of diseases or symptoms.
  • a completely preventive effective dose need not occur by administering one dose, and may only occur after administering a series of doses. Therefore, the prophylactically effective amount can be administered in one or more applications.
  • the term "patient” refers to a mammal, such as a human.
  • the present disclosure provides an antibody or antigen-binding portion thereof that binds CD38, which comprises an amino acid sequence SEQ ID NO: 2-4, 12-14, 22-24, 32-34, 42-44, 52- 54, 62-64, 72-74, 82-84, 92-94, 102-144, 112-114, 122-124, 132-34, 142-144, 152-154, 162-164, 172-174, 182-184, 187-189, 192-194, 197-199, 202-204, 207-209, 212-214, 217-219, 222-224, 227-229, 232-234 or any variants thereof Chain CDR, and/or selected from the amino acid sequence SEQ ID NO: 7-9, 17-19, 27-29, 37-39, 47-49, 57-59, 67-69, 77-79, 87-89, 97-99, 107-109, 117-119, 127-129, 137-139, 147-149, 157-159,
  • the antibody or antigen-binding portion thereof which comprises an amino acid sequence SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132,
  • the heavy chain CDR2 is selected from the amino acid sequence SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144,
  • the antibody or antigen-binding portion thereof which comprises a combination of CDRs selected from the following heavy and light chains:
  • the antibody or antigen-binding portion thereof which comprises an amino acid sequence SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131,
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 1 or any variant thereof, and the amino acid sequence SEQ ID NO: 6 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 11 or any variant thereof, and the amino acid sequence SEQ ID NO: 16 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 21 or any variant thereof, and the amino acid sequence SEQ ID NO: 26 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 31 or any variant thereof, and the amino acid sequence SEQ ID NO: 36 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 41 or any variant thereof, and the amino acid sequence SEQ ID NO: 46 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 51 or any variant thereof, and the amino acid sequence SEQ ID NO: 56 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 61 or any variant thereof, and the amino acid sequence SEQ ID NO: 66 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 71 or any variant thereof, and the amino acid sequence SEQ ID NO: 76 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence of SEQ ID NO: 81 or any variant thereof, and the amino acid sequence of SEQ ID NO: 86 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 91 or any variant thereof, and the amino acid sequence SEQ ID NO: 96 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 101 or any variant thereof, and the amino acid sequence SEQ ID NO: 106 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 111 or any variant thereof, and the amino acid sequence SEQ ID NO: 116 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence of SEQ ID NO: 121 or any variant thereof, and the amino acid sequence of SEQ ID NO: 126 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 131 or any variant thereof, and the amino acid sequence SEQ ID NO: 136 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 141 or any variant thereof, and the amino acid sequence SEQ ID NO: 146 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 151 or any variant thereof, and the amino acid sequence SEQ ID NO: 156 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 161 or any variant thereof, and the amino acid sequence SEQ ID NO: 166 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 171 or any variant thereof, and the amino acid sequence SEQ ID NO: 176 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 181 or any variant thereof, and the amino acid sequence SEQ ID NO: 236 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 181 or any variant thereof, and the amino acid sequence SEQ ID NO: 241 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 181 or any variant thereof, and the amino acid sequence SEQ ID NO: 246 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 181 or any variant thereof, and the amino acid sequence SEQ ID NO: 251 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 181 or any variant thereof, and the amino acid sequence SEQ ID NO: 256 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence of SEQ ID NO: 186 or any variant thereof, and the amino acid sequence of SEQ ID NO: 236 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 186 or any variant thereof, and the amino acid sequence SEQ ID NO: 241 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 186 or any variant thereof, and the amino acid sequence SEQ ID NO: 246 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence of SEQ ID NO: 186 or any variant thereof, and the amino acid sequence of SEQ ID NO: 251 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence of SEQ ID NO: 186 or any variant thereof, and the amino acid sequence of SEQ ID NO: 256 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 191 or any variant thereof, and the amino acid sequence SEQ ID NO: 236 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 191 or any variant thereof, and the amino acid sequence SEQ ID NO: 241 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence of SEQ ID NO: 191 or any variant thereof, and the amino acid sequence of SEQ ID NO: 246 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence of SEQ ID NO: 191 or any variant thereof, and the amino acid sequence of SEQ ID NO: 251 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 191 or any variant thereof, and the amino acid sequence SEQ ID NO: 256 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 196 or any variant thereof, and the amino acid sequence SEQ ID NO: 236 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 196 or any variant thereof, and the amino acid sequence SEQ ID NO: 241 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 196 or any variant thereof, and the amino acid sequence SEQ ID NO: 246 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 196 or any variant thereof, and the amino acid sequence SEQ ID NO: 251 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 196 or any variant thereof, and the amino acid sequence SEQ ID NO: 256 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 201 or any variant thereof, and the amino acid sequence SEQ ID NO: 236 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 201 or any variant thereof, and the amino acid sequence SEQ ID NO: 241 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 201 or any variant thereof, and the amino acid sequence SEQ ID NO: 246 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 201 or any variant thereof, and the amino acid sequence SEQ ID NO: 251 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 201 or any variant thereof, and the amino acid sequence SEQ ID NO: 256 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 206 or any variant thereof, and the amino acid sequence SEQ ID NO: 236 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 206 or any variant thereof, and the amino acid sequence SEQ ID NO: 241 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 206 or any variant thereof, and the amino acid sequence SEQ ID NO: 246 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 206 or any variant thereof, and the amino acid sequence SEQ ID NO: 251 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 206 or any variant thereof, and the amino acid sequence SEQ ID NO: 256 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 211 or any variant thereof, and the amino acid sequence SEQ ID NO: 261 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 211 or any variant thereof, and the amino acid sequence SEQ ID NO: 266 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 216 or any variant thereof, and the amino acid sequence SEQ ID NO: 261 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 216 or any variant thereof, and the amino acid sequence SEQ ID NO: 266 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 221 or any variant thereof, and the amino acid sequence SEQ ID NO: 261 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 221 or any variant thereof, and the amino acid sequence SEQ ID NO: 266 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 226 or any variant thereof, and the amino acid sequence SEQ ID NO: 261 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 226 or any variant thereof, and the amino acid sequence SEQ ID NO: 266 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 231 or any variant thereof, and the amino acid sequence SEQ ID NO: 261 or The light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD38, which comprises the heavy chain variable region of the amino acid sequence SEQ ID NO: 231 or any variant thereof, and the amino acid sequence SEQ ID NO: 266 or The light chain variable region of any variant thereof.
  • the present disclosure provides a bispecific or multispecific molecule comprising the antibody or antigen binding portion thereof of any one of the foregoing aspects.
  • nucleic acid molecule encoding an antibody or an antigen binding portion thereof or a bispecific or multispecific molecule according to any of the foregoing aspects.
  • the nucleic acid molecule comprises SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155, 165, 175,
  • the antibody or antigen-binding portion thereof that binds to human CD38 has at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% with the antibody or antigen-binding portion of any one of the foregoing , 96%, 97%, 98%, 99% or higher sequence identity.
  • a nucleic acid molecule encoding an antibody or antigen-binding portion thereof according to any one of the foregoing aspects, or at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% therewith. %, 98%, 99% or higher sequence identity nucleic acid molecules.
  • a vector containing a nucleic acid as in any one of the foregoing is provided.
  • a cell containing a vector as in any one of the preceding aspects is a cell containing a vector as in any one of the preceding aspects.
  • a pharmaceutical composition comprising the antibody or antigen-binding portion or nucleic acid encoding thereof according to any one of the foregoing aspects and a pharmaceutically acceptable carrier.
  • the antibody of the present invention can be used as a therapeutic or diagnostic tool in various diseases in which CD38 is unfavorably expressed or discovered.
  • the diseases and symptoms particularly suitable for treatment with the CD38 antibody of the present invention are tumors or autoimmune diseases.
  • the tumors are selected from multiple myeloma (MM) and chronic lymphocytic leukemia (CLL), chronic myeloid leukemia Leukemia (CML), T-cell acute lymphoblastic leukemia, acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL).
  • the autoimmune disease is selected from rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE).
  • the method for treating diseases and symptoms with the CD38 antibody of the present invention includes the following steps: administering to the mammal a therapeutically effective amount of the antibody or antigen-binding fragment or nucleic acid molecule or carrier or cell or pharmaceutical composition of any one of the foregoing aspects.
  • the antibody is conjugated to other drugs, such as a labeled or cytotoxic conjugate.
  • kits for example, the kits include the antibodies of the present disclosure, fragments, homologues, derivatives thereof, etc., such as labeled or cytotoxic conjugates, and instructions for use of the antibody , Conjugates that kill specific types of cells, etc.
  • the instructions may include instructions for the use of antibodies, conjugates, etc. in vitro, in vivo, or ex vivo.
  • Antibodies can be in liquid form or solid, and are usually lyophilized.
  • the kit may contain other suitable reagents, such as buffers, reconstitution solutions, and other necessary components for the intended use. A combination of reagents packaged in predetermined amounts and instructions for their use are considered, such as for therapeutic use or for performing diagnostic assays.
  • the kit may include a substrate and cofactors required for the enzyme (for example, a substrate precursor that provides a detectable chromophore or fluorophore).
  • a substrate precursor that provides a detectable chromophore or fluorophore for example, a substrate precursor that provides a detectable chromophore or fluorophore.
  • other additives such as stabilizers, buffers (for example, blocking buffer or lysis buffer), etc. may also be included.
  • the relative amount of multiple reagents can be changed to provide a concentrate of the reagent solution, which provides user flexibility, space saving, reagent saving, etc.
  • These reagents can also be provided in the form of a dry powder, usually in a lyophilized form, including excipients, which can provide a reagent solution with an appropriate concentration when dissolved.
  • antibodies of the present invention can also be used in immunoassays, purification methods, and other methods using immunoglobulins or fragments thereof. Such uses are well known in the art.
  • the present invention also provides a composition comprising the anti-CD38 antibody of the present invention or a fragment thereof, which is conveniently combined with a pharmaceutically acceptable carrier, diluent or excipient, which is a common practice in the art .
  • composition refers to a preparation of various preparations.
  • the preparation containing a therapeutically effective amount of the multivalent antibody is in a sterile liquid solution, liquid suspension or lyophilized form, and optionally contains stabilizers or excipients.
  • the antibody of the present invention can be used as a composition to be administered alone, or can be used in combination with other active agents.
  • the therapeutic agents according to the described embodiments will be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc.
  • suitable pharmaceutically acceptable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc.
  • suitable formulations can be found in the pharmacopoeia known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, Pa. (1975)), especially Chapter 87 by Blaug, Seymour.
  • These formulations include for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic) vector (e.g.
  • Lipofectin TM DNA conjugates, anhydrous absorption pulp, oil in water And water-in-oil emulsion, emulsion polyethylene glycol (polyethylene glycol of various molecular weight), semi-solid gel and semi-solid mixture containing polyethylene glycol.
  • emulsion polyethylene glycol polyethylene glycol of various molecular weight
  • semi-solid gel and semi-solid mixture containing polyethylene glycol.
  • the antibody can be used as a therapeutic agent.
  • Such agents will generally be used to treat, alleviate and/or prevent diseases or pathologies associated with abnormal CD38 expression, activity, and/or signal transduction in a subject.
  • Standard methods can be used to implement a treatment regimen by identifying a subject, such as a human patient suffering from (or at risk or developing) a disease or disorder associated with abnormal CD38 expression, activity, and/or signaling, such as a CD38-related disorder.
  • An antibody preparation preferably an antibody preparation with high specificity and high affinity for its target antigen, is administered to a subject and will usually have an effect due to its binding to the target.
  • the administered antibody can eliminate or inhibit or hinder the expression, activity, and/or signaling function of the target (eg, CD38).
  • the administered antibody can eliminate or inhibit or prevent the binding of the target (eg CD38) to the endogenous ligand to which it naturally binds.
  • the antibody binds to the target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, and/or otherwise interferes with CD38 expression, activity, and/or signaling.
  • antibodies with heavy and light chain CDRs can be administered to the subject.
  • antibodies against CD38 can be used in methods known in the art related to the localization and/or quantification of CD38 (for example, for determining the level of CD38 and/or CD38 in an appropriate physiological sample, for Diagnostic methods, used for protein imaging, etc.).
  • an antibody specific for CD38 or its derivatives, fragments, analogs or homologues and comprising an antigen-binding domain derived from an antibody is used as a pharmacologically active compound (hereinafter referred to as "Therapeutics").
  • CD38 polypeptides can be separated using antibodies specific for CD38 by standard techniques such as immunoaffinity, chromatography or immunoprecipitation.
  • Antibodies against CD38 protein (or fragments thereof) can be used to detect proteins in biological samples.
  • CD38 can be detected in a biological sample as part of a clinical testing process, for example, to determine the efficacy of a given treatment regimen. Coupling (ie, physically linking) the antibody to a detectable substance can facilitate detection. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/ Biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include Lu Minnow; Examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 H.
  • the antibodies according to the present disclosure can be used as reagents for detecting the presence of CD38 or protein fragments thereof in a sample.
  • the antibody comprises a detectable label.
  • the antibody is a polyclonal antibody, or more preferably a monoclonal antibody. Use whole antibodies or fragments thereof (e.g. Fab, scFv or F(ab') 2 ).
  • labeling with respect to an antibody is intended to include directly labeling the antibody by coupling (ie, physically connecting) a detectable substance to the antibody, and indirectly labeling the antibody by reacting with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of the first antibody using a fluorescently-labeled second antibody, and end-labeling of the antibody with biotin to enable detection with fluorescently-labeled streptavidin.
  • bio sample is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present in the subject. Therefore, the term “biological sample” is used to include blood and fractions or components in blood, including serum, plasma, or lymph.
  • the detection method of the described embodiment can be used to detect analyte mRNA, protein, or genomic DNA in a biological sample in vitro and in vivo.
  • in vitro detection techniques for analyte mRNA include Norhtern hybridization and in situ hybridization.
  • In vitro detection techniques for analyte proteins include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence.
  • In vitro detection techniques for analyte genomic DNA include Southern hybridization. The procedure for performing immunoassays is described in, for example, "ELISA: Theory and Practice: Methods in Molecular Biology", Volume 42, JRCrowther (Editor) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T.
  • analyte protein includes the introduction of labeled anti-analyte protein antibodies into the subject.
  • the antibody can be labeled with a radioactive label, and then the presence and location of the radioactive label in the subject can be detected by standard imaging techniques.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • the principles and considerations involved in preparing such compositions and guidelines for selecting components are well known in the art, for example, see Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy 19th edition (edited by Alfonso R. Gennaro et al.) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances Science, Parental Volume 4), 1991, M. Dekker, New York.
  • compositions generally include an antibody and a pharmaceutically acceptable carrier.
  • antibody fragments When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein may be preferred.
  • peptide molecules that retain the ability to bind the target protein sequence can be designed.
  • Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)).
  • the term "pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are compatible with drug administration .
  • Suitable pharmaceutically acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard bibliography in the field, which is incorporated herein by reference.
  • Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as immobilized oils can also be used.
  • the use of such media and reagents for pharmaceutically active substances is well known in the art. Except for the incompatibility of any conventional medium or reagent with the antibody, its use in the composition is envisaged.
  • the pharmaceutical composition of the described embodiments is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e. topical), transmucosal, and rectal administration.
  • Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: sterile diluents for injection such as water, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; Antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate, citrate Or phosphate, and an agent that regulates osmotic pressure, such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (herein water-soluble) or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions.
  • suitable pharmaceutically acceptable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability is achieved. It must be stable under the conditions of manufacture and storage and must be able to prevent the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.
  • polyol for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.
  • suitable fluidity can be maintained.
  • antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like.
  • isotonic agents such as sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride.
  • Prolonged absorption of the composition for injection can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.
  • a sterile injectable solution can be prepared by incorporating the antibody in the required amount into a suitable solvent having one or a combination of the ingredients listed above (as required), and then filter sterilized.
  • dispersions are prepared by incorporating the antibody into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above.
  • the preparation methods are vacuum drying and freeze drying to obtain powders containing active ingredients and any additional desired ingredients, which are derived from the aforementioned sterile filtered solutions of these ingredients .
  • the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser or nebulizer containing a suitable propellant such as a gas such as carbon dioxide.
  • transmucosal or transdermal administration penetrants suitable for penetrating the barrier are used in the formulation.
  • penetrants are generally generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration.
  • Transmucosal administration can be achieved through the use of nasal sprays or suppositories.
  • one or more of the antibodies can be formulated as an ointment, ointment, gel, or cream as commonly known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with a conventional suppository base such as cocoa butter or other glycerides) or retention enemas for transrectal delivery.
  • suppositories e.g., with a conventional suppository base such as cocoa butter or other glycerides
  • retention enemas for transrectal delivery.
  • the antibody can be prepared with a carrier that prevents it from being rapidly eliminated by the body, such as a sustained/controlled release formulation, including implants and microencapsulated delivery systems.
  • a sustained/controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. The methods for preparing such formulations will be obvious to those skilled in the art.
  • dosage unit form refers to a physically separable unit suitable as a unit dose for the subject to be treated; each unit contains a predetermined amount calculated in combination with the required pharmaceutical carrier to produce the desired therapeutic effect One or more of said antibodies.
  • the specifications of the dosage unit form of the embodiment are indicated by and directly depend on the unique characteristics of the antibody and the specific therapeutic effect to be achieved, and the limitations inherent in the field of formulation of such antibodies for the treatment of individuals.
  • the pharmaceutical composition can be placed in a container, package, or dispenser together with instructions for administration.
  • compositions described herein may also contain more than one of the antibodies, depending on the specific situation to be treated, preferably those with complementary activities but not negatively affecting each other.
  • the composition may, for example, comprise an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent.
  • cytotoxic agent such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent.
  • cytokine such as cytokine, chemotherapeutic agent, or growth inhibitory agent.
  • growth inhibitory agent such molecules are suitably present in combination in an amount effective for the intended purpose.
  • they can be co-existing in the kit, or they can be co-existing in use.
  • one or more of the antibodies can be administered in combination therapy, that is, with other agents such as therapeutic agents (which can be used to treat pathological conditions or disorders, such as various forms of cancer, autoimmune disorders And inflammatory diseases).
  • therapeutic agents which can be used to treat pathological conditions or disorders, such as various forms of cancer, autoimmune disorders And inflammatory diseases.
  • combined herein refers to the administration of agents substantially simultaneously, simultaneously or sequentially. If administered sequentially, when the second compound is initially administered, the first of the two compounds is still preferably detected at an effective concentration at the treatment site.
  • “combination” can also mean that the antibody of the present invention and other therapeutic agents are included in the kit.
  • the combination therapy may comprise one or more antibodies described herein and one or more additional therapeutic agents (e.g., one or more cytokines and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors , Enzyme inhibitors, and/or cytotoxins or cell growth inhibitors, as described in more detail below) co-formulated and/or co-administered.
  • additional therapeutic agents e.g., one or more cytokines and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors , Enzyme inhibitors, and/or cytotoxins or cell growth inhibitors, as described in more detail below.
  • Such combination therapies can advantageously utilize lower doses of administered therapeutic agents, thus avoiding possible toxicity or complications associated with various monotherapy.
  • lentiviral vector plasmid containing the full-length sequence of human CD38.
  • the lentivirus packaging kit instructions (Lenti-Pac HIV Expression Packaging Kit, Gene Copoeia, Cat: HPK-LvTR-20), the constructed lentiviral plasmid and packaging The plasmids were co-transfected into HEK293T cells for lentivirus packaging. After 48 hours of transfection, the medium was collected, centrifuged at 500*g for 10 minutes to remove cell debris, and the culture supernatant containing lentiviral particles was obtained. After filtration with a 0.45 ⁇ m PES membrane, the medium was aliquoted into 1.5mL EP tubes and 10 ⁇ L was used.
  • Human CD38 recombinant protein or CHO-CD38-1H7 cells were used as antigens and were mixed with an equal amount of immune adjuvant (Freund's adjuvant), and each group of 6 6-week female Balb/c mice was subcutaneously immunized. Two weeks after the initial immunization, a booster immunization was performed. After three immunizations, blood was taken from the orbit to test the serum titer.
  • mice Before fusion, 1x10 6 CHO-CD38-1H7 cells were injected into the tail vein for impulse immunization. Three days later, the mice were sacrificed by neck dissection. The mouse spleen and some peripheral lymph nodes were taken, ground in DMEM medium and centrifuged; after pouring the supernatant, gently disperse the spleen cell mass, add 5 mL of red blood cell lysate, and lyse for 50 seconds Afterwards, 40 mL of DMEM was added and centrifuged to obtain a spleen cell suspension without red blood cells. After mixing an appropriate amount of lymph node and spleen cell mixture with SP2/0, use a BTX electrofusion instrument for cell fusion.
  • the fusion cells were seeded in a 96-well plate, and cultured at 37°C under 5% CO 2 in DMEM complete medium containing HAT. Start to observe the growth of hybridoma cells in about a week, and take the supernatant for testing when it grows to more than 60%.
  • the supernatant of positive clones that bind to CD38 antibody detected by ELISA was further verified for binding to CD38 positive cells.
  • variable region sequence after sequencing, the candidate positive clone heavy chain and light chain variable region sequences obtained are:
  • the heavy chain variable region is cloned into a vector containing the human heavy chain constant region and regulatory elements to express the complete IgG heavy chain in mammalian cells.
  • the light chain variable region is cloned into a vector containing a human light chain constant region and regulatory elements to express a complete IgG light chain in mammalian cells. After being sequenced correctly, it was transfected into CHO-S mammalian cells, and IgG was expressed and secreted into the culture medium. The supernatant was collected, filtered and purified.
  • IgG was purified by Protein A chromatography, the culture supernatant was loaded on a protein A column of appropriate size, washed with 50mM Tris-HCl pH8.0, 250mM NaCl, and combined with 0.1M Glycine-HCl (pH3.0) The IgG elutes. The protein was concentrated by ultrafiltration using a concentration tube, OD280 was detected, and the concentration of IgG was determined by spectrophotometry.
  • Daudi cells were resuspended and spread into a 96-well plate at 50 ⁇ L/well, and then 50 ⁇ L of antibody was added to each well. Dilute the humanized antibody to 10 ⁇ g/mL, use this as the starting concentration, and dilute 3 times in a gradient for a total of 3 gradients.
  • CD38 catalyzes the synthesis and degradation of cyclic adenosine diphosphate ribose (cADPR, cyclic ADP-ribose), which can cyclize nicotinamide adenine dinucleotide (NAD+) into cyclic adenosine diphosphate ribose (cADPR) and generate The cADPR is hydrolyzed to ADPR. Because cADPR is unstable, in the study of CD38 enzyme activity, nicotinamide guanine dinucleotide (NGD+) is usually used as a substrate to detect the catalytically generated cGDPR.
  • cADPR cyclic adenosine diphosphate ribose
  • NGD+ nicotinamide guanine dinucleotide
  • Anti-CD38 antibody can bind to CD38, thereby inhibiting CD38 protease from catalyzing NGD+ to generate cGDPR.
  • the mouse antibody light chain of mAb48H1-B11 is mouse IMGT_mVK13-84.
  • the humanized framework region was screened.
  • IMGT_hVK_1_39, IMGT_hVK_2D_28, IMGT_hVK_3_7, IMGT_hVK_3_20 and IMGT_hVK_4_1 were selected for CDR grafting, and 5 humanized light chains were obtained.
  • h48H1-Vkv1-v5 the mouse antibody heavy chain is IGHV2-5
  • IMGT_hVH_1_69, IMGT_hVH_2_26, IMGT_hVH_3_23, IMGT_hVH_4_59, IMGT_hVH_5_51 and IMGT_hVH_7_4_1 were selected for transplantation of 48 human CDR heavy chains to obtain 6 humanized H6VHvHv1 variants. After combining and pairing the light and heavy chain variants, they were transiently expressed in CHO-S cells, and the affinity of each group and the antibody and CDC activity were detected.
  • the human IMGT_hVK3-20 with the highest homology to the light chain framework region of the murine antibody mAb48H1-B11 was selected for humanization sequence optimization, and the one with the highest homology was selected for FM4 Human IGKJ4*01; human heavy chain germline gene IMGT_hVH4-59 was selected for CDR transplantation, and human IGHJ4*01 with the highest homology was selected for FM4.
  • a total of 5 heavy chain variants h48H1-VHv7-v11 and 2 light chain variants h48H1-VKv7-v8 were designed. After the light chain and heavy chain were synthesized respectively, they were cloned into Ckappa or human containing the constant region of the antibody kappa chain.
  • the eukaryotic expression vector of IgG1 constant region CH1-CH3 after the light and heavy chain plasmids are combined and paired, they are transfected into CHO-S cells and expressed at 37°C for 5-6 days. The culture supernatant is collected and purified by a Protein A column.
  • the sequence of the humanized antibody heavy chain/light chain variable region is as follows:
  • the 96-well microtiter plate was coated with human CD38 protein and incubated overnight at 4°C. Then the solution in the well was discarded, washed 3 times with washing buffer, and blocked with a PBS solution containing 2% milk for 60 minutes.
  • FACS analysis was used to detect the expression level of CD38 on different tumor cells. 1*10 6 different tumor cells were collected by centrifugation and washed once with 3mL PBS; after blocking for 20 minutes, 50 ⁇ l of diluted primary antibody solution anti-human CD38hIgG1-biotin ( anti-human CD38hIgG1-biotin), incubate on ice for 40 minutes; wash twice, add 100 ⁇ l of diluted secondary antibody solution SA-PE (Jackson, catalog number: 016110084) to each well, incubate on ice for 30 minutes in the dark, wash twice Collect the cells by centrifugation; add 200 ⁇ l PI diluent to each well, gently pipette to mix, and at the same time take a standard BD Qusntibrite PE tube (BD, catalog number: 340495), add 500 ⁇ l 0.5% BSA/PBS solution in the dark, and place Incubate on ice and perform flow testing on the computer.
  • SA-PE Jointson, catalog
  • the results are shown in Figures 11-1, 11-2, 11-3, 11-4, 11-5, 11-6, 11-7, 11-8.
  • the humanized antibodies Ab031 and Ab033 have a positive effect on Ramos.2G6.4C10.
  • the cell killing rate was 90%, the killing rate of NALM-6 cells was 65%, the killing rate of RPMI-8226 cells was 67%, the killing rate of CRFF-CEM cells was 48%, and the killing rate of Daudi cells was 62%.
  • the killing rate for TALL-1 cells was 40%, the killing rate for MM.1S cells was 20%, the killing rate for NCI-H929 cells was 48%, and the killing rate for Raji cells was 75%.
  • the CD38 recombinant protein was mixed with the humanized CD38 antibodies Ab031 and Ab0337 after serial dilutions, and incubated at room temperature for 15 minutes; NGD substrate was added at different time points and mixed for detection; the result is shown in Figure 13, the chimeric antibody Both 48H1-B11 and humanized CD38 antibodies Ab031 and Ab033 have a significant inhibitory effect on CD38 enzyme activity.
  • Example 13 Apoptosis induced by humanized CD38 antibody
  • Example 14 Humanized CD38 antibody induces human red blood cell lysis
  • Example 15 Anti-tumor effect of humanized CD38 antibody in Daudi cell line subcutaneous xenograft NOD/SCID mouse model
  • Raji cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. Raji cells in logarithmic growth phase were collected, resuspended in PBS to a suitable concentration and mixed with matrigel 1:1, and used for subcutaneous tumor inoculation in NOD/SCID mice.
  • Female mice were inoculated subcutaneously with 1 ⁇ 10 7 Raji cells on the right side. When the average tumor volume was 100 mm 3 , they were randomly grouped according to the tumor size. There were 6 mice in each group. The mice in the treatment group were injected into the tail vein twice a week at a dose of 10 mg/kg for a total of two weeks, and the tumor volume was monitored.
  • CM313-1 is of human origin.
  • Antibody Ab031, CM313-2 is humanized antibody Ab033
  • CM313-3 is antibody-005 (US20150231235A1)
  • CM313-4 is 38SB19 (US20110293606A1).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Rheumatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Pain & Pain Management (AREA)
  • Oncology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Hematology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

