CN114409788A

CN114409788A - anti-CD 38 antibodies and uses thereof

Info

Publication number: CN114409788A
Application number: CN202210209470.3A
Authority: CN
Inventors: 龚艳萍; 杨敬; 龚日祥
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2022-03-04
Filing date: 2022-03-04
Publication date: 2022-04-29
Anticipated expiration: 2042-03-04
Also published as: CN114409788B

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 the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO.1, 2 and 3, and the amino acid sequences of the light chain variable region CDR1, CDR2 and CDR3 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-CD 38 antibodies and uses thereof

Technical Field

The invention belongs to the fields of cell biotechnology and immunology, and relates to an anti-CD 38 antibody and application thereof.

Background

Leukocyte Differentiation group surface antigen (CD) 38, CD38 for short, is a two-class transmembrane glycoprotein with a molecular weight of 46kD, including an Extracellular region (ECD Domain ) containing 257 amino acid residues, a single transmembrane Structure containing 23 amino acid residues, and a cytoplasmic region (Liu Qun, Kriksunov Irina. A, Graeff Richard, et a1.Crystal Structure 2015 of Human CD38 Extracellular Domain [ J ]. Structure, September,2005, 13: 1331-1339. CD38 gene spans about 80kb, is located on chromosome 4p15, contains 8 exons, wherein exon 1 encodes dyplasma amino acid, transmembrane region amino acid, and Extracellular region has about 33 amino acids (Extracellular fatty W.A., Monoclonal Antibody 635. C., My. molecular dynamics, My. 645J.: My & 15).

CD38 is a multifunctional transmembrane protein that is widely expressed in immune cells. The level of expression of cell surface CD38 in lymphocytes, monocytes, macrophages, dendritic Cells, granulocytes and natural killer Cells depends on the level of maturation and activation of the cell (Kar a, Mehrotra S, Chatterjee s.cd38: TCell Immuno-Metabolic Modulator [ J ] Cells,20209 (7)), and there is evidence that CD38 plays an important role in a variety of Cells in both physiological and pathological environments. Early studies showed that human CD38 can establish lateral association with a variety of membrane proteins/complexes, such as CD16 in NK cells, TCR complex and CD4 in T cells, membrane immunoglobulin (Ig) and B cell co-receptor complex (CD19/CD81) in B lymphocytes, and MHC class II complex in monocytes, and it was suggested that CD38 potentially contributes to cell signaling downstream of these complexes. The gene knockout mouse experiment proves that the deletion of CD38 can cause a series of physiological changes of the mouse, such as immune system damage, metabolic disorder and the like. In recent years, CD38 has been found to be involved in the pathogenesis of various autoimmune diseases (Partida-SanchezS, river-Naval, Shi G, et a1.CD38: an ecto enzyme at the cross of amino and adaptive immune responses. adv Exp Med biol. 2007; 590: 171-. Therefore, the research on the anti-CD 38 antibody is of great significance for realizing the detection and treatment of diseases.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention aims to provide a monoclonal antibody against CD38 and application thereof.

The specific scheme is as follows:

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

In some embodiments, the heavy chain variable region further comprises heavy chain variable region framework regions FR1, FR2, FR3, and FR 4; the light chain variable region further comprises light chain variable region framework regions FR1, FR2, FR3 and FR4, wherein the amino acid sequences of heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown as 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.

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

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

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

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

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

Further, the CDR1, CDR2, CDR3 nucleic acid molecule encoding the variable region of the heavy chain has a sequence having at least 90%, preferably 95% sequence identity with the nucleotide sequence shown in SEQ ID No.17, 18, 19;

the CDR1, CDR2, CDR3 nucleic acid molecules encoding the variable region of the light chain have a sequence which has at least 90%, preferably 95% sequence identity to the nucleotide sequences set forth in SEQ ID nos. 25, 26, 27.

Further, the nucleic acid molecules encoding framework regions FR1, FR2, FR3 and FR4 of the heavy chain variable region have a sequence which has at least 90%, preferably 95%, sequence identity to the nucleotide sequences set forth in SEQ ID nos. 20, 21, 22, 23; nucleic acid molecules encoding framework regions FR1, FR2, FR3 and FR4 of the light chain variable region have a sequence which has at least 90%, preferably 95%, sequence identity to the nucleotide sequences set forth in SEQ ID Nos. 28, 29, 30 and 31.

