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

anti-CD 38 antibodies and uses thereof Download PDF

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CN114409788B
CN114409788B CN202210209470.3A CN202210209470A CN114409788B CN 114409788 B CN114409788 B CN 114409788B CN 202210209470 A CN202210209470 A CN 202210209470A CN 114409788 B CN114409788 B CN 114409788B
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monoclonal antibody
variable region
chain variable
host cell
heavy chain
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CN114409788A (en
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龚艳萍
杨敬
龚日祥
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West China Hospital of Sichuan University
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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-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, comprising an Extracellular region (ECD Domain, single transmembrane Structure with 23 amino acid residues, and a cytoplasmic region with 20 amino acids (Liu Qun, kriksunov irina.a, graeff Richard, et a1.Crystal Structure 20152015 of Human CD38 excellar Domain [ J ]. Structure, september,2005, 13 1331-1339.) the CD38 gene span is approximately 80kb, is located on chromosome 4p15, contains 8 exons, wherein exon 1 encodes the plasma region amino acid, the transmembrane region, and approximately 33 amino acids of the Extracellular region (exon dynamics w. Uk, combo Raymond. Amino acid 38 Monoclonal antibodies 635. Loop [ c. F. C. Molecular dynamics, c..
CD38 is a multifunctional transmembrane protein that is widely expressed in immune cells. The level of expression of CD38 on the cell surface 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 complexes with CD4 in T cells, membrane immunoglobulin (Ig) and B cell co-receptor complexes (CD 19/CD 81) in B lymphocytes, and MHC class II complexes in monocytes, which suggest that CD38 potentially contributes to cellular signals 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, it has been found that CD38 may be involved in the pathogenesis of various autoimmune diseases (Partida-SanchezS, rivero-Naval, shi G, et a1.CD38: an ecto enzyme at the cross of amino and adaptive immune responses. Adv Exp Med biol.2007; 590. 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 overcome the defects in the prior art, the invention aims to provide an anti-CD 38 monoclonal antibody 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 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.
In some embodiments, 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.
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 amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.8, and the amino acid sequence of the light chain variable region is shown as 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 an 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 a monoclonal antibody according to the first aspect of the invention or a functional fragment thereof.
Further, the CDR1, CDR2, CDR3 nucleic acid molecules encoding the heavy chain variable region have a sequence with at least 90%, preferably 95% sequence identity to the nucleotide sequences shown in SEQ ID No.17, 18, 19;
the CDR1, CDR2, CDR3 nucleic acid molecules encoding the variable regions of the light chains have sequences with at least 90%, preferably 95% sequence identity to the nucleotide sequences shown 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 with the nucleotide sequence set forth in SEQ ID Nos. 20, 21, 22 and 23; nucleic acid molecules encoding the 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 with the nucleotide sequences shown 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 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 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 the anti-CD 38 monoclonal antibody of the first aspect with a sample to be detected; 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) 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 product according to the seventh aspect of the invention for the detection of 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, in the manufacture of a product for the diagnosis of a CD 38-associated disease;
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 CD 38-associated disease.
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 symptoms, 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 severe respiratory syndrome and choroid virus-related diseases, acute respiratory syndrome, acute herpes virus, and immune-induced host disease.
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 tumors, 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 testing for binding of antibodies to antigens;
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
Through extensive and intensive research, the invention discovers an anti-CD 38 monoclonal antibody which 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 to the molecule, particularly the immunogenicity and specificity of the antigen binding protein. However, in some cases greater variation may be tolerated. Amino acid substitutions are typically of a single base; the insertion 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, the 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 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 a 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, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027pCAG Kosak-Cherry (L45 a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, clopPV-1, pdBPVMneo, pRSVgpo, pRSVnefro, pGsubpidyVdhyV 2-35, bepcCMV-35, EPCMYO-DNA (pReXpIPsec-DNA), pRpIPcDNAs (LgP-expression vectors (pRepGE) and expression vectors (pRepGE-pIPcDNAs) (pRepGE-10). 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+.
