CN114763383A - Monoclonal antibody targeting human BCMA and application thereof - Google Patents

Monoclonal antibody targeting human BCMA and application thereof Download PDF

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Publication number
CN114763383A
CN114763383A CN202110044548.6A CN202110044548A CN114763383A CN 114763383 A CN114763383 A CN 114763383A CN 202110044548 A CN202110044548 A CN 202110044548A CN 114763383 A CN114763383 A CN 114763383A
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antibody
ser
thr
leu
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杨林
游凤涛
荆光军
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Persongen Biotherapeutics Suzhou Co ltd
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Persongen Biotherapeutics Suzhou Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Abstract

The invention provides a monoclonal antibody targeting human BCMA and application thereof. In particular, the invention provides antibodies targeting BCMA with high affinity and high specificity to human BCMA. The invention also provides a cell and a pharmaceutical composition containing the antibody, and a preparation method and application of the antibody.

Description

Monoclonal antibody targeting human BCMA and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a monoclonal antibody targeting human BCMA and application thereof.
Background
B-cell surface maturation antigen (BCMA) was first found on the surface of mature B lymphocytes and is hardly expressed in other tissue cells. It is highly expressed in malignantly proliferating B lymphocytes (e.g., myeloma cells, leukemia cells). Meanwhile, the polypeptide plays a key role in the survival, proliferation, metastasis and drug resistance of cells by mediating downstream signaling pathways, and the characteristics make the polypeptide a target point of immunotherapy, particularly for the treatment of multiple myeloma. BCMA, due to its limited expression in plasma cells, non-expression in native and memory B cells, has been used as a new drug target for the diagnosis and treatment of multiple myeloma. Currently, novel tumor Immunotherapy approaches to BCMA mainly include CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) therapy, Bispecific antibodies (BsAb), and Antibody-drug conjugates (ADC). Recent studies have also shown that BCMA-targeted CAR-T is able to efficiently clear myeloma cells in vivo.
Currently, there are many deficiencies in the research on BCMA antibodies and there is a need in the art to develop new BCMA antibodies and related applications.
Disclosure of Invention
The invention aims to provide a monoclonal antibody targeting human BCMA and application thereof.
The invention also aims to provide a BCMA humanized antibody and further aims to treat multiple myeloma by constructing a CAR-T or bispecific antibody or antibody coupling drug targeting BCMA.
In a first aspect of the present invention, there is provided a heavy chain variable region of an antibody, said heavy chain variable region comprising the following three Complementarity Determining Regions (CDRs):
CDR1 shown in SEQ ID NO. 1,
CDR2 shown in SEQ ID NO. 2, and
CDR3 shown in SEQ ID NO. 3.
In another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and capable of retaining BCMA binding affinity.
In another preferred embodiment, the heavy chain variable region further comprises a human FR region or a murine FR region.
In another preferred embodiment, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO. 7.
In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NOS 9-14.
In a second aspect of the invention, there is provided a heavy chain of an antibody, said heavy chain having a heavy chain variable region as described in the first aspect of the invention.
In another preferred embodiment, the heavy chain of said antibody further comprises a heavy chain constant region.
In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin.
In a third aspect of the present invention, there is provided an antibody light chain variable region comprising the following three complementarity determining regions CDR:
CDR 1' shown in SEQ ID NO. 4,
CDR 2' shown in SEQ ID NO. 5, and
CDR 3' shown in SEQ ID NO. 6.
In another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and capable of retaining BCMA binding affinity.
In another preferred embodiment, the light chain variable region further comprises a human FR region or a murine FR region.
In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID NO 8.
In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID NO 15-20.
In a fourth aspect of the invention, there is provided a light chain of an antibody, said light chain having a light chain variable region as described in the third aspect of the invention.
In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.
In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin.
In a fifth aspect of the invention, there is provided an antibody targeting BCMA, the antibody having:
(1) a heavy chain variable region according to the first aspect of the invention; and/or
(2) A light chain variable region according to the third aspect of the invention;
alternatively, the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.
In another preferred embodiment, the antibody binds to human BCMA protein, preferably wild-type, with an affinity constant kd (m) of (1-10) × 10-10Preferably (1-5). times.10-10。
In another preferred embodiment, the antibody is selected from the group consisting of: an antibody of animal origin, a chimeric antibody, a humanized antibody, or a combination thereof.
In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.
In another preferred embodiment, the antibody is a monoclonal antibody.
In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.
In another preferred embodiment, the heavy chain variable region sequence of said antibody is as set forth in SEQ ID NO:7 is shown in the specification; and/or the light chain variable region sequence of the antibody is shown as SEQ ID NO: shown in fig. 8.
In another preferred embodiment, the heavy chain variable region sequence of said antibody is as set forth in SEQ ID NO: 10 is shown in the figure; and the light chain variable region sequence of the antibody is shown as SEQ ID NO: shown at 15.
In another preferred embodiment, the heavy chain variable region sequence of said antibody is as set forth in SEQ ID NO: 12 is shown in the specification; and the light chain variable region sequence of the antibody is shown in SEQ ID NO: shown at 16.
In another aspect of the invention there is provided a bispecific antibody comprising a BCMA-targeting antibody according to the fifth aspect of the invention and a second antibody which targets a tumor antigen other than BCMA.
In another preferred embodiment, the tumor antigen comprises CD3 and CD 19.
In another preferred embodiment, the second antibody is a nanobody or an scFv.
In another preferred embodiment, the second antibody is linked to a region of the BCMA-targeting antibody selected from the group consisting of: a heavy chain variable region, a heavy chain constant region, or a combination thereof.
In a sixth aspect of the present invention, there is provided a recombinant protein having:
(i) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; and
(ii) optionally a tag sequence to facilitate expression and/or purification.
In another preferred embodiment, the tag sequence comprises a 6His tag.
In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.
In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
In a seventh aspect of the invention there is provided a CAR construct, wherein the scFV fragment of the antigen binding region of the monoclonal antibody of the CAR construct is a binding region that specifically binds to BCMA, and the scFV has a heavy chain variable region according to the first aspect of the invention and a light chain variable region according to the third aspect of the invention.
In another preferred embodiment, the chimeric antigen receptor has the following formula I:
L-scFv-H-TM-C-CD3ζ(I)
in the formula (I), the compound is shown in the specification,
each "-" is independently a linker peptide or a peptide bond;
l is a null or signal peptide sequence;
the scFv is a BCMA-targeted scFv;
h is an optional hinge region;
TM is a transmembrane domain;
c is a costimulatory signal molecule;
CD3 ζ is the CD3 ζ cytoplasmic signaling sequence.
In an eighth aspect of the invention there is provided a recombinant immune cell expressing an exogenous CAR construct according to the seventh aspect of the invention.
In another preferred embodiment, the immune cell is selected from the group consisting of: NK cells, T cells.
In another preferred embodiment, the immune cell is from a human or non-human mammal (e.g., a mouse).
In a ninth aspect of the present invention, there is provided an antibody drug conjugate comprising:
(a) an antibody moiety selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, or a combination thereof; and
(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
In another preferred embodiment, said antibody moiety is coupled to said coupling moiety by a chemical bond or a linker.
In a tenth aspect of the invention, there is provided the use of an active ingredient selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, an immune cell according to the eighth aspect of the invention, an antibody drug conjugate according to the ninth aspect of the invention, or a combination thereof, wherein the active ingredients are used for (a) preparing a detection reagent or a kit; and/or (b) preparing a medicament for preventing and/or treating BCMA positive tumors.
In another preferred embodiment, the tumor is selected from the group consisting of: a hematologic tumor, a solid tumor, or a combination thereof.
