CN114075285B

CN114075285B - Humanized monoclonal antibody against human CD38 and application thereof

Info

Publication number: CN114075285B
Application number: CN202010841602.5A
Authority: CN
Inventors: 万云超; 胡琛霏; 杨冬美; 崔文俊
Original assignee: Reyoung Suzhou Biology Science & Technology Co ltd
Current assignee: Reyoung Suzhou Biology Science & Technology Co ltd
Priority date: 2020-08-20
Filing date: 2020-08-20
Publication date: 2023-05-30
Anticipated expiration: 2040-08-20
Also published as: CN114075285A

Abstract

The invention provides a monoclonal antibody combined with CD38 antigen and application thereof. The humanized monoclonal antibody provided by the invention can specifically recognize CD38 antigen, has better affinity, and simultaneously has antibody-dependent cell-mediated cytotoxicity (ADCC) activity and complement-dependent cytotoxicity (CDC) activity, thereby killing CD38+ cells. The application also provides application of the monoclonal antibody or the variant in preventing and/or treating tumors.

Description

Humanized monoclonal antibody against human CD38 and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a humanized monoclonal antibody combined with human CD38 protein and application thereof.

Background

Multiple Myeloma (MM) is a malignant blood tumor with abnormal proliferation of plasma cells, and can affect the generation of leucocytes, erythrocytes and stem cells in bone marrow, resulting in anemia; bone destruction, bone damage and soft tissue damage; causing symptoms such as repeated infection and renal insufficiency. The incidence rate of the traditional Chinese medicine is increased year by year, the incidence rate of global multiple myeloma in 2019 accounts for 1.8 percent of all tumors, accounts for 10 to 15 percent of blood tumors, and is discharged at the 2 nd position of the blood tumors. As people continuously deepen cognition on multiple myeloma, research and development efforts are continuously increased, new medicines, diagnosis and treatment means are more and more abundant, especially, immune modulators (such as thalidomide and lenalidomide), proteasome inhibitors (such as bortezomib and carfilzomib), autologous hematopoietic stem cell transplantation (ASCT), monoclonal antibodies (such as Daratumumab) and other medicines are clinically applied, and the total survival time (OS) of MM patients is obviously improved in the past two decades, and compliance is also obviously improved; however, due to the presence of myeloma stem cells and minimal residual lesions in patients, multiple myeloma is not completely cured at present, and almost all patients eventually relapse. However, current treatment regimens for relapsed/refractory myeloma (RRMM) are limited and patient survival outcomes are poor; therefore, the treatment of RRMM has become a focus of attention for researchers at home and abroad.

The prior art shows that the immunophenotype of plasma cells includes: cd138+, cd38+, cd19+, cd56-. Typically, CD38 is expressed by hematopoietic cells and solid tissue, most of the medullary thymus cells are CD38+, resting and circulating T and B cells are CD38-, and activated cells are CD38+. CD38 protein is one of the antigens expressed on malignant plasma cells and is expressed in a variety of malignant blood disease cells including, but not limited to, multiple myeloma cells, B-cell chronic lymphocytic leukemia cells, B-cell acute lymphocytic leukemia cells.

CD38, a single-chain transmembrane glycoprotein, is 45kDa in length and comprises a short amino-terminal intracellular region, a transmembrane region and a long carboxy-terminal extracellular region, which has the functions of enzyme activity, receptor and adhesion in addition to being a phenotypic marker of cell differentiation. CD38, an extracellular enzyme, catalyzes the conversion of nicotinamide adenine dinucleotide (NAD+) to adenosine diphosphate ribose (ADPR) and nicotinic adenine dinucleotide phosphate (NAADP), a process involved in the mobilization of intracellular calcium ions. As a receptor, CD38 plays a role in the immune process, mediating cytokine production, proliferation of T lymphocytes and protecting mature B lymphocytes and dendritic cells from apoptosis. CD38 is also involved in cell-to-cell adhesion, which makes the pattern of migration of cells with different levels of CD38 expression into tissues different. In addition, the adhesion function of CD38 is tightly linked to the receptor function, CD31 is a natural ligand for CD38, and human T lymphocytes adhere to endothelial cells through CD38-CD31 interactions. CD38 is mainly expressed in T cells, dendritic cells, natural Killer (NK) cells, hematopoietic stem cells, and also expressed in pancreatic cells, cornea cells, and the like. Most importantly, the high expression of CD38 in myeloma cells makes CD38 an attractive therapeutic target for MM.

anti-CD 38 antibodies have been reported in the literature, for example, lande R, et al, cell immunol.220 (1), 30-8 (2002), ausiello CM, et al, tissue antibodies.56 (6), 539-47 (2000), and Cotner T, et al, lnt J immunol.3 (3), 255-68 (1981). Patent CN110144008A discloses an antibody capable of binding to CD38 protein, having a good affinity, capable of killing CD38 by antibody-dependent cell-mediated cytotoxicity ADCC, complement-dependent cytotoxicity CDC and/or apoptosis ⁺ And (3) cells. Patent WO2020/052546A1 also discloses a series of murine, chimeric or humanized antibodies comprising CDR regions of an anti-CD 38 antibody, as well as pharmaceutical compositions comprising an anti-CD 38 antibody or antigen binding fragment thereof and their use as medicaments.

