CN114409788B - anti-CD 38 antibodies and uses thereof - Google Patents

Abstract

The invention discloses an anti-CD 38 antibody and application thereof, wherein the antibody comprises a heavy chain variable region containing three CDRs and a light chain variable region containing three CDRs; wherein, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID No.1, 2 and 3, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID No.9, 10 and 11. The invention also discloses a nucleic acid molecule for coding the antibody, an expression vector, a host cell and application thereof.

Description

Anti-CD38 antibody and its application

technical field

The invention belongs to the fields of cell biotechnology and immunology, and relates to an anti-CD38 antibody and its application.

Background technique

Leukocyte differentiation group surface antigen (Cluster of Differentiation, CD) 38, referred to as CD38, is a second-class transmembrane glycoprotein with a molecular weight of 46kD, including an extracellular domain (Extracellular Domain, ECD domain, containing 23 amino acid residues) containing 257 amino acid residues. The single-pass transmembrane structure of amino acid residues and the cytoplasmic domain containing 20 amino acids (Liu Qun, Kriksunov Irina.A, Graeff Richard, et a1.Crystal Structure of Human CD38Extracellular Domain[J].Structure,September,2005, 13: 1331-1339). The CD38 gene spans about 80 kb and is located on chromosome 4p15, including 8 exons, of which exon 1 encodes cytoplasmic amino acids, transmembrane regions, and about 33 amino acids in the extracellular region (along Sandy W., Comenzo Raymond L. CD38 Monoclonal Antibody Therapies for Multiple Myeloma [J]. Clinical division mphom, Myeloma & Leukemia, 2015, 15, No. 1 l:635-645).

CD38 is a multifunctional transmembrane protein widely expressed in immune cells. In lymphocytes, monocytes, macrophages, dendritic cells, granulocytes and natural killer cells, the expression level of cell surface CD38 depends on the level of cell maturation and activation (Kar A, Mehrotra S, Chatterjee S. CD38 :TCell Immuno-MetabolicModulator[J].Cells,20209(7).), the existing evidence shows that CD38 plays an important role in various cells in both physiological and pathological environments. Early studies have shown that human CD38 can establish lateral associations with various membrane proteins/complexes, such as CD16 in NK cells, TCR complexes with CD4 in T cells, and membrane immunoglobulins (Ig) in B lymphocytes with B-cell co-receptor complexes (CD19/CD81), and MHC class II complexes in monocytes, and studies suggest that CD38 has a potential contribution to cellular signaling downstream of these complexes. Experiments in knockout mice have shown that CD38 deletion will lead to a series of physiological changes such as immune system damage and metabolic disorders in mice. In recent years, studies have found that CD38 may be involved in the pathogenesis of a variety of autoimmune diseases (Partida-SanchezS, Rivero-Naval, Shi G, et a1. CD38: an ecto enzyme at the crossroads of innate and adapive immune responses. Adv Exp Med Biol . 2007;590:171-183). Therefore, the study of anti-CD38 antibodies is of great significance for the detection and treatment of diseases.

SUMMARY OF THE INVENTION

In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide an anti-CD38 monoclonal antibody and its application.

The specific plans are as follows:

A first aspect of the present invention provides an anti-CD38 monoclonal antibody, the monoclonal antibody comprising a heavy chain variable region containing three CDRs and a light chain variable region containing three CDRs; wherein the heavy chain The amino acid sequences of the variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NOs. 1, 2, and 3, and the amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NOs.

In some embodiments, the heavy chain variable region further includes the heavy chain variable region framework regions FR1, FR2, FR3 and FR4; the light chain variable region further includes the light chain variable region framework regions FR1, FR2, FR3 and FR4, wherein the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NO.4, 5, 6, and 7; the light chain variable region framework regions FR1, FR2, FR3 and FR4 The amino acid sequences of s are shown in SEQ ID NO. 12, 13, 14, and 15.

In some embodiments, the monoclonal antibody comprises:

(a) a heavy chain variable region sequence having at least 90%, preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 8;

(b) a light chain variable region sequence having at least 90%, preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 16; or

(c) heavy chain variable region sequence as in (a) and light chain variable region sequence as in (b).

In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.16.

In a further embodiment, the monoclonal antibody comprises all or part of an antibody heavy chain constant region and/or an antibody light chain constant region.

In some embodiments, the subtype of the monoclonal antibody heavy chain is the IgM subtype.

In some embodiments, the subtype of the monoclonal antibody light chain is the kappa subtype.

The second aspect of the present invention provides a nucleic acid molecule encoding the monoclonal antibody or functional fragment thereof of the first aspect of the present invention.

Further, the CDR1, CDR2, CDR3 nucleic acid molecules encoding the variable region of the heavy chain have sequences that are at least 90%, preferably 95% identical to the nucleotide sequences shown in SEQ ID NO. 17, 18, and 19;

The CDR1, CDR2, CDR3 nucleic acid molecules encoding the light chain variable regions have sequences that are at least 90%, preferably 95% identical to the nucleotide sequences shown in SEQ ID NO. 25, 26, 27.