本公开涉及一种结合CD38蛋白的抗体或其抗原结合部分及其制备方法和应用。该抗体可高亲和性地结合人CD38，对CD38酶具有抑制作用，对不同肿瘤细胞具有CDC、ADCC和/或ADCP杀伤活性，具有抗肿瘤功能。并且，该抗体不会引起红细胞裂解。

Description

药物组合物及其制备方法和应用 技术领域
本发明涉及一种CD38的抗体及其制备方法和应用。
背景技术
CD38是一种46kDa的II型跨膜糖蛋白，N-末端含21个氨基酸残基，位于胞内，跨膜区含21个氨基酸，C-末端为258个氨基酸残基构成的胞外区。CD38通过与CD31或透明质酸的相互作用，在调节迁移和受体介导的粘附中发挥作用，此外，CD38还具有胞外酶活性并参与核苷酸代谢物的产生，在细胞内钙储存的控制中起作用。在一些正常组织中，如骨髓和淋巴样细胞，以及一些非造血细胞中，CD38以相对低的水平表达；相反，在浆细胞和一些恶性细胞中，通常具有高水平的CD38表达，包括多发性骨髓瘤(multiple myeloma，MM)和慢性淋巴细胞白血病(chronic lymphocytic leukemia，CLL)等，表达丰度的差异使得CD38成为靶向多发性骨髓瘤细胞治疗的靶标分子。
多发性骨髓瘤是一种浆细胞癌，其特征在于骨髓中恶性细胞的积累和单克隆免疫球蛋白(M蛋白)的产生。尽管近年来新的治疗方法的引入，患者的中位总生存率已显著提升，但通常在一年内复发并产生耐药。daratumumab是一种CD38靶向抗体，已批准上市用于复发/难治性多发性骨髓瘤，其他几种正在进行临床试验的单克隆抗体包括isatuximab，MOR202和TAK-079等，针对的适应症除了复发/难治性多发性骨髓瘤，还包括新诊断的多发性骨髓瘤。CD38抗体在其他恶性肿瘤，如：NK/T细胞淋巴瘤、T细胞急性淋巴细胞白血病和免疫球蛋白轻链淀粉样变性，也显示出良好的治疗效果。
类风湿关节炎(rheumatoidarthritis，RA)是一种自身免疫性疾病，病变滑膜内存在大量B淋巴细胞增生。流式结果显示RA患者外周血中CD38+、CD3+CD38+和CD56+CD38+亚群细胞表达率明显升高，且CD38+细胞的表达率与RA患者类风湿因子(RF)水平显著相关；免疫组织化学结果表明CD38在RA患者滑膜中特异性高表达；ELISA实验证实：对CD38基因进行siRNA干扰后，RASF培养基中的IL-1α和IL-β表达水平显著降低，CD38基因的表达增加可能参与RA患者的免疫激活(CD38在类风湿关节炎外周血与滑膜组织中的表达.岳龙涛，等.现代免疫学,第89-93页，2014年第2期)。CD38在其他自身免疫性疾病例如系统性红斑狼疮(systemic lupus erythematosus，SLE)外周血中明显升高，在SLE的发生发展过程中起到重要作用。尽管在体外或临床前动物实验中，多种CD38抗体均显示了非常显著的抑瘤效果，但在临床研究中发现，不同抗体的治疗效果和反应持续时间存在显着的异质性，而这种差异可能和抗体的作用机制相关。例如，在CD38抗体治疗后，患者血液中的NK细胞很快被耗竭，主要利用抗体依赖的细胞毒作用(ADCC)的抗体可能由于缺乏效应细胞，作用受到限制；同时兼具另一种作用机制的抗体(如daratumumab具有补体依赖的细胞毒作用，CDC)，显示在治疗总有效率和生存时间上，高于其他处于临床研究阶段的CD38抗体。有研究通过在IgG1的Fc上引入突变，或将IgG1铰链区置换为IgG3铰链区，提高了抗体的CDC效能。最近也有研究通过IgG1的Fc突变使得单抗形成六聚体，有效增强了CD38抗体的杀伤效能，但带来一些生产和开发上的困难。
发明内容
本发明人以CD38重组蛋白免疫小鼠，得到了多种新型高亲和力CD38抗体，其具有同类最强的CDC、ADCC和抗体依赖的细胞吞噬作用(ADCP)活性，同时还具有抑制CD38胞外酶活性，以及在Fc交联条件下引起细胞凋亡的功能。本发明人更进一步通过在抗体Fc段引入新的突变，所获得的新型CD38抗体的肿瘤杀伤效率远高于目前市场上的同类抗体。在一方面，本公开提供了结合CD38蛋白的抗体或其抗原结合部分。
在一方面，本公开提供了编码如前述方面的抗体或其抗原结合部分的核酸分子。
在一方面，本公开提供了含有前述方面的核酸的载体。
在一方面，本公开提供了含有前述方面的载体的细胞。
根据前述任一方面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是人源化的。
在一方面，本公开提供了包含如前述任一方面的抗体或其抗原结合部分或其编码核酸和药学上可接受的载体的药物组合物或试剂盒。
在一方面，本公开提供了治疗CD38相关病症的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的前述任一方面的抗体或其抗原结合片段、核酸分子、载体、细胞和/或药物组合物。
前述任一方面的抗体或其抗原结合片段、核酸分子、载体、细胞和/或药物组合物在制备用于治疗哺乳动物中CD38相关病症的药物或试剂盒中的用途。
所述抗体可高亲和性地结合人CD38，对CD38酶具有抑制作用，对不同肿瘤细胞具有CDC、ADCC和/或ADCP杀伤活性，具有抗肿瘤功能。并且，该抗体不会引起红细胞裂解。
附图说明
图1、HEK293细胞重组表达的人CD38胞外区蛋白
图2、流式细胞术检测人CD38/CHO稳转细胞
图3-1、嵌合抗体对Duadi细胞的补体依赖性细胞毒性(CDC)作用
图3-2、嵌合抗体对Duadi细胞的补体依赖性细胞毒性(CDC)作用
图4-1、嵌合抗体对CD38酶活性的抑制作用
图4-2、嵌合抗体对CD38酶活性的抑制作用
图5、嵌合抗体对Duadi细胞的抗体依赖性细胞毒性(ADCC)杀伤活性
图6、嵌合抗体对Duadi细胞的抗体依赖性细胞吞噬作用(ADCP)杀伤活性
图7-1、不同人源化抗体对Daudi细胞的补体依赖性细胞毒性(CDC)作用
图7-2、不同人源化抗体对Daudi细胞的补体依赖性细胞毒性(CDC)作用
图8-1、ELISA亲和力测定
图8-2、抗CD38人源化抗体与Daudi细胞的结合
图8-3、抗CD38人源化抗体与NALM-6细胞的结合
图8-4、抗CD38人源化抗体与Ramos.2G6.4C10细胞的结合
图8-5、抗CD38人源化抗体与TALL-1细胞的结合
图9、CD38在不同肿瘤细胞上的表达量
图10-1、抗CD38人源化抗体对Ramos.2G6.4C10的补体依赖性细胞毒性(CDC)作用
图10-2、抗CD38人源化抗体对Daudi的补体依赖性细胞毒性(CDC)作用
图10-3、抗CD38人源化抗体对TALL-1的补体依赖性细胞毒性(CDC)作用
图11-1、抗CD38人源化抗体对Ramos.2G6.4C10的抗体依赖性细胞毒性(ADCC)作用
图11-2、抗CD38人源化抗体对NALM-6的抗体依赖性细胞毒性(ADCC)作用
图11-3、抗CD38人源化抗体对RPMI-8226的抗体依赖性细胞毒性(ADCC)作用
图11-4、抗CD38人源化抗体对CCRF-CEM的抗体依赖性细胞毒性(ADCC)作用
图11-5、抗CD38人源化抗体对Daudi的抗体依赖性细胞毒性(ADCC)作用
图11-6、抗CD38人源化抗体对TALL-1的抗体依赖性细胞毒性(ADCC)作用
图11-7、抗CD38人源化抗体对MM.1S的抗体依赖性细胞毒性(ADCC)作用
图11-8、抗CD38人源化抗体对NCI-H929的抗体依赖性细胞毒性(ADCC)作用
图11-9、抗CD38人源化抗体对Raji的抗体依赖性细胞毒性(ADCC)作用
图12-1、抗CD38人源化抗体对Daudi的抗体依赖性细胞吞噬作用(ADCP)作用
图12-2、抗CD38人源化抗体对TALL-1的抗体依赖性细胞吞噬作用(ADCP)作用
图12-3、抗CD38人源化抗体对Ramos.2G6.4C10的抗体依赖性细胞吞噬作用(ADCP)作用
图12-4、抗CD38人源化抗体对Raji的抗体依赖性细胞吞噬作用(ADCP)作用
图12-5、抗CD38人源化抗体对NALM-6的抗体依赖性细胞吞噬作用(ADCP)作用
图13、人源化CD38抗体对CD38酶活抑制作用分析
图14、人源化CD38抗体诱导的Daudi细胞凋亡
图15、人源化CD38抗体诱导人红细胞裂解分析
图16、CD38抗体对人B细胞淋巴瘤Daudi皮下移植瘤生长的影响
具体实施方式
I.定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在一个方面，本文提供的是特异性结合CD38的抗体(例如，单克隆抗体)及其抗原结合片段。在具体的方面，本文提供的是特异性结合CD38的单克隆抗CD38抗体，其中所述抗CD38抗体包括亲本抗体的变体。在具体的方面，本文提供的是特异性结合CD38(例如，人CD38)的抗体。在特定的方面，本文提供的是包含一个或更多个氨基酸残基中的修饰的抗CD38抗体(例如，重链可变区的框架区中5-13个氨基酸取代)，与没有所述修饰的亲本抗体相比，其保持与抗原的亲和力。
如本文使用的和除非另作说明，术语“约”或“大约”是指在给定值或范围的加或减10％之内。在需要整数的情况下，该术语是指在给定值或范围的加或减10％之内、向上或向下舍入到最接近的整数。
就抗体链多肽序列而言，短语“基本相同”可理解为表现出与参照多肽序列至少60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更多的序列同一性的抗体链。就核酸序列而言，该术语可理解为表现出与参照核酸序列至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性的核苷酸序列。
序列“相同性”或“同一性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列相同性(参见，例如：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Pres,New York,1988；Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993；Computer Analysis of Sequence Data,Part I, Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994；Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987；and Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991)。虽然存在许多测量两个多核苷酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo,H.&Lipman,D.,SIAM J Applied Math 48:1073(1988))。
“取代型”变体是天然序列中至少一个氨基酸残基被除去并被不同的氨基酸插入其相同位置的变体。所述取代可为单个的，其中该分子中仅有一个氨基酸被取代；或可为多个的，其中该相同分子有两个或更多的氨基酸被取代。多个取代可位于连续的位点。同样，一个氨基酸可被多个残基取代，其中这样的变体包括取代和插入二者。“插入型”变体是一个或多个氨基酸被插入到紧邻一段天然序列某个特定位置处的氨基酸的变体。紧邻氨基酸意指与该氨基酸的α-羧基或α-氨基官能团连接。“缺失型”变体是天然氨基酸序列中一个或多个氨基酸被除去的变体。通常情况下，缺失型变体在其分子的特定区域内有一个或两个氨基酸被缺失。
就抗体的可变结构域而言，术语“可变”系指抗体之间有广泛序列差异的相关分子的某些部分，且被用于针对其特异靶的特定抗体的特异识别和结合。但是，可变性在抗体的整个可变结构域内不是均匀分布的。可变性集中在被称为互补决定区域(CDRs；即CDR1、CDR2和CDR3)或超变区的三个区段，它们均位于轻链和重链的可变结构域内。可变结构域内保守程度更高的部分被称为构架(FR)区或构架序列。天然重链和轻链的每个可变结构域均包括四个FR区，其主要采用β-折叠构型，它们籍三个CDRs连接起来，CDRs形成环，所述环连接β-折叠结构并在某些情形下形成部分的β-折叠结构。每条链的CDRs通常被FR区在邻近连接起来，并且借助于来自其它链的CDR，有助于抗体靶结合位点(表位或决定簇)的形成(参看Kabat等人Sequences of Proteins of Immunological Interest,NationalInstitute of Health,Bethesda,MD(1987))。正如本文所使用，免疫球蛋白氨基酸残基的编号是依据Kabat等人的免疫球蛋白氨基酸残基编号系统而进行的，除非另有说明。一个CDR可具有特异结合关联表位的能力。
如本文所用，抗体的“抗体片段”或“抗原结合片段”指全长抗体的任何部分，其少于全长，但是至少包含结合抗原的所述抗体的部分可变区(例如一个或多个CDR和/或一个或多个抗体结合位点)，并且因此保留结合特异性以及所述全长抗体的至少部分特异性结合能力。因此，抗原结合片段指包含与衍生抗体片段的抗体结合相同抗原的抗原结合部分的抗体片段。抗体片段包括通过酶促处理全长抗体所产生的抗体衍生物，以及合成产生的衍生物，例如重组产生的衍生物。抗体包括抗体片段。抗体片段的实例包括但不限于Fab、Fab'、F(ab') 2、单链Fv(scFv)、Fv、dsFv、双抗体、Fd和Fd'片段以及其他片段，包括修饰的片段(参见，例如，Methods in Molecular Biology，Vol 207:Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003)；Chapter 1；p 3-25,Kipriyanov)。所述片段可以包括连接在一起的多条链，例如通过二硫键和/或通过肽接头。抗体片段一般包含至少或约50个氨基酸，并且典型至少或约200个氨基酸。抗原结合片段包括任何抗体片段，其在被插入抗体框架(例如通过置换相应区域)时获得免疫特异性地结合(即表现出至少或至少约10 7-10 8M-1的Ka)抗原的抗体。“功能片段”或“抗CD38抗体的类似物”是可防止或实质降低所述受体结合配体或启动信号转导的能力的片段或类似物。正如本文所使用，功能片段一般与“抗体片段″含义相同，且就抗体而论，可指能防止或实质降低所述受体结合配体或启动信号转导的能力的片段，例如Fv、Fab、F(ab') 2等等。“Fv”片段由一条重链的可变结构域和一条轻链的可变结构域籍非共价结合方式而形成的二聚体(V H-V L二聚体)组成。在该构型中，每个可变结构域的三个CDRs相互作用，以确定V H-V L二聚体表面上的靶结合位点，与完整抗体的情况一样。所述六个CDRs共同赋予完整抗体的靶结合特异性。但是，即使是单个可变结构域(或仅包括3个靶特异的CDRs的Fv的一半)，仍可具有识别和结合靶的能力。
如本文中所用，术语“双特异性”(Bispecific antibody，BsAb)指抗体和/或抗原结合分子能够特异性结合两种不同的抗原性决定簇，通常，双特异性抗体和/或抗原结合分子包含两种抗原结合位点，其中每种特异于不同的抗原性决定簇。在某些实施方案中，所述双特异性抗体和/或抗原结合分子能够同时结合两种抗原决定簇，特别是在两种不同的细胞上表达的两种抗原性决定簇。
如本文所用，“单克隆抗体”指相同抗体的群体，表示单克隆抗体群体中的每个单独的抗体分子与其他抗体分子相同。这种特性与抗体的多克隆群体的特性相反，所述抗体的多克隆群体包含具有多种不同序列的抗体。单克隆抗体可以通过许多公知的方法来制备(Smith et al.(2004)J.Clin.Pathol.57,912-917；和Nelson et al.,J Clin Pathol(2000),53,111-117)。例如，单克隆抗体可以通过永生化B细胞来制备，例如通过与骨髓瘤细胞融合以产生杂交瘤细胞系或者通过用诸如EBV的病毒感染B细胞。重组技术还可以用来在体外通过用携带编码抗体的核苷酸的人工序列的质粒转化宿主细胞来从宿主细胞的克隆群体制备抗体。
如本文中所用，术语“杂交瘤”或“杂交瘤细胞”指由融合产抗体的淋巴细胞和不产抗体的癌细胞而产生的细胞或细胞系(通常为骨髓瘤或淋巴瘤细胞)。如本领域普通技术人员所知的，杂交瘤可增殖并持续供应产生特定单克隆抗体。用于产生杂交瘤的方法为本领域已知的(见例如，Harlow&Lane，1988)。当提及术语“杂交瘤”或“杂交瘤细胞”时，其还包括杂交瘤的亚克隆和后代细胞。
如本文所用，全长抗体是具有两条全长重链(例如VH-CH1-CH2-CH3或VH-CH1-CH2-CH3-CH4)和两条全长轻链(VL-CL)和铰链区的抗体，例如通过抗体分泌B细胞天然产生的抗体以及合成产生的具有相同结构域的抗体。
术语“嵌合抗体”是指这样的抗体，其中可变区序列源自一个物种，恒定区序列源自另一物种，如其中可变区序列源自小鼠抗体及恒定区序列源自人抗体的抗体。
“人源化”抗体是指非人(例如小鼠)抗体形式，其是嵌合的免疫球蛋白、免疫球蛋白链或者其片段(如Fv、Fab、Fab'、F(ab') 2或者抗体的其它抗原结合亚序列)，含有源自非人免疫球蛋白的最小序列。优选地，人源化抗体是人免疫球蛋白(接受者抗体)，其中接受者抗体的互补决定区(CDR)的残基由来自具有希望的特异性、亲和性和能力 的非人物种(供体抗体)如小鼠、大鼠或者兔的CDR残基置换。
此外，在人源化中，还可能对VH和/或VL的CDR1、CDR2和/或CDR3区内的氨基酸残基进行突变，由此改善抗体的一或多种结合特性(例如亲和性)。可进行例如PCR介导的突变引入突变，其对抗体结合或其它功能特性的影响可利用本文所述的体外或体内测试评估。通常，引入保守性突变。此类突变可为氨基酸取代、添加或缺失。另外，CDR内的突变通常不超过一个或两个。因此，本发明所述人源化抗体还涵盖CDR内包含1或2两个氨基酸突变的抗体。
如本文所用，术语“CDR”指互补决定区(complementarity-determining region)，已知抗体分子的每个重链和轻链具有3个CDR。CDR也称作高变区，且存在于抗体的每个重链和轻链的可变区中，在CDR的一级结构中具有非常高的变异性位点。本说明书中，重链的CDR由来自重链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示，轻链的CDR由来自轻链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示。这些位点在三级结构中彼此临近，并决定抗体所结合的抗原的特异性。
如本文所用，术语“表位”指抗体的互补位结合的抗原上的任何抗原决定簇。表位决定簇通常包含分子的化学活性表面分型，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。
如本文所用，关于抗体或其抗原结合片段的“特异性结合”或“免疫特异性地结合”在本文中可交换使用，并且指抗体或抗原结合片段通过抗体和抗原的抗体结合位点之间的非共价相互作用与同种抗原形成一个或多个非共价键的能力。所述抗原可以是分离的抗原或存在于肿瘤细胞。通常，免疫特异性地结合(或特异性结合)抗原的抗体是以约或1×10 7M -1或1x10 8M -1或更大的亲和常数Ka(或者1x10 -7M或1×10 -8M或更低的解离常数(Kd))结合所述抗原。亲和常数可以通过抗体反应的标准动力学方法来测定，例如，免疫测定、表面等离子共振(SPR)(Rich and Myszka(2000)Curr.Opin.Biotechnol 11:54；Englebienne(1998)Analyst.123:1599)、等温滴定量热法(ITC)或本领域已知的其他动力学相互作用测定(参见，例如，Paul,ed.,Fundamental Immunology,2nd ed.,Raven Press,New York,pages 332-336(1989)；还参见描述用于计算抗体的结合亲和力的示例性SPR和ITC方法的美国专利第7，229，619号)。用于实时检测和监测结合速率的仪器和方法是已知的，并且可商购(参见，BiaCore 2000,Biacore AB,Upsala,Sweden and GE Healthcare Life Sciences；Malmqvist(2000)Biochem.Soc.Trans.27:335)。
如本文所用，术语“多核苷酸”和“核酸分子”指包含至少两个连接的核苷酸或核苷酸衍生物的寡聚体或聚合物，包括通常通过磷酸二酯键连接在一起的脱氧核糖核酸(DNA)和核糖核酸(RNA)。如本文所使用，术语“核酸分子”意欲包括DNA分子及RNA分子。核酸分子可为单链或双链，且可为cDNA。
如本文所用，分离的核酸分子是从存在于核酸分子的天然来源中的其他核酸分子分离的核酸分子。诸如cDNA分子的“分离的”核酸分子可以在通过重组技术制备时基本上不含其他细胞物质或培养基，或者在化学合成时基本上不含化学前体或其他化学成分。本文所提供的示例性分离的核酸分子包括编码所提供的抗体或抗原结合片段的分离的核酸分子。
如本文所用，关于核酸序列、区域、元件或结构域的“可操作地连接”表示核酸区域互相功能相关。例如，启动子可以可操作地连接至编码多肽的核酸，从而所述启动子调控或介导所述核酸的转录。
