Antibodies that specifically bind to BCMA and their use

Abstract

An antibody that specifically binds to BCMA is disclosed. The antibody comprises a heavy chain variable domain VH, a light chain variable domain VL, and a chimeric antigen receptor that contains the sequence. In addition, the amino acid sequence, vector, host cell, and composition of the antibody or chimeric antigen receptor of the present invention are disclosed. Also disclosed is the use of the antibody or chimeric antigen receptor in the preparation of a drug for preventing or treating a disease.

Description

[Technical Field]

[0001] The present invention relates to an antibody that specifically binds to BCMA, and a chimeric antigen receptor constructed based on the antibody, and discloses an amino acid sequencer for the antibody, a cloning or expression vector, host cells, a method for producing the antibody, and the use of the antibody or chimeric antigen receptor. [Background technology]

[0002] Multiple myeloma (MM) is a malignant tumor characterized by the large-scale proliferation of clonal plasma cells. While treatment options for multiple myeloma have significantly advanced in recent years, the disease remains characterized by high morbidity and mortality. Therefore, new treatment methods for multiple myeloma are urgently needed.

[0003] In recent years, chimeric antigen receptor T cell (CAR-T) immunotherapy has achieved rapid advancements in the treatment of malignant hematological malignancies, and it is one of the most effective treatment methods for malignant tumors. A chimeric antigen receptor (CAR) is a fusion protein composed of a single-strand variable fragment (scFv), a hinge region, a transmembrane domain, and an intracellular signaling domain (costimulatory domain and T cell activation domain). The scFv of CAR-T cells is generally derived from a monoclonal antibody, which can circumvent the constraints of the major histocompatibility complex (MHC) for recognizing intact cell surface proteins.

[0004] A candidate target in multiple myeloma immunotherapy is B-cell maturation antigen (BCMA). BCMA is primarily expressed by plasma cells and some mature B cells, but not by most B cells or other tissues (B-cell Maturation Antigen Is a Promising Target for Adoptive T-cell Therapy of Multiple Myeloma, Clinical Cancer Research, April 15, 2013). Therefore, BCMA can be used as a target antigen for CAR-T cell therapy for MM. Currently, clinical trials both domestically and internationally have confirmed that CAR-T cell therapy has achieved favorable therapeutic effects in the treatment of multiple myeloma. However, most CAR-T cells currently used in clinical trials are mouse-derived single-chain antibody scFv, which can cause CAR-T cells to generate an in vivo anti-CAR immune response, leading to decreased proliferative capacity of CAR-T cells in vivo, unsustainable CAR-T cells in vivo, and unfortunately poor clinical efficacy (The novel anti-CD19 chimeric antigen receptors with humanized scFv (single-chain variable fragment) trigger leukemia cell killing), Cellular Immunology, March 14, 2016; Chimeric Antigen Receptor T-cell Therapies for Multiple Myeloma, blood, December 14, 2017). In addition, the affinity of scFv to the target antigen is also an important factor in determining whether CAR-T can effectively kill target cells.Therefore, the selection and removal of scFvs that can specifically and effectively recognize BCMA is extremely important for the construction of safe and effective CAR-T for the treatment of MM.

Summary of the Invention

Means for Solving the Problems

[0005] The present invention discloses an isolated antibody that specifically binds to BCMA and comprises a heavy chain variable domain (hereinafter abbreviated as VH) and a light chain variable domain (hereinafter abbreviated as VL). VH comprises VH-CDR1 containing the amino acid sequence of SEQ ID NO: 1 (GYTFX1X2YSMN), VH-CDR2 containing the amino acid sequence of SEQ ID NO: 2 (RINTX3SGX4PX5YADDFKG), and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 3 (SNDYX6YSLDX7), wherein X1 is selected from S, T, R; X2 is selected from R, S, H; X3 is selected from G, E, R; X4 is selected from A, T, V; X5 is selected from I, N; X6 is selected from N, L; X7 is selected from H, Y, F. VL comprises VL-CDR1 containing the amino acid sequence of SEQ ID NO: 4 (RASX8SVX9X 10 X 11 GX 12 X 13 X 14 X 15 Y), VL-CDR2 containing the amino acid sequence of SEQ ID NO: 5 (LASNVQT), and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 6 (X 16 QSRX 17 IPRT), wherein X8 is selected from R, E; X9 is selected from T, S; X 10 is selected from I, V; X 11 is selected from A, L; X 12 is selected from S, N; X 13 is selected from P, H; X 14 is selected from I, L; X 15 is selected from I, V; X 16 is selected from L, F; X 17 is selected from T, S.