Further, the nucleic acid molecule encoding the heavy chain variable region has a sequence with at least 90%, preferably 95% sequence identity to the nucleotide sequence shown in SEQ ID No. 24; the nucleic acid molecule encoding the variable region of the light chain has a sequence which has at least 90%, preferably 95% sequence identity with the nucleotide sequence shown in SEQ ID NO. 32.

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

In a third aspect, the present invention provides a vector comprising a nucleic acid molecule according to the second aspect of the invention.

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

In a fourth aspect, the invention provides a host cell comprising a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the 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 cell is a mammalian cell.

In a fifth aspect, the present invention provides a drug conjugate comprising a monoclonal antibody according to the first aspect of the invention.

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

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

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

In a seventh aspect the present invention provides a product for detecting or determining CD38 in a sample, said product comprising a monoclonal antibody according to the first aspect of the invention.

In some embodiments, the product further comprises a reagent for processing the sample.

An eighth aspect of the present invention provides a method of preparing a monoclonal antibody according to the first aspect of the invention, the method comprising the steps of:

culturing the cell of the first aspect of the invention and recovering the monoclonal antibody.

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

In a ninth aspect, the present invention provides a method for detecting CD38 in a sample, comprising contacting a test sample with an anti-CD 38 monoclonal antibody of the first aspect of the present invention; determining the presence or level of CD38 in the test sample.

A tenth aspect of the invention provides the use of any one of:

1) the monoclonal antibody of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, the vector of the third aspect of the invention, the host cell of the fourth aspect of the invention, the use of the product of the seventh aspect of the invention for detecting CD 38;

2) use of a monoclonal antibody according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, or a product according to the seventh aspect of the invention for the manufacture of a product for the diagnosis of a disease associated with CD 38;

3) use of a monoclonal antibody according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, a drug conjugate according to the fifth aspect of the invention, or a composition according to the sixth aspect of the invention for the manufacture of a medicament for the prevention and/or treatment of a disease associated with CD 38.

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

The immune disease is selected from: 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 deficiency disease, Raynaud's syndrome, Sjogren's syndrome, juvenile diabetes, Reiter's disease, Behcet's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia syndrome, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis, atopic dermatitis, pemphigus, Graves ' disease, Hashimoto's thyroiditis, Wegener's granuloma, Omenn's syndrome, chronic renal failure, acute mononucleosis, multiple sclerosis, HIV and herpesvirus-related diseases, Severe acute respiratory syndrome and chorioretinitis, graft versus host disease, and immune diseases caused by viral infections.

In further 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, graft-versus-host disease, immune-mediated thrombocytopenia.

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

The tumor is selected from: 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: mature B cell tumors, precursor B cell lymphoblastic leukemia/lymphoma, B cell non-hodgkin lymphoma, B cell hodgkin lymphoma.

In some embodiments, the tumor is selected from: acute lymphocytic leukemia, acute lymphoblastic leukemia, acute promyelocytic leukemia, chronic lymphocytic leukemia, acute or chronic myelogenous leukemia, multiple myeloma, promyelocytic tumor, light chain amyloidosis, B-cell chronic lymphocytic leukemia, small lymphocytic leukemia, B-cell acute lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytoid lymphoma, mantle cell lymphoma, follicular lymphoma, cutaneous follicular central lymphoma, marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, burkitt's lymphoma, plasmacytoma, plasma cell myeloma, plasma cell leukemia, post-transplant lymphoproliferative disorder, Waldenstrom macroglobulinemia, plasma cell leukemia and anaplastic large cell lymphoma, hairy cell lymphoma.

Drawings

FIG. 1 is a diagram of ELISA for detecting the binding ability of an antibody to an antigen;

FIG. 2 is a diagram of a flow cytometry technique for detecting the binding ability of an antibody to an antigen, wherein 2A is a control; 2B is the 70B5 antibody.

Detailed Description

The invention discovers a monoclonal antibody against CD38 through extensive and intensive research, and the monoclonal antibody has higher affinity activity.

The term "monoclonal antibody" as used herein 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 the same epitope, such variants typically being present in minor amounts, except for possible variants that may arise during the course of production of the monoclonal antibody. Such monoclonal antibodies typically include an antibody comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select unique clones from a collection of multiple clones, such as hybridoma clones, phage clones, or recombinant DNA clones. It will be appreciated that the selected target binding sequence may be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the invention. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are generally uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

In the present invention, a monoclonal antibody encompasses a sequence 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, and in the present invention, "homology" may be equivalent to "identity".