In another preferred embodiment, the hematological tumor is selected from the group consisting of: acute Myeloid Leukemia (AML), Multiple Myeloma (MM), Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.
In another preferred embodiment, the solid tumor is selected from the group consisting of: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphatic cancer, nasopharyngeal cancer, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumor, thyroid cancer, or a combination thereof.
In another preferred embodiment, the tumor is gastric cancer, breast cancer, osteosarcoma.
In another preferred embodiment, the antibody is in the form of A Drug Conjugate (ADC).
In another preferred embodiment, the detection reagent or kit is used for:
(1) detecting BCMA protein in the sample; and/or
(2) Detecting endogenous BCMA protein in tumor cells; and/or
(3) Detecting tumor cells expressing the BCMA protein.
In another preferred embodiment, the detection reagent is a detection sheet.
In an eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) an active ingredient selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, an immune cell according to the eighth aspect of the invention, an antibody drug conjugate according to the ninth aspect of the invention, or a combination thereof; and
(ii) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is a liquid preparation.
In another preferred embodiment, the pharmaceutical composition is an injection.
In a twelfth aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:
(1) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; or
(2) A recombinant protein according to the sixth aspect of the invention;
(3) a CAR construct according to the seventh aspect of the invention.
In another preferred embodiment, the nucleotide sequence is shown in SEQ ID NO 21-32.
In a thirteenth aspect of the invention, there is provided a vector comprising a polynucleotide according to the twelfth aspect of the invention.
In another preferred embodiment, the carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
In a fourteenth aspect of the invention, there is provided a genetically engineered host cell comprising a vector according to the thirteenth aspect of the invention or having a polynucleotide according to the twelfth aspect of the invention integrated into its genome.
In a fifteenth aspect of the invention, there is provided a method for in vitro detection (including diagnostic or non-diagnostic) of BCMA protein in a sample, said method comprising the steps of:
(1) contacting the sample in vitro with an antibody according to the fifth aspect of the invention or an antibody drug conjugate according to the ninth aspect of the invention;
(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex is indicative of the presence of BCMA protein in the sample.
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
In a sixteenth aspect of the present invention, there is provided a detection board comprising: a substrate (support plate) and a test strip comprising an antibody according to the fifth aspect of the invention or an antibody drug conjugate according to the ninth aspect of the invention.
In a seventeenth aspect of the present invention, there is provided a kit comprising:
(1) a first container comprising an antibody according to the fifth aspect of the invention; and/or
(2) A second container comprising a secondary antibody directed against the antibody according to the fifth aspect of the invention;
alternatively, the kit comprises a detection plate according to the sixteenth aspect of the invention.
In an eighteenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:
(a) culturing a host cell according to the fourteenth aspect of the invention under conditions suitable for expression;
(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.
In a nineteenth aspect of the present invention, there is provided a method for preventing and/or treating a BCMA positive tumor, the method comprising: administering to a subject in need thereof an antibody according to the fifth aspect of the invention, an antibody-drug conjugate of said antibody, or a CAR-T cell expressing said antibody, or a combination thereof.
In another preferred example, the method further comprises: administering to a subject in need thereof an additional agent or treatment for combination therapy.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
Figure 1 shows a mouse immunization protocol.
FIG. 2 shows a vector schematic diagram of the heavy chain expression vector pcDNA3.1-IgG1Fc and the light chain expression vector pcDNA3.1-IgKc.
FIG. 3 shows the results of FACS detection of immunized mice.
FIG. 4 shows the results of ELISA detection of hybridoma fusion plates.
FIG. 5 shows the results of FACS detection of hybridoma fusion plates.
FIG. 6 shows the results of ELISA assays of the first subcloned plates.
FIG. 7 shows the results of FACS detection of the first subclone.
FIG. 8 shows the results of ELISA assays on the second subcloned plate.
FIG. 9 shows the results of FACS detection of the chimeric antibody.
FIG. 10 shows the results of FACS detection of the humanized antibody.
FIG. 11 shows the SDS-PAGE results for humanized antibodies B1H2-B1L1 and B1H4-B1L 2.
FIG. 12 shows an alignment of humanized antibody B1H2-B1L1 with murine antibody.
FIG. 13 shows an alignment of humanized antibody B1H4-B1L2 with murine antibody.
FIG. 14 shows the ligand-coupled pre-enrichment results.
Figure 15 shows the ligand coupling results.
FIG. 16 shows the results of an affinity assay for MH18100901-B1 Chimeric.
FIG. 17 shows the results of affinity assay for MH18100901-B1H2-B1L 1.
FIG. 18 shows the results of affinity assays for MH18100901-B1H4-B1L 2.
Detailed Description
The present inventors have made extensive and intensive studies and, for the first time, have unexpectedly found an antibody targeting BCMA with high affinity and high specificity, and have produced a humanized antibody having an affinity comparable to that of a chimeric antibody. The present invention has been completed based on this finding.
Term(s) for
As used herein, the terms "administration" and "treatment" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells, and contacting the reagent with a fluid, and contacting the fluid with the cells. "administering" and "treating" also mean treating in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, animal or study subject refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnosis; including contacting a BCMA antibody with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.
As used herein, the term "treatment" refers to the administration of a therapeutic agent, either internally or externally, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect, comprising any one of the BCMA antibodies and compositions thereof of the present invention. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (therapeutically effective amount).
As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but need not, be 1, 2 or 3.
Antibodies
As used herein, the term "antibody" refers to an immunoglobulin, a tetrapeptide chain structure made up of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, or different classes called immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, and the heavy chain constant regions corresponding to the different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. IgG represents the most important class of immunoglobulins, which can be divided into 4 subclasses due to differences in chemical structure and biological function: IgG1, IgG2, IgG3, and IgG 4. Light chains are classified as kappa or lambda chains by differences in the constant regions. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable regions include 3 hypervariable regions (HVRs) and 4 FR Regions (FRs) which are relatively conserved in sequence. The amino acid sequences of the 4 FRs are relatively conserved and do not directly participate in the binding reaction. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) is composed of 3 CDR regions and 4 FR regions, which are sequentially arranged from amino terminus to carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain, the light chain hypervariable region (LCDR), designated LCDR1, LCDR2 and LCDR 3; the 3 CDR regions of the heavy chain, the hypervariable region of the Heavy Chain (HCDR), are referred to as HCDR1, HCDR2 and HCDR 3. The CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention are in number and position in accordance with known Kabat numbering convention (LCDR1-3, HCDR2-3), or in accordance with Kabat and chothia numbering convention (HCDR 1). The four FR regions in the native heavy and light chain variable regions are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held together tightly by the FR regions and form the antigen binding site of the antibody with the CDRs of the other chain. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.
The term "antigen-binding fragment," as used herein, refers to a Fab fragment, Fab 'fragment, F (ab') 2 fragment, or single Fv fragment having antigen-binding activity. Fv antibodies contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest antibody fragment of the entire antigen binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding.
As used herein, the term "antigenic determinant" refers to a three-dimensional spatial site on an antigen that is not contiguous and is recognized by an antibody or antigen-binding fragment of the invention.
The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.
As used herein, the term "monoclonal antibody" refers to an antibody secreted by a clone obtained from a single cell source. Monoclonal antibodies are highly specific, being directed against a single epitope. The cell may be a eukaryotic, prokaryotic, or phage clonal cell line.
As used herein, the term "chimeric antibody" is an antibody molecule expressed by a host cell transfected with a vector after splicing a V region gene of a murine antibody to a C region gene of a human antibody into a chimeric gene. Not only retains the high specificity and affinity of the parent mouse antibody, but also ensures that the humanized Fc segment can effectively mediate the biological effect function.
