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

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 its application

technical field

The present invention relates to the field of biotechnology, and more particularly to a monoclonal antibody targeting human BCMA and its application.

Background technique

B-cell maturation antigen (BCMA) was first found on the surface of mature B-lymphocytes and was hardly expressed in other tissue cells. It is highly expressed in malignantly proliferating B lymphocytes (eg myeloma cells, leukemia cells). At the same time, it plays a key role in cell survival, proliferation, metastasis and drug resistance by mediating downstream signaling pathways. These properties make it a target for immunotherapy, especially for the treatment of multiple myeloma. BCMA, due to its limited expression in plasma cells and not in naive and memory B cells, has been used as a new drug target for the diagnosis and treatment of multiple myeloma. At present, the new tumor immunotherapy methods for BCMA mainly include CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) therapy, bispecific antibody (Bispecific antibody, BsAb) and antibody-drug conjugate (Antibody-drug coupling, ADC). ). Recent studies have also shown that CAR-T targeting BCMA can effectively eliminate myeloma cells in vivo.

At present, there are still many deficiencies in the research on BCMA antibodies, and the field needs to develop new BCMA antibodies and related applications.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a monoclonal antibody targeting human BCMA and its application.

The purpose of the present invention is also to provide a BCMA humanized antibody, and further target the treatment of multiple myeloma by constructing a CAR-T or bispecific antibody or antibody conjugated drug targeting BCMA.

A first aspect of the present invention provides a heavy chain variable region of an antibody, the heavy chain variable region comprising the following three complementarity determining region 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 also includes at least one (such as 1-3, preferably 1-2, more preferably through addition, deletion, modification and/or substitution) 1) amino acids and derived sequences capable of retaining BCMA binding affinity.

In another preferred embodiment, the heavy chain variable region further includes a human FR region or a murine FR region.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:7.

In another preferred embodiment, the heavy chain variable region has the amino acid sequences shown in SEQ ID NOs: 9-14.

The second aspect of the present invention provides a heavy chain of an antibody, wherein the heavy chain has the variable region of the heavy chain described in the first aspect of the present invention.

In another preferred embodiment, the heavy chain of the antibody further includes a heavy chain constant region.

In another preferred embodiment, the heavy chain constant region is of human origin, mouse origin or rabbit origin.

A third aspect of the present invention provides a light chain variable region of an antibody, the light chain variable region comprising the following three complementarity determining region CDRs:

CDR1' shown in SEQ ID NO:4,

CDR2' shown in SEQ ID NO: 5, and

CDR3' shown in SEQ ID NO:6.

In another preferred embodiment, any one of the above amino acid sequences also includes at least one (such as 1-3, preferably 1-2, more preferably through addition, deletion, modification and/or substitution) 1) amino acids and derived sequences capable of retaining BCMA binding affinity.

In another preferred embodiment, the light chain variable region further includes 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 sequences shown in SEQ ID NOs: 15-20.

The fourth aspect of the present invention provides a light chain of an antibody, the light chain has the light chain variable region according to the third aspect of the present invention.

In another preferred embodiment, the light chain of the antibody further includes a light chain constant region.

In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin.

A fifth aspect of the present invention provides an antibody targeting BCMA, the antibody having:

(1) the heavy chain variable region according to the first aspect of the present invention; and/or

(2) the light chain variable region as described in the third aspect of the present invention;

Alternatively, the antibody has: a heavy chain as described in the second aspect of the invention; and/or a light chain as described in the fourth aspect of the invention.

In another preferred example, the affinity constant KD(M) of the antibody binding to human BCMA protein (preferably wild type) is (1-10)×10 ^-10 , preferably (1-5)×10 ^{- 10} .

In another preferred embodiment, the antibody is selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, or a combination thereof.

In another preferred embodiment, the antibody is a diabody 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 the antibody is shown in SEQ ID NO:7; and/or the light chain variable region sequence of the antibody is shown in SEQ ID NO:8.

In another preferred embodiment, the heavy chain variable region sequence of the antibody is shown in SEQ ID NO: 10; and the light chain variable region sequence of the antibody is shown in SEQ ID NO: 15.

In another preferred embodiment, the heavy chain variable region sequence of the antibody is shown in SEQ ID NO: 12; and the light chain variable region sequence of the antibody is shown in SEQ ID NO: 16.

In another aspect of the present invention, there is provided a bispecific antibody comprising the antibody targeting BCMA according to the fifth aspect of the present invention and a second antibody, wherein the second antibody targets tumors other than BCMA antigen.

In another preferred embodiment, the tumor antigens include CD3 and CD19.

In another preferred embodiment, the second antibody is a Nanobody or 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.

The sixth aspect of the present invention provides a recombinant protein, the recombinant protein has:

(i) the variable region of the heavy chain according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, the variable region of the light chain according to the third aspect of the present invention, the variable region of the light chain according to the fourth aspect of the present invention The light chain of the aspect, or the antibody of the fifth aspect of the invention; and

(ii) Optional tag sequences to facilitate expression and/or purification.

In another preferred embodiment, the tag sequence includes a 6His tag.