[0006] In a specific embodiment, the antibody VH includes VH-CDR1 presented in the amino acid sequence of SEQ ID NO: 1, VH-CDR2 presented in the amino acid sequence of SEQ ID NO: 2, and VH-CDR3 presented in the amino acid sequence of SEQ ID NO: 3. The VL of the antibody includes VL-CDR1, presented in the amino acid sequence of SEQ ID NO: 4; VL-CDR2, presented in the amino acid sequence of SEQ ID NO: 5; and VL-CDR3, presented in the amino acid sequence of SEQ ID NO: 6.

[0007] The present invention discloses isolated antibodies that specifically bind to BCMA, comprising VH and VL, wherein VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, VL comprises VL-CDR1, VL-CDR2, and VL-CDR3, VH-CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, and 9, VH-CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, and 13, VH-CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, and 16, VL-CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18, and 19, VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 20, and VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 22.

[0008] In a specific embodiment, VH-CDR1 is selected from the amino acid sequence of the group consisting of SEQ ID NOs: 7, 8, and 9; VH-CDR2 is selected from the amino acid sequence of the group consisting of SEQ ID NOs: 10, 11, 12, and 13; VH-CDR3 is selected from the amino acid sequence of the group consisting of SEQ ID NOs: 14, 15, and 16; VL-CDR1 is selected from the amino acid sequence of the group consisting of SEQ ID NOs: 17, 18, and 19; VL-CDR2 includes the amino acid sequence of SEQ ID NO: 20; and VL-CDR3 includes the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 22.

[0009] In a specific embodiment, the antibody is 1) VH including VH-CDR1 presented in SEQ ID NO: 7, VH-CDR2 presented in SEQ ID NO: 10, and VH-CDR3 presented in SEQ ID NO: 14, and VL including VL-CDR1 presented in SEQ ID NO: 17, VL-CDR2 presented in SEQ ID NO: 20, and VL-CDR3 presented in SEQ ID NO: 21, 2) VH including VH-CDR1 presented in SEQ ID NO: 8, VH-CDR2 presented in SEQ ID NO: 11, and VH-CDR3 presented in SEQ ID NO: 15, and VL including VL-CDR1 presented in SEQ ID NO: 18, VL-CDR2 presented in SEQ ID NO: 20, and VL-CDR3 presented in SEQ ID NO: 21, 3) VH including VH-CDR1 presented in SEQ ID NO: 8, VH-CDR2 presented in SEQ ID NO: 12, and VH-CDR3 presented in SEQ ID NO: 14, and VL including VL-CDR1 presented in SEQ ID NO: 17, VL-CDR2 presented in SEQ ID NO: 20, and VL-CDR3 presented in SEQ ID NO: 21, 4) VH including VH-CDR1 presented in SEQ ID NO: 7, VH-CDR2 presented in SEQ ID NO: 10, and VH-CDR3 presented in SEQ ID NO: 14, and VL including VL-CDR1 presented in SEQ ID NO: 17, VL-CDR2 presented in SEQ ID NO: 20, and VL-CDR3 presented in SEQ ID NO: 22, or 5) VH includes VH-CDR1 presented in SEQ ID NO: 9, VH-CDR2 presented in SEQ ID NO: 13, and VH-CDR3 presented in SEQ ID NO: 16, and VL includes VL-CDR1 presented in SEQ ID NO: 19, VL-CDR2 presented in SEQ ID NO: 20, and VL-CDR3 presented in SEQ ID NO: 21.

[0010] In a specific embodiment, the VH on the antibody contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32, or contains an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% identity thereto.

[0011] In a specific embodiment, the VL on the above antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 35, 36, 37, 38, 39, 40, 41, 42, and 43, or comprises an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99% identity thereto.