Although CAR-T and ADC products for multiple myeloma have shown some good results in terms of efficacy in recent years, their drawbacks are not negligible. Such as off-target effects, excessive T cell activation, induction of cytokine release syndrome, and neurotoxicity of CAR-T products; the ADC product has complex production process, poor in-vivo stability of molecules and off-target toxicity of small molecules; BCMA targets are less mature than CD38 targets. Combining clinical therapeutic effects of darimumab and clinical study data of sarcolisa and MOR202, monoclonal antibodies to the CD38 target remain a very good choice for the treatment of multiple myeloma and relapsed/refractory myeloma.

Therefore, the development of monoclonal antibodies with high selectivity, high affinity and high drug property aiming at CD38 targets continues to be a problem to be solved.

Disclosure of Invention

The invention provides a humanized monoclonal antibody combined with CD38 protein and application thereof. The humanized monoclonal antibody combined with the CD38 protein has higher affinity, and can kill tumor cells and inhibit tumor growth through antibody-dependent cell-mediated cytotoxicity ADCC and complement-dependent cytotoxicity CDC functions, thereby achieving the purposes of tumor prevention and treatment.

In one aspect, the invention provides a series of monoclonal antibodies that bind to CD38 protein.

Specifically, the monoclonal antibody binding to CD38 protein comprises:

(1) An HCDR variant comprising a heavy chain complementarity determining region having any one or more of heavy chains HCDR1, HCDR2 and HCDR3 of sequences SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively, or having a3, 2 or 1 amino acid difference from heavy chain HCDR1, HCDR2 or HCDR3 of SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3, respectively;

and/or (2) a light chain complementarity determining region comprising any one or more of the light chains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively, or an LCDR variant having a3, 2, or 1 amino acid difference from the light chain LCDR1, LCDR2, or LCDR3 shown in SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6, respectively.

Further specifically, the monoclonal antibody that binds to CD38 protein comprises:

(1) A heavy chain variable region variant comprising a heavy chain variable region as set forth in SEQ ID NO. 7 or SEQ ID NO. 8, or having a3, 2 or 1 amino acid difference from the heavy chain variable region set forth in SEQ ID NO. 7 and SEQ ID NO. 8;

and/or (2) a light chain variable region comprising a light chain variable region as set forth in SEQ ID NO. 9 or SEQ ID NO. 10, or a light chain variable region variant having a3, 2 or 1 amino acid difference from the light chain variable regions set forth in SEQ ID NO. 9 and SEQ ID NO. 10.

(1) A heavy chain with a sequence shown as SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO. 14;

and/or (2) a light chain having a sequence shown as SEQ ID NO. 15 or SEQ ID NO. 16.

(1) A heavy chain with a sequence shown as SEQ ID NO. 11 and a light chain with a sequence shown as SEQ ID NO. 15;

or (2) a heavy chain having a sequence shown as SEQ ID NO. 12 and a light chain having a sequence shown as SEQ ID NO. 15;

or (3) a heavy chain having a sequence shown as SEQ ID NO. 13 and a light chain having a sequence shown as SEQ ID NO. 16;

or (4) a heavy chain having a sequence shown as SEQ ID NO. 14 and a light chain having a sequence shown as SEQ ID NO. 16.

In another aspect, the invention provides a series of nucleic acid molecules encoding said monoclonal antibodies that bind to CD38 protein.

In particular, the nucleic acid molecules comprise one or more codon optimized nucleic acid molecules.

In another aspect, the invention provides a series of vectors comprising one or more nucleic acid molecules as described herein.

In particular, the vectors include, but are not limited to, plasmids, viruses, phages.

In another aspect, the invention provides a series of host cells comprising or producing said monoclonal antibodies that bind to CD38 protein.

In another aspect, the invention provides a method of producing the monoclonal antibody or variant thereof, comprising culturing a host cell as described herein under conditions such that the monoclonal antibody or variant thereof is expressed.

In particular, the host cells include, but are not limited to, microorganisms, plants or animal cells, into which the vectors of the present invention can be introduced by methods known to those skilled in the art, such as electroporation, lipofectine transfection, lipofectamine transfection, and the like.

In another aspect, the invention provides a pharmaceutical composition comprising a monoclonal antibody or variant thereof, the nucleic acid molecule, the vector and/or the cell of the invention, and optionally a pharmaceutically acceptable adjuvant.

Specifically, the pharmaceutically acceptable adjuvants include, but are not limited to: diluents, excipients, fillers, wetting agents, disintegrants, flavoring agents and binders.

In a further aspect, the invention provides the use of said monoclonal antibody or variant thereof, said nucleic acid molecule, said vector and/or said cell in the manufacture of a medicament, kit and/or device for the prevention and/or treatment of a CD38 positive disease or condition.

In particular, the CD38 positive disease or disorder includes an immune disease or hematological tumor.

Further specifically, the immune diseases include rheumatoid arthritis, systemic lupus erythematosus, immune-mediated thrombocytopenia symptoms, hemolytic anemia, ankylosing spondylitis, multiple sclerosis, psoriasis, crohn's disease, glomerulonephritis, asthma and the like.

Further specifically, the hematological neoplasm includes leukemia, B-cell lymphoma, T-cell lymphoma, NK cell lymphoma, plasma cell malignancy, and myeloma.

Preferably, the hematological neoplasm is multiple myeloma.

In another aspect, the invention provides a method of preventing and/or treating a tumor, said method comprising administering to a subject in need thereof the monoclonal antibody or variant thereof, the nucleic acid molecule, the vector, the host cell and/or the pharmaceutical composition, kit and/or device of the invention.

Compared with the prior art, the invention has the following positive and beneficial effects:

(1) The present invention provides a series of monoclonal antibodies that bind to the CD38 antigen, said monoclonal antibodies being humanized monoclonal antibodies.