Further, the nucleic acid molecules encoding the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 have at least 90%, preferably 95% sequence identity with the nucleotide sequences shown in SEQ ID NO. 20, 21, 22, 23 SEQ ID NO: 28, 29, 30, 31 The nucleic acid molecules encoding the light chain variable region framework regions FR1, FR2, FR3 and FR4 have at least 90%, preferably 95% of the nucleotide sequences shown in SEQ ID NO. 28, 29, 30, and 31. sequence of identity.

Further, the nucleic acid molecule encoding the variable region of the heavy chain has a sequence that is at least 90%, preferably 95% identical to the nucleotide sequence shown in SEQ ID NO. 24; the nucleic acid molecule encoding the variable region of the light chain has the same sequence as SEQ ID NO. The nucleotide sequence shown in ID NO. 32 has at least 90%, preferably 95% sequence identity.

Preferably, the nucleotide sequence encoding the variable region of the heavy chain is shown in SEQ ID NO.24, and the nucleotide sequence encoding the variable region of the light chain is shown in SEQ ID NO.32.

A third aspect of the present invention provides a vector comprising the nucleic acid molecule of the second aspect of the present invention.

In some embodiments, the carrier comprises a signal peptide operably linked to the antibody.

The fourth aspect of the present invention provides a host cell comprising the nucleic acid molecule of the second aspect of the present invention or the vector of the third aspect of the present invention.

In some embodiments, the host cell is selected from a prokaryotic cell or a eukaryotic cell.

In some embodiments, the host cell is a eukaryotic cell.

In some embodiments, the eukaryotic cells are mammalian cells.

The fifth aspect of the present invention provides a drug conjugate comprising the monoclonal antibody of the first aspect of the present invention.

In some embodiments, the drug conjugate further comprises a conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, or enzyme.

The sixth aspect of the present invention provides a composition comprising the monoclonal antibody of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the vector of the third aspect of the present invention, The host cell described in the fourth aspect of the present invention or the drug conjugate described in the fifth aspect of the present invention.

In further embodiments, the composition further comprises a pharmaceutically acceptable carrier.

The seventh aspect of the present invention provides a product for detecting or measuring CD38 in a sample, the product comprising the monoclonal antibody of the first aspect of the present invention.

In some embodiments, the product further includes reagents for processing the sample.

The eighth aspect of the present invention provides a method for preparing the monoclonal antibody of the first aspect of the present invention, the method comprising the steps of:

The cells according to the first aspect of the present invention are cultured, and monoclonal antibodies are recovered.

In further embodiments, the method further comprises purifying the monoclonal antibody.

The ninth aspect of the present invention provides a method for detecting CD38 in a sample, by contacting the anti-CD38 monoclonal antibody described in the first aspect of the present invention with the sample to be tested; and determining the presence or level of CD38 in the sample to be tested.

A tenth aspect of the present invention provides the application described in any one of the following:

1) The monoclonal antibody of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the vector of the third aspect of the present invention, the host cell of the fourth aspect of the present invention, and the seventh aspect of the present invention. The application of the product described in the aspect in the detection of CD38;

2) The monoclonal antibody of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the vector of the third aspect of the present invention, the host cell of the fourth aspect of the present invention, and the seventh aspect of the present invention. Use of the product described in the aspect in the preparation of a product for diagnosing CD38-related diseases;

3) The monoclonal antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, and the fifth aspect of the present invention. Use of the drug conjugate described in the aspect and the composition described in the sixth aspect of the present invention in the preparation of a medicament for preventing and/or treating CD38-related diseases.

In some embodiments, the disease is a tumor or an immune disease.

The immune disease is selected from the group consisting of: rheumatoid arthritis, psoriasis, ankylosing spondylitis, joint psoriasis, dermatitis, systemic scleroderma and sclerosis, inflammatory bowel disease, Crohn's disease, ulcerative Colitis, Respiratory Distress Syndrome, Meningitis, Encephalitis, Gastritis, Uveitis, Glomerulonephritis, Eczema, Asthma, Arteriosclerosis, Leukocyte Adhesion Deficient Disease, Raynaud's Syndrome, Sjogren's Syndrome, Juvenile Diabetes, Reiter's Disease, Behcet disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, symptoms of immune-mediated thrombocytopenia, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis, atopic dermatitis, Pemphigus, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, Omenn syndrome, chronic renal failure, acute infectious mononucleosis syndrome, multiple sclerosis, HIV and herpes virus-related diseases, severe acute respiratory disease Syndromes and chorioretinitis, graft-versus-host disease, and immune disorders caused by viral infections.