One skilled in the art will also appreciate that antibodies may be subjected to various post-translational modifications. The type and extent of these modifications often depends 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. Common modifications are the deletion of a basic residue at the carboxy terminus (such as lysine or arginine) due to the action of carboxypeptidase.

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

Generally, modification of one or more amino acids in a protein does not affect the function of the protein. One skilled in the art will recognize that individual amino acid changes or small percentage amino acids or individual additions, deletions, insertions, substitutions to an amino acid sequence are conservative modifications, wherein a change in a protein results 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 used to arrive at the final derivative or variant. Typically, these changes are made over several amino acids to minimize changes in the molecule, particularly the immunogenicity and specificity of the antigen binding protein. However, greater variations may 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 sequence of the variable region including, but not limited to, the framework region, the hypervariable region, and particularly the CDR3 region, is modified. Typically, the light or heavy chain region includes three hypervariable regions (including three CDRs) and more conserved regions (so-called Framework Regions (FRs)). The hypervariable region comprises amino acid residues from the CDRs and amino acid residues from the hypervariable loops. Computer algorithms known to those skilled in the art, such as Gap or Bestfit, can be used to optimally align the amino acid sequences for comparison, and to define similar or identical amino acid residues. The parental monoclonal antibody or a part thereof can be altered by general molecular biology methods known in the art, including PCR, oligonucleotide-directed mutagenesis (oligonucleotide-directed mutagenesis) and site-directed mutagenesis (site-directed mutagenesis), or functional variants can be obtained by organic synthesis methods.

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

Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments. "Fab" refers to the portion of an antibody molecule that contains one light chain variable and constant region and one heavy chain variable and constant region that are disulfide bonded; "Fab'" refers to a Fab fragment comprising part of the hinge region; "F (ab ') 2" refers to a dimer of Fab'; "Fv" refers to the smallest antibody fragment containing the heavy chain variable region, the light chain variable region of an antibody and having all antigen binding sites, and can be derived from any mammal, including but not limited to, human, mouse, rat, camelid, or rabbit. A functional portion of an antibody, such as one or more CDRs described herein, can be covalently linked to a secondary protein or small molecule compound and used as a target therapeutic agent for a particular target.

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

The expression vector of the present invention is not particularly limited, and its selection depends on the desired function. Non-limiting examples of vectors include plasmids, cosmids, viruses, bacteriophages and other vectors routinely used in, for example, 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 invention is capable of directing the replication and expression of the nucleic acid molecule of the invention in a host and thus ensuring the expression of the variable chain domain of the anti-IgG antibody of the invention encoded thereby in the selected host. In a further embodiment, the one or more vectors comprise further sequences to ensure that not only said variable chain domain of the invention is expressed, but also a full length IgG antibody comprising said variable chain domain of the invention is expressed.

The expression vector may be, for example, a cloning vector, a binary vector or an integrative vector. Expression includes transcription of the nucleic acid molecule, e.g., into translatable mRNA.

Non-limiting examples of vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), lambda gt11, pJOE, pBBR 1-MCS-series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13, E-027pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitroen), pMC1neo (Stratagene), pXT1(Stratagene), pSG5(Stratagene), EBO-V2 neo, pBPV-1, PDBPVMneo, SVgMTpttherein, pXT1(Stratagene), pSSVpGSV 6323-Pro-DNA (pRpCAP9), pRpCAPJpA-7-cDNA (pRpCpIVcDNA (pRpCmS 6326), pAK-7), pEPcDNAs (pRpIVcDNAs), pIcDNAs (pR-7) and pGpCmAGrDNA (L6335). Non-limiting examples of plasmid vectors suitable for Pichia pastoris (Pichia pastoris) include, for example, plasmids pAO815, pPIC9K and pPIC3.5K (all Invitrogen). Another vector suitable for expressing proteins in Xenopus (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 (e.g., antibiotic resistance), and one or more expression cassettes. In addition, the coding sequences contained in the vector may 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 initiation of transcription, an Internal Ribosome Entry Site (IRES), and optionally regulatory elements that ensure termination of transcription and stabilization of the transcript. Non-limiting examples of such regulatory elements that ensure initiation of transcription include promoters, translation initiation codons, enhancers, insulators, and/or regulatory elements that ensure termination of transcription, 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 which, depending on the expression system used, are capable of directing the expressed protein to a cell compartment 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, yet additional examples of suitable promoters include, but are not limited to, the Cytomegalovirus (CMV) promoter, SV 40-promoter, RSV-promoter (rous sarcoma virus), lacZ promoter, tetracycline promoter/operator (tetp/o), chicken β -actin promoter, CAG-promoter (a combination of chicken β -actin promoter and cytomegalovirus immediate early enhancer), gai10 promoter, human elongation factor 1 α -promoter, AOX1 promoter, GAL1 promoter CaM-kinase promoter, lac, trp or tac promoter, T7 or T5 promoter, lacUV5 promoter, Autographa californica (Autographa) polynedron promoter or globin intron in mammalian and other animal cells. An example of an enhancer is, for example, the SV 40-enhancer. Additional non-limiting examples of regulatory elements that ensure transcription termination include the SV 40-polyadenylation site, tk-polyadenylation site, rho factor-independent lpp terminator or AcMNPV polyhedric 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 paromomycin (paromycin), and hygro, which confers resistance to hygromycin. An additional selection gene has been described, trpB, which allows cells to use indole instead of tryptophan; hisD, which allows cells to utilize histidinol instead of histidine; mannose 6-phosphate isomerase, which allows the cell to utilise mannose and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor 2- (difluoromethyl) -DL-ornithine DFMO or confers resistance to blasticidin S a deaminase from Aspergillus terreus (Aspergillus terreus).