(2) The monoclonal antibody provided by the invention is a high-expression sequence, has high expression quantity and less impurities, is easy to purify and collect, reduces the purification and collection cost, and is favorable for large-scale production and application.

(3) The monoclonal antibody provided by the invention has good thermal stability and solution stability, and is convenient to store.

(4) The monoclonal antibody provided by the invention can specifically identify the human CD38 antigen, and has high affinity.

(5) The monoclonal antibody provided by the invention has better capability of combining with CD38 antigen, and has effective antibody-dependent cell-mediated cytotoxicity (ADCC) activity and complement-dependent cytotoxicity (CDC) activity.

(6) The monoclonal antibody provided by the invention has better anti-tumor activity in vivo.

Drawings

FIG. 1 shows SDS-PAGE 4-12% gradient gel electrophoresis of monoclonal antibodies RB0021-S1, RB0021-S2, RB0021-S3, and RB 0021-S4.

FIG. 2 is a SEC-HPLC chromatogram of monoclonal antibody RB 0021-S1.

FIG. 3 is a SEC-HPLC chromatogram of monoclonal antibodies RB 0021-S2.

FIG. 4 is a SEC-HPLC chromatogram of monoclonal antibodies RB 0021-S3.

FIG. 5 is a SEC-HPLC chromatogram of monoclonal antibodies RB 0021-S4.

FIG. 6 is a graph showing the results of detection of the binding capacity of monoclonal antibodies RB0021-S1, RB0021-S2, RB0021-S3, and RB0021-S4 to cell surface antigens.

FIG. 7 is a graph showing the results of detection of ADCC activities of monoclonal antibodies RB0021-S1, RB0021-S2, RB0021-S3, and RB 0021-S4.

FIG. 8 is a graph showing the results of detection of CDC activities of monoclonal antibodies RB0021-S1, RB0021-S2, RB0021-S3, and RB 0021-S4.

FIG. 9 is a statistical plot of the mean tumor volume results of mice tested for monoclonal antibody in vivo activity.

FIG. 10 is a statistical plot of the average tumor weight results of monoclonal antibody in vivo activity test mice.

FIG. 11 is a schematic diagram of tumor of mice with monoclonal antibody in vivo activity.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

Terminology

In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The three-letter and one-letter codes for amino acids used in the present invention are as described in J.biol. Chem,243, p3558 (1968).

The antibody disclosed by the invention refers to an immunoglobulin, and is a tetrapeptide chain structure formed by linking two identical heavy chains and two identical light chains through inter-chain disulfide bonds. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or isotypes, i.e., igM, igD, igG, igA, igE, with their respective heavy chains being μ, δ, γ, α, ε chains, respectively. The Ig of the same class can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, for example, igG can be classified into IgG1, igG2, igG3 and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each of the five classes of Ig may have either a kappa chain or a lambda chain.

The sequences of the heavy and light chains of antibodies, near the N-terminus, vary widely, being the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each light chain variable region (VL or LCVR) and heavy chain variable region (VH or HCVR) consists of 3 CDR regions and 4 FR regions, arranged in order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2 and LCDR3 and the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3.

The term "complementarity determining region" (CDR) refers to one of 6 hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding. Typically there are three CDRs (HCDR 1, HCDR2, HCDR 3) in each heavy chain variable region and three CDRs (LCDR 1, LCDR2, LCDR 3) in each light chain variable region. The amino acid sequence boundaries of the CDRs are determined using the "Kabat numbering convention" (see Kabat et al (1991)).

The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to identical epitopes, except for possible variant antibodies. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants, each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the method is applicable to a variety of applications. "monoclonal" refers to the properties of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. The monoclonal antibodies of the present invention can be prepared by a variety of techniques known to those skilled in the art, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, transgenic methods, and the like.

The monoclonal antibody disclosed by the invention is a humanized monoclonal antibody.

The term "murine monoclonal antibody" is herein a monoclonal antibody raised against human CD38 according to the knowledge and skill in the art. The preparation is performed by injecting a test subject with a CD38 antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties. In a preferred embodiment of the invention, the murine CD38 antibody or antigen binding fragment thereof may further comprise a murine kappa, lambda chain or variant light chain constant region or further comprise a murine IgG1, igG2, igG3 or variant heavy chain constant region.

The term "humanized monoclonal antibody" refers to an antibody produced by grafting murine CDR sequences into the framework of human antibody variable regions, i.e., different types of human germline antibody framework sequences, which overcomes the heterologous response induced by chimeric antibodies carrying a large number of murine protein components. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse mutation (or back mutation) to maintain activity.

The monoclonal antibodies that bind to CD38 protein of the present invention include combinations of heavy and light chains as set forth in table 1 below.

TABLE 1 sequence characterization of monoclonal antibodies that bind CD38 protein

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The terms "specific binding", "selective binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is present at about less than 10 ^-8 M, e.g. less than about 10 ^-9 M、10 ^-10 M、10 ^-11 Affinity (KD) binding of M or less. Where "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

The term "ADCC" is antibody-dependent cell-mediated cytotoxicity (anti bodydependent cell-mediated cytotoxicity, ADCC), generally refers to the recognition of an Fc fragment coated on a target antigen by cells with killing activity through its surface-expressed Fc receptor (FcR), whereby effector cells of the immune system actively lyse the target cells whose membrane surface antigens have been bound by specific antibodies.

The term "CDC" is complement dependent cytotoxicity (complement dependent cytotoxicity, CDC), generally refers to the cytotoxic effect of complement participation, i.e. activation of the classical pathway of complement by binding of specific antibodies to the corresponding antigen on the surface of the cell membrane, forming complexes which exert a lytic effect on the target cells.