In a further embodiment, the immune disease is selected from the group consisting of: rheumatoid arthritis, systemic lupus erythematosus, asthma, inflammatory bowel disease, multiple sclerosis, Crohn's disease, gastritis, Hashimoto's thyroid inflammation, ankylosing spondylitis, graft-versus-host disease, immune-mediated thrombocytopenia symptoms.

In some embodiments, the immune-mediated thrombocytopenia symptom is acute idiopathic thrombocytopenic purpura or chronic idiopathic thrombocytopenic purpura.

The tumor is selected from the group consisting of leukemia, B cell lymphoma, T cell lymphoma, NK cell lymphoma, plasma cell malignancy and myeloma.

In some embodiments, the B cell lymphoma is selected from the group consisting of mature B cell tumor, precursor B cell lymphoblastic leukemia/lymphoma, B cell non-Hodgkin lymphoma, B cell Hodgkin lymphoma.

In some embodiments, the tumor is selected from the group consisting of: acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute promyelocytic leukemia, chronic lymphocytic leukemia, acute or chronic myeloid leukemia, multiple myeloma, promyelocytic Neoplasms, light chain amyloidosis, B-cell chronic lymphocytic leukemia, small lymphocytic leukemia, B-cell acute lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, filtration Follicular lymphoma, cutaneous follicular center lymphoma, marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, plasmacytoma, plasma cell myeloma, plasma cell leukemia, Post-transplant lymphoproliferative disorders, Waldenstrom macroglobulinemia, plasma cell leukemia and anaplastic large cell lymphoma, hairy cell lymphoma.

Description of drawings

Fig. 1 is a graph showing the binding ability of ELISA detection antibody and antigen;

Fig. 2 is a graph showing the binding ability of the antibody to the antigen detected by flow cytometry, wherein 2A is the control; 2B is the 70B5 antibody.

Detailed ways

The present invention has found an anti-CD38 monoclonal antibody through extensive and in-depth research, and the monoclonal antibody has high affinity activity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical and/or bind the same epitope, except for those that may arise during the production of monoclonal antibodies. Outside of possible variants, such variants are generally present in very small amounts. Such monoclonal antibodies typically include antibodies comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence is obtained by a process involving the selection of a single target-binding polypeptide sequence from among a plurality of polypeptide sequences. For example, the selection process can be the selection of unique clones from a collection of numerous clones such as hybridoma clones, phage clones or recombinant DNA clones. It will be appreciated that the selected target-binding sequence can be further altered, eg, to increase affinity for the target, humanize the target-binding sequence, increase its yield in cell culture, decrease its immunogenicity in vivo , creating multispecific antibodies, etc., and antibodies comprising altered target-binding sequences are also monoclonal antibodies of the present invention. Unlike a typical polyclonal antibody preparation, which contains different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Beyond their specificity, monoclonal antibody preparations have the advantage that they are generally uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method.

In the present invention, monoclonal antibodies encompass sequences having a certain degree of sequence identity or sequence homology with the amino acid sequence of the antibody or any nucleotide sequence encoding the antibody, in the present invention, "homology" Can be equated with "consistency".

It will also be understood by those of skill in the art that antibodies can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line used to express the antibody and the culture conditions. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. A common modification is the deletion of carboxy-terminal basic residues such as lysine or arginine due to the action of carboxypeptidases.

As used herein, "identity" indicates that at any particular position in the sequences being aligned, the amino acid residues between the sequences are identical. As used herein, "similarity" indicates that at any particular position in the sequences being aligned, the amino acid residues between the sequences are of a similar type. For example, leucine can be substituted for isoleucine or valine. Other amino acids that are commonly substituted for each other include, but are not limited to: phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains), lysine, arginine, and histidine (with base amino acids with sexual side chains), aspartic acid and glutamic acid (amino acids with acidic side chains), asparagine and glutamine (amino acids with amide side chains), and cysteine and methionine (amino acids with sulfur-containing side chains).

Generally, modification of one or more amino acids in a protein does not affect the function of the protein. Those skilled in the art will recognize that changing a single amino acid or a small percentage of amino acids, or individual additions, deletions, insertions, substitutions to an amino acid sequence, are conservative modifications, where changes in a protein result in a protein with a similar function. Conservative substitution tables providing functionally similar amino acids are well known in the art.

Substitutions, deletions, insertions, or any combination thereof, may be utilized to effect final derivatives or variants. Typically, these changes are made in a few amino acids to minimize changes in the molecule, especially the immunogenicity and specificity of the antigen binding protein. However, larger changes can be tolerated in some cases. Amino acid substitutions are typically of a single base; insertions will typically be on the order of about one to about twenty amino acid residues, although significantly larger insertions may be tolerated. Deletions range from about one to about twenty amino acid residues, although in some cases deletions can be much larger.