亦提供本文所述序列表中所述序列的“保守序列修饰”，即不消除由核苷酸序列编码或含有氨基酸序列的抗体与抗原的结合的核苷酸及氨基酸序列修饰。这些保守序列修饰包括保守核苷酸及氨基酸取代以及核苷酸及氨基酸添加及缺失。例如，可通过本领域已知的标准技术(例如定点诱变及PCR介导的诱变)将修饰引入本文所述的序列表中。保守序列修饰包括保守氨基酸取代，其中氨基酸残基被替换为具有类似侧链的氨基酸残基。具有类似侧链的氨基酸残基的家族是本领域中已有定义的。这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、具有β分枝侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)及具有芳香族侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，抗CD38抗体中的预测的非必需氨基酸残基优选被来自同一侧链家族的另一氨基酸残基替代。鉴定不消除抗原结合的核苷酸及氨基酸保守取代的方法为本领域所熟知(例如，参见Brummell et al.,Biochem.32:1180-1187(1993)；Kobayashi et al.,Protein Eng.12(10):879-884(1999)；Burks et al.,Proc.Natl.Acad.Sci.USA 94:412-417(1997))。
作为另一选择，在另一实施方案中，可通过例如饱和诱变沿抗CD38抗体编码序列的全部或一部分随机引入突变，且可针对改良的结合活性筛选所得经修饰抗CD38抗体。
如本文所用，“表达”指通过多核苷酸的转录和翻译产生多肽的过程。多肽的表达水平可以利用本领域已知的任何方法来评价，包括例如测定从宿主细胞产生的多肽的量的方法。这类方法可以包括但不限于通过ELISA定量细胞裂解物中的多肽，凝胶电泳之后考马斯蓝染色，Lowry蛋白测定以及Bradford蛋白测定。
如本文所用，“宿主细胞”是用于接受、保持、复制和扩增载体的细胞。宿主细胞还可以用来表达载体所编码的多肽。当宿主细胞分裂时，载体中所含的核酸复制，从而扩增核酸。宿主细胞可以是真核细胞或原核细胞。合适的宿主细胞包括但不限于CHO细胞、各种COS细胞、HeLa细胞、HEK细胞例如HEK 293细胞。
如本文所用，“载体”是可复制的核酸，当载体转化入适当的宿主细胞时，可以从该载体表达一种或多种异源蛋白。关于载体包括那些通常通过限制酶切消化和连接可以将编码多肽或其片段的核酸引入其中的载体。关于载体还包括那些包含编码多肽的核酸的载体。载体用来将编码多肽的核酸引入宿主细胞，用于扩增核酸或者用于表达/展示核酸所编码的多肽。载体通常保持游离，但是可以设计为使基因或其部分整合入基因组的染色体。还考虑人工染色 体的载体，例如酵母人工载体和哺乳动物人工染色体。这类媒介物的选择和用途是本领域技术人员公知的。
如本文所用，载体还包括“病毒载体”或“病毒的载体”。病毒的载体是工程化的病毒，其可操作地连接至外源基因以将外源基因转移(作为媒介物或穿梭(shuttle))入细胞。
如本文所用，“表达载体”包括能够表达DNA的载体，所述DNA与诸如启动子区的能够影响这类DNA片段表达的调控序列可操作地连接。这类额外的片段可以包括启动子和终止子序列，并且任选地可以包括一个或多个复制起点、一个或多个选择标记、增强子、多腺苷酸化信号等。表达载体一般来源于质粒或病毒DNA，或者可以包含这两者的元件。因此，表达载体指重组DNA或RNA构建体，例如质粒、噬菌体、重组病毒或其他载体，当引入适当的宿主细胞时，导致克隆DNA的表达。适当的表达载体是本领域技术人员公知的，并且包括在真核细胞和/或原核细胞中可复制的表达载体以及保持游离的表达载体或者整合入宿主细胞基因组的表达载体。
如本文所用，“治疗”患有疾病或疾病状况的个体表示所述个体的症状部分或全部缓解，或者在治疗后保持不变。因此，治疗包括预防、治疗和/或治愈。预防指防止潜在疾病和/或防止症状恶化或疾病发展。治疗还包括所提供的任何抗体或其抗原结合片段以及本文所提供的组合物的任何药学用途。
如本文所用，“疗效”表示由个体的治疗所导致的效果，其改变、通常改良或改善疾病或疾病状况的症状，或者治愈疾病或疾病状况。
如本文所用，“治疗有效量”或“治疗有效剂量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或包含化合物的组合物的量。因此，其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。
如本文所用，“预防有效量”或“预防有效剂量”指在施用于对象时会具有预期的预防效果的物质、化合物、材料或包含化合物的组合物的量，例如，防止或延迟疾病或症状的发生或复发，减少疾病或症状发生或复发的可能性。完全预防有效剂量不必通过施用一个剂量发生，并且可以仅在施用一系列剂量之后发生。因此，预防有效量可以在一次或多次施用中施用。
如本文中所使用的，术语“患者”是指哺乳动物，例如人。
II.具体实施方式
在一方面，本公开提供了结合CD38的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:2-4、12-14、22-24、32-34、42-44、52-54、62-64、72-74、82-84、92-94、102-144、112-114、122-124、132-34、142-144、152-154、162-164、172-174、182-184、187-189、192-194、197-199、202-204、207-209、212-214、217-219、222-224、227-229、232-234或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO:7-9、17-19、27-29、37-39、47-49、57-59、67-69、77-79、87-89、97-99、107-109、117-119、127-129、137-139、147-149、157-159、167-169、177-179、237-239、242-244、247-249、252-254、257-259、262-264、267-269或任何变体的轻链CDR。
根据前一方面的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:2、12、22、32、42、52、62、72、82、92、102、112、122、132、142、152、162、172、182、187、192、197、202、207、212、217、222、227、232或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO:3、13、23、33、43、53、63、73、83、93、103、113、123、133、143、153、163、173、183、188、193、198、203、208、213、218、223、228、233或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO:4、14、24、34、44、54、64、74、84、94、104、114、124、134、144、154、164、174、189、194、199、204、209、214、219、224、229、234或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO:7、17、27、37、47、57、67、77、87、97、107、117、127、137、147、157、167、177、237、242、247、252、257、262、267或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO:8、18、28、38、48、58、68、78、88、98、108、118、128、138、148、158、168、178、238、243、248、253、258、263、268或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO:9、19、29、39、49、59、69、79、89、99、109、119、129、139、149、159、169、179、239、244、249、254、259、264、269或其任何变体的轻链CDR3。
根据前一方面的抗体或其抗原结合部分，其包含选自下列的重链和轻链的CDR组合：
(1)分别包含SEQ ID NO:2-4的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:7-9的轻链CDR1、CDR2及CDR3序列；
(2)分别包含SEQ ID NO:12-14的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:17-19的轻链CDR1、CDR2及CDR3序列；
(3)分别包含SEQ ID NO:22-24的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:27-29的轻链CDR1、CDR2及CDR3序列；
(4)分别包含SEQ ID NO:32-34的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:37-39的轻链CDR1、CDR2及CDR3序列；
(5)分别包含SEQ ID NO:42-44的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:47-49的轻链CDR1、CDR2及CDR3序列；
(6)分别包含SEQ ID NO:52-54的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:57-59的轻链CDR1、CDR2及CDR3序列；
(7)分别包含SEQ ID NO:62-64的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:67-69的轻链CDR1、CDR2及CDR3序列；
(8)分别包含SEQ ID NO:72-74的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:77-79的轻 链CDR1、CDR2及CDR3序列；
(9)分别包含SEQ ID NO:82-84的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:87-89的轻链CDR1、CDR2及CDR3序列；
(10)分别包含SEQ ID NO:92-94的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:97-99的轻链CDR1、CDR2及CDR3序列；
(11)分别包含SEQ ID NO:102-104的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:107-109的轻链CDR1、CDR2及CDR3序列；
(12)分别包含SEQ ID NO:112-114的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:117-119的轻链CDR1、CDR2及CDR3序列；
(13)分别包含SEQ ID NO:122-124的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:127-129的轻链CDR1、CDR2及CDR3序列；
(14)分别包含SEQ ID NO:132-134的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:137-139的轻链CDR1、CDR2及CDR3序列；
(15)分别包含SEQ ID NO:142-144的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:147-149的轻链CDR1、CDR2及CDR3序列；
(16)分别包含SEQ ID NO:152-154的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:157-159的轻链CDR1、CDR2及CDR3序列；
(17)分别包含SEQ ID NO:162-164的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:167-169的轻链CDR1、CDR2及CDR3序列；
(18)分别包含SEQ ID NO:172-174的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:177-179的轻链CDR1、CDR2及CDR3序列；
(19)分别包含SEQ ID NO:182-184的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:237-239的轻链CDR1、CDR2及CDR3序列；
(20)分别包含SEQ ID NO:182-184的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:242-244的轻链CDR1、CDR2及CDR3序列；
(21)分别包含SEQ ID NO:182-184的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:247-249的轻链CDR1、CDR2及CDR3序列；
(22)分别包含SEQ ID NO:182-184的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:252-254的轻链CDR1、CDR2及CDR3序列；
(23)分别包含SEQ ID NO:182-184的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:257-259的轻链CDR1、CDR2及CDR3序列；
(24)分别包含SEQ ID NO:187-189的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:237-239的轻链CDR1、CDR2及CDR3序列；
(25)分别包含SEQ ID NO:187-189的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:242-244的轻链CDR1、CDR2及CDR3序列；
(26)分别包含SEQ ID NO:187-189的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:247-249的轻链CDR1、CDR2及CDR3序列；
(27)分别包含SEQ ID NO:187-189的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:252-254的轻链CDR1、CDR2及CDR3序列；
(28)分别包含SEQ ID NO:187-189的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:257-259的轻链CDR1、CDR2及CDR3序列；
(29)分别包含SEQ ID NO:192-194的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:237-239的轻链CDR1、CDR2及CDR3序列；
(30)分别包含SEQ ID NO:192-194的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:242-244的轻链CDR1、CDR2及CDR3序列；
(31)分别包含SEQ ID NO:192-194的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:247-249的轻链CDR1、CDR2及CDR3序列；
(32)分别包含SEQ ID NO:192-194的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:252-254的轻链CDR1、CDR2及CDR3序列；
(33)分别包含SEQ ID NO:192-194的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:257-259的轻链CDR1、CDR2及CDR3序列；
(34)分别包含SEQ ID NO:197-199的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:237-239的轻链CDR1、CDR2及CDR3序列；
(35)分别包含SEQ ID NO:197-199的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:242-244的轻链CDR1、CDR2及CDR3序列；
(36)分别包含SEQ ID NO:197-199的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:247-249 的轻链CDR1、CDR2及CDR3序列；
(37)分别包含SEQ ID NO:197-199的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:252-254的轻链CDR1、CDR2及CDR3序列；
(38)分别包含SEQ ID NO:197-199的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:257-259的轻链CDR1、CDR2及CDR3序列；
(39)分别包含SEQ ID NO:202-204的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:237-239的轻链CDR1、CDR2及CDR3序列；
(40)分别包含SEQ ID NO:202-204的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:242-244的轻链CDR1、CDR2及CDR3序列；
(41)分别包含SEQ ID NO:202-204的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:247-249的轻链CDR1、CDR2及CDR3序列；
(42)分别包含SEQ ID NO:202-204的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:252-254的轻链CDR1、CDR2及CDR3序列；
(43)分别包含SEQ ID NO:202-204的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:257-259的轻链CDR1、CDR2及CDR3序列；
(44)分别包含SEQ ID NO:207-209的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:237-239的轻链CDR1、CDR2及CDR3序列；
(45)分别包含SEQ ID NO:207-209的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:242-244的轻链CDR1、CDR2及CDR3序列；
(46)分别包含SEQ ID NO:207-209的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:247-249的轻链CDR1、CDR2及CDR3序列；
(47)分别包含SEQ ID NO:207-209的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:252-254的轻链CDR1、CDR2及CDR3序列；
(48)分别包含SEQ ID NO:207-209的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:257-259的轻链CDR1、CDR2及CDR3序列；
(49)分别包含SEQ ID NO:212-214的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:262-264的轻链CDR1、CDR2及CDR3序列；
(50)分别包含SEQ ID NO:212-214的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:267-269的轻链CDR1、CDR2及CDR3序列；
(51)分别包含SEQ ID NO:217-219的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:262-264的轻链CDR1、CDR2及CDR3序列；
(52)分别包含SEQ ID NO:217-219的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:267-269的轻链CDR1、CDR2及CDR3序列；
(53)分别包含SEQ ID NO:222-224的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:262-264的轻链CDR1、CDR2及CDR3序列；
(54)分别包含SEQ ID NO:222-224的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:267-269的轻链CDR1、CDR2及CDR3序列；