As used herein, the term "humanized antibody", is a variable region engineered version of a murine antibody of the invention, having CDR regions derived from (or substantially derived from) a non-human antibody (preferably a mouse monoclonal antibody), and FR regions and constant regions substantially derived from human antibody sequences; that is, the CDR sequence of the mouse antibody is grafted to the framework sequences of different types of human germline antibodies. Because the CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing an expression vector.
In the present invention, the antibody may be monospecific, bispecific, trispecific, or more multispecific.
In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced with amino acids having similar or similar properties as compared to the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.
TABLE A
Initial residue(s) Representative substitutions Preferred substitutions
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
anti-BCMA antibodies
As used herein, the term "BCMA" generally refers to natural or recombinant human BCMA, as well as non-human homologs of human BCMA.
The present invention provides a highly specific and high affinity antibody against BCMA comprising a heavy chain variable region (VH) amino acid sequence and a light chain comprising a light chain variable region (VL) amino acid sequence.
Preferably, the CDRs of the heavy chain variable region (VH) are selected from the group consisting of:
CDR1 shown in SEQ ID NO. 1,
CDR2 shown in SEQ ID NO. 2, and
CDR3 shown in SEQ ID NO. 3; and/or
The CDRs of the light chain variable region (VL) are selected from the group consisting of:
CDR 1' shown in SEQ ID NO. 4,
CDR 2' of SEQ ID NO. 5, and
CDR 3' shown in SEQ ID NO. 6.
Wherein, any one of the above amino acid sequences further comprises a derivative sequence with BCMA binding affinity, which is added, deleted, modified and/or substituted with at least one (e.g., 1-5, 1-3, preferably 1-2, more preferably 1) amino acid.
In another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence is preferably an amino acid sequence with homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.
The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, and still more preferably a fully humanized antibody.
The antibody derivatives of the present invention may be single chain antibodies, and/or antibody fragments, such as: fab, Fab ', (Fab')2Or other known antibody derivatives in the art, and any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or antibodies of other subtypes.
Among them, the animal is preferably a mammal such as a mouse.
The antibodies of the invention may be murine, chimeric, humanized, CDR-grafted and/or modified antibodies targeting human BCMA.
Preparation of antibodies
Any method suitable for producing monoclonal antibodies can be used to produce the anti-BCMA antibodies of the invention. For example, an animal can be immunized with BCMA or a fragment thereof. Suitable immunization methods, including adjuvants, immunostimulants, repeated booster immunizations, can be used, and one or more routes can be used.
Any suitable form of BCMA (BCMA-Fc recombinant protein) may be used as immunogen (antigen) for the production of non-human antibodies specific for BCMA, which antibodies are screened for biological activity. The challenge immunogen may be full-length mature human BCMA, including native homodimers, or peptides containing single/multiple epitopes. The immunogen may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. Immunogens can be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic in origin (e.g., cDNA). DNA encoding the immunogen may be expressed using suitable genetic vectors including, but not limited to, adenoviral vectors, adeno-associated viral vectors, baculovirus vectors, plasmids and non-viral vectors.
Humanized antibodies may be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. In the present invention, the antibody is an IgG antibody, and an IgG1 subtype is used. Optimization of the sequence of the essential constant domains to produce the desired biological activity is readily achieved by screening antibodies using the biological assays described in the examples below.
Likewise, any type of light chain can be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the present invention.
The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.
The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Preferred animal cells include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.
The steps described in the present invention for transforming a host cell with a recombinant DNA can be performed by techniques well known in the art. The obtained transformant can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured in a conventional medium under suitable conditions.
Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. The antibody of the invention is then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, among others, which are well known to those skilled in the art.
The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
Antibody-drug conjugates (ADC)
The invention also provides an antibody-conjugated drug (ADC) based on the antibody of the invention.
Typically, the antibody-conjugated drug comprises the antibody, and an effector molecule to which the antibody is conjugated, and preferably chemically conjugated. Wherein the effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.
The antibody of the invention may be coupled to the effector molecule by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The non-selective coupling agent is a compound which enables effector molecules and antibodies to form covalent bonds, such as glutaraldehyde and the like. The coupling agent using carboxyl can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (coupling site is acylhydrazone).
Certain residues on the antibody (e.g., Cys or Lys, etc.) are used to attach to a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizing agents (e.g., ethylene glycol polymers) and therapeutic agents. The antibody may be conjugated to a functional agent to form an antibody-functional agent conjugate. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent may be attached to the antibody directly, or indirectly through a linker.
Antibodies may be conjugated to drugs to form Antibody Drug Conjugates (ADCs). Typically, the ADC comprises a linker between the drug and the antibody. The linker may be degradable or non-degradable. Degradable linkers are typically susceptible to degradation in the intracellular environment, e.g., the linker degrades at the site of interest, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. Peptidyl linkers may include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH sensitive linkers (e.g., linkers that hydrolyze at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by a protease.
Prior to attachment to the antibody, the linker has reactive groups capable of reacting with certain amino acid residues, and attachment is achieved by the reactive groups. Thiol-specific reactive groups are preferred and include: for example maleimide compounds, haloamides (for example iodine, bromine or chlorine); halogenated esters (e.g., iodo, bromo, or chloro); halomethyl ketones (e.g., iodo, bromo, or chloro), benzyl halides (e.g., iodo, bromo, or chloro); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3, 6-bis- (mercuric methyl) dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may comprise, for example, a maleimide linked to the antibody via a thiosuccinimide.
The drug may be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, the linker links the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or keto group that may form a bond with the linker. In the case of a drug directly attached to a linker, the drug has a reactive group prior to attachment to the antibody.
Useful classes of drugs include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. In the present invention, a drug-linker can be used to form an ADC in one simple step. In other embodiments, bifunctional linker compounds may be used to form ADCs in a two-step or multi-step process. For example, a cysteine residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker is reacted with a drug, thereby forming an ADC.
Generally, the functional group on the linker is selected to facilitate specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, an azide-based moiety may be used to specifically react with a reactive alkynyl group on a drug moiety. The drug is covalently bound to the linker by 1, 3-dipolar cycloaddition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other ligation strategies, such as those described in bioconjugation technology, second edition (Elsevier), are well known to those skilled in the art. It will be appreciated by those skilled in the art that for selective reaction of a drug moiety and a linker, each member of a complementary pair may be used for both the linker and the drug when the reactive functional group of the complementary pair is selected.
The present invention also provides a method of preparing an ADC, which may further comprise: the antibody is conjugated to a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
In certain embodiments, the methods of the invention comprise: the antibody is conjugated to the bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present invention further comprises: the antibody linker conjugate is bound to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.
In some embodiments, the antibody drug conjugate ADC has the formula:
Figure BDA0002897008700000151
wherein:
ab is an antibody, and Ab is an antibody,
LU is a joint;
d is a drug;
and subscript p is a value selected from 1 to 8.
Pharmaceutical composition
The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or an active fragment thereof or a fusion protein thereof or an ADC thereof or a corresponding CAR-T cell as described above, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.
The antibody of the present invention may also be used for cell therapy by intracellular expression of a nucleotide sequence, for example, for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
The pharmaceutical composition of the present invention can be directly used for binding BCMA protein molecules, and thus can be used for preventing and treating BCMA positive tumors. In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
When a pharmaceutical composition is used, a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases does not exceed about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the particular dosage will also take into account such factors as the route of administration, the health of the patient, and the like, which are within the skill of the skilled practitioner.
Detection use and kit
The antibodies of the invention are useful in detection applications, for example, for detecting a sample, thereby providing diagnostic information.
