CN114763383B

CN114763383B - Monoclonal antibody targeting human BCMA and application thereof

Info

Publication number: CN114763383B
Application number: CN202110044548.6A
Authority: CN
Inventors: 杨林; 游凤涛; 荆光军
Original assignee: Persongen Biotherapeutics Suzhou Co ltd
Current assignee: Persongen Biotherapeutics Suzhou Co ltd
Priority date: 2021-01-13
Filing date: 2021-01-13
Publication date: 2024-12-17
Anticipated expiration: 2041-01-13
Also published as: CN114763383A

Abstract

The invention provides a monoclonal antibody targeting human BCMA and application thereof. In particular, the present invention provides an antibody targeting BCMA with high affinity and high specificity for human BCMA. The invention also provides cells and pharmaceutical compositions comprising the antibodies, and methods of making and using the antibodies.

Description

Monoclonal antibody targeting human BCMA and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a monoclonal antibody targeting human BCMA and application thereof.

Background

B cell surface maturation antigens (B-cell maturation antigen, BCMA) were first found on the surface of mature B lymphocytes and were hardly expressed in other tissue cells. It is highly expressed in malignant proliferating B lymphocytes (e.g., myeloma cells, leukemia cells). At the same time it plays a critical role in cell survival, proliferation, metastasis and drug resistance by mediating downstream signaling pathways, which properties make it a target for immunotherapy, especially for the treatment of multiple myeloma. BCMA, due to its limited expression in plasma cells and non-expression in natural and memory B cells, has been used as a new drug target for diagnosis and treatment of multiple myeloma. Currently, novel tumor immunotherapy approaches against BCMA mainly include CAR-T (CHIMERIC ANTIGEN Receptor T-Cell Immunotherapy) therapy, bispecific antibodies (Bispecific Antibody, bsAb) and Antibody drug conjugates (antibodies-drug coupl ing, ADC). Recent studies have also shown that BCMA-targeted CAR-T is able to effectively clear myeloma cells in vivo.

Currently, there are many shortcomings 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.

It is also an object of the present invention to provide a BCMA humanized antibody and further targeted treatment of multiple myeloma by constructing BCMA-targeted CAR-T or bispecific antibody or antibody conjugated drugs.

In a first aspect of the invention, there is provided a heavy chain variable region of an antibody, said heavy chain variable region comprising the following three complementarity determining region CDRs:

CDR1 shown in SEQ ID NO.1,

CDR2 as shown in SEQ ID NO. 2, and

CDR3 as shown in SEQ ID NO. 3.

In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence optionally having at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid added, deleted, modified and/or substituted and capable of retaining BCMA binding affinity.

In another preferred embodiment, the heavy chain variable region further comprises an FR region of human origin or an FR region of murine origin.

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 sequence shown in SEQ ID NOS.9-14.

In a second aspect of the invention there is provided an antibody heavy chain having a heavy chain variable region according to the first aspect of the invention.

In another preferred embodiment, the heavy chain of the 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 invention, there is provided a light chain variable region of an antibody, said 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, the light chain variable region further comprises an FR region of human origin or an FR region of murine origin.

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 NOS.15-20.

In a fourth aspect of the invention there is provided an antibody light chain having a light chain variable region according to 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, said antibody having:

(1) The 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;

Or 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 embodiment, the antibody binds to human BCMA protein (preferably wild type) with an affinity constant KD (M) of (1-10) x 10 ^-10, preferably (1-5) x 10 ^-10.

In another preferred embodiment, the antibody is selected from the group consisting of an animal-derived antibody, 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 the antibody is shown as SEQ ID NO. 7 and/or the light chain variable region sequence of the antibody is shown as 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 an antibody targeting BCMA according to the fifth aspect of the present invention and a second antibody targeting a tumor antigen other than BCMA.

In another preferred embodiment, the tumor antigen comprises CD3, 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.

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 assist 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 whose scFV fragment of the monoclonal antibody antigen binding region is a binding region that specifically binds to BCMA and which 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 structure of formula I:

L-scFv-H-TM-C-CD3ζ(I)

in the formula,

Each "-" is independently a connecting peptide or peptide bond;

l is a none or signal peptide sequence;

scFv is BCMA-targeting scFv;

H is an optional hinge region;

TM is a transmembrane domain;

c is a costimulatory signaling molecule;

cd3ζ is a cd3ζ 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 cells are selected from the group consisting of NK cells, T cells.