In another preferred embodiment, the recombinant protein (or polypeptide) includes fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

The seventh aspect of the present invention provides 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 the scFv has the scFv segment as described in the first aspect of the present invention. A heavy chain variable region according to one aspect 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 structure of formula I:

L-scFv-H-TM-C-CD3ζ(I)

In the formula,

each "-" is independently a linking peptide or peptide bond;

L is none or a signal peptide sequence;

scFv is an scFv targeting BCMA;

H is an optional hinge region;

TM is the transmembrane domain;

C is a costimulatory signal molecule;

CD3ζ is the CD3ζ cytoplasmic signaling sequence.

The eighth aspect of the present invention provides a recombinant immune cell expressing the exogenous CAR construct according to the seventh aspect of the present invention.

In another preferred embodiment, the immune cells are selected from the group consisting of NK cells and T cells.

In another preferred embodiment, the immune cells are derived from human or non-human mammals (eg, mice).

The ninth aspect of the present invention provides an antibody-drug conjugate, the antibody-drug conjugate comprising:

(a) an antibody portion selected from the group consisting of a heavy chain variable region as described in the first aspect of the invention, a heavy chain as described in the second aspect of the invention, a heavy chain as described in the third aspect of the invention The light chain variable region of the present invention, the light chain of the fourth aspect of the present invention, or the antibody of the fifth aspect of the present invention, or a combination thereof; and

(b) a conjugation moiety conjugated to the antibody 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, the antibody moiety and the coupling moiety are coupled through chemical bonds or linkers.

The tenth aspect of the present invention provides the use of an active ingredient selected from the group consisting of the heavy chain variable region described in the first aspect of the present invention, the heavy chain variable region described in the second aspect of the present invention. chain, light chain variable region according to the third aspect of the invention, light chain according to the fourth aspect of the invention, or antibody according to the fifth aspect of the invention, recombinant according to the sixth aspect of the invention The protein, the immune cell according to the eighth aspect of the present invention, the antibody-drug conjugate according to the ninth aspect of the present invention, or a combination thereof, the active ingredient is used for (a) preparing a detection reagent or 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 hematological tumors, solid tumors, 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 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, colorectal cancer, cervical cancer , ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumor, thyroid cancer, or its combination.

In another preferred example, the tumor is gastric cancer, breast cancer, and 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) Detection of BCMA protein in the sample; and/or

(2) Detection of endogenous BCMA protein in tumor cells; and/or

(3) Detection of tumor cells expressing BCMA protein.

In another preferred embodiment, the detection reagent is a detection sheet.

The eleventh aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition contains:

(i) an active ingredient selected from the group consisting of a heavy chain variable region as described in the first aspect of the present invention, a heavy chain as described in the second aspect of the present invention, and a heavy chain as described in the third aspect of the present invention The light chain variable region of the present invention, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, or the eighth aspect of the present invention. the immune cells, the antibody drug conjugate of the ninth aspect of the present 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.

A twelfth aspect of the present invention provides a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) The heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, the light chain variable region according to the third aspect of the present invention, the fourth aspect of the present invention The light chain of the aspect, or the antibody of the fifth aspect of the invention; or

(2) the recombinant protein as described in the sixth aspect of the present invention;

(3) The CAR construct according to the seventh aspect of the present invention.

In another preferred example, the nucleotide sequences are shown in SEQ ID NOs: 21-32.

The thirteenth aspect of the present invention provides a vector, the vector containing the polynucleotide according to the twelfth aspect of the present invention.

In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

The fourteenth aspect of the present invention provides a genetically engineered host cell, wherein the host cell contains the vector according to the thirteenth aspect of the present invention or integrates the vector according to the twelfth aspect of the present invention in the genome polynucleotides.

A fifteenth aspect of the present invention provides a method for detecting BCMA protein in a sample (including diagnostic or non-diagnostic) in vitro, the method comprising the steps of:

(1) contacting the sample with the antibody according to the fifth aspect of the present invention or the antibody-drug conjugate according to the ninth aspect of the present invention in vitro;

(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of BCMA protein in the sample.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

The sixteenth aspect of the present invention provides a detection plate, the detection plate comprises: a substrate (support plate) and a test strip, the test strip contains the antibody according to the fifth aspect of the present invention or the The antibody drug conjugate of the ninth aspect of the present invention.

A seventeenth aspect of the present invention provides a kit comprising:

(1) a first container containing the antibody according to the fifth aspect of the present invention; and/or

(2) a second container containing a secondary antibody against the antibody according to the fifth aspect of the present invention;

Alternatively, the kit contains the detection plate according to the sixteenth aspect of the present invention.

An eighteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:

(a) culturing the host cell according to the fourteenth aspect of the present invention under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody according to the fifth aspect of the present invention or the recombinant protein according to the sixth aspect of the present invention.

The nineteenth aspect of the present invention provides a method for preventing and/or treating BCMA-positive tumors, the method comprising: administering to a subject in need the antibody of the fifth aspect of the present invention, the antibody of the antibody- A drug conjugate, or a CAR-T cell expressing the antibody, or a combination thereof.

In another preferred embodiment, the method further comprises: administering other drugs or therapeutic methods to the object in need for combined therapy.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Figure 1 shows the mouse immunization workflow.

Figure 2 shows the vector diagrams of the heavy chain expression vector pcDNA3.1-IgG1Fc and the light chain expression vector pcDNA3.1-IgKc.

Figure 3 shows the results of FACS detection of immunized mice.

Figure 4 shows the results of ELISA detection of hybridoma fusion plates.

Figure 5 shows the results of FACS detection of hybridoma fusion plates.

Figure 6 shows the results of the ELISA assay of the first subcloning plate.