The term "Daudi cell" generally refers to a cell line derived from Burkitt's lymphoma. Although Daudi cells have an intracellular heavy chain of class I, they have no expression of class I human leukocyte antigen (human leukocyte antigen, HLA) molecules on their surface because of the defect in the gene encoding the beta 2-microglobulin (beta 2 m) resulting in the protein lacking translatable mRNA.

The term "Jurkat-Lucia NFAT-CD16 cells" is genetically engineered from a human T lymphocyte Jurkat cell line. The cell line stably expressed the luciferases reporter gene from Lucia under the control of the minimal promoter and 6 NFAT response elements, as well as the cell surface Fc receptor CD16a (fcyriiia; V158 high affinity allograft). Jurkat cells naturally express the NFAT functional signaling pathway.

The term "NCI-H929 cell" refers to a cell line established in the malignant effusion of a myeloma patient, a human myeloma cell whose cell surface expresses the CD38 antigen.

The term "NOD/SCID mice", i.e., non-obese diabetic/severe combined immunodeficiency mice, is an immunodeficient mouse that is backcrossed with a non-obese diabetic mouse (NOD/Lt) strain (TL, BL, NK cell deficiency) on the basis of the SCID (severe combined immunodeficiency) mice. NOD/SCID mice have both innate immunity and T and B lymphocyte deficiencies, and various tumor cells can be implanted with less rejection and Graft Versus Host Disease (GVHD), so NOD/SCID mice are increasingly becoming a useful tool for hematological experimental research.

EXAMPLE 1 monoclonal antibody preparation

The DNA nucleotide sequence of the RB0021-S1 heavy chain is:

CAAGTGACACTGAAAGAGAGCGGCCCTACACTGGTGAAGCCCACCCAGACACTGACACTGACATGCACCACCAGCGGCTACACATTCACCTCCCACGGCATCAACTGGGTGAGACAGCCCCCCGGCAAGGCCCTCGAGTGGATCGGCTACATCTACATCGGCAACGGCTACACCGAGTACAACGAGAAGTTCAAGGGAAGAGCTACACTGACCAGCGACACCAGCAAGAATCAAGCCGTGCTGACCATGACCAACATGGACCCCGTGGATACCGCCACCTACTTCTGCGCTAGATCCCACTACGACAGCTCCAGCTGGTTTGCTTACTGGGGCCAAGGCACACTGGTGACCGTGAGCTCCGCCAGCACCAAGGGACCTAGCGTGTTTCCTCTGGCCCCTTCTAGCAAGAGCACAAGCGGAGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTTCCCGAGCCCGTGACCGTGTCTTGGAATTCAGGAGCCCTGACCAGCGGAGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGACTGTATAGCCTGAGCAGCGTGGTGACCGTGCCTTCTTCTTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGTCTTGCGACAAGACCCACACTTGCCCCCCTTGTCCAGCTCCAGAACTCCTGGGAGGACCTAGCGTGTTCCTGTTCCCTCCCAAGCCTAAGGACACCCTGATGATCAGCCGGACCCCAGAAGTGACTTGCGTGGTGGTGGACGTGTCCCACGAAGACCCCGAGGTCAAGTTCAATTGGTACGTGGACGGAGTGGAGGTGCACAACGCTAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCAGCCCCTATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCTCAGGTGTACACCCTGCCCCCTAGCAGAGACGAGCTGACCAAGAACCAGGTGTCCCTGACTTGCCTCGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAATCTAACGGTCAGCCAGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAAAGCCGCTGGCAGCAGGGCAACGTGTTCTCTTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCAGGAAAG

the DNA nucleotide sequence of the RB0021-S2 heavy chain is:

CAAGTGACACTGAAAGAGAGCGGCCCTACACTGGTGAAGCCCACCCAGACACTGACACTGACATGCACCACCAGCGGCTACACATTCACCTCCCACGGCATCAACTGGGTGAGACAGCCCCCCGGCAAGGCCCTCGAGTGGATCGGCTACATCTACATCGGCAACGGCTACACCGAGTACAACGAGAAGTTCAAGGGAAGAGCTACACTGACCAGCGACACCAGCAAGAATCAAGCCGTGCTGACCATGACCAACATGGACCCCGTGGATACCGCCACCTACTTCTGCGCTAGATCCCACTACGACAGCTCCAGCTGGTTTGCTTACTGGGGCCAAGGCACACTGGTGACCGTGAGCTCCGCCAGCACCAAGGGACCTAGCGTGTTTCCTCTGGCCCCTTCTAGCAAGAGCACAAGCGGAGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTTCCCGAGCCCGTGACCGTGTCTTGGAATTCAGGAGCCCTGACCAGCGGAGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGACTGTATAGCCTGAGCAGCGTGGTGACCGTGCCTTCTTCTTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGTCTTGCGACAAGACCCACACTTGCCCCCCTTGTCCAGCTCCAGAACTCCTGGGAGGACCTAGCGTGTTCCTGTTCCCTCCCAAGCCTAAGGACACCCTGATGATCAGCCGGACCCCAGAAGTGACTTGCGTGGTGGTGGACGTGTCCCACGAAGACCCCGAGGTCAAGTTCAATTGGTACGTGGACGGAGTGGAGGTGCACAACGCTAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCAGCCCCTATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCTAGAAAGCCTCAGGTGTACACCCTGCCCCCTAGCAGAGACGAGCTGACCAAGAACCAGGTGTCCCTGACTTGCCTCGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAATCTAACGGTCAGCCAGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAAAGCCGCTGGCAGCAGGGCAACGTGTTCTCTTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCAGGAAAG

the DNA nucleotide sequence of the RB0021-S3 heavy chain is:

CAAGTGCAGCTGGTGCAGAGCGGCGCTGAGGTGAAGAAACCCGGCAGCAGCGTGAAGGTGAGCTGCAAGACCAGCGGCTACACCTTCACCAGCCACGGCATCAACTGGGTGAGACAAGCCCCCGGCCAAGGACTGGAGTGGATCGGCTACATCTACATCGGCAACGGCTACACCGAGTACAACGAGAAGTTCAAGGGAAGAGCCACACTGACCTCCGACACAAGCACCAGCACCGCCTACATGGAGCTGAGCTCTCTGAGGAGCGAGGACACCGCCGTGTATTTCTGCGCTAGAAGCCACTACGACAGCAGCAGCTGGTTCGCTTACTGGGGACAAGGCACACTGGTGACCGTGTCCAGCGCCAGCACCAAGGGACCTAGCGTGTTTCCTCTGGCCCCTTCTAGCAAGAGCACAAGCGGAGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTTCCCGAGCCCGTGACCGTGTCTTGGAATTCAGGAGCCCTGACCAGCGGAGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGACTGTATAGCCTGAGCAGCGTGGTGACCGTGCCTTCTTCTTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGTCTTGCGACAAGACCCACACTTGCCCCCCTTGTCCAGCTCCAGAACTCCTGGGAGGACCTAGCGTGTTCCTGTTCCCTCCCAAGCCTAAGGACACCCTGATGATCAGCCGGACCCCAGAAGTGACTTGCGTGGTGGTGGACGTGTCCCACGAAGACCCCGAGGTCAAGTTCAATTGGTACGTGGACGGAGTGGAGGTGCACAACGCTAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCAGCCCCTATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCTCAGGTGTACACCCTGCCCCCTAGCAGAGACGAGCTGACCAAGAACCAGGTGTCCCTGACTTGCCTCGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAATCTAACGGTCAGCCAGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAAAGCCGCTGGCAGCAGGGCAACGTGTTCTCTTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCAGGAAAG

the DNA nucleotide sequence of the RB0021-S4 heavy chain is:

CAAGTGCAGCTGGTGCAGAGCGGCGCTGAGGTGAAGAAACCCGGCAGCAGCGTGAAGGTGAGCTGCAAGACCAGCGGCTACACCTTCACCAGCCACGGCATCAACTGGGTGAGACAAGCCCCCGGCCAAGGACTGGAGTGGATCGGCTACATCTACATCGGCAACGGCTACACCGAGTACAACGAGAAGTTCAAGGGAAGAGCCACACTGACCTCCGACACAAGCACCAGCACCGCCTACATGGAGCTGAGCTCTCTGAGGAGCGAGGACACCGCCGTGTATTTCTGCGCTAGAAGCCACTACGACAGCAGCAGCTGGTTCGCTTACTGGGGACAAGGCACACTGGTGACCGTGTCCAGCGCCAGCACCAAGGGACCTAGCGTGTTTCCTCTGGCCCCTTCTAGCAAGAGCACAAGCGGAGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTTCCCGAGCCCGTGACCGTGTCTTGGAATTCAGGAGCCCTGACCAGCGGAGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGACTGTATAGCCTGAGCAGCGTGGTGACCGTGCCTTCTTCTTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGTCTTGCGACAAGACCCACACTTGCCCCCCTTGTCCAGCTCCAGAACTCCTGGGAGGACCTAGCGTGTTCCTGTTCCCTCCCAAGCCTAAGGACACCCTGATGATCAGCCGGACCCCAGAAGTGACTTGCGTGGTGGTGGACGTGTCCCACGAAGACCCCGAGGTCAAGTTCAATTGGTACGTGGACGGAGTGGAGGTGCACAACGCTAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCAGCCCCTATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCTAGAAAGCCTCAGGTGTACACCCTGCCCCCTAGCAGAGACGAGCTGACCAAGAACCAGGTGTCCCTGACTTGCCTCGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAATCTAACGGTCAGCCAGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAAAGCCGCTGGCAGCAGGGCAACGTGTTCTCTTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCAGGAAAG

the nucleotide sequences of the RB0021-S1 and RB0021-S2 light chain DNA are:

GATGTGGTGATGACCCAGAGCCCCGACTCTCTGGCCGTGTCTCTGGGAGAGAGAGCCACCATCAACTGCAGATCCAGCCAGAGCCCCGAGCACAGCAACGGCAACACCTATCTGCACTGGTATCAGCAGAAGCCCGGCCAGCCTCCCAAGCTGCTGATCTACAAGGTGTCCAATAGATTCAGCGGCGTGCCCGACAGATTCAGCGGATCCGGCAGCGGCACCGATTTCACACTGACCATCAGCTCTCTGCAAGCCGAAGACGTGGCCGTGTACTTCTGCAGCCAAGGCACACACTTCCCTTTCACCTTCGGCCAAGGCACCAAGGTGGAGATTAAGAGAACCGTGGCCGCTCCTAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAAAGCGGAACAGCCAGCGTCGTCTGCCTGCTGAATAACTTCTACCCCCGGGAGGCCAAAGTCCAGTGGAAAGTGGACAACGCCCTGCAGAGCGGAAACTCTCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACACTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCTTGCGAGGTCACACACCAGGGACTGTCTAGCCCAGTGACCAAGAGCTTCAACCGCGGCGAGTGTTGA

the nucleotide sequences of the RB0021-S3 and RB0021-S4 light chain DNA are:

GACGTGCAGATGACCCAGTCCCCTAGCAGCGTGAGCGCTTCCGTGGGAGACAGAGTGACCATCACATGCAGAAGCAGCCAGAGCCCCGAGCATAGCAACGGCAACACCTATCTGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTCATCTACAAGGTGTCCAATAGATTCAGCGGCGTGCCCTCCAGATTCAGCGGATCCGGCAGCGGCACCGATTTCACACTGACCATCAGCTCTCTGCAGCCCGAGGACTTCGCCACCTACTTTTGCTCCCAAGGCACCCACTTCCCCTTCACCTTCGGCCAAGGCACCAAGGTGGAGATCAAGAGAACCGTGGCCGCTCCTAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAAAGCGGAACAGCCAGCGTCGTCTGCCTGCTGAATAACTTCTACCCCCGGGAGGCCAAAGTCCAGTGGAAAGTGGACAACGCCCTGCAGAGCGGAAACTCTCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACACTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCTTGCGAGGTCACACACCAGGGACTGTCTAGCCCAGTGACCAAGAGCTTCAACCGCGGCGAGTGTTGA

cloning different antibody nucleotide sequences into pcDNA3.1 expression vector, and preparing transfection by using large drawing plasmid of large drawing kit for controlling endotoxin, OPM-293 CD05 medium culture 293F cell. Cell count the day before transfection, density 2.59 x 10 ⁶ Per mL, 98.1% viability, cells were diluted to 1.5 x 10 with fresh medium ⁶ Culture was continued with/mL. Cell count on day of transfection, density 3.01 x 10 ⁶ Per mL, 98.8% viability, cells were diluted to 2.78 x 10 with fresh medium ⁶ /mL. 1/10 of the transfection volume of OPM-293 CD05 medium (Ao Pu Mai, cat# 81075-001) was taken, 1:1 heavy and light chain plasmids were added to 1.5. Mu.g/mL transfection volume, followed by 3-fold additionPlasmid amount of PEI, incubated at room temperature for 15min. Adding the incubation mixture into the treated cells, and mixing the cells uniformly while adding the incubation mixture. Placing into a shaking table for culturing at 37deg.C, 90rpm and 8% CO ₂ . OPM-CHO PFF05 feed (O Pu Mai, cat. FB 1279-001) was added on the first and fourth days after transfection, respectively, and supernatants were collected on day 6 after transfection.

EXAMPLE 2 purification and detection of monoclonal antibodies

1. Affinity chromatography

The filler Mabselect Sure was rinsed with 0.5M NaOH, 0.1M NaOH, each for 1 hour to time endotoxin, and the filler was equilibrated with 20mM PB,150mM NaCl (pH 7.2). The supernatant of transient expression of RB0021 PC (Daratumumab), RB0021-S1, RB0021-S2, RB0021-S3 and RB0021-S4 was filtered and loaded at a flow rate of 1mL/min, after loading, the supernatant was washed with 20mM PB,150mM NaCl to UV280 peak level, then 20mM PB,1M NaCl (pH 6.5) to peak level, finally 20mM Cit-Na3Cit (pH 3.5) was used for elution and 1M Tris (pH 9.0) was used for neutralization.

2. Cationic chromatography

The packing SP-HP was rinsed with 0.5M NaOH, and the endotoxin was equilibrated with 20mM PB (pH 6.0) at 2 hours of each treatment with 0.1M NaOH. Samples of RB0021 PC (Daratumumab), RB0021-S1, RB0021-S2, RB0021-S3 and RB0021-S4 obtained by affinity chromatography are subjected to pH adjustment to 6.0, the samples are subjected to dilution adjustment, the conductance is less than 3 mu S/cm, the sample loading is performed, the flow rate is controlled to be 1mL/min, 20mM PB (pH 6.0) buffer solution is used for washing to reach a UV280 baseline after the sample loading is finished, and then 20mM PB and 1M NaCl (pH 6.0) are used for performing linear gradient elution (0-30% B and 20 CV), and the eluted samples are collected according to peaks. Table 2 below shows the statistics of the final monoclonal antibodies.

Table 2 statistical table of monoclonal antibody data

As shown in Table 2, under the condition of low transient dye expression level, the monoclonal antibody with higher monomer quality and similar acid-base variant compared with the control group can be obtained, and the denaturation temperature, namely TM value of the monoclonal antibody obtained by the invention is higher than that of the control group, so that the monoclonal antibody has better thermal stability; meanwhile, the monoclonal antibody has higher isoelectric point, and is favorable for stable storage in buffer solution.

3. Detection and analysis

SDS-PAGE 4-12% gradient gel and SEC-HPLC analysis of protein purity.

Wherein, FIG. 1 is a gradient gel electrophoresis chart of monoclonal antibodies RB0021-S1, RB0021-S2, RB0021-S3, RB0021-S4 SDS-PAGE 4-12%, and FIG. 2, FIG. 3, FIG. 4, FIG. 5 are respectively RB0021-S1, RB0021-S2, RB0021-S3, RB0021-S4 SEC-HPLC chromatograms. As can be seen from FIGS. 1 to 5, the electrophoresis patterns of RB0021-S1, RB0021-S2, RB0021-S3 and RB0021-S4 are single bands and are single peaks, which indicates that the monoclonal antibody obtained by the invention has high expression level and less impurity.