In a further embodiment, the vector is a eukaryotic expression plasmid containing an expression cassette consisting of a5 '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 (e.g.BL 21(DE3), BL21(DE3) PlysS, BL21(DE3) RIL, BL21(DE3) PRARE) or Rosettaa.

The nucleic acid molecules and/or vectors of the invention can be designed to be introduced into cells by, for example, chemical-based methods (polyethyleneimine, calcium phosphate, liposomes, DEAE-dextran, nuclear transfection, non-chemical methods (electroporation, sonoporation, light transfection, gene electrotransfer, fluid delivery, or transformation that occurs naturally when cells are contacted with the nucleic acid molecules of the invention), particle-based methods (gene gun, magnetic transfection, transfections by puncture), phage-based vector methods, and viral methods.

To facilitate purification of the nucleic acid molecules of the invention, a tag (tag) sequence may be inserted into the expression vector. Examples of tags include, but are not limited to, a hexa-histidine tag, a myc tag, or a FLAG tag. Any tag known to those skilled in the art to facilitate purification may be used in the present invention.

In the present invention, any suitable host cell/vector system may be used for the expression of the DNA sequence encoding the antibody molecule of the present invention. Bacterial (e.g., E.coli) and other microbial systems may be used, or eukaryotic (e.g., 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 the mammalian cell, one selected from the group consisting of, but not limited to, CHO cell, F2N cell, CSO cell, BHK cell, Bowes melanoma cell, HeLa cell, 911 cell, AT1080 cell, a549 cell, HEK293 cell, and HEK293T cell can be preferably used as the host cell. Any cell known to those skilled in the art to be useful as a mammalian host cell may be used in the art.

The recombinant cells of the invention can then be used for expression as well as culture purposes for antibody expression for large scale drug production. Can also be used as active ingredient of pharmaceutical composition. Any suitable culture technique may be used, including but not limited to static culture, spinner flask culture, ascites fluid, hollow fiber-type bioreactor cartridges, modular mini-fermenters, stirred tanks, microcarrier culture, ceramic core perfusion, and the like.

As an alternative embodiment, the product of the invention comprises the antibody or functional fragment thereof prepared according to the invention. As another alternative embodiment, the article of manufacture of the invention comprises a diagnostic composition comprising at least one detectable label, such as a detectable moiety/agent. The tag may be non-covalently conjugated to a monoclonal antibody of the invention. The tag may also be conjugated directly to the monoclonal antibody by a covalent bond. Alternatively, the tag may be conjugated to the monoclonal antibody 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/agent as a label is preferably one selected from the group consisting of, but not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting materials and non-radioactive paramagnetic metal ions. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin, and biotin; suitable fluorescent substances include, but are not limited to, FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine-type labels, including TAMRA; dansyl; lissamine; cyanine; phycoerythrin; texas Red; and the like. Fluorescent labels can be conjugated to aldehyde groups contained in the target molecule using the techniques disclosed herein. Suitable luminescent substances include luminol, acridine compounds, coelenterazine and analogues, dioxetanes, systems based on peroxyoxalic acid and derivatives thereof; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radionuclides include 125I, 131I, 111In, and 99 Tc.