In the present invention, the specimen (sample) used includes cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. Thus, a biopsy as used in the present invention may comprise a tissue sample prepared, for example, by endoscopic methods or by needle or needle biopsy of an organ.
Samples for use in the present invention include fixed or preserved cell or tissue samples.
The invention also provides a kit containing the antibody (or fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.
The main advantages of the present invention include:
(a) a novel murine monoclonal antibody targeting human BCMA was developed.
(b) A humanized antibody sequence was developed that targets a human BCMA monoclonal antibody.
(c) Compared with the drug constructed by the murine antibody sequence, the bispecific antibody, or CAR-T cell, or antibody coupling drug constructed by the humanized antibody sequence developed by the invention can reduce the rejection reaction of the drug in vivo and further improve the curative effect.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
General materials and methods:
1. the main reagents used in the examples are as follows:
PBS(Gibco,CAT#14190-250)
DMEM(Gibco,CAT#41965-062)
F12K(Gibco,CAT#21127030)
FBS(Gibco,CAT#10099-141)
genome DNA purification kit (Thermo, CAT # K0512)
T4 DNA Ligase(Takara,CAT#2011B)
Recombinant human BCMA protein (His Tag) (prepared by iCarTab)
Recombinant human BCMA protein (Fc Tag) (prepared by iCarTab)
PrimeScriptTM 1st Strand cDNA Synthesis kit (Takara, Cat #6110A) Trizol RNA extraction reagent (Thermo, CAT #15596018)
Q5 PCR cloning kit (NEB, Cat # E0555S)
AxyPrep DNA gel recovery kit (Axygen, Cat # AP-GX-250G)
BamHI restriction enzyme (NEB, CAT # R3136M)
KpnI restriction enzyme (NEB, CAT # R3142M)
TOP10 competent (prepared by iCarTab)
pcDNA3.1-IgG1Fc vector (prepared by iCarTab)
pcDNA3.1-IgKc vector (prepared from iCarTab)
PE-Anti-human IgG (Biolegend, CAT #409304)
PE-Anti-murine IgG (Multisciences, CAT #70-GAM0041)
FreeStyleTM293 expression Medium (Thermo, CAT #12338018)
LVTransm transfection reagent (iCarTab, Cat # LVTrans 100)
Buffer solution: HBS-EP +10X (GE Cat # BR100669)
Amino coupling kit (GE, Cat # BR100050)
10mM Glycine 2.5(GE,Cat#BR100356)
S series CM5 chip (GE Cat #29149603)
2. The main equipment used in the examples is as follows:
ordinary optical inverted microscope
Desktop centrifuge Thermo ST41
High-speed centrifuge Thermo RC6+
Biological safety cabinet
Roche480 fluorescent quantitative PCR instrument
Thermo Attune Nxt flow cytometer
Thermo 3111 CO2 incubator
BiaCore T200
3. The main methods involved in the examples are as follows:
3.1 hybridoma selection
3.1.1 mouse immunization
The mouse immunization protocol is shown in FIG. 1.
All mice were housed in a barrier system using sterilized pelleted feed and autoclaved drinking water. 5 Balb/c mice (SPF grade) were labeled with ear tags and immunized with purified BCMA-Fc recombinant protein according to the immunization protocol described above.
3.1.2 detection of the immune potency
1) Taking out the mouse from the cage, disinfecting the tail of the mouse by using a 75% medical alcohol cotton ball, and pricking a small wound on the tail of the mouse by using a 5mm blood taking needle;
2) collecting blood drops (100 uL of plasma is required to be prepared) by using a capillary glass blood collection tube;
3) after blood collection, the mice are returned to the cage for slight observation after the blood collection point is slightly pressed by a dry sterile cotton ball to stop bleeding;
4) placing the centrifuge tube with the collected blood sample in an incubator at 37 ℃ for 1 hour; the blood samples were then transferred to 4 ℃ overnight.
5) Separating serum from blood clot, transferring into a new sterile centrifuge tube, centrifuging at 4 deg.C and 10000Xg for 10 min;
6) serum was transferred to a new sterile centrifuge tube and the immunotiter was measured by FACS.
3.1.3 FACS detection
1. CHO-K1 null cells and CHO-K1-BCMA recombinant cell lines were recovered from liquid nitrogen and the cell status was adjusted to logarithmic growth phase using F12K, 10% FBS complete medium.
2. Dividing two kinds of cells into several parts, each part having a number of 5 × 105The diluted mouse serum (1:1000 dilution) was added to each cell, mixed well and incubated at room temperature for 1 hour.
Centrifuging at 3.800Xg for 5 minutes at room temperature, removing the supernatant containing the antibody, and washing the cells 3 times with PBS;
4. adding 1uL PE-labeled Anti-mouse IgG, fully and uniformly mixing, and incubating for 30min at room temperature in a dark place;
5.800Xg at room temperature for 5 minutes, remove the secondary antibody containing supernatant, use PBS washing cells 3 times;
6. the cells were resuspended using 500uL PBS and flow analyzed.
3.1.4 hybridoma fusions
1) Taking the mouse with the best immune titer detection, dislocating and killing the neck of the mouse, obtaining the spleen of the mouse under the aseptic condition, preparing B cell single cell suspension, mixing the B cell single cell suspension with non-antibody secretory SP2/0 myeloma cells according to the ratio of 1:1, and carrying out cell fusion by using a Bio-rad Gene Pulser electroporation system.
2) Immediately after electrofusion, all cells were suspended in complete media (DMEM, 20% FBS and HAT) and seeded into 96-well plates.
3) The culture medium was changed to HT medium about 10 days after the fusion, and after two days, the supernatant was taken to detect the specific antibody.
3.1.5 hybridoma selection
120uL of the culture supernatant was removed from each well of the 96-well plate while each well was supplemented with fresh HT medium. The culture supernatant was incubated with ELISA plates precoated with the antigen of interest and identified according to standard ELISA procedures. Wells with higher o.d values were selected for the second and third rounds of subcloning. After each round of subcloning, ELISA identification was performed according to the same procedure until a single clone was formed.
3.1.6 ELISA assays
1) The BCMA-His recombinant protein was diluted with sterile PBS to a final concentration of 1 ug/mL. A new 96-well plate was added and coated overnight at 4 ℃ with 100 uL/well.
2) Antigen coating was removed and washed 3 times with PBST (containing 0.5% tween).
3) Adding 200 uL/hole of 3% BSA at 37 ℃ for blocking for 2 hours;
4) after removal of the blocking buffer, the well plates were washed 3 times with PBST;
5) adding 100ul hybridoma supernatant, incubating at room temperature for 1 hour, and taking PBS as a control hole;
6) remove the liquid in the wells and wash 3 times with PBST;
7) adding 100uL HRP-mouse IgG (diluted 1: 10000), and incubating for 1 hour at room temperature;
8) after removing the liquid in the wells, the well plates were washed 3 times with PBST;
9) adding 100 uL/hole TMB color development liquid;
10) incubating for 15 minutes at room temperature in dark;
11) adding 50 uL/hole stop solution;
12) o.d values in the wells were read using a microplate reader.
3.1.7 hybridoma clone flow assay
1) The CHO-K1-BCMA cell line was recovered from liquid nitrogen and the cell status was adjusted to logarithmic growth phase using F12K, 10% FBS complete medium.
2) The CHO-K1-BCMA cell line is divided into several parts, and the number of each part of cells is 5 multiplied by 105Each cell was incubated with 100uL of hybridoma supernatant for 1 hour at room temperature after mixing well.
3) After centrifugation at 800Xg for 5 minutes at room temperature, the supernatant containing the antibody was removed, and the cells were washed 3 times with PBS.