Table 3 below shows the dispersion index of the monoclonal antibodies obtained by SEC detection.

TABLE 3 dispersion index of monoclonal antibodies

As shown in Table 3, compared with the control, the monoclonal antibody of the invention has better dispersion index at three different concentrations of large, medium and small, which indicates that the monoclonal antibody has better solution stability.

EXAMPLE 3 monoclonal antibody affinity assay

Biacore affinity assay was performed using GE 8K, HBS-EP+ as assay buffer, at 25℃and ligand dilution to 5. Mu.g/mL, immobilization at a flow rate of 10. Mu.L/min for 20s onto Protein A chip. Analyte CD38 was diluted gradient to 0,0.111,0.333,1,3,9 (. Times.3), 27, 81nM at 30. Mu.L/min, bound for 300s, dissociated for 300s, regenerated with 10mM Glycine-HCl (pH 1.5) at 100. Mu.L/min for 30s and repeated once. The measured affinity data are shown in table 4 below:

table 4 affinity data

Antibodies to	affinity(human)
		RB0021 PC(Daratumumab)	8.86E-08
RB0021-S1	1.17E-09
		RB0021-S2	1.17E-09
RB0021-S3	1.04E-09
		RB0021-S4	1.04E-09

As can be seen from Table 4, the monoclonal antibodies of the present invention specifically recognize human CD38 protein, and have higher affinity for CD38 protein than the monoclonal antibodies of the control group.

Example 4 binding to cell surface antigen

Daudi cells cultured in RPMI1640+10% FBS medium were added to 96-well plates, the supernatant was discarded, 100. Mu.L/well of gradient diluted antibody-resuspended cell pellet (positive control for RB0021 PC, negative control for Isotype control) was added, the supernatant was discarded, 100. Mu.L/well of pre-diluted secondary antibody (FITC) resuspended cells was added, and after incubation at 2-8℃for 0.5 hours, detection was performed on a flow cytometer.

TABLE 5 binding data to cell surface antigens

As can be seen from Table 5, the monoclonal antibodies of the present invention have a better ability to bind to cell surface CD38 antigen.

Example 5 antibody-dependent cell-mediated cytotoxicity (ADCC) Activity

Will be 1 x 10 ⁵ NCI-H929 cells were added to 96-well plates, 40. Mu.L of gradient diluted antibody (positive control RB0021 PC, negative control Isotype control) was added, 37℃and 5% CO ₂ Incubating for 1 hour; and then 2 x 10 ⁵ Jurkat-Lucia-NFAT-CD16 cells were plated in 96-well plates. 37 ℃,5% CO ₂ After 6 hours of incubation, 50. Mu.L of Quanti-LUC was added, and after 5 minutes of reaction at room temperature, the fluorescent signal was read.

TABLE 6 ADCC Activity assay data

Sample ID	EC50(μg/mL)	Top MFI
			RB0021 PC(Daratumumab)	0.017	57161
RB0021-S1	0.018	65759
			RB0021-S2	0.011	45150
RB0021-S3	0.011	47332
			RB0021-S4	0.014	56729
Istype Control	0.375	2118

As can be seen from Table 6, the EC50 values of the monoclonal antibodies RB0021-S2, RB0021-S3 and RB0021-S4 of the invention are obviously lower than those of the control group, and the EC50 values of the monoclonal antibodies RB0021-S1 are basically the same as those of the control group, so that the ADCC activity of the monoclonal antibodies RB0021-S2, RB0021-S3 and RB0021-S4 EC50 of the invention is superior to that of the control group, and the ADCC activity of the RB0021-S1 is similar to that of the control group. Therefore, the monoclonal antibody of the present invention has potent ADCC activity.

EXAMPLE 6 complement dependent cytotoxin Activity (CDC)

Daudi cells cultured in RPMI1640+10% FBS medium were added to 96-well plates, 40. Mu.L of gradient diluted antibody was added, and 40. Mu.L of pre-diluted complement, 37℃and 5% CO were added ₂ Culturing for 6 hours. RB0021 PC (Daratumumab) was used as positive control and Isotype control was used as negative controlControl, with Cell Titer-Glo ^TM Reagent detects cell killing.

TABLE 7 CDC Activity assay data

As can be seen from Table 7, the monoclonal antibodies RB0021-S2 and RB0021-S4 of the invention were significantly superior to the control for Complement Dependent Cytotoxin (CDC) activity of Daudi cells.