Further, the amino acid sequences of variable regions including but not limited to framework regions, hypervariable regions, especially CDR3 regions are modified. Typically, a light or heavy chain region includes three hypervariable regions (including three CDRs) and more conserved regions (so-called framework regions (FRs)). Hypervariable regions include amino acid residues from CDRs and amino acid residues from hypervariable loops. Computer algorithms known to those skilled in the art such as Gap or Bestfit can be used to optimally align amino acid sequences to be compared and define similar or identical amino acid residues. The parental monoclonal antibody, or portion thereof, can be altered by general molecular biology methods known in the art, including PCR, oligonucleotide-directed mutagenesis, and site-directed mutagenesis, Or obtain functional variants by organic synthesis methods.

The antibodies and fragments disclosed herein are expressed at good levels from host cells. Accordingly, the properties of the antibodies and/or binding fragments are suitable for commercial scale expression.

Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. "Fab" refers to a part of an antibody molecule that contains the variable and constant regions of a light chain and the variable and constant regions of a heavy chain through disulfide bonds; "Fab'" refers to a part of the hinge region "F(ab')2" refers to the dimer of Fab'; "Fv" refers to the smallest antibody containing the variable region of the heavy chain, the variable region of the light chain and having all antigen-binding sites Antibody fragments, and can be derived from any mammal including, but not limited to, human, mouse, rat, camelid or rabbit. Functional portions of antibodies, such as one or more of the CDRs described herein, can be covalently linked to secondary proteins or small molecule compounds and used as targeted therapeutics against specific targets.

The term "nucleic acid molecule" as used in the present invention refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

The present invention is not particularly limited to the expression vector, the choice of which depends on the desired function. Non-limiting examples of vectors include plasmids, cosmids, viruses, phages, and other vectors conventionally used, eg, in genetic engineering. Methods well known to those skilled in the art can be used to construct various plasmids and vectors.

In one embodiment, the vector is an expression vector. The expression vector according to the present invention is capable of directing the replication and expression of the nucleic acid molecule of the present invention in a host, and thus ensures expression in the host of choice of the variable chain domain of the anti-IgG antibody of the present invention encoded thereby. In a further embodiment, one or more vectors comprise further sequences to ensure that not only the variable chain domains of the invention are expressed, but also full-length IgG comprising the variable chain domains of the invention are expressed Antibody.

The expression vector can be, for example, a cloning vector, a binary vector or an integrating vector. Expression includes transcription of a nucleic acid molecule, eg, into translatable mRNA.

Non-limiting examples of vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), λgt11, pJOE, pBBR1-MCS-series, pJB861, pBSMuL, pBC2, pUCPKS , pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027pCAG Kosak-Cherry(L45a) vector system, pREP(Invitrogen), pCEP4(Invitrogen), pMC1neo(Stratagene), pXT1(Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (EdgeBiosystems) pTriEx-Hygro (Novagen) and pCINeo (Promega). Non-limiting examples of plasmid vectors suitable for Pichia pastoris include, eg, plasmids pAO815, pPIC9K and pPIC3.5K (all Invitrogen). Another vector suitable for expressing proteins in Xenopus embryos, zebrafish embryos and a wide variety of mammalian and avian cells is the multipurpose expression vector pCS2+.

In general, a vector may contain one or more origins of replication (ori) and genetic systems for cloning or expression, one or more markers for selection in a host (eg, antibiotic resistance), and one or more an expression cassette. In addition, the coding sequences contained in the vector can be linked to transcriptional regulatory elements and/or to other amino acid coding sequences using established methods. Such regulatory sequences are well known to those skilled in the art and include, but are not limited to, regulatory sequences that ensure transcription initiation, internal ribosome entry sites (IRES), and optional regulatory elements that ensure transcription termination and transcript stability. Non-limiting examples of such regulatory elements that ensure transcription initiation include promoters, translation initiation codons, enhancers, insulators, and/or regulatory elements that ensure transcription termination, which are included downstream of the nucleic acid molecules of the invention. Further examples include Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing, nucleotide sequences encoding secretion signals, or signal sequences depending on the expression system used, which enable expression directed proteins to cellular compartments or culture medium. The vector may also contain additional expressible polynucleotides encoding one or more chaperones to facilitate proper protein folding.