4) Adding 0.5uL PE-labeled Anti-mouse IgG, fully and uniformly mixing, and incubating for 30 minutes at room temperature in a dark place;
5) centrifuging at 800Xg for 5 minutes at room temperature, removing the supernatant containing the secondary antibody, and washing the cells 3 times with PBS;
6) the cells were resuspended using 500uL PBS and flow analyzed.
3.2 sequencing of hybridomas
1) Take 5x106The resulting hybridoma cells were finally selected, lysed using Trizol and total RNA extracted from the hybridoma cells according to standard methods.
2) After the total RNA is reversely transcribed into a cDNA sample by using a reverse transcription kit, a hybridoma sequencing primer is used for amplifying heavy chain and light chain variable regions of an antibody by adopting a PCR method, TA cloning is carried out, and a fragment obtained by the PCR is subcloned into a pMD-19T vector. After blue white screening, 10 clones were picked for sequencing for each strand. And analyzing the finally obtained antibody sequence.
3.3 murine anti-humanization
3.3.1 humanized antibody sequence design
According to the amino acid sequence information of the heavy chain and the light chain of the target antibody obtained by the sequencing, carrying out humanized design, keeping the CDR region sequence of the original antibody unchanged, respectively selecting different humanized antibody templates for the heavy chain and the light chain according to the germline al-alignment result and the antibody structure simulation result, and carrying out back mutation on the humanized framework region to design a candidate humanized antibody sequence.
3.3.2 humanized antibody Gene Synthesis and expression vector construction
The heavy chain and the light chain of the humanized antibody designed above were subjected to gene synthesis, respectively, and the heavy chain was subcloned into pcDNA3.1-IgG1Fc expression vector and the light chain was subcloned into pcDNA3.1-IgKc expression vector (the schematic vector diagram is shown in FIG. 2). After the vector was verified to be free of errors by sequencing, endotoxin-free plasmids were prepared using a Qiagen plasmid macrodrawer.
3.3.3 humanized antibody expression and purification
1. And taking the LVTransm transfection reagent and the antibody expression vector out of the refrigerator, thawing at room temperature, and blowing and beating the LVTransm transfection reagent and the antibody expression vector up and down by using a liquid transfer gun to be completely and uniformly mixed. Remove PBS or HBSS buffer and warm to room temperature. 2mL of PBS is taken to be put into one hole of a 6-hole plate, 2 mu g of pcDNA3.1-IgG1Fc and 2 mu g of pcDNA3.1-IgKc are respectively added, a liquid transfer gun is blown up and down to be fully mixed, 12 mu L of LVTransm is added, a liquid transfer gun is immediately blown up and down to be mixed, and the mixture is kept stand for 10 minutes at room temperature.
2. The DNA/LVTransm complex was added to 1.5mL of 293F-SVP16 cells and mixed well by gentle shaking. The cells were incubated at 37 ℃ with 5% CO2Culturing in an incubator at 130RPM for 6-8 hours, adding 1.5mL of fresh FreeStyleTM293 expression medium, and the cells are returned to the incubator for further culture.
3. After continuous culture for 3 days, the culture medium supernatant was collected by centrifugation, filtered through a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube for flow assay.
3.3.4 flow assay of binding of humanized antibodies to target proteins
1. CHO-K1 null cells and CHO-K1-BCMA recombinant cell lines were recovered from liquid nitrogen and the cell status was adjusted to logarithmic growth phase using F12K, 10% FBS complete medium.
2. Dividing two kinds of cells into several parts, each part having a number of 5 × 105And respectively adding humanized antibodies into each cell, wherein each antibody needs to be incubated with CHO-K1 empty cells and CHO-K1-BCMA recombinant cell strains, fully mixing the mixture, and then incubating the mixture at room temperature for 1 hour.
Centrifugation at 3.800Xg for 5 minutes at room temperature, removal of antibody-containing supernatant, washing cells 3 times with PBS;
4. adding 2uL of Anti-human IgG labeled by PE, fully and uniformly mixing, and incubating for 30min at room temperature in a dark place;
5.800Xg at room temperature for 5 minutes, remove the secondary antibody containing supernatant, use PBS washing cells 3 times;
6. the cells were resuspended using 500uL PBS and flow analyzed.
3.3.5 humanized antibody affinity assays
The BCMA-Fc recombinant protein was immobilized on a CM5 chip using 10mM acetic acid coupling buffer, and the binding ability of the antibody to the target protein BCMA before and after humanization was examined using the positive flow-identified humanized antibody and human-mouse chimeric antibody prepared above as mobile phases.
Example 1 mouse immunoTiter FACS assay
Mice were immunized as described above, and sera were isolated from the tail vein of all immunized mice, diluted 1:1000, and FACS-detected with CHO-K1 and CHO-K1-BCMA cells, respectively.
Results of FACS detection are shown in fig. 3, and based on the experimental results, 29 mice were selected for shock immunization and subsequent hybridoma fusion.
Example 2 fusion plate ELISA assay
100uL of supernatant was taken from the fused well plate and ELISA was performed using a 96-well plate precoated with the target antigen.
The ELISA assay results for the fusion clones are shown in FIG. 4.
According to the ELISA detection result of the fusion clone, selecting the clone with high OD value and better growth and CHO-K1-BCMA cell strain to perform FACS verification and first subcloning.
The results of FACS detection of the fused clones are shown in FIG. 5,
EXAMPLE 3 first subcloning
100uL of supernatant was taken from the well plate after the first subcloning, and ELISA detection was performed using a 96-well plate precoated with the target antigen.
The results of the ELISA assay for the first subclone are shown in FIG. 6.
According to the result of the first subclone ELISA detection, the clone with high OD value and better growth is selected to be subjected to FACS verification and second subcloning with a CHO-K1-BCMA cell strain.
The results of FACS measurements are shown in FIG. 7.
Example 4 second subcloning
100uL of supernatant was taken from the well plate after the second subcloning, and ELISA detection was performed using a 96-well plate precoated with the target antigen.
The results of the second subclone ELISA test are shown in FIG. 8.
Example 5 sequencing of hybridomas
The clone (1-D2-C8-E5, named B1) with good FACS identification result is selected and amplified, the clone is cracked by TRIZOL reagent, RNA is extracted and is reversely transcribed into cDNA, then a hybridoma sequencing primer is used for sequencing the heavy chain and light chain variable regions corresponding to the hybridoma antibody, and the CDR region and the Framework region are analyzed, and the result is as follows.
Heavy chain amino acid sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
EVQLQQSGPGLVKPSQSLSLTCTVSGYSITSDYVWNWIRQFPGNKLEWMAYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATFYCAVYNYDGIFAYWGQGTLVTVSA(SEQ ID NO.:7)
Wherein the underlined amino acid sequences are CDR regions, including:
VH CDR1：SDYVWN(SEQ ID NO.:1)
VH CDR2：YISYSGSTSYNPSLKS(SEQ ID NO.:2)
VH CDR3：YNYDGIFAY(SEQ ID NO.:3)
light chain amino acid sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
DVQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLKICT(SEQ ID NO.:8)
Wherein the underlined amino acid sequences are CDR regions, including:
VL CDR1：RASQDISNYLN(SEQ ID NO.:4)
VL CDR2：YTSRLHS(SEQ ID NO.:5)
VL CDR3：QQGNTLPWT(SEQ ID NO.:6)
example 6 construction of chimeric antibody expression vector and FACS verification
And constructing a chimeric expression vector by using the sequence of the antibody obtained by sequencing, and verifying the expressed chimeric antibody. The heavy chain variable region obtained by hybridoma sequencing is subcloned into pcDNA3.1-IgG1Fc expression vector, and the light chain variable region is subcloned into pcDNA3.1-IgKc expression vector. And combining the constructed light chain expression vector and the constructed heavy chain expression vector in pairs respectively. 293F cells were transiently co-transfected and 48 hours after transfection, the expressed supernatant was collected for FACS detection.