Figure 7 shows the results of FACS detection of the first subclone.

Figure 8 shows the results of the ELISA assay of the second subcloning plate.

Figure 9 shows the results of FACS detection of chimeric antibodies.

Figure 10 shows the results of FACS detection of humanized antibodies.

Figure 11 shows the SDS-PAGE results of humanized antibodies B1H2-B1L1 and B1H4-B1L2.

Figure 12 shows the alignment results of humanized antibodies B1H2-B1L1 and murine antibodies.

Figure 13 shows the alignment results of humanized antibodies B1H4-B1L2 and murine antibodies.

Figure 14 shows ligand-conjugated pre-enrichment results.

Figure 15 shows the ligand coupling results.

Figure 16 shows the results of affinity determination of MH18100901-B1Chimeric.

Figure 17 shows the results of affinity determination of MH18100901-B1H2-B1L1.

Figure 18 shows the results of affinity determination of MH18100901-B1H4-B1L2.

Detailed ways

After extensive and in-depth research, the inventors unexpectedly discovered a BCMA-targeting antibody with high affinity and high specificity for the first time, and prepared a humanized antibody with an affinity comparable to that of a chimeric antibody. The present invention has been completed on this basis.

the term

As used herein, the terms "administer" and "treating" 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. "Administering" and "treatment" can refer to therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contact of reagents with cells, as well as contact of reagents with fluids, and contact of fluids with cells. "Administering" and "treating" also mean in vitro and ex vivo treatment by an agent, diagnostic, binding composition, or by another cell. "Treatment" when applied to a human, animal, or research subject, means therapeutic treatment, prophylactic or preventive measures, research and diagnosis; or contact with physiological fluids.

As used herein, the term "treating" refers to the administration of an internal or external therapeutic agent, comprising any one of the BCMA antibodies and compositions thereof of the invention, to a patient with one or more disease symptoms for which the therapeutic agent is known Treat these symptoms. Typically, a patient is administered to a patient in an amount of the therapeutic agent effective to alleviate one or more symptoms of the disease (therapeutically effective amount).

As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance can, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a specific sequence may, but does not necessarily have, one, two or three.

Antibody

As used herein, the term "antibody" refers to an immunoglobulin, a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or different types of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and the heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, respectively. , δ, ε, γ, and μ. IgG represents the most important class of immunoglobulins. Due to differences in chemical structure and biological function, it can be divided into 4 subclasses: IgG1, IgG2, IgG3 and IgG4. Light chains are classified into kappa or lambda chains by differences in the constant region. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those in the art.

The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region). The variable region includes 3 hypervariable regions (HVR) and 4 FR regions (FR) with relatively conserved sequences. The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions, and the sequence from the amino terminus to the carboxy terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain, namely the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2 and LCDR3; the three CDR regions of the heavy chain, namely the heavy chain hypervariable region (HCDR), refer to HCDR1, HCDR2 and HCDR3. The number and position of CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen-binding fragment of the invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3), or the numbering rules of Kabat and Chothia (HCDR1). ). The four FR regions in the variable regions of native heavy and light chains are roughly in a β-sheet configuration, connected by three CDRs that form a linking loop, and in some cases can form part of a β-sheet structure. The CDRs in each chain are brought together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody. Which amino acids make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type. The constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.

As used herein, the term "antigen-binding fragment" refers to a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a single Fv fragment having antigen-binding activity. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and are the smallest antibody fragment with all antigen-binding sites. Typically, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.

As used herein, the term "antigenic determinant" refers to a discrete three-dimensional site on an antigen that is recognized by an antibody or antigen-binding fragment of the invention.

The present invention includes not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of said antibodies.

In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by 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 a cloned secreted antibody derived from a single cell. Monoclonal antibodies are highly specific, directed against a single epitope. The cells may be eukaryotic, prokaryotic or phage clonal cell strains.

As used herein, the term "chimeric antibody" is an antibody molecule expressed by a murine antibody V region gene and a human antibody C region gene spliced into a chimeric gene, which is then inserted into a vector and transfected into a host cell. It not only retains the high specificity and affinity of the parental mouse antibody, but also enables its human Fc segment to effectively mediate biological effector functions.

As used herein, the term "humanized antibody" is a variable region engineered form of the murine antibody of the present invention having CDR regions derived from (or substantially derived from) a non-human antibody, preferably a mouse monoclonal antibody , and FR and constant regions derived substantially from human antibody sequences; that is, grafting the CDR region sequences of the murine antibody onto different types of human germline antibody framework sequences. Because the CDR sequences are responsible for most antibody-antigen interactions, expression vectors can be constructed to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies.

In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.

In the present invention, the antibody of the present invention also includes its conservative variants, which means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, most preferably Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservatively variant polypeptides are best produced by amino acid substitutions according to Table A.

Table A

initial residue representative substitution Preferred substitution 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 antibody

As used herein, the term "BCMA" generally refers to native or recombinant human BCMA, as well as non-human homologues of human BCMA.

The present invention provides a highly specific and high affinity antibody against BCMA, comprising a heavy chain and a light chain, the heavy chain containing a heavy chain variable region (VH) amino acid sequence, and the light chain containing 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:

CDR1' shown in SEQ ID NO:4,

CDR2' shown in SEQ ID NO: 5, and

CDR3' shown in SEQ ID NO:6.