[0012] In a specific embodiment, the antibody 1) VH presented by SEQ ID NO: 23 and VL presented by SEQ ID NO: 33, 2) VH presented by SEQ ID NO: 24 and VL presented by SEQ ID NO: 33, 3) VH presented by SEQ ID NO: 25 and VL presented by SEQ ID NO: 35, 4) VH presented by SEQ ID NO: 26 and VL presented by SEQ ID NO: 36, 5) VH presented by SEQ ID NO: 27 and VL presented by SEQ ID NO: 37, 6) VH presented by SEQ ID NO: 28 and VL presented by SEQ ID NO: 38, 7) VH presented by SEQ ID NO: 29 and VL presented by SEQ ID NO: 33, 8) VH presented by SEQ ID NO: 30 and VL presented by SEQ ID NO: 39, 9) VH presented by SEQ ID NO: 31 and VL presented by SEQ ID NO: 40, 10) VH presented by SEQ ID NO: 32 and VL presented by SEQ ID NO: 41, 11) VH presented by SEQ ID NO: 28 and VL presented by SEQ ID NO: 42, or 12) VH presented by SEQ ID NO: 29 and VL presented by SEQ ID NO: 43.

[0013] In specific embodiments, the antibody contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, and 56, or contains an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% identity thereto, or Antibodies are composed of, or essentially composed of, the above sequence.

[0014] In a specific embodiment, the antibody of the present invention is a single-chain antibody scFv, in which the VH and VL are linked by a linker peptide chain rich in glycine and serine, and the order of VH and VL is interchangeable.

[0015] In specific embodiments, the antibody of the present invention is a chimeric or humanized antibody.

[0016] In another embodiment, the present invention further discloses a chimeric antigen receptor that targets BCMA, comprising 1) the scFv of the present invention, 2) a hinge region, 3) a transmembrane region, 4) a costimulatory domain, and 5) a CD3ζ intracellular signaling domain. Herein, the costimulatory domain is selected from CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD-2, CD7, LIGHT, NKG2C, B7-H3, or any combination thereof, preferably the costimulatory domain is selected from 4-1BB, where 4-1BB comprises the amino acid sequence of SEQ ID NO: 57, and the CD3ζ intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 58.

[0017] In specific embodiments, the hinge region and transmembrane domain are selected from the hinge region and transmembrane domain of IgG1, IgG4, CD8α, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1, or CD34.

[0018] The example antibody sequences are shown in Table 1 and the sequence listing.

[0019] [Table 1]

[0020] The CDR sequences of the exemplary antibodies are shown in Table 2 and the sequence listing.

[0021] [Table 2]

[0022] [Table 3]

[0023] [Table 4]

[0024] [Table 5]

[0025] [Table 6]

[0026] [Table 7]

[0027] [Table 8]

[0028] The present invention discloses isolated nucleic acid molecules encoding the aforementioned antibodies or chimeric antigen receptors.

[0029] The present invention also discloses cloning or expression vectors comprising the aforementioned nucleic acid molecules, wherein the vector is selected from one or more of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, and retroviral vectors.

[0030] The present invention also discloses host cells comprising the aforementioned nucleic acid molecules or vectors, where the host cell is a T lymphocyte, B lymphocyte, natural killer cell, dendritic cell, macrophage, cytotoxic T cell, tumor-infiltrating T cell, or Treg (regulatory T cell).

[0031] The present invention also discloses compositions or kits comprising the aforementioned antibody, the aforementioned chimeric antigen receptor, the aforementioned nucleic acid molecule, the aforementioned vector, at least one of the aforementioned host cells, and pharmaceutically acceptable excipients(s), diluents(s), or carriers.

[0032] The present invention also discloses the use of the aforementioned antibodies, the aforementioned chimeric antigen receptors, the aforementioned nucleic acid molecules, the aforementioned vectors, the aforementioned host cells, or the aforementioned compositions in the preparation of pharmaceuticals for the prevention or treatment of B cell-associated neoplasms, autoimmune diseases, and infectious diseases caused by viruses or bacteria.