(55)分别包含SEQ ID NO:227-229的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:262-264的轻链CDR1、CDR2及CDR3序列；
(56)分别包含SEQ ID NO:227-229的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:267-269的轻链CDR1、CDR2及CDR3序列；
(57)分别包含SEQ ID NO:232-234的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:262-264的轻链CDR1、CDR2及CDR3序列；
(58)分别包含SEQ ID NO:232-234的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:267-269的轻链CDR1、CDR2及CDR3序列。
根据前一方面的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:1、11、21、31、41、51、61、71、81、91、101、111、121、131、141、151、161、171、181、186、191、196、201、206、211、216、221、226、231或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:6、16、26、36、46、56、66、76、86、96、106、116、126、136、146、156、166、176、236、241、246、251、256、261、266或其任何变体的轻链可变区；
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:1或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:6或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:11或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:16或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:21或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:26或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:31或其任 何变体的重链可变区，和氨基酸序列SEQ ID NO:36或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:41或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:46或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:51或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:56或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:61或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:66或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:71或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:76或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:81或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:86或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:91或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:96或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:101或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:106或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:111或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:116或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:121或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:126或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:131或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:136或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:141或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:146或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:151或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:156或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:161或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:166或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:171或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:176或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:181或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:236或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:181或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:241或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:181或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:246或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:181或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:251或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:181或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:256或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:186或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:236或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:186或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:241或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:186或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:246或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:186或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:251或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:186或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:256或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:191或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:236或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:191或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:241或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:191或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:246或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:191或其任 何变体的重链可变区，和氨基酸序列SEQ ID NO:251或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:191或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:256或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:196或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:236或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:196或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:241或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:196或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:246或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:196或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:251或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:196或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:256或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:201或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:236或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:201或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:241或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:201或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:246或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:201或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:251或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:201或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:256或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:206或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:236或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:206或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:241或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:206或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:246或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:206或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:251或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:206或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:256或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:211或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:261或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:211或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:266或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:216或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:261或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:216或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:266或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:221或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:261或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:221或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:266或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:226或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:261或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:226或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:266或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:231或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:261或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD38的抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO:231或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:266或其任何变体的轻链可变区。
在一方面，本公开提供了一种双特异性或多特异性分子，其包含前述任一方面的抗体或其抗原结合部分。
编码根据前述任一方面的抗体或其抗原结合部分或双特异性或多特异性分子的核酸分子。优选地，所述核酸分子包含选自SEQ ID NO:5、15、25、35、45、55、65、75、85、95、105、115、125、135、145、155、165、175、 185、190、195、200、205、210、215、220、225、230、235或其任何变体的抗体重链核酸序列，和/或选自SEQ ID NO:10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、240、245、250、255、260、265、270或其任何变体的抗体轻链核酸序列。
结合人CD38的抗体或其抗原结合部分，其与前述任一方面的抗体或其抗原结合部分具有至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性。
编码如前述任一方面的抗体或其抗原结合部分的核酸分子，或与其具有至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性的核酸分子。
含有如前述任一方面的核酸的载体。
含有如前述任一方面的载体的细胞。
包含如前述任一方面的抗体或其抗原结合部分或其编码核酸和药学上可接受的载体的药物组合物。
本发明的抗体在各种CD38被不利地表达或发现的疾病中可用作治疗或诊断工具。特别适合用本发明的CD38抗体治疗的疾病和症状是肿瘤或自身免疫性疾病，优选地，所述肿瘤选自多发性骨髓瘤(MM)和慢性淋巴细胞性白血病(CLL)、慢性髓细胞性白血病(CML)、T细胞急性淋巴细胞白血病、急性骨髓性白血病(AML)和急性淋巴细胞性白血病(ALL)。优选地，所述自身免疫性疾病选自类风湿性关节炎(RA)或系统性红斑狼疮(SLE)。
用本发明的CD38抗体治疗的疾病和症状方法包括下述步骤：向所述哺乳动物施用治疗有效量的前述任一方面的抗体或其抗原结合片段或核酸分子或载体或细胞或药物组合物。
前述任一方面的抗体或其抗原结合片段或核酸分子或载体或细胞或药物组合物在制备用于治疗哺乳动物中CD38相关病症的药物中的用途。
根据前述任一方面，任选地，所述抗体偶联其他药物，例如带标记或具有细胞毒性的缀合物。
在一方面，本公开还包括试剂盒，例如所述试剂盒包括本公开的抗体、其片段、同源物、其衍生物等，例如带标记或具有细胞毒性的缀合物，以及抗体使用说明书、杀死特定类型细胞的缀合物等等。该说明书可包括在体外、体内或离体使用抗体、缀合物等的指导。抗体可以是液体形式或固体，通常是冻干的。该试剂盒可包含其它适宜的试剂，如缓冲液、重构溶液以及为了预定用途的其它必要成分。考虑了以预定量包装好的试剂组合与用于其用途的说明书，所述用途例如用于治疗用途或用于进行诊断测定。当抗体是带标记的时，例如用酶标记的，那么该试剂盒可包括底物和酶所需的辅因子(例如提供可检测生色团或荧光团的底物前体)。此外，其它添加剂，如稳定剂、缓冲液(例如封闭缓冲液或裂解缓冲液)等也可包括在内。多种试剂的相对量可以改变而提供试剂溶液的浓缩物，这就提供了用户灵活性、节省空间、节省试剂等。这些试剂也可以干粉形式提供，通常是冻干形式，包括赋形剂，它在溶解时可提供具有适当浓度的试剂溶液。
前述任一方面的抗体或其功能片段或核酸分子或载体或细胞或药物组合物或试剂盒在制备用于制备抑制CD38与CD38R结合的试剂中的用途。
此外，本发明的抗体还可用于免疫测定、纯化方法以及其它用到免疫球蛋白或其片段的方法。此类用途在本领域为人所熟知。
相应地，本发明还提供包含本发明的抗CD38的抗体或其片段的组合物，所述抗体方便地和药学上可接受的载体、稀释剂或赋形剂组合，这是本领域的常规做法。
本发明所使用的术语“药物组合物”系指多种制备物的制剂。含有治疗有效量的多价抗体的制剂为无菌液体溶液、液体悬浮剂或冻干形式，任选地包含稳定剂或赋形剂。
本发明的抗体可以作为单独施用的组合物使用，或可与其它活性剂联合使用。