In another preferred embodiment, the immune cells are derived 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, the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.

According to a tenth aspect of the present 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 present invention, a heavy chain variable region according to the second aspect of the present invention, a light chain variable region according to the third aspect of the present invention, an antibody according to the fourth aspect of the present invention, a recombinant protein according to the sixth aspect of the present invention, an immune cell according to the eighth aspect of the present invention, an antibody drug conjugate according to the ninth aspect of the present invention, or a combination thereof, for (a) the preparation of a detection reagent or kit, and/or (b) the preparation of a medicament for the prevention and/or treatment of BCMA positive tumors.

In another preferred embodiment, the tumor is selected from the group consisting of a hematological tumor, a solid tumor, or a combination thereof.

In another preferred embodiment, the hematological neoplasm is selected from the group consisting of Acute Myelogenous 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 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 example, 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 for:

(1) Detecting BCMA protein in the sample, and/or

(2) Detecting endogenous BCMA proteins in tumor cells, and/or

(3) Detecting tumor cells expressing BCMA protein.

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

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 formulation.

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 NOS.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 vector comprises a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vector.

In a fourteenth aspect of the invention there is provided a genetically engineered host cell comprising a vector or genome according to the thirteenth aspect of the invention having incorporated therein a polynucleotide according to the twelfth aspect of the invention.

In a fifteenth aspect of the present invention, there is provided a method of detecting BCMA protein in an in vitro sample (including diagnostic or non-diagnostic), 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 whether an antigen-antibody complex is formed, wherein the formation of the complex indicates 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 invention there is provided a test plate 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 second antibody against an antibody according to the fifth aspect of the invention;

or the kit contains 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 the 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 invention there is provided a method of preventing and/or treating a BCMA positive tumor 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 embodiment, the method further comprises administering to a subject in need thereof an additional agent or treatment regimen for combination therapy.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Figure 1 shows the mouse immunization protocol.

FIG. 2 shows a schematic vector diagram of heavy chain expression vector pcDNA3.1-IgG1Fc and light chain expression vector pcDNA3.1-IgKc.

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

FIG. 4 shows ELISA detection results of hybridoma fusion plates.

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

FIG. 6 shows ELISA detection results of the first subclone plate.

FIG. 7 shows the results of FACS detection of the first subclones.

FIG. 8 shows ELISA detection results of the second subclone plate.

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

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

FIG. 11 shows SDS-PAGE results of humanized antibodies B1H2-B1L1 and B1H4-B1L 2.

FIG. 12 shows the results of the comparison of humanized antibody B1H2-B1L1 with murine antibody.

FIG. 13 shows the results of the comparison of humanized antibody B1H4-B1L2 with murine antibody.

FIG. 14 shows ligand-coupled pre-enrichment results.

FIG. 15 shows ligand coupling results.

FIG. 16 shows the affinity assay results of MH18100901-B1 Chimeric.

FIG. 17 shows the affinity assay results of MH18100901-B1H2-B1L 1.

FIG. 18 shows the affinity assay results for MH18100901-B1H4-B1L 2.

Detailed Description

The present inventors have studied extensively and intensively, and have unexpectedly found a high affinity and high specificity BCMA-targeting antibody for the first time, and prepared a humanized antibody having affinity comparable to that of a chimeric antibody. The present invention has been completed on the basis of this finding.

Terminology

As used herein, the terms "administering" and "treating" refer to the application of an exogenous drug, therapeutic, diagnostic, 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. Treatment of a cell includes contacting a reagent with the cell, contacting a reagent with a fluid, and contacting a fluid with the cell. "administration" and "treatment" also mean in vitro and ex vivo treatment by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, animal or study subject refers to therapeutic treatment, prophylactic or preventative measures, study and diagnosis, including exposure of BCMA antibodies to human or animal, subject, cell, tissue, physiological compartment or physiological fluid.

As used herein, the term "treatment" refers to the administration of an internally or externally used therapeutic agent, including any one of the BCMA antibodies of the present invention and compositions thereof, to a patient having one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the patient is administered an amount of the therapeutic agent (therapeutically effective amount) effective to alleviate one or more symptoms of the disease.

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 there may be, but need not be, 1, 2, or 3 antibody heavy chain variable regions of a particular sequence.