[0033] The present invention discloses a method for treating a disease, wherein an effective amount of the aforementioned antibody, the aforementioned chimeric antigen receptor, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned host cell, or the aforementioned composition is administered to a subject in need of treatment, the subject suffering from at least one of B cell-associated neoplasms, autoimmune diseases, or infectious diseases caused by viruses or bacteria.

[0034] In one embodiment, B-cell related neoplasms are at least one selected from, for example, acute leukemias such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia; chronic leukemias such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia; polycythemia vera; lymphoma; mantle cell lymphoma; diffuse large B-cell lymphoma; Hodgkin's disease; non-Hodgkin lymphoma; multiple myeloma; Waldenström macroglobulinemia; heavy chain disease; myelodysplastic syndrome; hairy cell leukemia; and myelodysplasia. [Brief explanation of the drawing]

[0035] [Figure 1] Figure 1 shows a schematic diagram of a CAR molecule. [Figure 2] Figure 2 shows a schematic diagram of the plasmid vector. The plasmid used for lentivirus preparation is the pLenti plasmid, with the upstream promoter of the target gene being EF-1α, and downstream of the promoter being, in order, the signal peptide, the linker region, and the CAR structural sequence. [Figure 3] Figure 3 shows the transduction efficiency in T cells of lentiviruses possessing a CAR with an scFv that specifically recognizes BCMA. [Figure 4] Figure 4 shows flow assay plots of BCMA expression on the surface of NCI-H929 and K562 cells. [Figure 5] Figure 5 shows a graph of the cell death effect of CAR-T cells containing scFv-01 (01-CAR-T) against target cells. 01-CAR-T cells refer to CAR-T cells containing scFv-01, C-CAR-T cells are the positive control, and the T cell therapy group is the negative control. [Figure 6] Figure 6 shows a graph of the cell-killing effect of CAR-T (04-CAR-T) containing scFv-04 on target cells. [Figure 7] Figure 7 shows a graph of the cell-killing effect of CAR-T (05-CAR-T) containing scFv-05 on target cells. [Figure 8] Figure 8 shows a graph of the cell death effect of CAR-T (06-CAR-T) containing scFv-06 on target cells. [Figure 9] Figure 9 shows a graph of the cell-killing effect of CAR-T (07-CAR-T) containing scFv-07 on target cells. [Figure 10] Figure 10 shows a graph of the cell death effect of CAR-T (08-CAR-T) containing scFv-08 on target cells. [Figure 11] Figure 11 shows a graph of the cell death effect of CAR-T (09-CAR-T) containing scFv-09 on target cells. [Figure 12] Figure 12 shows a graph of the cell death effect of CAR-T (10-CAR-T) cells containing scFv-10 on target cells. [Figure 13] Figure 13 shows a graph of the cell-killing effect of CAR-T (11-CAR-T) containing scFv-11 on target cells. [Figure 14] Figure 14 shows a graph of the cell death effect of CAR-T (12-CAR-T) containing scFv-12 on target cells. [Figure 15] Figure 15 shows a graph of the cell-killing effect of CAR-T (13-CAR-T) containing scFv-13 on target cells. [Figure 16] Figure 16 shows a graph of the cell death effect of CAR-T (14-CAR-T) containing scFv-14 on target cells. [Figure 17] Figure 17 shows a graph of the cell-killing effect of CAR-T (15-CAR-T) containing scFv-15 on target cells. [Figure 18] Figure 18 shows a normalized graph of the CAR-T cell death effect of the present invention on target cells. [Figure 19] Figure 19 shows a graph of cytokine expression of CAR-T (01-CAR-T) cells containing scFv-01 in response to target cell stimulation. [Modes for carrying out the invention]

[0036] The present invention is further described below by specific embodiments. Unless otherwise specified, terms used herein have the same meaning as commonly understood by those skilled in the art.