Additional examples of suitable origins of replication include, for example, full-length ColE1, truncated ColEI, SV40 virus, and M13 origins of replication, while additional examples of suitable promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, SV40-promoter, RSV-promoter (Rous sarcoma virus), lacZ promoter, tetracycline promoter/operator (tetp/o), chicken β-actin promoter, CAG-promoter (chicken β-muscle combination of kinesin promoter and cytomegalovirus immediate early enhancer), gai10 promoter, human elongation factor 1α-promoter, AOX1 promoter, GAL1 promoter, CaM-kinase promoter, lac, trp or tac promoter, T7 Or T5 promoter, lacUV5 promoter, Autographa californica polynuclear polyhedrosis virus (AcMNPV) polyhedron promoter or globin introns in mammalian and other animal cells. An example of an enhancer is eg the SV40-enhancer. Non-limiting additional examples of regulatory elements that ensure transcription termination include the SV40-polyadenylation site, the tk-polyadenylation site, the p-factor independent lpp terminator or the AcMNPV polyhedron polyadenylation signal. Further non-limiting examples of selectable markers include dhfr, which confers resistance to methotrexate, npt, which confers resistance to the aminoglycosides neomycin, kanamycin, and paromycin sex and hygro, which confers resistance to hygromycin. Additional selection genes have been described, namely trpB, which allows cells to utilize indole instead of tryptophan; hisD, which allows cells to utilize histinol instead of histidine; mannose 6-phosphate isomerase, which allows Cells utilize mannose and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor 2-(difluoromethyl)-DL-ornithine DFMO or confers resistance to blasticidin S Resistant deaminase from Aspergillus terreus.

In a further embodiment, the vector is a eukaryotic expression plasmid containing an expression cassette consisting of a 5'CMV promoter including intron A and a 3'BGH polyadenylation sequence.

Suitable bacterial expression hosts include, for example, strains derived from JM83, W3110, KS272, TG1, K12, BL21 (eg, BL21(DE3), BL21(DE3)PlysS, BL21(DE3)RIL, BL21(DE3)PRARE) or Rosettaa.

Nucleic acid molecules and/or vectors of the invention can be designed to be processed by, for example, chemical-based methods (polyethylenimine, calcium phosphate, liposomes, DEAE-dextran, nucleofection, non-chemical methods (electroporation, sonoporation) effect, phototransfection, gene electrotransfer, fluid delivery or transformation that naturally occurs when cells are in contact with the nucleic acid molecules of the invention), particle-based methods (gene gun, magnetic transfection, puncture transfection), phage vector-based methods and viral methods into cells. For example, expression vectors derived from, for example, retroviruses, vaccinia virus, adeno-associated virus, herpes virus, Semliki virus, or bovine papilloma virus can be used to deliver nucleic acid molecules into targeted cell populations. In addition, the baculovirus system can also be used as a vector in the eukaryotic expression system of the nucleic acid molecules of the invention.

To facilitate purification of the nucleic acid molecules of the invention, tag sequences can be inserted into expression vectors. Examples of tags include, but are not limited to, six histidine tags, myc tags, or FLAG tags. Any tag known to those skilled in the art to facilitate purification can be used in the present invention.

In the present invention, any suitable host cell/vector system can be used for the expression of DNA sequences encoding the antibody molecules of the present invention. Bacterial (eg, E. coli) and other microbial systems may be used, or eukaryotic (eg, mammalian) host cell expression systems may also be used. Such cells include, but are not limited to, mammalian cells, plant cells, insect cells, fungal cells or cells of bacterial origin. As mammalian cells, it can be preferably used selected from (but not limited to) cells selected from CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK293 cells and HEK293T cells One of the group consisting of cells serves as a host cell. Any cell known to those skilled in the art that can be used as a mammalian host cell can be used in the art.

The recombinant cells of the present invention can then be used for expression as well as culture purposes for antibody expression for mass drug production. It can also be used as an active ingredient in pharmaceutical compositions. Any suitable culture technique can be used including, but not limited to, stationary culture, spinner flask culture, ascites fluid, hollow fiber type bioreactors, modular mini-fermenters, stirred tanks, microcarrier culture, ceramic core perfusion, and the like.

As an optional embodiment, the product of the present invention includes the antibody or functional fragment thereof prepared by the present invention. As another alternative embodiment, the product of the present invention includes a diagnostic composition comprising at least one detectable label, such as a detectable moiety/agent. Tags can be non-covalently conjugated to the monoclonal antibodies of the invention. Tags can also be directly conjugated to monoclonal antibodies via covalent bonds. Alternatively, tags can be conjugated to the monoclonal antibodies described above using one or more linking compounds. Techniques for conjugating tags to monoclonal antibodies are well known to those skilled in the art. The detectable moiety/reagent as a label is preferably selected from, but not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting materials and non-radioactive paramagnetic metal ions one of the groups. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent substances include but not limited to FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine type labels including TAMRA; dansyl; lissamine; cyanine; phycoerythrin; Texas red; and the like thing. Fluorescent labels can be conjugated to aldehyde groups contained in target molecules using the techniques disclosed herein. Suitable luminescent substances include luminol, acridine compounds, coelenterazine and analogs, dioxetane, peroxyoxalic acid based systems and derivatives thereof; suitable bioluminescent substances include luciferase, luciferin and aequorin; and suitable radionuclides include 125I, 131I, 111In and 99Tc.

In the present invention, the method for detecting or measuring the amount of the target antigen (eg, CD38) may be any known method. For example, it includes immunodetection or assay methods.