Antibodies to

As used herein, the term "antibody" refers to an immunoglobulin that is a tetrapeptide chain structure formed from two identical heavy chains and two identical light chains joined by an interchain disulfide bond. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or different types of immunoglobulins, igM, igD, igG, igA and IgE, the heavy chain constant regions corresponding to the different classes of immunoglobulins being 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, igG1, igG2, igG3 and IgG4, due to differences in chemical structure and biological function. Light chains are classified as either kappa or lambda chains by the difference in constant regions. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (V region), and the remaining amino acid sequences near the C-terminus are relatively stable, being the constant region (C region). The variable region includes 3 hypervariable regions (HVRs) and 4 FR Regions (FR) that 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) consists of 3 CDR regions and 4 FR regions, arranged in sequence from amino-to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain, namely the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2 and LCDR3, and the 3 CDR regions of the heavy chain, namely the heavy chain hypervariable region (HCDR), refer to HCDR1, HCDR2 and HCDR3. The CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention are in numbers and positions that meet the known Kabat numbering convention (LCDR 1-3, HCDR 2-3), or that meet the numbering convention of Kabat and chothia (HCDR 1). The four FR regions in the natural heavy and light chain variable regions are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and form together with the CDRs of the other chain an antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody. The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

As used herein, the term "antigen binding fragment" refers to a Fab fragment, fab 'fragment, F (ab') 2 fragment, or single Fv fragment having antigen binding activity. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal 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 "epitope" refers to a discrete, three-dimensional spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the invention.

The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues 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 DNA recombination techniques well known in the art.

As used herein, the term "monoclonal antibody" refers to an antibody secreted from a clone derived from a single cell source. Monoclonal antibodies are highly specific, being directed against a single epitope. The cells may be eukaryotic, prokaryotic or phage clonal cell lines.

As used herein, the term "chimeric antibody" is an antibody molecule expressed by a host cell by splicing the V region gene of a murine antibody to the C region gene of a human antibody into a chimeric gene, followed by insertion into a vector. The high specificity and affinity of the parent mouse antibody are maintained, and the human Fc segment of the parent mouse antibody can effectively mediate biological effect functions.

As used herein, the term "humanized antibody", a variant, engineered version of the murine antibody of the present invention, has CDR regions derived (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, i.e., by grafting the murine antibody CDR region sequences onto different types of human germline antibody framework sequences. Because 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 expression vectors.

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

In the present invention, the antibodies of the invention also include conservative variants thereof, meaning that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the antibodies of the invention to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.

Table A

Initial residues	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 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 high specificity 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 as shown in SEQ ID NO. 2, and

CDR3 as shown in SEQ ID NO 3, and/or

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 one of the amino acid sequences described above further includes a derivative sequence having BCMA binding affinity that has undergone addition, deletion, modification and/or substitution of at least one (e.g., 1 to 5, 1 to 3, preferably 1 to 2, more preferably 1) amino acids.

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 having a homology of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%.

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

The antibody derivatives of the present invention may be single chain antibodies, and/or antibody fragments such as Fab, fab ', (Fab') ₂ or other known antibody derivatives in the art, etc., as well as IgA, igD, igE, igG and any one or more of IgM antibodies or other subclasses of antibodies.

Wherein 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 that target human BCMA.

Preparation of antibodies

Any method suitable for producing monoclonal antibodies may be used to produce the anti-BCMA antibodies of the invention. For example, animals may be immunized with BCMA or fragments thereof. Suitable immunization methods may be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may 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 may be full length mature human BCMA, including natural homodimers, or a peptide containing single/multiple epitopes. The immunogens may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. The immunogen may be purified from a natural source or produced in genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic (e.g., cDNA) in origin. DNA encoding the immunogen may be expressed using suitable genetic vectors including, but not limited to, adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, prime and non-viral vectors.

The humanized antibody 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 necessary constant domain sequences to produce the desired biological activity is readily achieved by screening antibodies using the biological assays described in the examples below.

Also, any type of light chain may 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 a conventional technique such as amplification by PCR or screening of a genomic library. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. 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.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.

The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include, but are not limited to, CHO-S, CHO-K1, HEK-293 cells.