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

The immunoassay or measurement method is a method of detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of the immunological detection or measurement method include a radioactive substance-labeled immune antibody method (RIA), an enzyme immunoassay (EIA or ELISA), a Fluorescence Immunoassay (FIA), a luminescence immunoassay, a western immunoblotting method, a flow cytometry, a physicochemical method, and the like.

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 a target antigen (e.g., CD38) is not particularly limited as long as it has a possibility of containing cells expressing the target antigen (e.g., CD38), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture fluid.

The pharmaceutical composition of the present invention comprises 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. In addition, 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 the patient.

Suitable administration forms include forms suitable for parenteral administration, for example by injection or infusion, for example by bolus injection or continuous infusion, intravenous, inhalable or subcutaneous forms. Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents such as suspending, preservative, stabilising and/or dispersing agents. Alternatively, the antibody or antigen-binding fragment thereof according to the invention may be in dry form for reconstitution with a suitable sterile liquid prior to use. Solid forms suitable for solution or suspension in a liquid vehicle 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 may be an animal. Preferably, the pharmaceutical composition according to the invention is adapted for administration to a human subject.

In the present invention, a "CD 38-associated disease" is a disease or disorder in which cells expressing CD38 are present. Such as, without limitation, immunological diseases involving B cells, plasma cells, monocytes and T cells expressing CD38, one of the disease features neoplastic diseases with the presence of tumor cells expressing CD38, such as CD38 expressing leukemias, B cell lymphomas, plasma cell malignancies, T/NK cell lymphomas and myelomas. In some embodiments of the invention, the leukemia is selected from: acute lymphocytic leukemia, acute lymphoblastic leukemia, acute promyelocytic leukemia, chronic lymphocytic leukemia, acute and chronic myelogenous leukemia. In some embodiments, the myeloma is selected from: multiple myeloma, promyelocytic tumors, and light chain amyloidosis. In some embodiments, the lymphoma is a non-hodgkin's lymphoma or a 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, mature B-cell tumor. In some embodiments, the tumor is selected from: b-cell Chronic Lymphocytic Leukemia (CLL), Small Lymphocytic Leukemia (SLL), B-cell acute lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytoid lymphoma, Mantle Cell Lymphoma (MCL), follicular lymphoma (including low, intermediate or high grade FL), cutaneous follicular central lymphoma, marginal zone B-cell lymphoma (including MALT type, lymph node MZBL type, spleen MZBL type), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma (Burkitt lymphoma), plasmacytoma, plasma cell myeloma, plasma cell leukemia, post-transplant lymphoproliferative disorder, Waldenstrom macroglobulinemia, 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: 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's syndrome, Sjogren's syndrome, juvenile diabetes, Reiter's disease, Behcet's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia symptoms (such as 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' thyroiditis, Wegener's granulomatosis, Omenn syndrome, chronic renal failure, acute infectious mononucleosis, multiple sclerosis, HIV and herpes virus-related diseases, severe acute respiratory syndrome, chorioretinitis (choreoretinitis), graft-versus-host disease, and immune diseases caused by viral infection (e.g., diseases caused or mediated by B cell infection with ibovirus (EBV)). In some embodiments, the immune disease is selected from rheumatoid arthritis, systemic lupus erythematosus, asthma, inflammatory bowel disease, multiple sclerosis, crohn's disease, gastritis, hashimoto's thyroiditis, ankylosing spondylitis, and graft-versus-host disease. In some embodiments, wherein the immune disease is rheumatoid arthritis.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the 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 sequences of antibody 70B5

The method comprises the following specific steps:

1. enzyme digestion experiment:

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

TABLE 2 digestion system and reaction conditions

2. Gel recovery of the cleavage products

Running the carrier subjected to enzyme digestion on the surface by using 1% agarose gel for electrophoresis, then cutting a gel block of a target strip, cutting the gel block as small as possible, and recovering the target DNA by using a full-type gold kit.

3. Amplification of PCR products

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

TABLE 3 amplification System

TABLE 4 amplification conditions

4. PCR product purification

The PCR product is purified by using a full-type gold kit, and the specific operation is shown in the kit specification.

5. Connection of

According to the Ligation Reaction in Table 5, the corresponding system was obtained by inputting the corresponding value, and water bath was carried out at 37 ℃ for 30 min.