An immunodetection or assay method is a method for detecting or measuring the amount of antibody or antigen by using a labeled antigen or antibody. Examples of immunodetection or assay methods include radiolabeled immunoantibody assays (RIA), enzyme immunoassays (EIA or ELISA), fluorescent immunoassays (FIA), luminescence immunoassays, Western blotting, flow cytometry technology, physical and chemical methods, etc.

Diseases associated with CD38 can be diagnosed by detecting or assaying cells expressing CD38 with the antibodies or antibody fragments of the invention.

In the present invention, the sample for detecting or measuring the target antigen (eg CD38) is not particularly limited as long as it has the possibility to contain cells expressing the target antigen (eg CD38), such as tissue cells, blood, plasma, Serum, pancreatic juice, urine, feces, tissue fluid or culture fluid.

The pharmaceutical composition of the present invention includes the monoclonal antibody of the present invention, and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may additionally contain liquids such as water, physiological saline, glycerol and ethanol. Additionally, auxiliary substances such as wetting or emulsifying agents or pH buffering substances may be present in the composition. These carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions for ingestion by a patient.

Suitable forms of administration include those suitable for parenteral administration, eg, by injection or infusion, eg, by bolus injection or continuous infusion, intravenous, inhalable or subcutaneous forms. Where the product is for injection or infusion, it may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and it may contain formulatory agents such as suspending agents, preservatives, stabilizers and/or Dispersant. Alternatively, the antibodies or antigen-binding fragments thereof according to the present invention may be in dry form for reconstitution with a suitable sterile liquid prior to use. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.

Once formulated, the compositions of the present invention can be administered directly to a subject. Accordingly, provided herein is the use of an antibody or antigen-binding fragment thereof according to the invention for the manufacture of a medicament.

The subject to be treated can be an animal. Preferably, the pharmaceutical composition according to the present invention is adapted for administration to human subjects.

In the present invention, a "CD38-related disease" is a disease or disorder in which cells expressing CD38 are present. Without limitation, such as immune diseases involving CD38-expressing B cells, plasma cells, monocytes and T cells, one of the characteristics of the disease is the presence of CD38-expressing tumor cells. Tumor diseases, such as CD38-expressing leukemia, B cell lymphomas, plasma cell malignancies, T/NK cell lymphomas and myeloma. In some embodiments of the invention, the leukemia is selected from the group consisting of: acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute promyelocytic leukemia, chronic lymphocytic leukemia, acute and chronic myeloid leukemia. In some embodiments, the myeloma is selected from the group consisting of multiple myeloma, promyeloma, and light chain amyloidosis. In some embodiments, the lymphoma is non-Hodgkin's lymphoma or Hodgkin's lymphoma. In some embodiments, the tumor may be selected from B-cell lymphomas/leukemias including, but not limited to, precursor B-cell lymphoblastic leukemia/lymphoma, B-cell non-Hodgkin's lymphoma, or B-cell Hodgkin's lymphoma Lymphoma, mature B-cell tumor. In some embodiments, the tumor is selected from the group consisting of: B-cell chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), B-cell acute lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic leukemia like lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (including low-, intermediate- or high-grade FL), skin follicular center lymphoma, marginal zone B-cell lymphoma (including MALT type, lymph node MZBL type, spleen MZBL type), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, plasmacytoma, plasma cell myeloma, plasma cell leukemia, post-transplant lymphoproliferative disease, Waldenstrom macrosphere Proteinemia, plasma cell leukemia and anaplastic large cell lymphoma (ALCL), hairy cell lymphoma. In some embodiments, the tumor is multiple myeloma. In some embodiments, the immune disease is selected from the group consisting of: rheumatoid arthritis, psoriasis, ankylosing spondylitis, joint psoriasis, dermatitis, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD) , Crohn's disease (Crohn's disease), ulcerative colitis, respiratory distress syndrome, meningitis, gastritis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, arteriosclerosis, leukocyte adhesion deficiency , Raynaud syndrome, Sjogren syndrome, juvenile diabetes, Reiter disease, Behcet disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, symptoms of immune-mediated thrombocytopenia (eg, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura), hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves disease, Hashimoto's thyroiditis, Wegener Granuloma, Omenn syndrome, chronic renal failure, acute infectious mononucleosis syndrome, multiple sclerosis, HIV and herpes virus-related diseases, severe acute respiratory syndrome, choreoretinitis, graft-versus-host Diseases, and immune diseases caused by viral infections (eg, diseases caused or mediated by B-cell infection by Epstein-Barr virus (EBV)). In some embodiments, the immune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, asthma, inflammatory bowel disease, multiple sclerosis, Crohn's disease, gastritis, Hashimoto's thyroiditis , ankylosing spondylitis, and graft-versus-host disease. In some embodiments, wherein the immune disease is rheumatoid arthritis.

The present invention will be described in further detail below with reference to the accompanying drawings and embodiments. The following examples are only used to illustrate the present invention and not to limit the scope of the present invention.