The results of FACS measurements are shown in FIG. 9.
Example 7 flow cytometry of humanized antibodies
The humanized antibody sequences were designed according to the general procedures described above, and a total of 6 humanized heavy chain variable regions and 6 humanized light chain variable regions were designed.
Wherein, the nucleic acid sequence information of the humanized candidate antibody is as follows:
>H1
CAGGTTCAGCTGCAAGAGTCTGGCCCTGGCCTGGTCAAGCCTAGCCAAACACTGAGCCTGACCTGTACCGTGTCCGGCTACAGCATCACCAGCGACTACGTGTGGAACTGGATCAGACAGCCTCCTGGCAAAGGCCTGGAATGGATCGGCTACATCAGCTACAGCGGCAGCACCAGCTACAACCCCAGCCTGAAGTCCAGAGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCTCCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACAGCCGTGTACTACTGCGCCAGATACAACTACGACGGCATCTTCGCCTATTGGGGCCAGGGCACACTGGTCACAGTTAGC(SEQ ID NO.:21)
>H2
CAGGTTCAGCTGCAAGAGTCTGGCCCTGGCCTGGTCAAGCCTAGCCAAACACTGAGCCTGACCTGTACCGTGTCCGGCTACAGCATCACCAGCGACTACGTGTGGAACTGGATCAGACAGCCTCCTGGCAAAGGCCTGGAATGGATCGGCTACATCAGCTACAGCGGCAGCACCAGCTACAACCCCAGCCTGAAGTCCAGAGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCTCCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACAGCCGTGTACTACTGCGCCGTGTACAACTACGACGGCATCTTCGCCTATTGGGGCCAGGGCACACTGGTCACAGTTAGC(SEQ ID NO.:22)
>H3
CAGGTTCAGCTGCAAGAGTCTGGCCCTGGCCTGGTCAAGCCTAGCGAAACACTGAGCCTGACCTGTACCGTGTCCGGCTACAGCATCACCAGCGACTACGTGTGGAACTGGATCAGACAGCCTCCTGGCAAAGGCCTGGAATGGATCGGCTACATCAGCTACAGCGGCAGCACCAGCTACAACCCCAGCCTGAAGTCCAGAGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCTCCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACAGCCGTGTACTACTGCGCCAGATACAACTACGACGGCATCTTCGCCTATTGGGGCCAGGGCACACTGGTCACAGTTAGC(SEQ ID NO.:23)
>H4
CAGGTTCAGCTGCAAGAGTCTGGCCCTGGCCTGGTCAAGCCTAGCGAAACACTGAGCCTGACCTGTACCGTGTCCGGCTACAGCATCACCAGCGACTACGTGTGGAACTGGATCAGACAGCCTCCTGGCAAAGGCCTGGAATGGATCGGCTACATCAGCTACAGCGGCAGCACCAGCTACAACCCCAGCCTGAAGTCCAGAGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCTCCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACAGCCGTGTACTACTGCGCCGTGTACAACTACGACGGCATCTTCGCCTATTGGGGCCAGGGCACACTGGTCACAGTTAGC(SEQ ID NO.:24)
>H5
CAGATCACCCTGAAAGAGTCTGGCCCCACACTGGTCAAGCCCACACAGACCCTGACACTGACCTGCACCTTTAGCGGCTACAGCATCACCAGCGACTACGTGTGGAACTGGATCAGACAGCCTCCTGGCAAGGCCCTGGAATGGCTGGCCTACATCAGCTACAGCGGCAGCACCAGCTACAACCCCAGCCTGAAAAGCCGGCTGACCATCACCAAGGACACCAGCAAGAACCAGGTGGTGCTGACCATGACAAACATGGACCCCGTGGACACCGCCACCTACTACTGCGCCCACTACAACTACGACGGCATCTTCGCCTATTGGGGCCAGGGCACACTCGTGACAGTTAGC(SEQ ID NO.:25)
>H6
CAGATCACCCTGAAAGAGTCTGGCCCCACACTGGTCAAGCCCACACAGACCCTGACACTGACCTGCACCTTTAGCGGCTACAGCATCACCAGCGACTACGTGTGGAACTGGATCAGACAGCCTCCTGGCAAGGCCCTGGAATGGCTGGCCTACATCAGCTACAGCGGCAGCACCAGCTACAACCCCAGCCTGAAAAGCCGGCTGACCATCACCAAGGACACCAGCAAGAACCAGGTGGTGCTGACCATGACAAACATGGACCCCGTGGACACCGCCACCTACTACTGCGCCGTGTACAACTACGACGGCATCTTCGCCTATTGGGGCCAGGGCACACTCGTGACAGTTAGC(SEQ ID NO.:26)
>L1
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGGACATCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACTACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCTTCACCATATCTAGCCTGCAGCCTGAGGATATCGCCACCTACTACTGCCAGCAGGGCAACACCCTGCCTTGGACATTTGGCGGCGGAACAAAGGTG(SEQ ID NO.:27)
>L2
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGGACATCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACTACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCTTTACAATCAGCAGCCTGCAGCAAGAGGATATCGCCACCTACTACTGCCAGCAGGGCAACACCCTGCCTTGGACATTTGGCGGCGGAACAAAGGTG(SEQ ID NO.:28)
>L3
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGGACATCAGCAACTACCTGAACTGGTATCAGCAGAAACCCGGCAAGGTGCCCAAGCTGCTGATCTACTACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACGTGGCCACCTACTATTGCCAGCAGGGCAATACCCTGCCTTGGACCTTTGGCGGCGGAACAAAACTG(SEQ ID NO.:29)
>L4
GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGGACATCAGCAACTACCTGAACTGGTATCAGCAGAAACCTGGCGGCGTGCCCAAGCTGCTGATCTACTACACAAGCAGACTGCACAGCGGAGTGCCCAGCAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACGTGGCCACCTACTATTGCCAGCAGGGCAATACCCTGCCTTGGACCTTTGGCGGCGGAACAAAACTG(SEQ ID NO.:30)
>L5
GACATCCAGATGACACAGAGCCCTAGCAGCGTGTCCGCCTCTGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGGACATCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACTACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTCGCCACCTACTATTGCCAGCAGGGCAACACCCTGCCTTGGACATTTGGCCAGGGCACCAAACTG(SEQ ID NO.:31)
>L6
GACATCCAGATGACACAGAGCCCTAGCAGCGTGTCCGCCTCTGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCCAGGACATCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACTACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCCTGACAATCTCTAGCCTGCAGCAAGAGGACTTCGCCACCTACTACTGCCAGCAGGGCAATACCCTGCCTTGGACATTTGGCCAGGGCACCAAACTG(SEQ ID NO.:32)
humanized candidate antibody amino acid sequence information:
>H1
QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDYVWNWIRQPPGKGLEWIGYISYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARYNYDGIFAYWGQGTLVTVS(SEQ ID NO.:9)
>H2
QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDYVWNWIRQPPGKGLEWIGYISYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAVYNYDGIFAYWGQGTLVTVS(SEQ ID NO.:10)
>H3
QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYVWNWIRQPPGKGLEWIGYISYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARYNYDGIFAYWGQGTLVTVS(SEQ ID NO.:11)
>H4
QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYVWNWIRQPPGKGLEWIGYISYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAVYNYDGIFAYWGQGTLVTVS(SEQ ID NO.:12)
>H5
QITLKESGPTLVKPTQTLTLTCTFSGYSITSDYVWNWIRQPPGKALEWLAYISYSGSTSYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCAHYNYDGIFAYWGQGTLVTVS(SEQ ID NO.:13)
>H6
QITLKESGPTLVKPTQTLTLTCTFSGYSITSDYVWNWIRQPPGKALEWLAYISYSGSTSYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCAVYNYDGIFAYWGQGTLVTVS(SEQ ID NO.:14)
>L1
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGGGTKV(SEQ ID NO.:15)
>L2
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQQEDIATYYCQQGNTLPWTFGGGTKV(SEQ ID NO.:16)
>L3
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKL(SEQ ID NO.:17)
>L4
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGVPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGNTLPWTFGGGTKL(SEQ ID NO.:18)
>L5
DIQMTQSPSSVSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKL(SEQ ID NO.:19)
>L6
DIQMTQSPSSVSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQQEDFATYYCQQGNTLPWTFGQGTKL(SEQ ID NO.:20)
after the humanized antibody expression vector is transiently transfected into 293F cells, culture medium supernatant is collected, and the binding condition of the humanized antibody and the antigen on the cell membrane surface of a recombinant cell CHO-K1-BCMA is detected by a flow cytometer. As a control, a human murine chimeric antibody constructed from the original murine antibodies VH and VL was used.