The steps described herein for transforming a host cell with recombinant DNA may be performed using techniques well known in the art. The transformant obtained 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 cultivated in conventional medium under suitable conditions.

Typically, the transformed host cell is cultured under conditions suitable for expression of the antibodies of the invention. The antibodies of the invention are 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, using conventional separation and purification means 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 using immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).

Antibody-drug conjugates (ADC)

The invention also provides an antibody-conjugated drug (ADC) based on the antibody.

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 nonselective coupling agent refers to a compound such as glutaraldehyde or the like that forms a covalent bond between the effector molecule and the antibody. The coupling agent using carboxyl can be any one or more of cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and acyl hydrazone coupling agent (the coupling site is acyl hydrazone).

Certain residues on antibodies (e.g., cys or Lys, etc.) are useful in connection with a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizers (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 directly attached to the antibody, or indirectly attached through a linker.

Antibodies can be conjugated to drugs to form Antibody Drug Conjugates (ADCs). Typically, an ADC comprises a linker between the drug and the antibody. The linker may be degradable or non-degradable. Degradable linkers typically degrade readily in the intracellular environment, e.g., the linker degrades at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers including peptide-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. The peptidyl linker may comprise, for example, a dipeptide, 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 bonds). The non-degradable linker typically releases the drug under conditions where the antibody is hydrolyzed by the protease.

Prior to attachment to the antibody, the linker has reactive groups capable of reacting with certain amino acid residues, the attachment being accomplished through the reactive groups. Thiol-specific reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodine, bromine, or chlorinated), haloesters (e.g., iodine, bromine, or chlorinated), halomethyl ketones (e.g., iodine, bromine, or chlorinated), benzyl halides (e.g., iodine, bromine, or chlorinated), vinyl sulfones, pyridyl disulfides, mercury derivatives such as 3, 6-bis- (mercury methyl) dioxane, with the counter ion being acetate, chloride, or nitrate, and polymethylene dimethyl sulfide thiosulfate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.

The drug may be any cytotoxic, cytostatic or immunosuppressive drug. In embodiments, the linker connects the antibody and the drug, and the drug has a functional group that can bond to the linker. For example, the drug may have an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, or a ketone group that may be bonded to the linker. In the case of a drug directly attached to a linker, the drug has reactive groups 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 a single step. In other embodiments, the bifunctional linker compounds may be used to form ADCs in two or more step processes. 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.

Typically, the functional groups on the linker are selected to facilitate specific reaction with the appropriate reactive groups 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 by1, 3-dipolar cycloaddition between the azide and the alkyne 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 attachment strategies, such as described in bioconjugate techniques, second edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that for selective reaction of a drug moiety with a linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used for both the linker and the drug.

The invention also provides methods of making ADCs, which may further comprise combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

In certain embodiments, the methods of the invention comprise binding an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further comprise binding the antibody linker conjugate 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 is of the formula:

Wherein:

ab is an antibody that is conjugated to a polypeptide,

LU is the linker;

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 active fragment thereof or fusion protein thereof or ADC thereof or corresponding CAR-T cell as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may 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 antibodies of the invention may also be used for cellular therapy where the nucleotide sequence is expressed intracellularly, e.g., for chimeric antigen receptor T cell immunotherapy (CAR-T), etc.

The pharmaceutical composition of the present invention can be directly used for binding to 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 compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the monoclonal antibodies (or conjugates thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The 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.

Where 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 typically at least about 10 micrograms per kilogram of body weight and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

Detection application and kit

The antibodies of the invention may be used in detection applications, for example for detecting samples, thereby providing diagnostic information.

In the present invention, the samples (specimens) used include cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to a person skilled in the art. Thus biopsies used in the present invention may include tissue samples prepared, for example, by endoscopic methods or by puncture 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 comprising an antibody (or fragment thereof) of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, etc. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.

The main advantages of the invention include:

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

(B) A humanized antibody sequence was developed that targets human BCMA monoclonal antibodies.

(C) Compared with the medicine constructed by the murine antibody sequence, the bispecific antibody constructed by the humanized antibody sequence, or the CAR-T cell or the antibody coupling medicine can reduce the rejection reaction of the medicine in vivo and further improve the curative effect.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Sambrook et al, molecular cloning, a laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations. Percentages and parts are weight percentages and parts unless otherwise indicated.