Example 1 Preparation of monoclonal antibody 70B5

The sequence of the 70B5 antibody shown in Table 1 was synthesized and cloned into a eukaryotic expression vector.

Table 1 Sequence of antibody 70B5

Specific steps are as follows:

1. Enzyme digestion experiment:

The enzyme cleavage reaction was carried out using the reagents and conditions shown in Table 2.

Table 2 Enzyme cleavage reaction system and reaction conditions

2. Gel recovery of enzyme digestion products

The vector digested above was run on a 1% agarose gel for electrophoresis, and then the gel block of the target band was cut out.

3. PCR product amplification

The systems and conditions for PCR amplification are shown in Table 3 and Table 4, respectively.

Table 3 Amplification system

Table 4 Amplification conditions

4. PCR product purification

The PCR product was purified using the full gold kit. For the specific operation, see the kit instructions.

5. Connection

According to the Ligation Reaction in Table 5, input the corresponding value to obtain the corresponding system, and water bath at 37°C for 30min.

Table 5 ligation reaction system

6. Conversion of ligation product to StbⅠ3

Take out the competent cells from the -80°C freezer and thaw on ice, transfer 50μl to a pre-cooled EP tube, add 2.5μl of the ligation product, gently shake, ice bath for 30min, heat shock in a 42°C water bath for 60s, and quickly put back on ice on and let stand for 2min. Add 200 μl of LB liquid medium (without Amp) to the centrifuge tube, incubate at 37° C., 180 rpm on a shaker for 1 h, pipette an appropriate amount of the transformation product onto LB (Amp) agar plate, and spread evenly. The plate was placed upside down in a 37°C biochemical incubator overnight.

7. Colony PCR identification

Pick 4-8 single colonies into 100μl LB (Amp) liquid medium, cultivate at 37°C, 220rpm shaker for 1-3h, take the bacterial solution for PCR identification and agarose gel electrophoresis after the reaction, the PCR product size is about 600bp , pick 1 positive colony for sequencing

8. Plasmid extraction and preservation

After the sequencing is correct, the colonies are amplified to 15ml of medium and cultured overnight to make glycerol bacteria, and the plasmids are extracted with an endotoxin-free plasmid extraction kit. concentration for antibody production.

9. Transfect the plasmid into cells to produce and purify the antibody.

Example 2 ELISA detection of monoclonal antibodies

1. Experimental materials

1. Main reagents and consumables

2. Main equipment

name company model

Microplate reader LabServ 3530917540

2. Experimental method

1. Dilute the unlabeled antigen (human CD38 Protein (ECD, His Tag)) in Elisa Coating Buffer (1x) to 0.5μg/ml and add 100μl Transfer to Nunc ^(TM) Maxisorp ^(TM) ELISA Plates Uncoated single wells, seal the plate to prevent evaporation, and incubate overnight at 4[deg.]C.

2. Place Nunc ^TM Maxisorp ^TM ELISA Plates Uncoated at room temperature, shake off the capture antibody solution, wash three times with Elisa Wash Buffer (1x) (diluted Elisa Wash Buffer (20x) with DL water), and add 200 μl of blocking solution to each well (the BSA was used to block non-specific binding sites with Elisa Wash Buffer (1x) at a concentration of 1%, and incubated at room temperature for 1 h.

3. Wash three times with Elisa Wash Buffer (1x) and dry firmly with a clean paper towel.

4. Set up several concentration gradients for the standard (standard Purified anti-human CD38 diluted with blocking solution) and sample (diluted with monoclonal antibody blocking solution) and take a duplicate well for each group. Nunc ^TM Maxisorp ^TM ELISA Plates Uncoated is added to each well. 100μl, incubated at room temperature for 2h.

5. Wash three times with Elisa Wash Buffer (1x) and dry firmly with a clean paper towel.

6. Dilute the biomarker detection antibodies HRP Goat anti-mouse IgG and HRP Goat anti-mouseIgM in blocking solution to 0.4μg/ml (0.25-2μg/ml), and add 100μl per well to standard wells and sample wells respectively Incubate at room temperature for 1 h.

7. Wash three times with Elisa Wash Buffer (1x) and dry firmly with a clean paper towel.

8. Mix 2ml of TMB Substrate A and 2ml of TMB Substrate B, transfer 100μl to a single well of Nunc ^TM Maxisorp ^TM ELISA Plates Uncoated, and incubate at room temperature for 10min (4-60min), after the incubation, add 100μl of Stop solution to stop the reaction .

9. Measure the OD value of each well with a microplate reader at 450 nm, and draw a standard curve by reading the standard wells to calculate the concentration of the sample antibody.

3. Results

The results are shown in Figure 1. The detection degree of the antibody in the experimental group is much greater than that in the negative control group, indicating that the antibody has a good affinity for CD38.