Wherein, any amino acid sequence in the above-mentioned amino acid sequence also includes at least one (such as 1-5, 1-3, preferably 1-2, more preferably 1) after addition, deletion, modification and/or substitution Derivative sequences of amino acids with BCMA binding affinity.

In another preferred embodiment, the sequence formed by the addition, deletion, modification and/or substitution of at least one amino acid sequence preferably has a homology of at least 80%, preferably at least 85%, more preferably at least 90% %, optimally at least 95% of the amino acid sequence.

The antibodies of the present invention may be double-chain or single-chain antibodies, and may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully human Antibody.

The antibody derivatives of the present invention can be single-chain antibodies, and/or antibody fragments, such as: Fab, Fab', (Fab') ₂ or other known antibody derivatives in the field, etc., as well as IgA, IgD, IgE , IgG and IgM antibodies or any one or more of other subtypes of antibodies.

Among them, the animal is preferably a mammal, such as a mouse.

The antibodies of the invention may be murine antibodies, chimeric antibodies, humanized antibodies, 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, animals can be immunized with BCMA or a fragment thereof. Appropriate methods of immunization can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.

Any suitable form of BCMA (BCMA-Fc recombinant protein) can be used as an immunogen (antigen) for generating non-human antibodies specific for BCMA and screening the antibodies for biological activity. The priming immunogen can be full-length mature human BCMA, including native homodimers, or peptides containing single/multiple epitopes. The immunogens can be used alone or in combination with one or more immunogenicity enhancers known in the art. Immunogens can be purified from natural sources, or produced in genetically modified cells. The DNA encoding the immunogen can be genomic or non-genomic in origin (eg, cDNA). The DNA encoding the immunogen can 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 can 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 the IgG1 subtype is used. Optimization of the necessary constant domain sequences to produce the desired biological activity is readily accomplished 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 sequences of the DNA molecules of the antibodies or fragments thereof of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences for the light and heavy chains can be fused together to form single chain antibodies.

Once the relevant sequences have been obtained, recombinant methods can be used to obtain the relevant sequences in bulk. 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, synthetic methods can also be used to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.

The present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.

Host cells can be prokaryotic cells, such as bacterial cells; 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 of transforming host cells with recombinant DNA described in the present invention can be performed using techniques well known in the art. The obtained transformants can be cultured by conventional methods, and the transformants express the polypeptides encoded by the genes of the present invention. Depending on the host cell used, it is cultured with conventional media under appropriate conditions.

Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc. skilled in the art The antibody of the present invention can be obtained by conventional separation and purification methods 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 in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

Antibody-Drug Conjugates (ADCs)

The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

Typically, the antibody-drug conjugate includes the antibody, and an effector molecule, and the antibody is conjugated to the effector molecule, preferably chemically conjugated. Wherein, the effector molecule is preferably a drug with therapeutic activity. Additionally, the effector molecule can be one or more of a toxin, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

The antibody of the present invention and the effector molecule can be coupled through a coupling agent. Examples of the coupling agent may be any one or more of non-selective coupling agents, coupling agents utilizing carboxyl groups, peptide chains, and coupling agents utilizing disulfide bonds. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde and the like. The coupling agent utilizing the carboxyl group may be any one or more of a cis-aconitic anhydride type coupling agent (such as cis-aconitic anhydride) and an acyl hydrazone type coupling agent (the coupling site is an acyl hydrazone).

Certain residues on antibodies (such as Cys or Lys, etc.) are used to link with various functional groups, including imaging reagents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes) , stabilizers (eg, ethylene glycol polymers) and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (eg, drugs, detection reagents, stabilizers) are conjugated (covalently linked) to the antibody. The functional agent can be attached to the antibody either directly or indirectly through a linker.

Antibodies can be conjugated to drugs to form antibody drug conjugates (ADCs). Typically, the ADC contains a linker between the drug and the antibody. Linkers can be degradable or non-degradable linkers. Degradable linkers are typically susceptible to degradation in the intracellular environment, eg, at the target site, where the linker is degraded, 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 (eg, lysosomal or endosomal proteases), or sugar linkers that, for example, can be degraded by glucuronides Enzymatically degraded glucuronide-containing linkers. Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH sensitive linkers (eg, linkers that hydrolyze at pH less than 5.5, eg, hydrazone linkers) and linkers that degrade under reducing conditions (eg, disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

Before being attached to the antibody, the linker has a reactive reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive reactive group. Sulfhydryl-specific reactive groups are preferred and include, for example, maleimides, haloamides (eg, iodo, bromo, or chloro); haloesters (eg, iodo, bromo, or chloro) ); halogenated methyl ketones (eg iodo, bromo or chloro), benzyl halides (eg iodo, bromo or chloro); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6- bis-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers can include, for example, maleimide linked to the antibody via a thiosuccinimide.

The drug can be any cytotoxic, cytostatic or immunosuppressive drug. In an embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker. In the case where the drug is directly attached to the linker, the drug has a reactive reactive group prior to attachment to the antibody.

Useful drug classes include, for example, antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors , Vinca alkaloids, etc. In the present invention, drug-linkers can be used to form ADCs in one simple step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two- or multi-step process. For example, a cysteine residue reacts with the reactive moiety of the linker in the first step, and in a subsequent step, the functional group on the linker reacts with the drug to form the ADC.

Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on drug moieties. The drug is covalently bound to the linker through a 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). 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", 2nd edition (Elsevier), are well known to those skilled in the art. Those skilled in the art can understand that for the selective reaction between the drug moiety and the linker, when a reactive functional group of a complementary pair is selected, each member of the complementary pair can be used for both the linker and the drug.

The present invention also provides a method for preparing an ADC, which may further include: combining the antibody with the drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

In certain embodiments, the methods of the invention comprise binding the antibody to a bifunctional linker compound under conditions sufficient to form the antibody-linker conjugate. In these embodiments, the methods of the invention further comprise: conjugating the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

In some embodiments, the antibody drug conjugate ADC is represented by the following molecular formula:

in:

Ab is an antibody,

LU is the connector;

D is a drug;

And the subscript p is a value selected from 1 to 8.

pharmaceutical composition

The present invention also provides a composition. In a preferred example, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding CAR-T cells, and a pharmaceutically acceptable carrier. In general, these materials can be formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably about pH 6-8, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated. The formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.

The antibody of the present invention can also be expressed in a cell by a nucleotide sequence for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

The pharmaceutical composition of the present invention can be directly used to bind BCMA protein molecules, and thus can be used to prevent and treat BCMA positive tumors. In addition, other therapeutic agents may also be used concomitantly.

The pharmaceutical composition of the present invention contains the above-mentioned monoclonal antibody (or its conjugate) of the present invention in a safe and effective amount (eg, 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) and a pharmaceutical an acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The drug formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, eg, about 1 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, the polypeptides of the present 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 per kilogram of body weight, and in most cases no more than about 50 mg per kilogram of body weight, compared to Preferably the dose is about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.

Assay Uses and Kits

Antibodies of the invention can be used in detection applications, eg, in detection of samples, to provide diagnostic information.

In the present invention, the sample (sample) used includes cells, tissue samples and biopsy specimens. The term "biopsy" as used in the present invention shall include all kinds of biopsies known to those skilled in the art. Biopsy used in the present invention may thus include tissue samples prepared, for example, by endoscopic methods or needle or needle biopsy of an organ.

Samples used in the present invention include fixed or preserved cell or tissue samples.

The present invention also provides a kit containing the antibody (or its fragment) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffers, and the like. In a preferred example, the antibody of the present invention can be immobilized on a detection plate.

The main advantages of the present invention include:

(a) A new murine monoclonal antibody targeting human BCMA was developed.

(b) A humanized antibody sequence targeting human BCMA monoclonal antibody was developed.

(c) Bispecific antibodies constructed with humanized antibody sequences developed in the present invention, or CAR-T cells, or antibody-conjugated drugs, can reduce the rejection of drugs in vivo compared with drugs constructed with mouse-derived antibody sequences , to further improve the efficacy.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, usually according to conventional conditions, such as people such as Sambrook, molecular cloning: conditions described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to manufacturer the proposed conditions. Percentages and parts are weight percentages and parts unless otherwise specified.

General Materials and Methods:

1. The main reagents used in the embodiment are as follows:

PBS (Gibco, CAT#14190-250)

DMEM (Gibco, CAT#41965-062)

F12K (Gibco, CAT#21127030)

FBS (Gibco, CAT#10099-141)

Genomic 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)

PrimeScript™ 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 endonuclease (NEB, CAT#R3136M)

KpnI restriction endonuclease (NEB, CAT#R3142M)

TOP10 competent (prepared by iCarTab)

pcDNA3.1-IgG1Fc vector (prepared by iCarTab)

pcDNA3.1-IgKc vector (prepared by iCarTab)

PE-Anti-human IgG (Biolegend, CAT#409304)

PE-Anti-mouse IgG (MultiSciences, CAT#70-GAM0041)

FreeStyle ^™ 293 Expression Medium (Thermo, CAT#12338018)

LVtransm transfection reagent (iCarTab, Cat#LVTran100)

Buffer: 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 embodiment is as follows:

Ordinary optical inverted microscope

desktop centrifuge Thermo ST41

High-speed centrifuge Thermo RC6+

biological safety cabinet

Roche480 fluorescence quantitative PCR instrument

Thermo Attune Nxt Flow Cytometer

Thermo 3111 CO2 Incubator

BiaCore T200

3. The main methods involved in the embodiment are as follows:

3.1 Hybridoma screening

3.1.1 Mouse immunization

The immunization process of mice is shown in Figure 1.

All mice were housed in a barrier system with sterile pelleted feed and autoclaved drinking water. 5 Balb/c mice (SPF grade) were marked with ear tags and immunized with purified BCMA-Fc recombinant protein according to the above immunization procedure.

3.1.2 Immune titer detection

1) Take out the mouse from the cage, use 75% medical alcohol cotton balls to sterilize the tail of the mouse, and use a 5mm blood collection needle to prick a small wound on the tail of the mouse;

2) Use capillary glass blood collection tubes to collect blood droplets (100uL of plasma needs to be prepared);

3) After collecting blood, use a dry sterile cotton ball to gently press the blood collection point to stop bleeding, and then return the mouse to the cage for a little observation;

4) Place the centrifuge tube with the blood sample collected in a 37°C incubator for 1 hour; then transfer the blood sample to 4°C overnight.

5) Separate the serum from the blood clot, transfer it to a new sterile centrifuge tube, and centrifuge at 10000×g for 10 min at 4°C;

6) Transfer the serum to a new sterile centrifuge tube, and use FACS to detect the immune titer.