Generally, 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 transcription initiation, an Internal Ribosome Entry Site (IRES), and optionally regulatory elements that ensure transcription termination and transcript stability. 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 origin of replication, while additional examples of suitable promoters include, but are not limited to, cytomegalovirus (CMV) promoter, SV 40-promoter, RSV-promoter (rous sarcoma virus), lacZ promoter, tetracycline promoter/operator (tetp/o), chicken beta-actin promoter, CAG-promoter (combination of chicken beta-actin promoter and cytomegalovirus immediate early enhancer), gai10 promoter, human elongation factor 1 alpha-promoter, AOX1 promoter, GAL1 promoter CaM-kinase promoter, lac, trp or tac promoter, T7 or T5 promoter, lacUV5 promoter, autographa californica (Autographa) type polynuclear polyhedrosis virus (AcMNPV) polyhedrin promoter, or globin introns 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., BL21 (DE 3) Pluss, BL21 (DE 3) RIL, BL21 (DE 3) 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, transfunctions), 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 cells, F2N cells, CSO cells, BHK cells, bowes melanoma cells, heLa cells, 911 cells, AT1080 cells, a549 cells, HEK293 cells, and HEK293T cells may 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 a tag to a monoclonal antibody 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 99Tc.
In the present invention, the method for detecting or determining the amount of the target antigen (e.g., CD 38) 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 the target antigen (e.g., CD 38) is not particularly limited as long as it has a possibility of containing cells expressing the target antigen (e.g., CD 38), 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 such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and 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 CD 38-expressing B-cells, plasma cells, monocytes and T-cells, one of the disease features is a neoplastic disease in which CD 38-expressing tumor cells are present, such as CD 38-expressing leukemia, B-cell lymphoma, plasma cell malignancy, T/NK cell lymphoma and myeloma. 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 lymphoma or B cell Hodgkin 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 disease, raynaud's syndrome, sjogren's syndrome, juvenile diabetes, reiter's disease, behcet's disease, immune complex nephritis, igA nephropathy, igM polyneuropathy, immune-mediated thrombocytopenic symptoms (such as acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura), hemolytic anemia, myasthenia gravis, lupus, systemic lupus erythematosus, rheumatoid Arthritis (RA), atopic dermatitis, pemphigus, graves ' disease, hashimoto's thyroiditis, wegener's granuloma, enn failure, kidney function syndrome, acute herpes simplex virus, infectious herpes simplex virus, multiple sclerosis (HIV-mediated), and host cell-induced diseases such as acute infectious herpesvirus infection, chronic infectious herpesvirus infection, and acute infectious herpesvirus infection (e-induced host cell disease), and infectious host cell-induced diseases such as acute infectious herpesvirus infection, chronic infectious herpesvirus infection, acute infectious herpesvirus infection, and acute infectious host disease (e-induced retinopathy). In some embodiments, the immune disorder 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 70B5 antibody sequences shown in table 1 were synthesized and cloned into eukaryotic expression vectors.
TABLE 1 sequences of antibody 70B5
Figure BDA0003532568580000141
Figure BDA0003532568580000151
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
Figure BDA0003532568580000152
Figure BDA0003532568580000161
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
Figure BDA0003532568580000162
TABLE 4 amplification conditions
Figure BDA0003532568580000163
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 30min.
TABLE 5 ligation reaction System
Figure BDA0003532568580000171
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 precooled EP tube, 2.5. Mu.l of the ligation product was added, the mixture was gently flicked, ice-cooled for 30min, heat-shocked in a water bath at 42 deg.C for 60s, quickly returned to ice and allowed to stand for 2min. Add 200. Mu.l LB liquid medium (without Amp) into the centrifuge tube, shake-culture at 37 ℃ and 180rpm for 1h, aspirate the appropriate amount of the transformation product, add to LB (Amp) agar plate, spread 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, amplifying the bacterial colony to 15ml of culture medium for overnight culture, preparing glycerol bacteria, extracting plasmids by using an endotoxin-free plasmid extraction kit, and determining the concentration of the plasmids for antibody production by referring to the specification of the endotoxin-free plasmid extraction kit (Tiangen).
9. The plasmid was transfected into cells to produce and purify the antibody.
EXAMPLE 2 ELISA detection of monoclonal antibodies
1. Experimental materials
1. Main reagent and consumable
Figure BDA0003532568580000181
2. Main instrument equipment
Name company model number
Enzyme-linked immunosorbent assay (LabServ) 3530917540
2. Experimental method
1. Unlabeled antigen (human CD38 Protein (ECD, his Tag)) was diluted to 0.5. Mu.g/ml in Elisa Coating Buffer (1X) (Elisa Coating Buffer (5X) diluted with D.L water), and 100. Mu.l was transferred to Nunc TM Maxisorp TM ELISA Plates Uncoated Single well, sealed plate, prevent evaporation, incubated overnight at 4 ℃.