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)

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)

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 endonuclease (NEB, CAT#R3142M)

TOP10 competence (prepared from iCarTab)

PcDNA3.1-IgG1Fc vector (prepared by iCarTab)

PcDNA3.1-IgKc vector (prepared from iCarTab)

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

PE-Anti-mouse IgG (MultiSciences, CAT #70-GAM 0041)

FreeStyle ^TM expression Medium (Thermo, CAT# 12338018)

LVtransm transfection reagent (iCarTab Cat # LVTran 100)

Buffer solution HBS-EP+10X (GE, cat#BR 100669)

Amino coupling reagent kit (GE, cat#BR 100050)

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

Desk centrifuge Thermo ST41

High-speed centrifuge ThermoRc6+

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 screening

3.1.1 Immunization of mice

The mouse immunization protocol is shown in FIG. 1.

All mice were housed in a barrier system and housed using sterilized pellet 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 Immunotiter detection

1) Taking out the mice from the cages, sterilizing the tail parts of the mice by using 75% medical alcohol cotton balls, and puncturing a small wound on the tail parts of the mice by using a blood taking needle with the length of 5 mm;

2) Blood drops were collected using capillary glass blood collection tubes (100 uL plasma was prepared);

3) After blood collection, the mice are returned to the cage for slightly observing after the blood collection points are lightly pressed by using dry sterile cotton balls to stop bleeding;

4) The centrifuge tube with the collected blood sample was placed in a 37 ℃ incubator for 1 hour, and then the blood sample was transferred to 4 ℃ overnight.

5) Separating serum from blood clot, transferring to a new sterile centrifuge tube, and centrifuging at 4deg.C and 10000Xg for 10min;

6) Serum was transferred to a new sterile centrifuge tube and immunotiters were detected using FACS.

3.1.3 FACS detection

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

2. The two cells are divided into a plurality of parts, the number of each part of cells is 5×10 ⁵ cells, diluted mouse serum (1:1000 dilution) is added respectively, and after fully mixing, the cells are incubated for 1 hour at room temperature.

3.800Xg room temperature centrifugation for 5 minutes, removing the antibody-containing supernatant, using PBS washing cells 3 times;

4. Adding 1uL of PE-labeled Anti-mouse IgG, fully mixing, and incubating for 30min at room temperature in a dark place;

5.800Xg room temperature centrifugation for 5 minutes, removing the second antibody containing supernatant, using PBS washing cells 3 times;

6. Cells were resuspended using 500uL PBS and flow analyzed.

3.1.4 Hybridoma fusions

1) The best mouse neck dislocation for immune titer detection was sacrificed, the mouse spleen was obtained under aseptic conditions, a single cell suspension of B cells was prepared, and mixed with non-antibody secreting SP2/0 myeloma cells in a ratio of 1:1, and cell fusion was performed using a Bio-rad Gene Pulser 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) After about 10 days after fusion, HT medium was changed, and after two days of incubation, supernatants were taken to detect specific antibodies.

3.1.5 Hybridoma screening

120UL of medium supernatant was removed from each well of the 96-well plate while fresh HT medium was replenished into each well. The removed culture supernatant was incubated with ELISA plates pre-coated 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 assays were performed with reference to the same procedure until monoclonal formation.

3.1.6 ELISA detection

1) BCMA-His recombinant protein was diluted with sterile PBS to a final concentration of 1ug/mL. A new 96-well plate was taken and coated overnight at 100 uL/well at 4 ℃.

2) The antigen coating was removed and washed 3 times with PBST (0.5% tween).

3) Blocking was performed for 2 hours at 37℃with 200 uL/well of 3% BSA;

4) After removal of the blocking buffer, the well plate was washed 3 times with PBST;

5) 100ul of hybridoma supernatant was added and incubated for 1 hour at room temperature, with PBS as control well;

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

7) 100uL of HRP-mouse IgG (1:10000 dilution) was added and incubated for 1 hour at room temperature;

8) After removing the liquid from the wells, the well plate was washed 3 times with PBST;

9) Adding 100 uL/hole TMB color development liquid;

10 Incubation for 15 minutes at room temperature in dark;

11 Adding 50 uL/Kong Zhongzhi liquid;

12 O.d. values within the wells were read using a microplate reader.

3.1.7 Flow detection of hybridoma clones

1) CHO-K1-BCMA cell lines were recovered from liquid nitrogen and cell status was adjusted to logarithmic growth phase using F12K, 10% fbs complete medium.