应当理解，根据所述实施方案的治疗剂将与合适的药学上可接受的载体、赋形剂、以及其它被掺入制剂中以提供改善的转移、递送、耐受性等的试剂一同施用。大量适当的制剂可见于所有药物化学工作者已知的药典中：Remington's Pharmaceutical Sciences(第15版，Mack Publishing Company，Easton，Pa.(1975))，特别是其中Blaug、Seymour的第87章。这些制剂包括例如粉末、糊剂、膏剂、凝胶剂、蜡、油、脂质、含脂质(阳离子或阴离子)载体(例如Lipofectin TM)、DNA缀合物、无水吸浆、水包油和油包水乳液、乳液聚乙二醇(各种分子量的聚乙二醇)、半固态凝胶以及含有聚乙二醇的半固态混合物。任何前述混合物均可适用于根据本发明的治疗或疗法，条件是制剂中的活性成分不被制剂灭活并且制剂在生理学上是相容的并耐受给药途径。
在一个实施方案中，可将所述抗体用作治疗剂。此类试剂将通常用于治疗、缓解和/或预防受试者的与异常CD38表达、活性和/或信号传导相关的疾病或病理。可使用标准方法通过鉴定受试者，例如患有(或处于风险或发展)与异常CD38表达、活性和/或信号传导相关的疾病或障碍，例如CD38相关病症的人患者来实施治疗方案。将抗体制剂，优选对其靶抗原有高特异性和高亲和性的抗体制剂施用给受试者并且将通常因其与靶标结合而产生效应。施用的抗体可消除或抑制或妨碍靶标(例如CD38)的表达、活性和/或信号传导功能。施用的抗体可消除或抑制或妨碍靶标(例如CD38)与其所天然结合的内源性的配体结合。例如，抗体与靶标结合并调节、阻断、抑制、减少、拮抗、中和/或以其它方式妨碍CD38表达、活性和/或信号传导。在一些实施方案中，为治疗与异常CD38表达相关的疾病或障碍，可将具有重链和轻链CDR的抗体施用给受试者。
在另一个实施方案中，针对CD38的抗体可用于本领域中已知的与CD38定位和/或定量相关的方法(例如，用于测定适当生理样品中的CD38和/或CD38的水平，用于诊断方法，用于蛋白成像等等)。在一个给定实施方案中，对CD38或其衍生物、片段、类似物或同系物具有特异性的、包含源于抗体的抗原结合结构域的抗体，被用作药物学活性化合物(下文称为“治疗剂”)。
在另一个实施方案中，可通过标准技术例如免疫亲和、色谱或免疫沉淀，使用对CD38具有特异性的抗体来分离CD38多肽。针对CD38蛋白质的抗体(或其片段)可用于检测生物样品中的蛋白质。在一些实施方案中，在生物样品中可检测CD38作为临床测试过程的一部分，例如，用于确定给定治疗方案的功效。将抗体偶联(即物理连接)到可检测物质可有利于检测。可检测物质的示例包括各种酶、辅基、荧光材料、发光材料、生物发光材料和放射性材料。合适的酶的示例包括辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶；合适的辅基复合物的示例包括链霉亲和素/生物素和亲和素/生物素；合适的荧光材料的示例包括伞形酮、荧光素、异硫氰酸荧光素、罗丹明、二氯三嗪氨荧光素、丹磺酰氯或藻红蛋白；发光材料的一个示例包括鲁米诺；生物发光材料的示例包括荧光素酶、荧光素和水母蛋白，并且合适放射性材料的示例包括 125I、 131I、 35S或 3H。
在另一个实施方案中，根据本公开的抗体可用作检测样品中CD38或其蛋白质片段存在的试剂。在一些实施方案中，抗体包含可检测标记。抗体为多克隆抗体，或更优选单克隆抗体。使用完整的抗体或其片段(例如Fab、scFv或F(ab') 2)。关于抗体的术语“标记”旨在包括通过将可检测物质偶联(即物理连接)到该抗体来直接标记该抗体，以及通过与直接标记的另一种试剂反应来间接标记该抗体。间接标记的示例包括使用荧光标记的第二抗体检测第一抗体，以及用生物素进行末端标记抗体，以便能够用荧光标记的链霉亲和素进行检测。术语“生物样品”旨在包括从受试者分离的组织、细胞和生物学流体，以及受试者体内存在的组织、细胞和流体。因此，使用的术语“生物样品”包括血液和血液中的级分或组分，包括血清、血浆、或淋巴液。换言之，所述实施方案的检测方法可用于在体外及体内检测生物样品中的分析物mRNA、蛋白质或基因组DNA。例如，分析物mRNA体外检测技术包括Norhtern杂交和原位杂交。分析物蛋白质体外检测技术包括酶联免疫吸附测定(ELISA)、Western印迹、免疫沉淀、以及免疫荧光。分析物基因组DNA体外检测技术包括Southern杂交。用于进行免疫测定的过程描述于例如“ELISA:Theory and Practice:Methods in Molecular Biology”，第42卷，J.R.Crowther(编辑)Human Press，Totowa，N.J.，1995；“Immunoassay”，E.Diamandis和T.Christopoulus，Academic Press，Inc.，San Diego，Calif.，1996；以及“Practice and Theory of Enzyme Immunoassays”，P.Tijssen，Elsevier Science Publishers，Amsterdam，1985。此外，分析物蛋白质的体内检测技术包括向受试者体内导入标记的抗分析物蛋白抗体。例如，可以用放射性标记标记抗体，然后可以通过标准成像技术检测受试者体内该放射性标记物的存在和位置。
可将本文所述抗体和其衍生物、片段、类似物和同系物掺入适于施用的药物组合物中。制备此类组合物所涉及的原理和考虑事项以及选择组分的指南在本领域中是熟知的，例如参见Remington's Pharmaceutical Sciences:The Science And Practice Of Pharmacy第19版(Alfonso R.Gennaro等人编辑)Mack Pub.Co.，Easton，Pa.:1995；Drug Absorption Enhancement:Concepts，Possibilities，Limitations，And Trends，Harwood Academic Publishers，Langhorne，Pa.，1994；以及Peptide And Protein Drug Delivery(Advances In Parenteral Sciences，第4卷)，1991，M.Dekker，New York。
此类组合物通常包含抗体和药学上可接受的载体。当使用抗体片段时，与靶蛋白结合结构域特异性结合的最小抑制片段可为优选的。例如，基于抗体的可变区序列，可以设计保留结合靶蛋白质序列能力的肽分子。此类肽可化学合成和/或通过重组DNA技术产生(参见例如Marasco等人，Proc.Natl.Acad.Sci.USA，90:7889-7893(1993))。
如本文所用，术语“药学上可接受的载体”旨在包括与药物给药相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延缓剂等。合适的药学上可接受的载体描述于最新版的Remington's Pharmaceutical Sciences中，这是本领域的标准参考书目，其以引用方式并入本文。此类载体或稀释剂的优选示例包括但不限于水、盐水、林格氏溶液、葡萄糖溶液和5％的人血清白蛋白。也可以使用脂质体和非水性载体，例如固定化油。将此类介质和试剂用于药物活性物质是本领域熟知的。除去任何常规的介质或试剂与抗体不相容之外，设想其在组合物中的用途。
将所述实施方案的药物组合物配制成与其预期施用途径相容。给药途径的示例包括肠胃外，例如静脉内、皮内、皮下、经口(例如吸入)、经皮(即局部的)、经粘膜和直肠给药。用于肠胃外、皮内或皮下施用的溶液或悬浮液可包括以下组分：注射用无菌稀释剂例如水、盐溶液、固定油、聚乙二醇类、甘油、丙二醇或其它合成溶剂；抗细菌剂，例如苄醇或对羟基苯甲酸甲酯；抗氧化剂，例如抗坏血酸或亚硫酸氢钠；螯合剂，例如乙二胺四乙酸(EDTA)；缓冲剂，例如乙酸盐、柠檬酸盐或磷酸盐、以及调节渗透压的试剂，例如氯化钠或右旋糖。pH可用酸或碱进行调节，例如盐酸或氢氧化钠。可将肠胃外制剂包装在安瓿、一次性注射器或玻璃或塑料制多剂量小瓶内。
适于注射用途的药物组合物包括无菌水性溶液(在此是水溶性的)或分散体以及用于即时制备无菌注射液或分散体的无菌粉末。对于静脉内施用，合适的药学上可接受的载体包括生理盐水、抑菌水、Cremophor EL TM(BASF，Parsippany，N.J.)或磷酸盐缓冲盐水(PBS)。在所有情况下，组合物必须是无菌的并且应当为流动性达到易于注射的程度。其在制造和储存条件下必须是稳定的并且必须能防止微生物例如细菌和真菌的污染作用。载体可以是含有例如水、乙醇、多元醇(例如，甘油、丙二醇和液体聚乙二醇等)的溶剂或分散介质，及其适宜的混合物。例如通过利用涂层例如卵磷脂，在分散体情况下维持所需颗粒尺寸，以及利用表面活性剂，可以保持适宜的流动性。对微生物作用的防止可以通过各种抗细菌剂和抗真菌剂例如对羟基苯甲酸酯、氯代丁醇、苯酚、抗坏血酸、硫柳汞等来实现。在许多情况下，将优选在组合物中包含等渗剂，例如糖、多元醇(诸如甘露糖醇、山梨醇)、氯化钠。注射用组合物的延长吸收可通过在所述组合物中包含延缓吸收的试剂例如单硬脂酸铝和明胶来达到。
根据需要，可以通过将抗体以所需量掺入具有上文所列成分中的一种或组合(按需要)的合适溶剂中来制备无菌注射溶液，然后过滤消毒。一般来讲，通过将抗体掺入含有碱性分散介质和上文所列那些中的所需其它成分的无菌载体中来制备分散体。就用于制备无菌注射溶液的无菌粉末而言，制备方法是获得粉末的真空干燥和冷冻干燥， 该粉末包含活性成分和任何另外的期望成分，它们来自前述的这些成分的无菌过滤溶液。
对于吸入给药，从包含合适推进剂如二氧化碳等气体的加压容器或分配器或者喷雾器以气溶胶喷雾形式递送化合物。
还可以通过经粘膜或透皮方式全身给药。对于经粘膜或透皮给药，在制剂中使用适于渗透屏障的渗透剂。此类渗透剂通常在本领域是通常所知的，并且包括如用于经粘膜给药的去污剂、胆盐和夫西地酸衍生物。经粘膜给药可以通过使用喷鼻剂或栓剂来实现。对于透皮给药，可将一种或多种所述抗体配制成如本领域通常所知的膏剂、软膏、凝胶、或霜膏。
还可将化合物以栓剂(例如，具有常规栓剂基质，如可可脂或其它甘油酯)或滞留性灌肠剂形式进行制备以用于经直肠递送。
在一个实施方案中，所述抗体可用防止其不被身体迅速消除的载体制备，例如缓释/控释制剂，包括植入体和微胶囊化递送体系。可使用可生物降解、可生物相容的聚合物，例如乙烯-乙酸乙烯酯、聚酐、聚乙醇酸、胶原、聚原酸酯和聚乳酸。用于制备此类制剂的方法对于本领域技术人员而言是显而易见的。
尤其有利的是以剂量单位形式配制肠胃外组合物以易于施用和剂量的一致性。如本文所用，剂量单位形式是指用于待治疗的受试者，适合作为单位剂量的物理上可分离的单位；每个单位含有经计算与所需药物载体结合产生期望治疗效果的预定量的一种或多种所述抗体。所述实施方案的剂量单位形式的规格由以下指示并直接取决于：抗体的独特特征和待实现的具体治疗效果，和用于治疗个体的此类抗体的调配领域中固有的局限性。
所述药物组合物可与给药说明书一起放于容器、包装、或分配器中。
本文所述制剂还可根据要治疗的具体情况而包含多于一种所述抗体，优选具有互补活性但对彼此无负面影响的那些。另选地或除此之外，组合物可例如包含增强其功能的试剂，诸如细胞毒素试剂、细胞因子、化学治疗剂、或生长抑制剂。此类分子以对预期目的有效的量适当地联合存在。例如，可以在试剂盒中联合存在，也可以在使用中联合存在。
在一个实施方案中，一种或多种所述抗体可在联合治疗中施用，即与其它试剂例如治疗剂(其可用于治疗病理学病症或障碍，例如各种形式的癌症、自身免疫性障碍和炎性疾病)联合。术语“联合”在本文中是指将试剂基本上同步地，同时地或顺次地给予。如果顺次给予，则在开始施用第二种化合物时，两种化合物中的第一种仍优选在治疗位点处以有效浓度被检测到。在一种情况下，“联合”也可以是在试剂盒中同时包含本发明的抗体和其他治疗剂。
例如，联合治疗可包含本文所述一种或多种抗体与一种或多种附加治疗剂(例如一种或多种细胞因子和生长因子抑制剂、免疫抑制剂、抗炎剂、代谢抑制剂、酶抑制剂、和/或细胞毒素或细胞生长抑制剂，如下更详述的)共同配制和/或共同施用。此类联合治疗可有利地利用较低剂量的施用的治疗剂，因而避免了与各种单一疗法相关的可能毒性或并发症。
为了达到清楚和简洁描述的目的，本文中作为相同的或分开的一些实施方案的一部分来描述特征，然而，将要理解的是，本发明的范围可包括具有所描述的所有或一些特征的组合的一些实施方案。
实施例
实施例1、CD38重组蛋白的制备
合成人CD38(UniProt KB：P28907)胞外区(Val 43-Ile 300)核苷酸序列，N端融合IgG kappa信号肽和6×his标签，酶切克隆到真核表达质粒CMV启动子下游，得到pSect-Nhis-CD38质粒，以此质粒转染HEK293.6E细胞，培养7天后，收集培养基上清液，经镍螯合柱纯化得到人CD38胞外区重组蛋白，SDS-PAGE见图1。
实施例2、构建人CD38/CHO稳转细胞株
构建含有人CD38全长序列的慢病毒载体质粒，按照慢病毒包装试剂盒说明书(Lenti-Pac HIV Expression Packaging Kit，Gene Copoeia，Cat：HPK-LvTR-20)，将构建好的慢病毒质粒和包装质粒共转染HEK293T细胞，进行慢病毒包装。转染48小时后收集培养基，以500*g离心10分钟去除细胞碎片，获得含慢病毒颗粒的培养上清，0.45μm的PES膜过滤后，分装至1.5mL EP管中，取10μL用于感染1x10 6CHO细胞，在培养基中加入10μg/mL嘌呤霉素(puromycin)进行筛选。获得阳性克隆后，通过有限稀释，最终得到表达人全长CD38的稳转细胞CHO-CD38-1H7，结果见图2。
实施例3、抗人CD38单克隆抗体的制备
1)免疫小鼠
将人CD38重组蛋白或CHO-CD38-1H7细胞作为抗原与等量免疫佐剂(弗氏佐剂)混合，取每组6只6周雌性Balb/c小鼠进行皮下免疫。在初次免疫两周后，进行加强免疫。三次免疫后，眼眶取血检测血清效价。
2)细胞融合与杂交瘤细胞筛选
融合前，以1x10 6个CHO-CD38-1H7细胞进行尾静脉注射冲击免疫。三天后，断颈处死小鼠，取小鼠脾和部分外周淋巴结，在DMEM培养基中研磨后离心；倾倒上清后，轻轻将脾细胞团打散，加入5mL红细胞裂解液，裂解50秒后，加入40mL DMEM离心，得到不含红细胞的脾细胞悬浮液。取适量的淋巴结和脾细胞的混合液和SP2/0混合后，采用BTX电融合仪进行细胞融合。将融合细胞接种于96孔板中，在含有HAT的DMEM完全培养基中置 于5％CO 2，37℃条件下培养。一周左右开始观察杂交瘤细胞的生长情况，待其长至60％以上时取上清进行检测。
实施例4、抗人CD38单克隆抗体的筛选和抗体序列的获得
1)酶联免疫吸附实验(ELISA)筛选
在96孔酶标板上包被1μg/mL的人CD38重组蛋白，4℃孵育过夜。次日PBS洗涤3次，用200μL 2％脱脂奶粉/PBS室温封闭2小时，PBS洗涤1次，加入50μL杂交瘤上清，室温孵育1小时后，用PBST和PBS重复洗涤3次，加入100μL/孔HRP标记的抗小鼠IgG Fc二抗，室温孵育60分钟。用PBST和PBS各洗三遍后，加入100μL显色液(TMB溶液，Sigma货号T2885)，37℃放置5分钟后加入50μL 2M浓硫酸溶液终止反应，立即放于酶标仪中读取OD450的值。
2)细胞筛选抗CD38特异性抗体(FACS)
将ELISA检测到的结合CD38抗体的阳性克隆上清，进一步验证与CD38阳性细胞的结合。准备CHO细胞与CHO-CD38稳转细胞，分别按5×10 4/孔细胞数，加入96孔板中，每孔再加入50μL杂交瘤上清，4℃孵育60分钟，离心吸弃上清，用0.5％BSA/PBS洗涤，再加入50μL二抗溶液(抗-mouse IgG-Fc-AF647，Jackson ImmunoResearch，Cat：115-606-071)，4℃孵育45分钟，之后用0.5％BSA/PBS洗去多余的二抗，加入PBS重悬细胞，上流式细胞仪检测。
3)抗人CD38嵌合抗体的基因克隆及表达
选择CD38阳性杂交瘤单克隆，采用TRNzol裂解法提取细胞总RNA，然后利用逆转录试剂盒(Invitrogen，货号18080051)合成单链cDNA后，以此为模板扩增杂交瘤单克隆细胞中抗体的可变区序列，经测序后，得到的候选阳性克隆重链和轻链可变区序列分别为：
克隆：21H12-G12 SEQ ID Nos:1-10
重链VH
Figure PCTCN2020128820-appb-000001
核酸序列
Figure PCTCN2020128820-appb-000002
轻链VK
Figure PCTCN2020128820-appb-000003
核酸序列
Figure PCTCN2020128820-appb-000004
克隆：7C10-G5 SEQ ID Nos:11-20
重链VH
Figure PCTCN2020128820-appb-000005
核酸序列
Figure PCTCN2020128820-appb-000006
轻链VK
Figure PCTCN2020128820-appb-000007
核酸序列
Figure PCTCN2020128820-appb-000008
克隆：15A1-G1 SEQ ID Nos:21-30
重链VH
Figure PCTCN2020128820-appb-000009
核酸序列
Figure PCTCN2020128820-appb-000010
轻链VK
Figure PCTCN2020128820-appb-000011
核酸序列
Figure PCTCN2020128820-appb-000012
克隆：51G7-D3 SEQ ID Nos:31-40
重链VH
Figure PCTCN2020128820-appb-000013
核酸序列
Figure PCTCN2020128820-appb-000014
轻链VK
Figure PCTCN2020128820-appb-000015
核酸序列
Figure PCTCN2020128820-appb-000016
克隆：17A4-H9 SEQ ID Nos:41-50
重链VH
Figure PCTCN2020128820-appb-000017
核酸序列
Figure PCTCN2020128820-appb-000018
轻链VK
Figure PCTCN2020128820-appb-000019
Figure PCTCN2020128820-appb-000020
核酸序列
Figure PCTCN2020128820-appb-000021
克隆：25C5-E9 SEQ ID Nos:51-60
重链VH
Figure PCTCN2020128820-appb-000022
核酸序列
Figure PCTCN2020128820-appb-000023
轻链VK
Figure PCTCN2020128820-appb-000024
核酸序列
Figure PCTCN2020128820-appb-000025
克隆：25F8-C4 SEQ ID Nos:61-70
重链VH
Figure PCTCN2020128820-appb-000026
核酸序列
Figure PCTCN2020128820-appb-000027
轻链VK
Figure PCTCN2020128820-appb-000028
核酸序列
Figure PCTCN2020128820-appb-000029
克隆：40A12-D2 SEQ ID Nos:71-80
重链VH
Figure PCTCN2020128820-appb-000030
核酸序列
Figure PCTCN2020128820-appb-000031
轻链VK
Figure PCTCN2020128820-appb-000032
核酸序列
Figure PCTCN2020128820-appb-000033
克隆：36G11-E10 SEQ ID Nos:81-90
重链VH
Figure PCTCN2020128820-appb-000034
核酸序列
Figure PCTCN2020128820-appb-000035
轻链VK
Figure PCTCN2020128820-appb-000036
核酸序列
Figure PCTCN2020128820-appb-000037
克隆：26C12-G9 SEQ ID Nos:91-100
重链VH
Figure PCTCN2020128820-appb-000038
核酸序列
Figure PCTCN2020128820-appb-000039
轻链VK
Figure PCTCN2020128820-appb-000040
核酸序列
Figure PCTCN2020128820-appb-000041
克隆：20E12-C6 SEQ ID Nos:101-110
重链VH
Figure PCTCN2020128820-appb-000042
核酸序列
Figure PCTCN2020128820-appb-000043
Figure PCTCN2020128820-appb-000044
轻链VK
Figure PCTCN2020128820-appb-000045
核酸序列
Figure PCTCN2020128820-appb-000046
克隆：22D3-F9 SEQ ID Nos:111-120
重链VH
Figure PCTCN2020128820-appb-000047
核酸序列
Figure PCTCN2020128820-appb-000048
轻链VK
Figure PCTCN2020128820-appb-000049
核酸序列
Figure PCTCN2020128820-appb-000050