3.1.3 FACS detection

1. Resuscitate CHO-K1 empty cells and CHO-K1-BCMA recombinant cell lines from liquid nitrogen, and use F12K and 10% FBS complete medium to adjust the cell state to logarithmic growth phase.

2. Divide the two types of cells into several parts, each with 5×10 ⁵ cells, add diluted mouse serum (1:1000 dilution), mix well, and incubate at room temperature for 1 hour.

3. Centrifuge at 800xg for 5 minutes at room temperature, remove the supernatant containing the antibody, and wash the cells 3 times with PBS;

4. Add 1uL PE-labeled Anti-mouse IgG, mix well, and incubate at room temperature for 30 minutes in the dark;

Centrifuge at 5.800xg for 5 minutes at room temperature, remove the supernatant containing the secondary antibody, and wash the cells 3 times with PBS;

6. Resuspend cells in 500uL PBS for flow analysis.

3.1.4 Hybridoma fusion

1) The mice with the best immune titer were sacrificed by cervical dislocation, and the spleen of the mice was obtained under sterile conditions to prepare a B cell single cell suspension, which was mixed with non-antibody-secreting SP2/0 bone marrow at a ratio of 1:1. Tumor cells were mixed and cell fusion was performed using the Bio-rad GenePulser Electroporation System.

2) Immediately after electrofusion, all cells were suspended in complete medium (DMEM, 20% FBS and HAT) and seeded into 96-well plates.

3) Change to HT medium about 10 days after fusion, and after culturing for two days, take the supernatant to detect specific antibodies.

3.1.5 Hybridoma screening

120 uL of medium supernatant was removed from each well of the 96-well plate, and each well was supplemented with fresh HT medium. The removed medium supernatant was incubated with the ELISA plate pre-coated with the target antigen, and the identification was carried out according to the standard ELISA procedure. Select wells with higher O.D values for the second and third rounds of subcloning. After each round of subcloning, ELISA identification was carried out with reference to the same steps until the formation of a single clone.

3.1.6 ELISA detection

1) Dilute the BCMA-His recombinant protein with sterile PBS to a final concentration of 1 ug/mL. Take a new 96-well plate and add 100uL/well to coat overnight at 4°C.

2) Remove the antigen coating solution and wash three times with PBST (containing 0.5% Tween).

3) Add 200uL/well of 3% BSA and block for 2 hours at 37°C;

4) After removing the blocking buffer, wash the plate 3 times with PBST;

5) Add 100ul hybridoma supernatant, incubate at room temperature for 1 hour, and the control well is PBS;

6) Remove the liquid in the well and wash 3 times with PBST;

7) Add 100uL HRP-mouse IgG (1:10000 dilution) and incubate at room temperature for 1 hour;

8) After removing the liquid in the well, wash the well plate 3 times with PBST;

9) Add 100uL/well TMB color developing solution;

10) Incubate at room temperature for 15 minutes in the dark;

11) Add 50uL/well stop solution;

12) Use a microplate reader to read the O.D value in the well.

3.1.7 Flow detection of hybridoma clones

1) The CHO-K1-BCMA cell line was recovered from liquid nitrogen, and the cell state was adjusted to the logarithmic growth phase using F12K and 10% FBS complete medium.

2) Divide the CHO-K1-BCMA cell line into several parts, the number of each cell is 5×10 ⁵ cells, respectively use 100uL hybridoma supernatant to incubate the target cells, and after fully mixing, incubate at room temperature for 1 hour.

3) Centrifuge at 800×g for 5 minutes at room temperature, remove the supernatant containing the antibody, and wash the cells three times with PBS.

4) Add 0.5uL PE-labeled Anti-mouse IgG, mix well, and incubate at room temperature for 30 minutes in the dark;

5) Centrifuge at 800×g for 5 minutes at room temperature, remove the supernatant containing the secondary antibody, and wash the cells 3 times with PBS;

6) Resuspend the cells in 500uL PBS for flow analysis.

3.2 Hybridoma sequencing

1) Take 5× ^{10 6} hybridoma cells obtained by final screening, use Trizol to lyse, and extract the total RNA of hybridoma cells with reference to standard methods.

2) After the total RNA was reverse transcribed into cDNA samples using a reverse transcription kit, the variable regions of the heavy and light chains of the antibody were amplified by PCR using hybridoma sequencing primers, followed by TA cloning, and the fragments obtained by PCR were Subcloned into pMD-19T vector. After blue-white screening, 10 clones were picked for each chain for sequencing. And the final antibody sequence was analyzed.

3.3 Mouse anti-humanization

3.3.1 Humanized Antibody Sequence Design

According to the amino acid sequence information of the heavy chain and light chain of the target antibody obtained by the above sequencing, the humanization design is carried out, and the CDR region sequence of the original antibody remains unchanged. According to the results of germline alignment and the results of antibody structure simulation, the heavy chain and light chain Different humanized antibody templates are selected for the chains respectively, and back-mutation is performed in the framework region after humanization to design candidate humanized antibody sequences.

3.3.2 Humanized antibody gene synthesis and expression vector construction

The heavy chain and light chain of the humanized antibody designed above were separately synthesized, the heavy chain was subcloned into the pcDNA3.1-IgG1Fc expression vector, and the light chain was subcloned into the pcDNA3.1-IgKc expression vector (the schematic diagram of the vector is shown in Figure 2). shown). After the vector was verified by sequencing, an endotoxin-free plasmid was prepared using the Qiagen Plasmid Extraction Kit.