2) The CHO-K1-BCMA cell line was divided into several parts, the number of each cell was 5X 10 ⁵ cells, the target cells were incubated with 100uL of hybridoma supernatant, and after thoroughly mixing, they were incubated at room temperature for 1 hour.

3) Centrifugation at 800Xg for 5min at room temperature, the supernatant containing the antibody was removed and the cells were washed 3 times with PBS.

4) Adding 0.5uL PE marked Anti-mouse IgG, fully mixing, and incubating for 30 minutes at room temperature in a dark place;

5) Centrifugation at 800Xg for 5min at room temperature, removal of the supernatant containing the secondary antibody, washing the cells 3 times with PBS;

6) Cells were resuspended using 500uL PBS and flow analyzed.

3.2 Hybridoma sequencing

1) The hybridoma cells obtained by 5×10 ⁶ final screens were lysed using Trizol and total RNA of the hybridoma cells was extracted with reference to standard methods.

2) After reverse transcription of total RNA into cDNA samples using a reverse transcription kit, heavy and light chain variable regions of antibodies were amplified by PCR using hybridoma sequencing primers, followed by TA cloning, and subcloning of PCR-derived fragments into pMD-19T vectors. After blue-white screening, 10 clones were individually picked for sequencing for each strand. And the antibody sequences finally obtained are analyzed.

3.3 Murine anti-humanization

3.3.1 Humanized antibody sequence design

And (3) carrying out humanized design according to the amino acid sequence information of the heavy chain and the light chain of the target antibody obtained by the sequencing, keeping the CDR region sequence of the original antibody unchanged, respectively selecting different humanized antibody templates according to the result of GERMLINE AL IGNMENT and the result of the structural simulation of the antibody, carrying out back mutation on the humanized framework region, and designing candidate humanized antibody sequences.

3.3.2 Humanized antibody Gene Synthesis and expression vector construction

The heavy chain and the light chain of the humanized antibody designed above are respectively subjected to gene synthesis, the heavy chain is subcloned into a pcDNA3.1-IgG1Fc expression vector, and the light chain is subcloned into a pcDNA3.1-IgKc expression vector (the schematic diagram of the vector is shown in figure 2). After the vector was verified by sequencing to be error-free, the Qiagen plasmid megapump kit was used to prepare endotoxin-free plasmids.

3.3.3 Expression and purification of humanized antibodies

1. Taking out LVTransm transfection reagent and antibody expression vector from refrigerator, thawing at room temperature, and blowing with pipetting gun. The PBS or HBSS buffer was removed and warmed to room temperature. 2mL of PBS was taken into one well of a 6-well plate, 2. Mu.g of pcDNA3.1-IgG1Fc and 2. Mu.g of pcDNA3.1-IgKc were added respectively, and after thoroughly mixing by up-and-down blowing with a pipette, 12. Mu. L LVTRANSM was added, immediately mixed by up-and-down blowing with a pipette, and left standing at room temperature for 10 minutes.

2. The DNA/LVTransm complex was added to 1.5mL of 293F-SVP16 cells and thoroughly mixed with gentle shaking. After placing the cells in a 37℃5% CO ₂ incubator at 130RPM for 6-8 hours, 1.5mL of fresh FreeStyle ^TM 293 expression medium was added and the cells were returned to the incubator for continued culture.

3. After 3 days of continuous culture, the culture 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 identification of humanized antibody binding to target protein

2. Dividing the two cells into a plurality of parts, wherein the number of each part of cells is 5 multiplied by 10 ⁵ cells, adding humanized antibodies respectively, incubating each antibody with CHO-K1 empty cells and CHO-K1-BCMA recombinant cell strain, fully mixing, and incubating at room temperature for 1 hour.

4. adding 2uL of PE marked Anti-human IgG, fully and uniformly mixing, and incubating for 30min at room temperature in a dark place;

6. Cells were resuspended using 500uL PBS and flow analyzed.

3.3.5 Humanized antibody affinity detection

The BCMA-Fc recombinant protein is fixed on a CM5 chip by using 10mM acetic acid coupling buffer solution, and the humanized antibody and the human-mouse chimeric antibody which are positive in flow identification prepared above are used as mobile phases, so that the binding capacity of the humanized antibody and the target protein BCMA is detected.