EXAMPLE 7 in vivo Activity assay

Will be 1 x 10 ⁷ Each mL of NCI-H929 cells was inoculated subcutaneously into the right armpit of 5-6 week NOD/SCID mice at 0.2 mL/after mixing matrigel in equal volumes. The tumor volume to be averaged was 150mm ³ When the tumor is left and right, tumor-bearing animals with vigorous tumor growth, no ulcer and good health condition are selected for grouping and starting administration. Mice were divided into 11 groups (n=13) such that the average tumor sizes of all groups were similar and dosing was started by tail vein injection (day 0), twice weekly. Group 1 (FIGS. 11G1-22, G1-32, G1-47, G1-48, G1-73, G1-93) received 45mg/kg of negative control antibody treatment twice weekly; group 2 (FIGS. 11G2-9, G2-10, G2-39, G2-74, G2-88, G2-92) received 5mg/kg of positive control antibody (Daratumumab) twice weekly; group 3 (FIG. 11G3-36, G3-38, G3-76, G3-77, G3-85, G3-86) received 45mg/kg of positive control antibody (Daratumumab) twice weekly. Group 4 (FIG. 11G4-1, G4-17, G4-43, G4-64, G4-66, G4-82) received 5mg/kg of RB0021-S1 antibody twice a week; group 5 (FIG. 11G5-3, G5-4, G5-19, G5-30, G5-44, G5-91) received 45mg/kg of RB0021-S1 antibody twice a week. Group 6 (FIG. 11G6-5, G6-50, G6-51, G6-60, G6-80, G6-100) received 5mg/kg of RB0021-S2 antibody, administered twice weekly; group 7 (FIG. 11G7-6, G7-18, G7-23, G7-28, G7-67, G7-75) received 45mg/kg of RB0021-S2 antibody twice a week. Group 8 (FIG. 11G8-27, G8-40, G8-54, G8-55, G8-57, G8-78) received 5mg/kg of RB0021-S3 antibody twice a week; group 9 (FIGS. 11G9-21, G9-24, G9-37, G9-83, G9-84, G9-90) received 45mg/kg of RB0021-S3 antibody, administered twice weekly. Group 10 (FIG. 11G10-16, G10-42, G10-61, G10-63, G10-69, G10-79) received 5mg/kg of RB0021-S4 antibody twice a week; group 11 (FIGS. 11G11-7, G11-12, G11-13, G11-26, G11-70, G11-81) received 45mg/kg of RB0021-S4 antibody, administered twice a week. Tumor volumes at different time points were determined using the following formula: tumor volume (mm) ³ ) =1/2×long diameter×short diameter ² The anti-tumor efficacy is judged according to the tumor growth condition. Body weight was measured twice weekly to monitor toxicity and all treatments were well tolerated.

The inhibition of tumor growth by the different treatments is different, fig. 9 shows the average tumor volume statistics of mice, fig. 10 shows the average tumor weight statistics of mice, and as can be seen from fig. 9 and 10, the anti-CD 38 monoclonal antibodies of the invention show potent anti-tumor efficacy in vivo, both superior to that of the control monoclonal antibody, wherein the RB0021-S1 antibody given 45mg/kg and the RB0021-S4 antibody given 45mg/kg can significantly inhibit tumor growth (P < 0.05).

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Sequence listing

<110> Ruiyang (su state) biotechnology Co., ltd

<120> anti-human CD38 humanized monoclonal antibody and use thereof

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Gly Ile Asn Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Ile

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Lys Gly Arg Ala Thr Leu Thr Ser Asp Thr Ser Lys Asn Gln Ala Val

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Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Phe Cys

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Gly Tyr Ile Tyr Ile Gly Asn Gly Tyr Thr Glu Tyr Asn Glu Lys Phe

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Lys Gly Arg Ala Thr Leu Thr Ser Asp Thr Ser Thr Ser Thr Ala Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

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Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

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Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

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Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

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Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

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Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

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Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

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Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

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Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

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Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

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Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

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Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

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Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

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Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

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Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

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Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

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Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

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Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

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Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

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Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

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Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

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Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

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Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

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Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

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Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

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Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

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Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

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Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

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Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

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Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

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Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Lys Pro Gln Val Tyr

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Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

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Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

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Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

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Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

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Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

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Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

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Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

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Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

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Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

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Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

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Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

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Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

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Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

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Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

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Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

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Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

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Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

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Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

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Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Tyr Ile Tyr Ile Gly Asn Gly Tyr Thr Glu Tyr Asn Glu Lys Phe

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Lys Gly Arg Ala Thr Leu Thr Ser Asp Thr Ser Thr Ser Thr Ala Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

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Ala Arg Ser His Tyr Asp Ser Ser Ser Trp Phe Ala Tyr Trp Gly Gln

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Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

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Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

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Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

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Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

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Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

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Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

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Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

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Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

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Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

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Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

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Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

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Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

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Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Lys Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 15

<211> 219

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 15

Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Pro Glu His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Ser Gln Gly

85 90 95

Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 16

<211> 219

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 16

Asp Val Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Pro Glu His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Ser Gln Gly

85 90 95

Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

Claims

1. A monoclonal antibody that binds to CD38 protein, said monoclonal antibody that binds to CD38 protein comprising:

(1) Heavy chain variable regions comprising heavy chains HCDR1, HCDR2 and HCDR3 having sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively;

and (2) light chain variable regions comprising light chains LCDR1, LCDR2 and LCDR3 having sequences as set forth in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

2. The monoclonal antibody of claim 1, wherein the monoclonal antibody comprises:

(1) A heavy chain variable region variant comprising a heavy chain variable region as set forth in SEQ ID NO. 7 or SEQ ID NO. 8, or a heavy chain variable region variant having a3, 2 or 1 amino acid difference in the non-CDR region from the heavy chain variable region set forth in SEQ ID NO. 7 and SEQ ID NO. 8;

and (2) a light chain variable region variant comprising a light chain variable region as set forth in SEQ ID NO. 9 or SEQ ID NO. 10, or having a3, 2 or 1 amino acid difference in the non-CDR regions from the light chain variable region as set forth in SEQ ID NO. 9 and SEQ ID NO. 10.

3. The monoclonal antibody of claim 1 or2, wherein the monoclonal antibody comprises:

and (2) a light chain having a sequence shown as SEQ ID NO. 15 or SEQ ID NO. 16.

4. The monoclonal antibody of claim 3, wherein the monoclonal antibody comprises:

5. A nucleic acid molecule encoding the monoclonal antibody of any one of claims 1-4.

6. A vector comprising the nucleic acid molecule of claim 5.

7. A cell comprising or producing the monoclonal antibody of any one of claims 1-4.

8. Use of the monoclonal antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the vector of claim 6 and the cell of claim 7 in the manufacture of a medicament or kit for treating multiple myeloma.