Example 3 Flow cytometric detection of monoclonal antibodies

1. Experimental materials

APC Goat anti-mouse IgM Antibody (biolegend: 406509), PBS (Gibco: C20012500BT), RPMI1640 (Gibco: C11875500BT), penicillin-streptomycin dual antibody solution (Hyclone: SV30010), special grade fetal bovine serum (Mason: CTCC- 002-001-50_10), mono-Mac-6 cells, 1.5 mL EP tubes (Titan: TS018-032)

2. Experimental method

1. Cultivate mono-Mac-6 cells, count the cells, then take 5×10 ⁵ cells into a 1.5ml EP tube, centrifuge at 1000rpm for 5min, and remove the supernatant;

2. Add 500 μl PBS to resuspend the cells, then centrifuge at 1000 rpm for 5 min, and remove the supernatant;

3. Resuspend the cells to be tested with the blank cell culture supernatant and the antibody production supernatant respectively, place at 4°C, and react in the dark for 30 minutes, then centrifuge at 1000 rpm for 5 minutes to remove the supernatant;

4. Add 500 μl PBS to resuspend the cells, then centrifuge at 1000 rpm for 5 min, and remove the supernatant;

5. Add 100 μl of secondary antibody (APC anti-human IgM Fc Antibody, the stock solution is diluted 1:600 with PBS) to resuspend the cells, place at 4°C, and react in the dark for 30 minutes, then centrifuge at 1000 rpm for 5 minutes, and remove the supernatant;

6. Add 500μl PBS to resuspend the cells, then centrifuge at 1000rpm for 5min, and remove the supernatant;

7. Add 400 μl PBS to resuspend the cells, and then test on the machine.

3. Results

The results are shown in Figure 2. Compared with the blank cell culture supernatant (2A), the antibody-producing supernatant could bind to CD38 on the surface of mono-Mac-6 cells (2B).

The description of the above embodiment is only for understanding the method and the core idea of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

sequence listing

<110> West China Hospital of Sichuan University

<120> Anti-CD38 antibody and its application

<141> 2022-03-04

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Claims (21)

1. An anti-CD 38 monoclonal antibody comprising a three CDR heavy chain variable region and a three CDR light chain variable region; wherein, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO.1, 2 and 3, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO.9, 10 and 11.

2. The monoclonal antibody of claim 1, wherein the heavy chain variable region further comprises heavy chain variable region framework regions FR1, FR2, FR3 and FR4; the light chain variable region also comprises light chain variable region framework regions FR1, FR2, FR3 and FR4, wherein the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NO.4, 5, 6 and 7; the amino acid sequences of framework regions FR1, FR2, FR3 and FR4 of the light chain variable region are shown in SEQ ID NO.12, 13, 14 and 15.

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

(a) A heavy chain variable region as shown in the amino acid sequence of SEQ ID NO. 8;

(b) A light chain variable region as shown by the amino acid sequence of SEQ ID No. 16; or

(c) A heavy chain variable region as in (a) and a light chain variable region as in (b).

4. The monoclonal antibody of claim 1 or 2, wherein the heavy chain of the monoclonal antibody is of the IgM subtype.

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 host cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6.

8. The host cell of claim 7, wherein the host cell is selected from the group consisting of prokaryotic cells and eukaryotic cells.

9. The host cell of claim 8, wherein the host cell is a eukaryotic cell.

10. The host cell of claim 9, wherein the eukaryotic cell is a mammalian cell.

11. A drug conjugate comprising the monoclonal antibody of any one of claims 1-4.

12. The drug conjugate of claim 11, further comprising a coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, or enzyme.

13. A composition comprising the monoclonal antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the vector of claim 6, the host cell of any one of claims 7-10, or the drug conjugate of any one of claims 11-12.

14. The composition of claim 13, wherein the composition further comprises a pharmaceutically acceptable carrier.

15. A product for detecting or determining CD38 in a sample, said product comprising a monoclonal antibody according to any one of claims 1-4.

16. The product of claim 15, further comprising a reagent for processing the sample.

17. A method for producing the monoclonal antibody of any one of claims 1-4, comprising the steps of:

culturing the host cell of any one of claims 7-10 and recovering the monoclonal antibody.

18. The method of claim 17, further comprising purifying the monoclonal antibody.

19. A method for detecting CD38 in a sample for non-diagnostic purposes,

contacting the anti-CD 38 monoclonal antibody of any one of claims 1-4 with a test sample; determining the presence or level of CD38 in the test sample.

20. Use according to any one of the following:

1) Use of the monoclonal antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the vector of claim 6, the host cell of any one of claims 7-10, the product of any one of claims 15-16 for the detection of CD38 for non-diagnostic purposes;

2) Use of the monoclonal antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the vector of claim 6, the host cell of any one of claims 7-10, the product of any one of claims 15-16 for the manufacture of a product for the diagnosis of a CD 38-associated disease.

21. The use according to claim 20, wherein the disease is a tumor or an immune disease.

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