3.3.3 Humanized antibody expression and purification

1. Take out the LVTransm transfection reagent and the antibody expression vector from the refrigerator. After thawing at room temperature, mix thoroughly by pipetting up and down with a pipette. Remove PBS or HBSS buffer and warm to room temperature. Take 2mL of PBS to one well of a 6-well plate, add 2μg pcDNA3.1-IgG1Fc and 2μg pcDNA3.1-IgKc respectively, pipette up and down to mix well, add 12μLLVTransm, immediately pipette up and down to mix well, Let stand at room temperature for 10 minutes.

2. Add the above DNA/LVTransm complex to 1.5mL of 293F-SVP16 cells, shake gently to mix well. The cells were placed in a 37°C, 5% CO ₂ incubator, and after culturing at 130 RPM for 6-8 hours, 1.5 mL of fresh FreeStyle ^TM 293 expression medium was added, and the cells were put back into the incubator to continue culturing.

3. After 3 days of continuous culture, the medium supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube for flow detection.

3.3.4 Flow cytometric identification of the binding of humanized antibodies to target proteins

1. Resuscitate CHO-K1 empty cells and CHO-K1-BCMA recombinant cell lines from liquid nitrogen, and use F12K and 10% FBS complete medium to adjust the cell state to logarithmic growth phase.

2. Divide the two types of cells into several parts, and the number of each cell is 5×10 ⁵ cells, and add humanized antibodies respectively. Each antibody needs to be reconstituted with CHO-K1 empty cells and CHO-K1-BCMA. Incubate the cell line, mix well, and incubate at room temperature for 1 hour.

3. Centrifuge at 800xg for 5 minutes at room temperature, remove the supernatant containing the antibody, and wash the cells 3 times with PBS;

4. Add 2uL PE-labeled Anti-human IgG, mix well, and incubate at room temperature for 30min in the dark;

Centrifuge at 5.800xg for 5 minutes at room temperature, remove the supernatant containing the secondary antibody, and wash the cells 3 times with PBS;

6. Resuspend cells in 500uL PBS for flow analysis.

3.3.5 Humanized Antibody Affinity Detection

The BCMA-Fc recombinant protein was immobilized on the CM5 chip using 10mM acetic acid coupling buffer, and the positive humanized antibody and human-mouse chimeric antibody prepared above were used as the mobile phase to detect the antibody and target before and after humanization. Binding capacity of the protein BCMA.

Example 1 FACS detection of mouse immune titer

Mice were immunized according to the above method, and serum was isolated from the tail vein of all immunized mice, diluted 1:1000, and subjected to FACS detection with CHO-K1 and CHO-K1-BCMA cells, respectively.

The FACS test results are shown in Figure 3. According to the experimental results, mouse No. 29 was selected for shock immunization and subsequent hybridoma fusion.

Example 2 Fusion plate ELISA detection

Take 100uL of supernatant from the fused well plate, and use the 96-well plate pre-coated with the target antigen for ELISA detection.

ELISA detection results of fusion clones are shown in FIG. 4 .

According to the ELISA results of fusion clones, the clones with high OD value and better growth were selected for FACS verification and the first subcloning with the CHO-K1-BCMA cell line.

The FACS detection results of the fusion clones are shown in Figure 5.

Example 3 Subcloning for the first time

Take 100uL of supernatant from the well plate after the first subcloning, and use the 96-well plate pre-coated with the target antigen for ELISA detection.

The ELISA test results of the first subcloning are shown in FIG. 6 .

According to the results of the first subcloning ELISA, the clones with high OD value and better growth were selected for FACS verification and the second subcloning with the CHO-K1-BCMA cell line.

The FACS test results are shown in Figure 7.

Example 4 Second subcloning

Take 100uL of supernatant from the well plate after the second subcloning, and use the 96-well plate pre-coated with the target antigen for ELISA detection.

The ELISA test results of the second subclone are shown in FIG. 8 .

Example 5 Hybridoma sequencing

A clone (1-D2-C8-E5, named B1) with a good FACS identification result was selected for amplification, cleaved with TRIZOL reagent, RNA was extracted and reverse transcribed into cDNA, and hybridoma sequencing primers were used. The heavy chain and light chain variable regions were sequenced, and the CDR regions and Framework regions were analyzed, and the results were as follows.

Heavy chain amino acid sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

EVQLQQSGPGLVKPSQSLSLTCTVSGYSIT SDYVWN WIRQFPGNKLEWMA YISYSGSTSYNPSLKS RISITRDTSKNQFFLQLNSVTTEDTATFYCAV YNYDGIFAYWGQGTLVTVSA (SEQ ID NO.:7)

Wherein, the amino acid sequence shown in the underline is the CDR region, and specifically includes:

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

DVQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGGTKLKICT ( SEQ ID NO.:8 )

Wherein, the amino acid sequence shown in the underline is the CDR region, and specifically includes:

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

The antibody sequence obtained by sequencing was used to construct a chimeric expression vector, and the chimeric antibody was expressed for verification. The heavy chain variable region obtained by hybridoma sequencing was subcloned into pcDNA3.1-IgG1Fc expression vector, and the light chain variable region was subcloned into pcDNA3.1-IgKc expression vector. The constructed light chain and heavy chain expression vectors were combined in pairs. 293F cells were transiently co-transfected, and 48 hours after transfection, the supernatant of the expression was collected for FACS detection.