克隆：12C7-A11 SEQ ID Nos:121-130
重链VH
Figure PCTCN2020128820-appb-000051
核酸序列
Figure PCTCN2020128820-appb-000052
轻链VK
Figure PCTCN2020128820-appb-000053
核酸序列
Figure PCTCN2020128820-appb-000054
克隆：11F12-E1 SEQ ID Nos:131-140
重链VH
Figure PCTCN2020128820-appb-000055
核酸序列
Figure PCTCN2020128820-appb-000056
轻链VK
Figure PCTCN2020128820-appb-000057
核酸序列
Figure PCTCN2020128820-appb-000058
克隆：23C4-D1 SEQ ID Nos:141-150
重链VH
Figure PCTCN2020128820-appb-000059
核酸序列
Figure PCTCN2020128820-appb-000060
轻链VK
Figure PCTCN2020128820-appb-000061
核酸序列
Figure PCTCN2020128820-appb-000062
克隆：29F3-G8 SEQ ID Nos:151-160
重链VH
Figure PCTCN2020128820-appb-000063
核酸序列
Figure PCTCN2020128820-appb-000064
轻链VK
Figure PCTCN2020128820-appb-000065
核酸序列
Figure PCTCN2020128820-appb-000066
克隆：27F8 SEQ ID Nos:161-170
重链VH
Figure PCTCN2020128820-appb-000067
核酸序列
Figure PCTCN2020128820-appb-000068
轻链VK
Figure PCTCN2020128820-appb-000069
核酸序列
Figure PCTCN2020128820-appb-000070
克隆：48H1-B11 SEQ ID Nos:171-180
重链VH
Figure PCTCN2020128820-appb-000071
核酸序列
Figure PCTCN2020128820-appb-000072
轻链VK
Figure PCTCN2020128820-appb-000073
核酸序列
Figure PCTCN2020128820-appb-000074
将重链可变区克隆入含有人重链恒定区和调节元件的载体，以在哺乳动物细胞中表达完整的IgG重链。类似地，将轻链可变区克隆入含有人轻链恒定区和调节元件的载体，以在哺乳动物细胞中表达完整的IgG轻链。经测序正确后转染入CHO-S哺乳动物细胞中，IgG经表达分泌入培养基中，合并收集上清，过滤后纯化。采用Protein A层析纯化IgG，将培养上清液加载于大小合适的Protein A柱子上，用50mM Tris-HCl pH8.0，250mM NaCl洗涤，用0.1M Glycine-HCl(pH3.0)将结合的IgG洗脱下来。利用浓缩管将蛋白超滤浓缩，检测OD280，通过分光光度法测定IgG的浓度。
4)嵌合抗体对Daudi细胞的CDC活性
Daudi细胞重悬后按照50μL/孔铺入96孔板中，然后每孔加入50μL抗体。将人源化抗体稀释至10μg/mL，以此为起始浓度，3倍梯度稀释，共3个梯度。每孔加入50μL补体混合液，将96孔板放至37℃5％CO 2培养箱中培养2小时。然后每孔加入50μL 40％CCK-8溶液，震荡10秒，放于5％CO 2培养箱培养3小时。将96孔板置于酶标仪(BioTek，Synergy HT)中，读取OD450的值，收集数据使用GraphPad Prism 5软件计算结果。结果如图3-1及3-2所示。
5)嵌合抗体对CD38酶活性的抑制作用
CD38催化环腺苷二磷酸核糖(cADPR，cyclic ADP-ribose)的合成与降解，可以将烟酰胺腺嘌呤二核苷酸(NAD+)环化为环腺苷二磷酸核糖(cADPR)，并将生成的cADPR水解为ADPR。由于cADPR不稳定，CD38酶活性研究中，通常使用烟酰胺鸟嘌呤二核苷酸(NGD+)作为底物，检测催化生成的cGDPR。anti-CD38抗体可以与CD38结合，进而抑制CD38蛋白酶催化NGD+生成cGDPR。将Daudi细胞与梯度稀释后的嵌合抗体混匀，室温下孵育15分钟；在不同时间点加入NGD底物，混匀进行检测；结果如表1和图4-1及4-2所示。
表1：CD38嵌合抗体生物学活性筛选
Figure PCTCN2020128820-appb-000075
6)嵌合抗体对Duadi细胞的ADCC杀伤活性
按照每孔50μL将密度为1×10 6个/mL Daudi细胞铺至U型96孔板中，每孔加入50μL抗体，放至37℃、5％CO 2培养箱培养30分钟。从液氮灌中取出PBMC细胞，37℃水浴迅速解冻，快速转移细胞至15mL离心管中，加入RPMI1640培养基，计数，300*g离心10分钟。弃上清，RPMI-1640培养基重悬细胞密度至1×10 7/mL；取出96孔板，每孔加入50μL PBMC细胞至孔板中，置于5％CO 2培养箱中培养4小时。检测前30分钟，向靶细胞最大孔加入2μL细胞裂解液(100×)，继续37℃培养。300*g离心3分钟，取50μL上清至黑色酶标板，每孔加入50μL LDH检测底物(放置室温提前溶解)，轻轻震荡混匀；10分钟后，每孔加入25μL终止液进行终止，震荡10秒，选择荧光(激发波长560nm，发射波长590nm)进行读板。结果如图5所示，嵌合抗体48H1-B11对Daudi细胞有明显的ADCC杀伤作用，并且呈剂量依赖关系。
7)嵌合抗体对Duadi细胞的ADCP杀伤活性
采用被1μM CFSE标记的Daudi细胞作为CD38+靶细胞，按照1x10 5/孔接种50μL至96孔板中，将抗体稀释4倍，取50μL至孔板中，最后加入100μl密度为2.5x10 5/ml用GM-CSF1诱导7天成熟的巨噬细胞，macrophage:Daudi＝1:4，37℃5％CO 2培养箱孵育4小时，最后上流式细胞仪检测收集细胞。结果如图6所示，嵌合抗体48H1-B11对Daudi细胞有明显的ADCP吞噬作用，并且呈剂量依赖关系。
实施例5、Anti-CD38抗体的人源化
mAb48H1-B11鼠源抗体轻链为小鼠IMGT_mVK13-84，首先进行了人源化框架区的筛选，分别选择IMGT_hVK_1_39、IMGT_hVK_2D_28、IMGT_hVK_3_7、IMGT_hVK_3_20和IMGT_hVK_4_1进行CDR移植，得到5个人源化轻链变体h48H1-Vkv1-v5；鼠源抗体重链为IGHV2-5，分别选择IMGT_hVH_1_69、IMGT_hVH_2_26、IMGT_hVH_3_23、IMGT_hVH_4_59、IMGT_hVH_5_51和IMGT_hVH_7_4_1进行CDR移植，得到6个人源化重链变体h48H1-VHv1-v6。将轻重链变体进行组合配对后，在CHO-S细胞中瞬时表达，检测各组和抗体的亲和力以及CDC活性。
在完整保留嵌合抗体CDC活性的轻重链变体中，选择与鼠源抗体mAb48H1-B11轻链框架区同源性最高的人IMGT_hVK3-20进行人源化序列优化，FM4选用同源性最高的人IGKJ4*01；选择人重链胚系基因IMGT_hVH4-59进行CDR移植，FM4选用同源性最高的人IGHJ4*01。利用计算机进行同源建模，分析CDR区及其周边框架氨基酸序列，避免分子表面电荷或疏水区域集中分布。设计共得到5个重链变体h48H1-VHv7-v11和2个轻链变体h48H1-VKv7-v8，将轻、重链分别进行全序列合成后，克隆到含有抗体kappa链恒定区Ckappa或人IgG1恒定区CH1-CH3的真核表达载体，将轻、重链质粒进行组合配对后，转染CHO-S细胞，37℃表达5-6天，收取培养上清，通过Protein A柱进行纯化。
人源化抗体重链/轻链可变区序列如下：
VH v1：
Figure PCTCN2020128820-appb-000076
核酸序列
Figure PCTCN2020128820-appb-000077
VH v2:
Figure PCTCN2020128820-appb-000078
核酸序列
Figure PCTCN2020128820-appb-000079
核酸序列
Figure PCTCN2020128820-appb-000080
VH v4:
Figure PCTCN2020128820-appb-000081
核酸序列
Figure PCTCN2020128820-appb-000082
VH v5:
Figure PCTCN2020128820-appb-000083
核酸序列
Figure PCTCN2020128820-appb-000084
VH v6:
Figure PCTCN2020128820-appb-000085
核酸序列
Figure PCTCN2020128820-appb-000086
VH v7:
Figure PCTCN2020128820-appb-000087
核酸序列
Figure PCTCN2020128820-appb-000088
Figure PCTCN2020128820-appb-000089
VH v8:
Figure PCTCN2020128820-appb-000090
核酸序列
Figure PCTCN2020128820-appb-000091
VH v9:
Figure PCTCN2020128820-appb-000092
核酸序列
Figure PCTCN2020128820-appb-000093
VH v10:
Figure PCTCN2020128820-appb-000094
核酸序列
Figure PCTCN2020128820-appb-000095
VH v11:
Figure PCTCN2020128820-appb-000096
核酸序列
Figure PCTCN2020128820-appb-000097
VK v1:
Figure PCTCN2020128820-appb-000098
核酸序列
Figure PCTCN2020128820-appb-000099
VK v2:
Figure PCTCN2020128820-appb-000100
核酸序列
Figure PCTCN2020128820-appb-000101
VK v3:
Figure PCTCN2020128820-appb-000102
核酸序列
Figure PCTCN2020128820-appb-000103
VK v4:
Figure PCTCN2020128820-appb-000104
核酸序列
Figure PCTCN2020128820-appb-000105
VK v5:
Figure PCTCN2020128820-appb-000106
核酸序列
Figure PCTCN2020128820-appb-000107
VK v7:
Figure PCTCN2020128820-appb-000108
核酸序列
Figure PCTCN2020128820-appb-000109
VK v8:
Figure PCTCN2020128820-appb-000110
核酸序列
Figure PCTCN2020128820-appb-000111
实施例6、不同人源化CD38抗体对肿瘤细胞Daudi的CDC杀伤
接种不同细胞至U型96孔板中，每孔加入不同浓度的抗体，放至CO 2培养箱培养半小时；然后每孔加入50μl补体混合液，置于CO 2培养箱中培养2小时。然后每孔加入50μl CCK-8溶液，震荡混匀，继续培养3小时；选择光吸收OD450，进行读板。结果如图7-1、7-2所示，在不同人源化CD38抗体中，人源化变体Ab031、Ab033对Dauai细胞的最大杀伤率最高。
表2：不同人源化CD38抗体对肿瘤细胞Daudi的CDC杀伤
Figure PCTCN2020128820-appb-000112
Figure PCTCN2020128820-appb-000113
实施例7、人源化CD38抗体的亲和力测定
1)ELISA亲和力测定
96孔酶标板包被人CD38蛋白，4℃孵育过夜。然后弃去孔内溶液，用洗涤缓冲液洗3次，加入含有2％牛奶的PBS溶液封闭60分钟。用洗涤缓冲液洗3次后100μL每孔加入不同稀释倍数的抗体，37℃孵育1小时，经洗涤缓冲液冲洗三次后，用0.5％BSA以1：5000倍稀释HPR标记的山羊抗-人IgG Fc(goat anti-human IgG Fc)，室温孵育1小时，经洗涤缓冲液冲洗3次后，加入100μL TMB底物溶液显色，37℃反应10分钟后，以50μL 2M的硫酸溶液终止反应并在450nm处读出吸光度。结果如图8-1和表3所示，ELISA检测到人源化CD38抗体Ab031(EC50＝0.51nM)、Ab033(EC50＝4.24nM)高亲和力结合CD38。
表3：ELISA亲和力测定
Figure PCTCN2020128820-appb-000114
2)Biacore亲和力测定
采用1:1单价结合模式，将0.5μg/ml的人源化变体捕获在protein A芯片的表面，捕获信号200RU左右，不同浓度的人CD38重组蛋白作为分析物流过芯片表面，通过记录抗体结合、解离过程中的SPR信号变化，与Langmuir1:1kinetics理论模型进行拟合，分析与模型的吻合度，并提供亲和力数据。如表4所示，Biacore检测到人源化抗体Ab031亲和力为0.28nM、Ab033亲和力为1.43nM。
表4：Biacore亲和力测定
Figure PCTCN2020128820-appb-000115
Figure PCTCN2020128820-appb-000116
3)FACS细胞亲和力测定
取对数生长期的不同细胞，3％BSA封闭30分钟以5×10 4细胞/100μL铺U型96孔板，1100rpm离心3分钟，弃上清，轻轻拍散细胞，每孔加入50μL梯度稀释的抗体(抗体浓度从30μg/mL起，3倍稀释5个梯度)，4℃孵育1小时。孵育结束后，每孔加入180μL 0.5％BSA洗3次，加30μL/孔二抗Alexa Fluro 647抗人IgG(Jackson ImmunoResearch，货号：109-606-170)，4℃孵育45分钟。孵育结束后，每孔加入180μL 0.5％BSA洗3次，最后每孔重悬于50μL PBS进行iQue(Intellicyt，USA)检测。如图8-2和表5所示，FACS检测到人源化抗体Ab031、Ab033高亲和力结合Daudi上表达的CD38分子；同时，人源化变体Ab031、Ab033也识别其他肿瘤细胞NALM-6、Ramos.2G6.4C10和TALL-1上表达的CD38分子(图8-3、8-4、8-5)。
表5：FACS细胞亲和力测定
Figure PCTCN2020128820-appb-000117
实施例8、CD38在不同肿瘤细胞上的表达量
FACS分析检测不同肿瘤细胞上CD38的表达水平，离心收集1*10 6个不同肿瘤细胞，加入3mL PBS洗涤一次；封闭20分钟后，加入50μl稀释后的一抗溶液抗-人CD38hIgG1-生物素(anti-human CD38hIgG1-biotin)，冰上孵育40分钟；洗涤两次，每孔各加入100μl稀释后的二抗溶液SA-PE(Jackson，货号：016110084)，冰上避光孵育30分钟，洗涤两次，离心收集细胞；每孔加入200μl PI稀释液，轻轻吹打混匀，同时取一支标准品BD Qusntibrite PE tube(BD，货号：340495)，避光加入500μl 0.5％BSA/PBS溶液，置于冰上孵育，上机流式检测。结果如图9所示，Daudi和Ramos.2G6.4C10(ATCC CRL-1923)细胞上CD38高表达；RPMI-8226(ATCC CRM-CCL-155)、TALL-1(上海斯信生物)和Raji(中国食品药品检定研究院)细胞上CD38中表达；在NCI-H929(南京科佰)、CCRF-CEM和NALM-6(上海子实生物)细胞上检测到CD38低表达，而细胞MM.1S(ATCC CRL-2974)、MOLP-8和WSU-DLCL-2(上海斯信生物)细胞上CD38表达微弱。
实施例9、人源化CD38抗体对不同肿瘤细胞的CDC杀伤
接种不同细胞至U型96孔板中，每孔加入不同浓度的抗体，放至CO 2培养箱培养半小时；然后每孔加入50μl补体混合液，置于CO 2培养箱中培养2小时。然后每孔加入50μl CCK-8溶液，震荡混匀，继续培养3小时；选择光吸收OD450，进行读板。结果如图10-1、10-2、10-3所示，人源化抗体Ab031、Ab033对Ramos.2G6.4C10细胞最大杀伤为98％，对Daudi细胞最大杀伤为65％，对TALL-1细胞大杀伤为40％。
实施例10、人源化CD38抗体对不同肿瘤细胞的ADCC杀伤
接种不同细胞至U型96孔板中，每孔加入不同浓度的抗体，放至CO 2培养箱培养半小时；然后每孔加入5×10 5个PBMC细胞，置于CO 2培养箱中培养4小时。检测前30分钟，加入细胞裂解液，继续培养；离心收集上清至黑色酶标板中，加入LDH检测底物，震荡混匀。10分钟后，加入终止液进行终止反应，震荡混匀；选择荧光，进行读板，并计算杀伤率。杀伤率计算公式如下：
Figure PCTCN2020128820-appb-000118
结果如图11-1、11-2、11-3、11-4、11-5、11-6、11-7、11-8所示，人源化抗体Ab031、Ab033对Ramos.2G6.4C10细胞杀伤率为90％，对NALM-6细胞杀伤率为65％，对RPMI-8226细胞杀伤率为67％，对CRFF-CEM细胞杀伤率为48％；而且对Daudi细胞杀伤率为62％，对TALL-1细胞杀伤率为40％，对MM.1S细胞杀伤率为20％，对NCI-H929细胞杀伤率为48％，对Raji细胞杀伤率为75％。
实施例11、人源化CD38抗体对不同肿瘤细胞的ADCP杀伤
用1μM CFSE标记的Daudi细胞作为CD38 +靶细胞，接种至96孔板中，加入梯度稀释的抗体，置于CO 2培养箱中培养，最后加入GM-CSF1诱导成熟的巨噬细胞，比例为巨噬细胞:Daudi＝1:4，CO 2培养箱中孵育4小时；加入抗人CD14APC抗体稀释溶液，于冰上避光20分钟；流式细胞仪检测收集细胞，以单独巨噬细胞孔为对照，圈出CD14单阳性细胞群，实验孔圈出CD14单阳性细胞及CD14和CSFE双阳性细胞群，统计两群细胞数量。结果如图12-1、12-2、12-3、12-4、12-5所示，人源化CD38抗体Ab031、Ab033对CD38不同表达细胞Daudi、TALL-1、 Ramos.2G6.4C10、Raji、NALM-6均有不同程度的ADCP吞噬作用。
实施例12、人源化CD38抗体对CD38酶活性抑制作用
将CD38重组蛋白与梯度稀释后的人源化CD38抗体Ab031、Ab0337混匀，室温下孵育15分钟；在不同时间点加入NGD底物，混匀进行检测；结果如图13所示，嵌合抗体48H1-B11和人源化CD38抗体Ab031、Ab033均对CD38酶活有明显的抑制作用。
实施例13、人源化CD38抗体诱导的细胞凋亡作用
接种2*10 5个Daudi细胞于24孔板中，37℃培养过夜；每孔加入100μl已稀释混合好的人源化抗体，置于CO 2培养箱中继续培养；细胞诱导刺激24小时或者48小时后，收集细胞，加入100μl Annexin V-FITC孵育液，轻轻混匀，再加入碘化丙啶染色液，轻轻混匀。室温避光孵育15分钟，随后流式细胞仪检测，Annexin V-FITC为绿色荧光，阳性为早期及晚期凋亡细胞；PI为红色荧光，为晚期凋亡或死亡细胞。结果如图14所示，人源化CD38抗体Ab031、Ab033均能检测到对Daudi细胞的凋亡作用。
实施例14、人源化CD38抗体诱导人红细胞裂解作用
取人红细胞铺板于96孔板中，并加入50μl梯度稀释后的不同浓度抗体(10μg/mL和1μg/mL)，再加入50μl稀释的补体，以灭活补体作为对照，置于CO 2培养箱中培养2小时，然后显微镜下观察细胞的状态。结果如图15所示，在对照组和实验组中，人源化CD38抗体Ab031、Ab033均不引起红细胞的裂解。
实施例15、人源化CD38抗体在Daudi细胞株皮下异种移植NOD/SCID小鼠模型中的抗肿瘤作用
在含10％胎牛血清的RPMI1640培养液中培养Raji细胞。收集对数生长期的Raji细胞，PBS重悬至适合浓度并与matrigel 1:1混合后用于NOD/SCID小鼠皮下肿瘤接种。雌性小鼠右侧皮下接种1×10 7Raji细胞，待肿瘤平均体积为100mm 3时，根据肿瘤大小随机分组。每组6只小鼠，治疗组的小鼠以10mg/kg的剂量每周尾静脉注射二次，共给药两周，监控肿瘤体积。肿瘤体积计算方法为：肿瘤体积(mm 3)＝1/2×(a×b 2)(其中a表示长径，b表示短径)。
结果如图16所示，CD38抗体(3/10mg/kg，IP，D0，3)均明显抑制人B细胞淋巴瘤Daudi小鼠皮下移植瘤的生产，引起肿瘤部分消退，其中CM313-1为人源化抗体Ab031，CM313-2为人源化抗体Ab033，CM313-3为antibody-005(US20150231235A1)，CM313-4为38SB19(US20110293606A1)。