TABLE 5 ligation reaction System

6. Ligation product transformation of StbI 3

The competent cells were thawed on ice from a freezer at-80 deg.C, 50. mu.l was pipetted into a pre-cooled EP tube, 2.5. mu.l of the ligation product was added, the mixture was gently flicked, ice-washed for 30min, heat-shocked in a water bath at 42 deg.C for 60s, quickly returned to ice and allowed to stand for 2 min. The tubes were added with 200. mu.l LB liquid medium (without Amp), shaken at 37 ℃ and 180rpm for 1h, and the appropriate amount of the transformant was pipetted onto LB (Amp) agar plates and spread out evenly. The plate was placed upside down in a 37 ℃ biochemical incubator overnight.

7. colony PCR identification

Selecting 4-8 single colonies to 100 μ l LB (Amp) liquid culture medium, shake culturing at 37 deg.C and 220rpm for 1-3h, subjecting the bacterial solution to PCR identification reaction, performing agarose gel electrophoresis to obtain PCR product with size of about 600bp, selecting 1 positive colony, and sequencing

8. Plasmid extraction and preservation

After the sequencing is correct, the bacterial colony is amplified to 15ml of culture medium to be cultured overnight, glycerol bacteria are prepared, an endotoxin-free plasmid extraction kit is used for extracting plasmids, the specific steps are carried out according to the specification of the endotoxin-free plasmid extraction kit (Tiangen), and the concentration of the plasmids is measured for antibody production.

9. The plasmid was transfected into cells to produce and purify the antibody.

EXAMPLE 2 ELISA detection of monoclonal antibodies

First, experimental material

1. Main reagent and consumable

2. Main instrument equipment

Name company model number

LabServ 3530917540 microplate reader

Second, Experimental methods

1. Unlabeled antigen (human CD38 Protein (ECD, His Tag)) was diluted to 0.5. mu.g/ml in Elisa Coating Buffer (1X) Coating Buffer (Elisa Coating Buffer (5X) diluted with D.L water), and 100. mu.l was transferred to Nunc^TM Maxisorp^TMELISA Plates Uncoated Single well, sealed plate, prevent evaporation, incubated overnight at 4 ℃.

2. Will Nunc^TM Maxisorp^TMELISA Plates Uncloated were placed at room temperature, the capture antibody solution was spun off, washed three times with Elisa Wash Buffer (1X) (Elisa Wash Buffer (20X) diluted with D.L water), and non-specific binding sites were blocked by adding 200. mu.l of blocking solution per well (BSA was made 1% concentration with Elisa Wash Buffer (1X)), and incubated for 1h at room temperature.

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

4. Several concentration gradients were set for standards (standards Purified anti-human CD38 diluted with blocking solution) and samples (monoclonal antibody blocking solution diluted) and one replicate well was taken per set, Nunc^TM Maxisorp^TM ELISA Plates Uncoated 100. mu.l per well were incubated for 2h at room temperature.

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

6. The biomarker detection antibodies HRP Goat anti-mouse IgG and HRP Goat anti-mouse IgM were diluted to 0.4. mu.g/ml (0.25-2. mu.g/ml) in blocking solution, added to the standard wells and the sample wells at 100. mu.l per well, respectively, and incubated at room temperature for 1 h.

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

8. 2ml of TMB Substrate A and 2ml of TMB Substrate B were mixed and 100. mu.l was transferred to Nunc^TMMaxisorp^TMELISA Plates Uncoated single wells and incubated at room temperature for 10min (4-60min), after incubation was complete 100. mu.l of Stop solution was added to Stop the reaction.

9. The OD value of each well is measured by a microplate reader under the condition of 450nm, and a standard curve is drawn by reading the standard wells, so that the concentration of the sample antibody is calculated.

Three, result in

As shown in FIG. 1, the detection degree of the antibodies in the experimental group is much greater than that in the negative control group, indicating that the antibodies have good affinity for CD 38.