EXAMPLE 1 mouse immunotiter FACS detection

Mice were immunized as described above, serum was isolated from the tail vein of all immunized mice, diluted 1:1000, and FACS detected with CHO-K1 and CHO-K1-BCMA cells, respectively.

The FACS detection results are shown in FIG. 3, and mouse No. 29 was selected for impact immunization and subsequent hybridoma fusion according to the experimental results.

Example 2 fusion plate ELISA detection

100UL of supernatant was taken from the fused well plate and ELISA was performed using 96 well plates pre-coated with the antigen of interest.

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

According to the ELISA detection result of the fusion clone, selecting clone with high OD value and better growth and CHO-K1-BCMA cell strain to perform FACS verification and first subcloning.

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

EXAMPLE 3 first subcloning

100UL of supernatant was removed from the well plate after the first subcloning and ELISA was performed using 96 well plates pre-coated with the antigen of interest.

The ELISA detection results of the first subclones are shown in FIG. 6.

According to the detection result of the first subclone ELISA, clones with high OD value and better growth and CHO-K1-BCMA cell strains are selected for FACS verification and the second subclone.

The FACS detection results are shown in FIG. 7.

EXAMPLE 4 second subcloning

100UL of supernatant was removed from the well plate after the second subcloning and ELISA was performed using 96 well plates pre-coated with the antigen of interest.

The ELISA detection results of the second subclones are shown in FIG. 8.

EXAMPLE 5 hybridoma sequencing

Clones (1-D2-C8-E5, designated as B1) with good FACS identification results were selected for amplification, cleaved with TRIZOL reagent, RNA was extracted and reverse transcribed into cDNA, and the heavy and light chain variable regions corresponding to the hybridoma antibodies were sequenced using hybridoma sequencing primers, and the CDR and frame regions were analyzed as follows.

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

EVQLQQSGPGLVKPSQSLSLTCTVSGYSITSDYVWNWIRQFPGNKLEWMAYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATFYCAVYNYDGIFAYWGQGTLVTVSA(SEQ ID NO.:7)

Wherein the amino acid sequence shown under the dash is a CDR region, specifically comprising:

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)

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 validation

And constructing a chimeric expression vector by using the antibody sequences obtained by sequencing, and verifying the expression chimeric antibody. 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. And respectively combining the constructed light chain expression vector and the constructed heavy chain expression vector in pairs. 293F cells were transiently co-transfected for 48 hours and the expressed supernatants were collected for FACS detection.

The FACS detection results are shown in FIG. 9.

EXAMPLE 7 humanized antibody flow cytometric detection

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

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)

amino acid sequence information of humanized candidate antibody:

>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 transiently transfecting 293F cells with the humanized antibody expression vector, collecting culture medium supernatant, and detecting the antigen binding condition of the humanized antibody and the recombinant cell CHO-K1-BCMA cell membrane surface by using a flow cytometer. Human murine chimeric antibodies constructed with the original murine antibodies VH and VL served as controls.

As shown in FIG. 10, the humanized antibodies to BCMA bound to recombinant CHO-K1-BCMA cells. The purified antibodies expressed in combination with B1H2-B1L1 and B1H4-B1L2 were selected for subsequent affinity detection.

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

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

Example 8 humanized antibody affinity assay

8.1 Experimental materials and reagents according to the present example

S series CM5 chip manufacturer GE, product number 29149603

Buffer solution, HBS-EP+10X, manufacturer GE, cat# BR100669. Before use, it was diluted 10-fold with deionized water.

Amino coupling kit GE, cat# BR100050

Regeneration reagent 10mM Glycine 2.5, manufacturer GE, cat# BR100356

8.2 Ligand coupled Pre-enrichment

Ligand BCMA recombinant protein

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

Flow rate 10. Mu.l/min

Buffer solution HBS-EP+buffer

As a result, as shown in FIG. 14, 10mM Acetate 5.5 was selected as the coupling buffer.

8.3 Ligand coupling

Ligand BCMA recombinant protein

Coupling buffer 10mM Acetate 5.5

Target coupling amount 200RU

The result is shown in FIG. 15, and the final coupling amount was 205.7RU.