Claims (12)

  1. 一种结合CD38的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:2-4、12-14、22-24、32-34、42-44、52-54、62-64、72-74、82-84、92-94、102-144、112-114、122-124、132-34、142-144、152-154、162-164、172-174、182-184、187-189、192-194、197-199、202-204、207-209、212-214、217-219、222-224、227-229、232-234或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO:7-9、17-19、27-29、37-39、47-49、57-59、67-69、77-79、87-89、97-99、107-109、117-119、127-129、137-139、147-149、157-159、167-169、177-179、237-239、242-244、247-249、252-254、257-259、262-264、267-269或其任何变体的轻链CDR。
  2. 根据权利要求1的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:2、12、22、32、42、52、62、72、82、92、102、112、122、132、142、152、162、172、182、187、192、197、202、207、212、217、222、227、232或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO:3、13、23、33、43、53、63、73、83、93、103、113、123、133、143、153、163、173、183、188、193、198、203、208、213、218、223、228、233或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO:4、14、24、34、44、54、64、74、84、94、104、114、124、134、144、154、164、174、184、189、194、199、204、209、214、219、224、229、234或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO:7、17、27、37、47、57、67、77、87、97、107、117、127、137、147、157、167、177、237、242、247、252、257、262、267或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO:8、18、28、38、48、58、68、78、88、98、108、118、128、138、148、158、168、178、238、243、248、253、258、263、268或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO:9、19、29、39、49、59、69、79、89、99、109、119、129、139、149、159、169、179、239、244、249、254、259、264、269或其任何变体的轻链CDR3。
  3. 根据前述任一项所述的抗体或其抗原结合部分，其包含选自下列的重链和轻链的CDR组合：
    (1)分别包含SEQ ID NO:2-4的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:7-9的轻链CDR1、CDR2及CDR3序列；
    (2)分别包含SEQ ID NO:12-14的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:17-19的轻链CDR1、CDR2及CDR3序列；
    (3)分别包含SEQ ID NO:22-24的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:27-29的轻链CDR1、CDR2及CDR3序列；
    (4)分别包含SEQ ID NO:32-34的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:37-39的轻链CDR1、CDR2及CDR3序列；
    (5)分别包含SEQ ID NO:42-44的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:47-49的轻链CDR1、CDR2及CDR3序列；
    (6)分别包含SEQ ID NO:52-54的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:57-59的轻链CDR1、CDR2及CDR3序列；
    (7)分别包含SEQ ID NO:62-64的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:67-69的轻链CDR1、CDR2及CDR3序列；
    (8)分别包含SEQ ID NO:72-74的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:77-79的轻链CDR1、CDR2及CDR3序列；
    (9)分别包含SEQ ID NO:82-84的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:87-89的轻链CDR1、CDR2及CDR3序列；
    (10)分别包含SEQ ID NO:92-94的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:97-99的轻链CDR1、CDR2及CDR3序列；
    (11)分别包含SEQ ID NO:102-104的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:107-109的轻链CDR1、CDR2及CDR3序列；
    (12)分别包含SEQ ID NO:112-114的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:117-119的轻链CDR1、CDR2及CDR3序列；
    (13)分别包含SEQ ID NO:122-124的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:127-129的轻链CDR1、CDR2及CDR3序列；
    (14)分别包含SEQ ID NO:132-134的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:137-139的轻链CDR1、CDR2及CDR3序列；
    (15)分别包含SEQ ID NO:142-144的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:147-149的轻链CDR1、CDR2及CDR3序列；
    (16)分别包含SEQ ID NO:152-154的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:157-159的轻链CDR1、CDR2及CDR3序列；
    (17)分别包含SEQ ID NO:162-164的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:167-169的轻链CDR1、CDR2及CDR3序列；
    (18)分别包含SEQ ID NO:172-174的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:177-179的轻链CDR1、CDR2及CDR3序列；
    (19)分别包含SEQ ID NO:182-184的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:237-239 的轻链CDR1、CDR2及CDR3序列；
    (20)分别包含SEQ ID NO:182-184的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:242-244的轻链CDR1、CDR2及CDR3序列；
    (21)分别包含SEQ ID NO:182-184的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:247-249的轻链CDR1、CDR2及CDR3序列；
    (22)分别包含SEQ ID NO:182-184的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:252-254的轻链CDR1、CDR2及CDR3序列；
    (23)分别包含SEQ ID NO:182-184的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:257-259的轻链CDR1、CDR2及CDR3序列；
    (24)分别包含SEQ ID NO:187-189的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:237-239的轻链CDR1、CDR2及CDR3序列；
    (25)分别包含SEQ ID NO:187-189的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:242-244的轻链CDR1、CDR2及CDR3序列；
    (26)分别包含SEQ ID NO:187-189的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:247-249的轻链CDR1、CDR2及CDR3序列；
    (27)分别包含SEQ ID NO:187-189的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:252-254的轻链CDR1、CDR2及CDR3序列；
    (28)分别包含SEQ ID NO:187-189的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:257-259的轻链CDR1、CDR2及CDR3序列；
    (29)分别包含SEQ ID NO:192-194的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:237-239的轻链CDR1、CDR2及CDR3序列；
    (30)分别包含SEQ ID NO:192-194的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:242-244的轻链CDR1、CDR2及CDR3序列；
    (31)分别包含SEQ ID NO:192-194的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:247-249的轻链CDR1、CDR2及CDR3序列；
    (32)分别包含SEQ ID NO:192-194的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:252-254的轻链CDR1、CDR2及CDR3序列；
    (33)分别包含SEQ ID NO:192-194的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:257-259的轻链CDR1、CDR2及CDR3序列；
    (34)分别包含SEQ ID NO:197-199的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:237-239的轻链CDR1、CDR2及CDR3序列；
    (35)分别包含SEQ ID NO:197-199的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:242-244的轻链CDR1、CDR2及CDR3序列；
    (36)分别包含SEQ ID NO:197-199的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:247-249的轻链CDR1、CDR2及CDR3序列；
    (37)分别包含SEQ ID NO:197-199的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:252-254的轻链CDR1、CDR2及CDR3序列；
    (38)分别包含SEQ ID NO:197-199的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:257-259的轻链CDR1、CDR2及CDR3序列；
    (39)分别包含SEQ ID NO:202-204的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:237-239的轻链CDR1、CDR2及CDR3序列；
    (40)分别包含SEQ ID NO:202-204的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:242-244的轻链CDR1、CDR2及CDR3序列；
    (41)分别包含SEQ ID NO:202-204的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:247-249的轻链CDR1、CDR2及CDR3序列；
    (42)分别包含SEQ ID NO:202-204的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:252-254的轻链CDR1、CDR2及CDR3序列；
    (43)分别包含SEQ ID NO:202-204的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:257-259的轻链CDR1、CDR2及CDR3序列；
    (44)分别包含SEQ ID NO:207-209的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:237-239的轻链CDR1、CDR2及CDR3序列；
    (45)分别包含SEQ ID NO:207-209的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:242-244的轻链CDR1、CDR2及CDR3序列；
    (46)分别包含SEQ ID NO:207-209的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:247-249的轻链CDR1、CDR2及CDR3序列；
    (47)分别包含SEQ ID NO:207-209的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:252-254 的轻链CDR1、CDR2及CDR3序列；
    (48)分别包含SEQ ID NO:207-209的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:257-259的轻链CDR1、CDR2及CDR3序列；
    (49)分别包含SEQ ID NO:212-214的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:262-264的轻链CDR1、CDR2及CDR3序列；
    (50)分别包含SEQ ID NO:212-214的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:267-269的轻链CDR1、CDR2及CDR3序列；
    (51)分别包含SEQ ID NO:217-219的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:262-264的轻链CDR1、CDR2及CDR3序列；
    (52)分别包含SEQ ID NO:217-219的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:267-269的轻链CDR1、CDR2及CDR3序列；
    (53)分别包含SEQ ID NO:222-224的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:262-264的轻链CDR1、CDR2及CDR3序列；
    (54)分别包含SEQ ID NO:222-224的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:267-269的轻链CDR1、CDR2及CDR3序列；
    (55)分别包含SEQ ID NO:227-229的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:262-264的轻链CDR1、CDR2及CDR3序列；
    (56)分别包含SEQ ID NO:227-229的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:267-269的轻链CDR1、CDR2及CDR3序列；
    (57)分别包含SEQ ID NO:232-234的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:262-264的轻链CDR1、CDR2及CDR3序列；
    (58)分别包含SEQ ID NO:232-234的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:267-269的轻链CDR1、CDR2及CDR3序列。
  4. 根据权利要求1或2所述的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:1、11、21、31、41、51、61、71、81、91、101、111、121、131、141、151、161、171、181、186、191、196、201、206、211、216、221、226、231或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:6、16、26、36、46、56、66、76、86、96、106、116、126、136、146、156、166、176、236、241、246、251、256、261、266或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:1或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:6或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:11或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:16或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:21或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:26或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:31或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:36或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:41或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:46或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:51或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:56或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:61或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:66或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:71或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:76或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:81或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:86或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:91或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:96或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:101或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:106或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:111或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:116或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:121或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:126或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:131或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:136或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:141或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:146或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:151或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:156或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:161或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:166或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:171或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:176或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:181或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:236或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:181或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:241或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:181或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:246或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:181或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:251或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:181或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:256或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:186或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:236或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:186或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:241或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:186或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:246或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:186或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:251或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:186或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:256或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:191或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:236或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:191或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:241或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:191或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:246或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:191或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:251或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:191或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:256或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:196或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:236或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:196或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:241或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:196或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:246或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:196或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:251或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:196或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:256或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:201或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:236或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:201或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:241或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:201或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:246或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:201或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:251或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:201或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:256或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:206或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:236或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:206或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:241或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:206或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:246或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:206或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:251或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:206或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:256或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:211或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:261或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:211或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:266或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:216或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:261或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:216或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:266或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:221或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:261或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:221或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:266或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:226或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:261或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:226或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:266或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:231或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:261或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:231或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:266或其任何变体的轻链可变区。
  5. 一种双特异性或多特异性分子，其包含权利要求1-4任一项所述的抗体或其抗原结合部分。
  6. 根据权利要求1-4任一项所述的抗体或其抗原结合部分或权利要求5所述的双特异性或多特异性分子，所述抗体或其抗原结合部分是人源化的。
  7. 编码权利要求1-4任一项所述的抗体或其抗原结合部分或权利要求5所述的双特异性或多特异性分子的核酸分子；优选地，所述核酸分子包含选自SEQ ID NO:5、15、25、35、45、55、65、75、85、95、105、115、125、135、145、155、165、175、185、190、195、200、205、210、215、220、225、230、235或其任何变体的抗体重链核酸序列，和/或选自SEQ ID NO:10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、240、245、250、255、260、265、270或其任何变体的抗体轻链核酸序列。
  8. 含有权利要求7所述核酸的载体。
  9. 含有权利要求7所述的核酸或权利要求8所述载体的细胞。
  10. 一种组合物，其包含权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的双特异性或多特异性分子、权利要求6所述的抗体或其抗原结合部分或双特异性或多特异性分子、权利要求7所述的核酸、权利要求8所述的载体和/或权利要求9所述的细胞。
  11. 一种试剂盒，其包含权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的双特异性或多特异性分子、权利要求6所述的抗体或其抗原结合部分或双特异性或多特异性分子、权利要求7所述的核酸、权利要求8所述的载体、权利要求9所述的细胞和/或权利要求10所述的组合物。
  12. 权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的双特异性或多特异性分子、权利要求6所述的抗体或其抗原结合部分或双特异性或多特异性分子、权利要求7所述的核酸子、权利要求8所述的载体、权利要求9所述的细胞和/或权利要求10所述的组合物在制备用于治疗CD38相关病症的药物或试剂盒中的用途，优选地，CD38相关病症选自肿瘤和自身免疫性疾病；优选地，所述肿瘤选自多发性骨髓瘤(MM)、NK/T细胞淋巴瘤、免疫球蛋白轻链淀粉样变性、慢性淋巴细胞性白血病(CLL)、慢性髓细胞性白血病(CML)、T细胞急性淋巴细胞白血病、急性骨髓性白血病(AML)和急性淋巴细胞性白血病(ALL)；优选地，所述自身免疫性疾病选自类风湿性关节炎(RA)或系统性红斑狼疮(SLE)。
PCT/CN2020/128820 2019-11-29 2020-11-13 药物组合物及其制备方法和应用 WO2021104052A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
EP20892076.9A EP4071170A4 (en) 2019-11-29 2020-11-13 PHARMACEUTICAL COMPOSITION, ITS PREPARATION METHOD AND ITS USE
US17/780,852 US20230132604A1 (en) 2019-11-29 2020-11-13 Pharmaceutical composition, preparation method therefor and use thereof
KR1020227022100A KR20220119393A (ko) 2019-11-29 2020-11-13 약학 조성물, 이의 제조 방법 및 이의 용도
AU2020390028A AU2020390028A1 (en) 2019-11-29 2020-11-13 Pharmaceutical composition, preparation method therefor and use thereof
JP2022532596A JP2023505123A (ja) 2019-11-29 2020-11-13 医薬組成物、その製造方法及び用途
JP2024005374A JP2024026820A (ja) 2019-11-29 2024-01-17 医薬組成物、その製造方法及び用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911207085.XA CN112876563B (zh) 2019-11-29 2019-11-29 药物组合物及其制备方法和应用
CN201911207085.X 2019-11-29