Example 3 flow cytometry detection of monoclonal antibodies

First, experimental material

APC Goat anti-mouse IgM Antibody (biolegend:406509), PBS (Gibco: C20012500BT), RPMI1640(Gibco: C11875500BT), penicillin-streptomycin double Antibody solution (Hyclone: SV30010), Special grade fetal bovine serum (Meisen: CTCC-002-

Second, Experimental methods

1. Mono-Mac-6 cells were cultured, cell counting was performed, and then 5X 10 cells were taken⁵Placing each cell in a 1.5ml EP tube, centrifuging at 1000rpm for 5min, and removing supernatant;

2. adding 500 μ l PBS to resuspend the cells, then centrifuging at 1000rpm for 5min, and removing the supernatant;

3. respectively resuspending the blank cell culture supernatant and the antibody production supernatant into cells to be detected, placing the cells at 4 ℃ for reaction in a dark place for 30min, then centrifuging the cells at 1000rpm for 5min, and removing the supernatant;

4. adding 500 μ l PBS to resuspend the cells, then centrifuging at 1000rpm for 5min, and removing the supernatant;

5. adding 100 μ l of second Antibody (APC anti-human IgM Fc Antibody, stock solution diluted with PBS at a ratio of 1:600 for use), resuspending cells, standing at 4 deg.C, reacting in dark for 30min, centrifuging at 1000rpm for 5min, and removing supernatant;

6. adding 500 μ l PBS to resuspend the cells, then centrifuging at 1000rpm for 5min, and removing the supernatant;

7. cells were resuspended in 400. mu.l PBS and then tested on the machine.

Three, result in

The results are shown in FIG. 2, where the antibody-producing supernatant binds to CD38 on the surface of mono-Mac-6 cells compared to the blank cell culture supernatant (2A) (2B).

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

<110> Sichuan university Hospital in western China

<120> anti-CD 38 antibody and use thereof

<141> 2022-03-04

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ggcacaggga ccacggtcac cgtctcctca g 31

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gaggtccagc tgcaacaatc tggacctgag ctggtgaagc ctggggcttc agtgaagata 60

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aaccagaagt tcaagggcaa ggccacattg actgtagaca agtcctccag cacagcctac 240

atggagctcc gcagcctgac atctgaggac tctgcagtct attactgtgc aagaggctac 300

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gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggacattggt agtagcttaa actggcttca gcaggaacca 120

gatggaacta ttaaacgcct gatctacgcc acatccagtt tagattctgg tgtccccaaa 180

aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240

gaagattttg tagactatta ctgtctacaa tatgctagtt ctccgctcac gttcggtgct 300

gggaccaagc tggagctgaa ac 322

Claims

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 the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO.1, 2 and 3, and the amino acid sequences of the light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO.9, 10 and 11.

Preferably, the heavy chain variable region further comprises heavy chain variable region framework regions FR1, FR2, FR3 and FR 4; the light chain variable region further comprises light chain variable region framework regions FR1, FR2, FR3 and FR4, wherein the amino acid sequences of heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown as 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;

preferably, the monoclonal antibody comprises:

Preferably, the heavy chain of the monoclonal antibody is of the IgM subtype.

2. A nucleic acid molecule encoding the monoclonal antibody or functional fragment thereof of claim 1.

3. A vector comprising the nucleic acid molecule of claim 2.

4. A host cell comprising the nucleic acid molecule of claim 2 or the vector of claim 3;

preferably, the host cell is selected from a prokaryotic cell or a eukaryotic cell;

preferably, the host cell is a eukaryotic cell;

preferably, the eukaryotic cell is a mammalian cell.

5. A drug conjugate comprising the monoclonal antibody of claim 1;

preferably, the drug conjugate further comprises a coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, or enzyme.

6. A composition comprising the monoclonal antibody of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, the host cell of claim 4, or the drug conjugate of claim 5;

preferably, the composition further comprises a pharmaceutically acceptable carrier.

7. A product for detecting or determining CD38 in a sample, the product comprising the monoclonal antibody of claim 1;

preferably, the product further comprises reagents for processing the sample.

8. A method of producing the monoclonal antibody of claim 1, comprising the steps of:

culturing the cell of claim 1 and recovering the monoclonal antibody;

preferably, the method further comprises purifying the monoclonal antibody.

9. A method for detecting CD38 in a sample,

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

10. Use according to any one of the following:

1) use of the monoclonal antibody of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, the host cell of claim 4, the product of claim 7 for detecting CD 38;

2) use of the monoclonal antibody of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, the host cell of claim 4, the product of claim 7 for the preparation of a product for the diagnosis of a CD 38-related disease;

3) use of the monoclonal antibody of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, the host cell of claim 4, the drug conjugate of claim 5, the composition of claim 6 for the preparation of a medicament for the prevention and/or treatment of a disease associated with CD 38;

preferably, the disease is a tumor or an immune disease.