The FACS detection results are shown in Figure 9.

Example 7 Humanized Antibody Flow Cytometry Detection

The humanized antibody sequence design was carried out according to the description in the general method, and a total of 6 humanized heavy chain variable regions and 6 humanized light chain variable regions were designed.

Among them, 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 was transiently transfected into 293F cells, the culture supernatant was collected, and flow cytometry was used to detect the binding of the humanized antibody to the antigen on the surface of the recombinant cell CHO-K1-BCMA cell membrane. The human-mouse chimeric antibody constructed with the original mouse-derived antibodies VH and VL was used as a control.

The FACS detection results are shown in Figure 10, and the BCMA humanized antibodies were all bound to recombinant CHO-K1-BCMA cells. A combination of B1H2-B1L1 and B1H4-B1L2 expressed and purified antibodies was selected for subsequent affinity detection.

The SDS-PAGE results of the humanized antibodies B1H2-B1L1 and B1H4-B1L2 are shown in FIG. 11 .

The sequences of the antibodies before and after humanization were compared, and the alignment results of B1H2-B1L1 are shown in FIG. 12 , and the alignment results of B1H4-B1L2 are shown in FIG. 13 .

Example 8 Humanized Antibody Affinity Determination

8.1 Experimental materials and reagents involved in this example

S series CM5 chip: manufacturer GE, article number 29149603

Buffer: HBS-EP+10X, manufacturer GE, catalog number BR100669. Dilute 10-fold with deionized water before use.

Amino Coupling Kit: GE, Cat. No. BR100050

Regeneration reagent: 10mM Glycine 2.5, manufacturer GE, Cat. No. BR100356

8.2 Ligand coupling pre-enrichment

Ligand: BCMA recombinant protein

Test pH: 10mM Acetate 5.5/5.0/4.5/4.0

Flow rate: 10μl/min

Buffer: HBS-EP+buffer

The results are shown in 14, and 10 mM Acetate 5.5 was chosen as the coupling buffer.

8.3 Ligand conjugation

Ligand: BCMA recombinant protein

Coupling buffer: 10mM Acetate 5.5

Target coupling amount: 200RU

The results are shown in Figure 15, and the final coupling amount was 205.7RU.

8.4 Affinity determination of MH18100901-B1Chimeric, MH18100901-B1H2-B1L1, MH18100901-B1H4-B1L2 and BCMA recombinant protein

Experimental conditions:

Antibody concentration: 0.15625/0.3125/0.625/1.25/2.5/5.0ug/ml

Flow rate: 30μl/min

Combination time: 300s

Dissociation time: 400s

Regeneration reagent: Glycine 1.5, 30S, 2 times

Figure 16 shows the affinity measurement results of MH18100901-B1Chimeric, Figure 17 shows the affinity measurement results of MH18100901-B1H2-B1L1, and Figure 18 shows the affinity measurement results of MH18100901-B1H4-B1L2.

The results of each antibody affinity test 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 test results showed that the humanized antibodies MH18100901-B1H2-B1L1 and MH18100901-B1H4-B1L2 had the same affinity as the chimeric antibodies.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> Boshengji Pharmaceutical Technology (Suzhou) Co., Ltd.

<120> Monoclonal antibody targeting human BCMA and its application

<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

<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 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

<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 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

<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 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

<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 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

<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 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

<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 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

<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

<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

<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

<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

<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

<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

<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

<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

<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

<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

<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

<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

<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

<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

<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

<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

<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

<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. the heavy chain variable region of an antibody is characterized in that, described heavy chain variable region comprises following three complementarity determining region CDRs: CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO:3. 2 . The heavy chain of an antibody, wherein the heavy chain has the heavy chain variable region of claim 1 . 3 . 3. a light chain variable region of an antibody, wherein the light chain variable region comprises the following three complementarity determining region CDRs: CDR1' shown in SEQ ID NO:4, CDR2' shown in SEQ ID NO: 5, and CDR3' shown in SEQ ID NO:6. 4. A light chain of an antibody, wherein the light chain has the light chain variable region of claim 3. 5. an antibody targeting BCMA, characterized in that the antibody has: (1) the heavy chain variable region of claim 1; and/or (2) light chain variable region as claimed in 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 the antibody is selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, or a combination thereof. 7. The antibody of claim 5, wherein the heavy chain variable region sequence of the antibody is shown in SEQ ID NOs: 7, 9-14; and/or The light chain variable region sequences of the antibodies are shown in SEQ ID NOs: 8, 15-20. 8. A CAR construct, characterized in that the scFV segment of the monoclonal antibody antigen-binding region of the CAR construct is a binding region that specifically binds to BCMA, and the scFv has the scFv as claimed in claim 1. Heavy chain variable region and light chain variable region as claimed in claim 3. 9. A recombinant immune cell expressing the exogenous CAR construct of claim 8. 10. the purposes of a kind of active ingredient, described active ingredient is selected from following group: heavy chain variable region as claimed in claim 1, heavy chain as claimed in claim 2, light chain as claimed in claim 3 variable region, 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 in (a ) preparation of detection reagents or kits; and/or (b) preparation of medicaments for preventing and/or treating BCMA-positive tumors.

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