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

Experimental conditions:

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

Flow rate of 30. Mu.l/min

Bonding time 300s

Dissociation time 400s

Regeneration reagent of Glycine 1.5,30S,2 times

The affinity measurement results of MH18100901-B1Chimeric are shown in FIG. 16, the affinity measurement results of MH18100901-B1H2-B1L1 are shown in FIG. 17, and the affinity measurement results of MH18100901-B1H4-B1L2 are shown in FIG. 18.

The results of each antibody affinity assay 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 results show that the affinities of the humanized antibodies MH18100901-B1H2-B1L1 and MH18100901-B1H4-B1L2 are consistent with those of the chimeric antibodies.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Sequence listing

<110> Bosheng Ji medicine technology (Suzhou) Co., ltd

<120> Monoclonal antibody targeting human BCMA and use thereof

<130> P2020-0532

<160> 32

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6

<212> PRT

<213> Mice (Mus musculus)

<400> 1

Ser Asp Tyr Val Trp Asn

1 5

<210> 2

<211> 16

<212> PRT

<213> Mice (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> Mice (Mus musculus)

<400> 3

Tyr Asn Tyr Asp Gly Ile Phe Ala Tyr

1 5

<210> 4

<211> 11

<212> PRT

<213> Mice (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> Mice (Mus musculus)

<400> 5

Tyr Thr Ser Arg Leu His Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Mice (Mus musculus)

<400> 6

Gln Gln Gly Asn Thr Leu Pro Trp Thr

1 5

<210> 7

<211> 118

<212> PRT

<213> Mice (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> Mice (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

1. An antibody targeting BCMA, said antibody comprising a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region comprises the following three complementarity determining region CDRs:

CDR1 shown in SEQ ID NO.1,

CDR2 as shown in SEQ ID NO. 2, and

CDR3 shown in SEQ ID NO. 3,

The light chain variable region includes 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.

2. The antibody of claim 1, wherein the antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, and combinations thereof.

3. The antibody of claim 1, wherein the heavy chain variable region sequence of said antibody is set forth in any one of SEQ ID NOs 7, 9-14, and

The light chain variable region sequence of the antibody is shown in any one of SEQ ID NOs 8 and 15-20.

4. The antibody of claim 1, wherein the heavy chain variable region sequence of said antibody is shown in SEQ ID NO. 7 and the light chain variable region sequence of said antibody is shown in SEQ ID NO. 8.

5. The antibody of claim 1, wherein the heavy chain variable region sequence of said antibody is shown in SEQ ID NO. 10 and the light chain variable region sequence of said antibody is shown in SEQ ID NO. 15.

6. The antibody of claim 1, wherein the heavy chain variable region sequence of said antibody is shown in SEQ ID NO. 12 and the light chain variable region sequence of said antibody is shown in SEQ ID NO. 16.

7. The antibody of claim 1, wherein said antibody is a partially or fully humanized monoclonal antibody.

8. A CAR construct, wherein the scFV fragment 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 and the light chain variable region of the antibody of claim 1.

9. A recombinant immune cell that expresses the CAR construct of claim 8 exogenously.

10. Use of the antibody of claim 1 for the preparation of a detection reagent or kit for detecting BCMA protein.

11. An antibody drug conjugate, said antibody drug conjugate comprising:

(a) An antibody moiety which is an antibody according to claim 1, and

12. A pharmaceutical composition, said pharmaceutical composition comprising:

(i) An active ingredient selected from the group consisting of an antibody according to claim 1, an immune cell according to claim 9, an antibody drug conjugate according to claim 11, or a combination thereof, and

(Ii) A pharmaceutically acceptable carrier.

13. A polynucleotide encoding a polypeptide selected from the group consisting of:

(1) The antibody of claim 1, or

(2) The CAR construct of claim 8.

14. A vector comprising the polynucleotide of claim 13.

15. A method for the non-diagnostic in vitro detection of BCMA protein in a sample comprising the steps of:

(1) Contacting the sample with the antibody of claim 1 or the antibody drug conjugate of claim 11 in vitro;

16. A test plate comprising a substrate and a test strip comprising the antibody of claim 1 or the antibody drug conjugate of claim 11.

17. A kit of parts for the manufacture of a kit, the kit comprises the following components:

(1) A first container containing the antibody according to claim 1, and

(2) A second container containing a second antibody against the antibody of claim 1;

or the kit contains the assay plate of claim 16.