As shown in FIG. 10, the results of FACS detection show that the BCMA humanized antibodies were bound to recombinant CHO-K1-BCMA cells. The purified antibodies expressed in combination B1H2-B1L1 and B1H4-B1L2 were selected for subsequent affinity testing.
The SDS-PAGE results of humanized antibodies B1H2-B1L1 and B1H4-B1L2 are shown in FIG. 11.
The sequences before and after antibody humanization were compared, and the results of B1H2-B1L1 alignment are shown in FIG. 12, and the results of B1H4-B1L2 alignment are shown in FIG. 13.
Example 8 humanized antibody affinity assay
8.1 Experimental materials and reagents according to this example
S series CM5 chip: manufacturer GE, Cat number 29149603
Buffer solution: HBS-EP +10X, manufacturer GE, cat # BR 100669. Diluted 10-fold with deionized water before use.
Amino coupling kit: GE, cat # BR100050
Regeneration reagent: 10mM Glycine 2.5, manufacturer GE, cat # BR100356
8.2 ligand coupling Pre-enrichment
Ligand: BCMA recombinant proteins
Test pH: 10mM Acetate 5.5/5.0/4.5/4.0
Flow rate: 10 mul/min
Buffer solution: HBS-EP + buffer
Results are shown in 14, with 10mM Acetate 5.5 selected as the coupling buffer.
8.3 ligand coupling
Ligand: BCMA recombinant proteins
Coupling buffer: 10mM Acetate 5.5
Target coupling amount: 200RU
The result is shown in FIG. 15, the final coupling amount is 205.7 RU.
Affinity assay of MH18100901-B1Chimeric, MH18100901-B1H2-B1L1, MH18100901-B1H4-B1L2 and BCMA recombinant protein
The experimental conditions are as follows:
antibody concentration: 0.15625/0.3125/0.625/1.25/2.5/5.0ug/ml
Flow rate: 30 mul/min
Binding time: 300s
Dissociation time: 400s
Regeneration reagent: glycine 1.5, 30S, 2 times
The results of the affinity assay for MH18100901-B1Chimeric are shown in FIG. 16, the results of the affinity assay for MH18100901-B1H2-B1L1 are shown in FIG. 17, and the results of the affinity assay for MH18100901-B1H4-B1L2 are shown in FIG. 18.
The results of the affinity detection of each antibody are shown in table 1.
TABLE 1
Ka(M-1s-1) Kd(s-1) KD(M)
MH18100901-B1 Chimeric 3.942×105 1.736×10-4 4.404×10-10
MH18100901-B1H2-B1L1 4.649×105 7.363×10-5 1.584×10-10
MH18100901-B1H4-B1L2 2.681×105 8.436×10-5 3.147×10-10
The affinity detection result shows that the affinity of the humanized antibodies MH18100901-B1H2-B1L1 and MH18100901-B1H4-B1L2 is consistent with that of the chimeric antibody.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Bosheng Ji pharmaceutical technology (Suzhou) Co., Ltd
<120> monoclonal antibody targeting human BCMA and application thereof
<130> P2020-0532
<160> 32
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213> mouse (Mus musculus)
<400> 1
Ser Asp Tyr Val Trp Asn
1 5
<210> 2
<211> 16
<212> PRT
<213> mouse (Mus musculus)
<400> 2
Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys Ser
1 5 10 15
<210> 3
<211> 9
<212> PRT
<213> mouse (Mus musculus)
<400> 3
Tyr Asn Tyr Asp Gly Ile Phe Ala Tyr
1 5
<210> 4
<211> 11
<212> PRT
<213> mouse (Mus musculus)
<400> 4
Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn
1 5 10
<210> 5
<211> 7
<212> PRT
<213> mouse (Mus musculus)
<400> 5
Tyr Thr Ser Arg Leu His Ser
1 5
<210> 6
<211> 9
<212> PRT
<213> mouse (Mus musculus)
<400> 6
Gln Gln Gly Asn Thr Leu Pro Trp Thr
1 5
<210> 7
<211> 118
<212> PRT
<213> mouse (Mus musculus)
<400> 7
Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Val Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45
Met Ala Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Phe Tyr Cys
85 90 95
Ala Val Tyr Asn Tyr Asp Gly Ile Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
<210> 8
<211> 108
<212> PRT
<213> mouse (Mus musculus)
<400> 8
Asp Val Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Lys Ile Cys Thr
100 105
<210> 9
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Val Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Asn Tyr Asp Gly Ile Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser
115
<210> 10
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Val Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Tyr Asn Tyr Asp Gly Ile Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser
115
<210> 11
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Val Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Asn Tyr Asp Gly Ile Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser
115
<210> 12
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Val Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Tyr Asn Tyr Asp Gly Ile Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser
115
<210> 13
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Val Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp
35 40 45
Leu Ala Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val Val
65 70 75 80
Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala His Tyr Asn Tyr Asp Gly Ile Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser
115
<210> 14
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Val Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp
35 40 45
Leu Ala Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val Val
65 70 75 80
Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Val Tyr Asn Tyr Asp Gly Ile Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser
115
<210> 15
<211> 104
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val
100
<210> 16
<211> 104
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val
100
<210> 17
<211> 104
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu
100
<210> 18
<211> 104
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Val Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu
100
<210> 19
<211> 104
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu
100
<210> 20
<211> 104
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Gln
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu
100
<210> 21
<211> 351
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagccaaac actgagcctg 60
acctgtaccg tgtccggcta cagcatcacc agcgactacg tgtggaactg gatcagacag 120
cctcctggca aaggcctgga atggatcggc tacatcagct acagcggcag caccagctac 180
aaccccagcc tgaagtccag agtgaccatc agcgtggaca ccagcaagaa ccagttctcc 240
ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgcgc cagatacaac 300
tacgacggca tcttcgccta ttggggccag ggcacactgg tcacagttag c 351
<210> 22
<211> 351
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagccaaac actgagcctg 60
acctgtaccg tgtccggcta cagcatcacc agcgactacg tgtggaactg gatcagacag 120
cctcctggca aaggcctgga atggatcggc tacatcagct acagcggcag caccagctac 180
aaccccagcc tgaagtccag agtgaccatc agcgtggaca ccagcaagaa ccagttctcc 240
ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgcgc cgtgtacaac 300
tacgacggca tcttcgccta ttggggccag ggcacactgg tcacagttag c 351
<210> 23
<211> 351
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagcgaaac actgagcctg 60
acctgtaccg tgtccggcta cagcatcacc agcgactacg tgtggaactg gatcagacag 120
cctcctggca aaggcctgga atggatcggc tacatcagct acagcggcag caccagctac 180
aaccccagcc tgaagtccag agtgaccatc agcgtggaca ccagcaagaa ccagttctcc 240
ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgcgc cagatacaac 300
tacgacggca tcttcgccta ttggggccag ggcacactgg tcacagttag c 351
<210> 24
<211> 351
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagcgaaac actgagcctg 60
acctgtaccg tgtccggcta cagcatcacc agcgactacg tgtggaactg gatcagacag 120
cctcctggca aaggcctgga atggatcggc tacatcagct acagcggcag caccagctac 180
aaccccagcc tgaagtccag agtgaccatc agcgtggaca ccagcaagaa ccagttctcc 240
ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgcgc cgtgtacaac 300
tacgacggca tcttcgccta ttggggccag ggcacactgg tcacagttag c 351
<210> 25
<211> 351
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
cagatcaccc tgaaagagtc tggccccaca ctggtcaagc ccacacagac cctgacactg 60
acctgcacct ttagcggcta cagcatcacc agcgactacg tgtggaactg gatcagacag 120
cctcctggca aggccctgga atggctggcc tacatcagct acagcggcag caccagctac 180
aaccccagcc tgaaaagccg gctgaccatc accaaggaca ccagcaagaa ccaggtggtg 240
ctgaccatga caaacatgga ccccgtggac accgccacct actactgcgc ccactacaac 300
tacgacggca tcttcgccta ttggggccag ggcacactcg tgacagttag c 351
<210> 26
<211> 351
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
cagatcaccc tgaaagagtc tggccccaca ctggtcaagc ccacacagac cctgacactg 60
acctgcacct ttagcggcta cagcatcacc agcgactacg tgtggaactg gatcagacag 120
cctcctggca aggccctgga atggctggcc tacatcagct acagcggcag caccagctac 180
aaccccagcc tgaaaagccg gctgaccatc accaaggaca ccagcaagaa ccaggtggtg 240
ctgaccatga caaacatgga ccccgtggac accgccacct actactgcgc cgtgtacaac 300
tacgacggca tcttcgccta ttggggccag ggcacactcg tgacagttag c 351
<210> 27
<211> 312
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60
atcacctgta gagccagcca ggacatcagc aactacctga actggtatca gcagaagccc 120
ggcaaggccc ctaagctgct gatctactac accagcagac tgcacagcgg cgtgcccagc 180
agattttctg gctctggcag cggcaccgac ttcaccttca ccatatctag cctgcagcct 240
gaggatatcg ccacctacta ctgccagcag ggcaacaccc tgccttggac atttggcggc 300
ggaacaaagg tg 312
<210> 28
<211> 312
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60
atcacctgta gagccagcca ggacatcagc aactacctga actggtatca gcagaagccc 120
ggcaaggccc ctaagctgct gatctactac accagcagac tgcacagcgg cgtgcccagc 180
agattttctg gctctggcag cggcaccgac ttcaccttta caatcagcag cctgcagcaa 240
gaggatatcg ccacctacta ctgccagcag ggcaacaccc tgccttggac atttggcggc 300
ggaacaaagg tg 312
<210> 29
<211> 312
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60
atcacctgta gagccagcca ggacatcagc aactacctga actggtatca gcagaaaccc 120
ggcaaggtgc ccaagctgct gatctactac accagcagac tgcacagcgg cgtgcccagc 180
agattttctg gctctggcag cggcaccgac ttcaccctga ccatatctag cctgcagcct 240
gaggacgtgg ccacctacta ttgccagcag ggcaataccc tgccttggac ctttggcggc 300
ggaacaaaac tg 312
<210> 30
<211> 312
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60
atcacctgta gagccagcca ggacatcagc aactacctga actggtatca gcagaaacct 120
ggcggcgtgc ccaagctgct gatctactac acaagcagac tgcacagcgg agtgcccagc 180
agattttctg gcagcggctc tggcaccgac ttcaccctga ccatatctag cctgcagcct 240
gaggacgtgg ccacctacta ttgccagcag ggcaataccc tgccttggac ctttggcggc 300
ggaacaaaac tg 312
<210> 31
<211> 312
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
gacatccaga tgacacagag ccctagcagc gtgtccgcct ctgtgggaga cagagtgacc 60
atcacctgta gagccagcca ggacatcagc aactacctga actggtatca gcagaagccc 120
ggcaaggccc ctaagctgct gatctactac accagcagac tgcacagcgg cgtgcccagc 180
agattttctg gctctggcag cggcaccgac ttcaccctga ccatatctag cctgcagcct 240
gaggacttcg ccacctacta ttgccagcag ggcaacaccc tgccttggac atttggccag 300
ggcaccaaac tg 312
<210> 32
<211> 312
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gacatccaga tgacacagag ccctagcagc gtgtccgcct ctgtgggaga cagagtgacc 60
atcacctgta gagccagcca ggacatcagc aactacctga actggtatca gcagaagccc 120
ggcaaggccc ctaagctgct gatctactac accagcagac tgcacagcgg cgtgcccagc 180
agattttctg gctctggcag cggcaccgac ttcaccctga caatctctag cctgcagcaa 240
gaggacttcg ccacctacta ctgccagcag ggcaataccc tgccttggac atttggccag 300
ggcaccaaac tg 312

Claims (10)

1. An antibody heavy chain variable region comprising the following three Complementarity Determining Regions (CDRs):
CDR1 shown in SEQ ID NO. 1,
CDR2 shown in SEQ ID NO. 2, and
CDR3 shown in SEQ ID NO. 3.
2. An antibody heavy chain having the heavy chain variable region of claim 1.
3. An antibody light chain variable region comprising the following three Complementarity Determining Regions (CDRs):
CDR 1' shown in SEQ ID NO. 4,
CDR 2' of SEQ ID NO. 5, and
CDR 3' shown in SEQ ID NO. 6.
4. An antibody light chain having the variable light chain region of claim 3.
5. An antibody targeting BCMA, wherein said antibody has:
(1) the heavy chain variable region of claim 1; and/or
(2) The light chain variable region of claim 3;
alternatively, the antibody has: the heavy chain of claim 2; and/or the light chain of claim 4.
6. The antibody of claim 5, wherein said antibody is selected from the group consisting of: an antibody of animal origin, a chimeric antibody, a humanized antibody, or a combination thereof.
7. The antibody of claim 5, wherein the heavy chain variable region sequence of said antibody is as set forth in SEQ ID NO: 7. 9-14; and/or
The light chain variable region sequence of the antibody is shown as SEQ ID NO: 8. 15-20.
8. A CAR construct wherein the scFV segment of the monoclonal antibody antigen binding region of the CAR construct is a binding region that specifically binds to BCMA, and wherein the scFV has the heavy chain variable region of claim 1 and the light chain variable region of claim 3.
9. A recombinant immune cell expressing an exogenous CAR construct of claim 8.
10. Use of an active ingredient selected from the group consisting of: the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5, the immune cell of claim 9, or a combination thereof, wherein the active ingredient is used (a) in the preparation of a detection reagent or kit; and/or (b) preparing a medicament for preventing and/or treating BCMA positive tumors.
CN202110044548.6A 2021-01-13 2021-01-13 Monoclonal antibody targeting human BCMA and application thereof Pending CN114763383A (en)

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