Publications (1)

Publication Number Publication Date
WO2021104052A1 true WO2021104052A1 (zh) 2021-06-03

Family

ID=76039150

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/128820 WO2021104052A1 (zh) 2019-11-29 2020-11-13 药物组合物及其制备方法和应用

Country Status (7)

Country Link
US (1) US20230132604A1 (zh)
EP (1) EP4071170A4 (zh)
JP (2) JP2023505123A (zh)
KR (1) KR20220119393A (zh)
CN (1) CN112876563B (zh)
AU (1) AU2020390028A1 (zh)
WO (1) WO2021104052A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2022350815A1 (en) * 2021-09-23 2024-05-02 Sound Biopharmaceuticals Co. Ltd. Cd38 monoclonal antibody and application thereof
WO2024131849A1 (zh) * 2022-12-21 2024-06-27 非同（成都）生物科技有限公司 Cd38单克隆抗体及其应用

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7229619B1 (en) 2000-11-28 2007-06-12 Medimmune, Inc. Methods of administering/dosing anti-RSV antibodies for prophylaxis and treatment
CN102264386A (zh) * 2008-11-28 2011-11-30 赛诺菲 含特异性识别cd38的抗体和阿糖胞苷的抗肿瘤组合
US20110293606A1 (en) 2008-11-28 2011-12-01 Sanofi-Aventis Antitumor combinations containing antibodies recognizing specifically cd38 and vincristine
US20150231235A1 (en) 2006-09-26 2015-08-20 Genmab A/S Combination treatment of cd38-expressing tumors
CN105873953A (zh) * 2013-11-04 2016-08-17 格兰马克药品股份有限公司 重靶向t细胞的异源二聚免疫球蛋白的产生
CN108495641A (zh) * 2015-08-11 2018-09-04 塞勒克提斯公司 用于靶向cd38抗原和用于cd38基因失活的工程化的用于免疫疗法的细胞
WO2019140410A1 (en) * 2018-01-12 2019-07-18 Takeda Pharmaceutical Company Limited Subcutaneous dosing of anti-cd38 antibodies
CN110144008A (zh) * 2018-02-12 2019-08-20 杭州尚健生物技术有限公司 Cd38蛋白抗体及其应用
CN110229230A (zh) * 2019-04-26 2019-09-13 上海科棋药业科技有限公司 一种靶向cd38的单域抗体及其应用
US20190330363A1 (en) * 2015-11-03 2019-10-31 Janssen Biotech, Inc. Subcutaneous Formulations Of Anti-CD38 Antibodies And Their Uses

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1914242A1 (en) * 2006-10-19 2008-04-23 Sanofi-Aventis Novel anti-CD38 antibodies for the treatment of cancer
JOP20210044A1 (ar) * 2010-12-30 2017-06-16 Takeda Pharmaceuticals Co الأجسام المضادة لـ cd38
NZ719784A (en) * 2013-10-31 2022-02-25 Sanofi Sa Specific anti-cd38 antibodies for treating human cancers
KR20200061402A (ko) * 2017-10-10 2020-06-02 사노피 항-cd38 항체 및 항-cd3 및 항-cd28 항체와의 조합

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7229619B1 (en) 2000-11-28 2007-06-12 Medimmune, Inc. Methods of administering/dosing anti-RSV antibodies for prophylaxis and treatment
US20150231235A1 (en) 2006-09-26 2015-08-20 Genmab A/S Combination treatment of cd38-expressing tumors
CN102264386A (zh) * 2008-11-28 2011-11-30 赛诺菲 含特异性识别cd38的抗体和阿糖胞苷的抗肿瘤组合
US20110293606A1 (en) 2008-11-28 2011-12-01 Sanofi-Aventis Antitumor combinations containing antibodies recognizing specifically cd38 and vincristine
CN105873953A (zh) * 2013-11-04 2016-08-17 格兰马克药品股份有限公司 重靶向t细胞的异源二聚免疫球蛋白的产生
CN108495641A (zh) * 2015-08-11 2018-09-04 塞勒克提斯公司 用于靶向cd38抗原和用于cd38基因失活的工程化的用于免疫疗法的细胞
US20190330363A1 (en) * 2015-11-03 2019-10-31 Janssen Biotech, Inc. Subcutaneous Formulations Of Anti-CD38 Antibodies And Their Uses
WO2019140410A1 (en) * 2018-01-12 2019-07-18 Takeda Pharmaceutical Company Limited Subcutaneous dosing of anti-cd38 antibodies
CN110144008A (zh) * 2018-02-12 2019-08-20 杭州尚健生物技术有限公司 Cd38蛋白抗体及其应用
CN110229230A (zh) * 2019-04-26 2019-09-13 上海科棋药业科技有限公司 一种靶向cd38的单域抗体及其应用

Non-Patent Citations (18)

* Cited by examiner, † Cited by third party
Title
"Drug Absorption Enhancement: Concepts, Possibilities, Limitations, and Trends", 1994, HARWOOD ACADEMIC PUBLISHERS
"Fundamental Immunology", 1989, RAVEN PRESS, pages: 332 - 336
"Peptide and Protein Drug Delivery (Advances In Parenteral Sciences", vol. 4, 1991, M STOCKTON PRESS
"Remington's Pharmaceutical Sciences", 1975, MACK PUBLISHING COMPANY
"Remington's Pharmaceutical Sciences: The Science and Practice of Pharmacy", vol. 42, 1995, MACK PUB. CO.
BRUMMELL ET AL., BIOCHEM., vol. 32, 1993, pages 1180 - 1187
BURKS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 94, 1997, pages 412 - 417
CARRILLO, HLIPMAN, D., SIAM J APPLIED MATH, vol. 48, 1988, pages 1073
E. DIAMANDIST. CHRISTOPOULUS: "Immunoassay", 1996, ACADEMIC PRESS, INC.
KOBAYASHI ET AL., PROTEIN ENG, vol. 12, no. 10, 1999, pages 879 - 884
MALMQVIST, BIOCHEM.SOC.TRANS., vol. 27, 2000, pages 335
MARASCO ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 7889 - 7893
NELSON ET AL., J CLIN PATHOL, vol. 53, 2000, pages 111 - 117
P. TIJSSEN: "Practice and Theory of Enzyme Immunoassays", 1985, ELSEVIER SCIENCE PUBLISHERS
See also references of EP4071170A4
SMITH ET AL., J.CLIN.PATHOL., vol. 57, 2004, pages 912 - 917
VON HEINJE, G.: "Sequence Analysis in Molecular Biology", 1987, ACADEMIC PRESS
YUE LONGTAO ET AL., CURRENT IMMUNOLOGY, no. 2, 2014, pages 89 - 93

Also Published As

Publication number Publication date
EP4071170A1 (en) 2022-10-12
CN112876563B (zh) 2022-08-16
AU2020390028A1 (en) 2022-07-21
EP4071170A4 (en) 2024-05-01
JP2024026820A (ja) 2024-02-28
CN112876563A (zh) 2021-06-01
KR20220119393A (ko) 2022-08-29
JP2023505123A (ja) 2023-02-08
US20230132604A1 (en) 2023-05-04

Similar Documents

Publication Publication Date Title
US11820820B2 (en) Antibody capable of blocking CD47-SIRPa interaction and application thereof
WO2021027674A1 (zh) 一种含有抗体的肿瘤治疗剂的开发和应用
US20220162331A1 (en) Anti-cd73 monoclonal antibody and application thereof
WO2021104053A1 (zh) 一种tslp相关病症治疗剂的开发和应用
JP2018502060A (ja) 抗ｃｄ１４７抗体、方法及び使用
JP7547581B2 (ja) 腫瘍治療薬及びその応用
JP2024026820A (ja) 医薬組成物、その製造方法及び用途
WO2022194201A1 (zh) 一种靶向cldn18.2的抗体或其抗原结合片段及其应用
WO2023174396A1 (zh) 一种新型免疫调节剂的开发和应用
WO2022143951A1 (zh) 一种功能增强型抗体阻断剂的开发及其应用
WO2022117050A1 (zh) 一种新型肿瘤衔接器治疗药物的开发和应用
WO2022117045A1 (zh) 一种t细胞衔接器治疗剂的开发和应用
WO2024002308A1 (zh) 一种新型多特异肿瘤抑制剂的开发和应用
WO2024022478A1 (zh) 结合大麻素受体cb1的抗体及其用途
TW202423987A (zh) 抗ccr8抗體及其應用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20892076

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022532596

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020892076

Country of ref document: EP

Effective date: 20220629

ENP Entry into the national phase

Ref document number: 2020390028

Country of ref document: AU

Date of ref document: 20201113

Kind code of ref document: A