2. Will Nunc TM Maxisorp TM ELISA Plates Uncoated were placed at RT, the capture antibody solution was spun off and washed with Elisa Wash Buffer (1 ×) (using D.L water)Dilution Elisa Wash Buffer (20X)) three times, each well was added 200. Mu.l of blocking solution (1% concentration of BSA with Elisa Wash Buffer (1X)) to block non-specific binding sites, 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. The standard (standard Purified anti-human CD38 diluted with blocking solution) and the sample (monoclonal antibody blocking solution diluted) were set up with several concentration gradients and each set was taken as one duplicate well, 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 down firmly with clean paper towels.
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 1h.
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 TM Maxisorp TM ELISA Plates Uncoated single wells and incubated at room temperature for 10min (4-60 min), after incubation was complete 100. Mu.l 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.
3. Results
The results are shown in fig. 1, and the detection degree of the antibodies in the experimental group is far greater than that in the negative control group, which indicates that the antibodies have good affinity for CD 38.
Example 3 flow cytometry detection of monoclonal antibodies
1. Experimental materials
APC Goat anti-mouse IgM Antibody (biolegend: 406509), PBS (Gibco: C20012500 BT), RPMI1640 (Gibco: C11875500 BT), penicillin-streptomycin double Antibody solution (Hyclone: SV 30010), extra-grade fetal bovine serum (Meisen: CTCC-002-001-50 xu 10), mono-Mac-6 cells, 1.5mL EP tube (Tantan: TS 018-032)
2. Experimental method
1. Mono-Mac-6 cells were cultured, counted and then 5X 10 cells were taken 5 Placing the cells in a 1.5ml EP tube, centrifuging at 1000rpm for 5min, and removing supernatant;
2. adding 500 mu l of PBS for resuspending the cells, then centrifuging at 1000rpm for 5min, and removing supernatant;
3. respectively resuspending the blank cell culture supernatant and the antibody production supernatant for cells to be detected, placing at 4 ℃, reacting in a dark place for 30min, then centrifuging at 1000rpm for 5min, and removing the supernatant;
4. adding 500. Mu.l PBS to resuspend the cells, then centrifuging at 1000rpm for 5min, and removing the supernatant;
5. adding 100 μ l of secondary Antibody (APC anti-human IgM Fc Antibody, stock solution diluted with PBS at 1;
6. adding 500. Mu.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.
3. Results
As a result, as shown in FIG. 2, the antibody-producing supernatant was able to bind to CD38 on the surface of mono-Mac-6 cells, as compared with 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 it would be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit of the invention, and these modifications and variations also fall within the scope of the claims of the present invention.
Sequence listing
<110> Sichuan university Hospital in Huaxi
<120> anti-CD 38 antibody and use thereof
<141> 2022-03-04
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<213> Artificial Sequence (Artificial Sequence)
<400> 28
gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60
ctcacttgt 69
<210> 29
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tggcttcagc aggaaccaga tggaactatt aaacgcctga tctac 45
<210> 30
<211> 93
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
ggtgtcccca aaaggttcag tggcagtagg tctgggtcag attattctct caccatcagc 60
agccttgagt ctgaagattt tgtagactat tac 93
<210> 31
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ggtgctggga ccaagctgga gctgaaac 28
<210> 32
<211> 322
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
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 (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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WO2011154453A1 (en) * 2010-06-09 2011-12-15 Genmab A/S Antibodies against human cd38
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Publication number Priority date Publication date Assignee Title
BR112020026432A2 (en) * 2018-07-13 2021-03-23 Genmab A/S antibody variant, isolated nucleic acid, expression vector, nucleic acid, combination of nucleic acids, dispensing vehicle, recombinant host cell, methods for producing an antibody variant, to increase at least one effector function of a parental antibody and to treat a disease, antibody, composition, pharmaceutical composition, and, antibody variant for use

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CN112513087A (en) * 2018-09-11 2021-03-16 江苏恒瑞医药股份有限公司 anti-CD 38 antibody, antigen binding fragment thereof and medical application
CN113939537A (en) * 2019-03-29 2022-01-14 索伦托药业有限公司 Engineered variant antibodies that bind CD38

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