[0037] As used herein, the term “antigen” refers to a molecule that elicits an immune response that may involve antibody production or activation of specific immune cells. Those skilled in the art will understand that any macromolecule, encompassing all proteins or peptides, can be used as an antigen. Antigens may be recombinant or derived from genomic DNA. Those skilled in the art will understand that any DNA encompassing a nucleotide sequence or portion of a nucleotide sequence that codes for a protein that elicits an immune response and is used herein as “antigen.” Furthermore, those skilled in the art will understand that antigens do not necessarily have to be exclusively encoded by a full-length nucleotide sequence of a gene. It will be immediately apparent that this disclosure encompasses, but is not limited to, the use of more than one partial nucleotide sequence of a gene, and that these nucleotide sequences are arranged in different combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that antigens do not necessarily have to be encoded by a “gene,” and that antigens may be generated, synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0038] As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be intact immunoglobulins of natural or recombinant origin, or they may be the immunoreactive portion of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies of the present invention may exist in various forms, including, for example, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid, mutant, and graft antibodies; the forms of antibodies of the present invention also include full-length antibodies, antibody fragments, e.g., Fab, Fab', F(ab')2, Fv, scFv, di-scFv, tri-scFv, Fd, and other antibody fragments that retain antigen-binding function; they may also be dimeric (diabody) or trimer (tribody) structures. Typically, a fragment should include an antigen-binding fragment, which typically includes VL and VH, but does not necessarily have to include both. For example, a so-called Fd antibody fragment consists only of the VH and CH1 domains, but still retains some of the antigen-binding function of an intact antibody. The term “antibody,” as an immunoglobulin or a fragment or derivative thereof, encompasses any polypeptide containing an antigen-binding site, whether produced in vitro or in vivo. The VH or VL region can be further subdivided into a hypervariable region called the complementarity-determining region (CDR) and a more conserved region called the scattered framework region (FWR). The variable regions of the heavy and light chains contain binding domains that interact with the antigen. CDRs on the heavy chain are abbreviated as VH-CDR, e.g., VH-CDR1, VH-CDR2, VH-CDR3, and CDRs on the light chain are abbreviated as VL-CDR, e.g., VL-CDR1, VL-CDR2, VL-CDR3. The CDRs of the antibodies and antigen-binding fragments disclosed in this invention are determined or identified by Kabat numbering.

[0039] As used herein, the terms “single-chain variable region fragment,” “single-chain antibody,” or “scfv” refer to antibodies formed by recombinant DNA technology in which the heavy and light chain variable regions of immunoglobulins are linked by amino acid peptide segments (linkers). Various methods for producing single-chain antibodies are known, including those described in U.S. Patent No. 4,694,778; Bird (1988), Science, Vol. 242: pp. 423–442; Huston et al. (1988), Proceedings of the National Academy of Sciences, Vol. 85: pp. 5879–5883; Ward et al. (1989), Nature, Vol. 334: pp. 54454; and Skerra et al. (1988), Science, Vol. 242: pp. 1038–1041. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. CDRs on the heavy chain are abbreviated as VH-CDRs, e.g., VH-CDR1, VH-CDR2, VH-CDR3, and CDRs on the light chain are abbreviated as VL-CDRs, e.g., VL-CDR1, VL-CDR2, VL-CDR3. The CDRs of the antibodies and antigen-binding fragments disclosed in this invention are determined or identified by Kabat numbering.

[0040] As used in this disclosure, the term “chimera” means an antibody or antibody polypeptide in which a portion of the heavy chain originates from one species and another portion of the heavy chain originates from another species. In exemplary examples, a chimeric antibody may include a constant region derived from humans and a variable region derived from a non-human animal such as a camelid. In certain embodiments, the non-human animal is a mammal, such as a camelid, mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0041] As used in this disclosure, the term “humanized” means an antibody or antibody polypeptide comprising a CDR derived from a non-human animal, a FWR region derived from a human, and, where applicable, a constant region derived from a human.

[0042] As used in this disclosure, "BCMA" may be derived from humans, and exemplary sequences of human BCMA include the human BCMA protein (Genbank accession No.: BAB60895.1).

[0043] As used in this invention, the term "BCMA" is intended to encompass any form of BCMA. For example, 1) a naturally occurring, unprocessed BCMA molecule, a "full-length" BCMA chain, or a naturally occurring BCMA variant, including, for example, splice variants or alleles; 2) any form of BCMA produced after intracellular processing; or 3) the full-length, fragment (e.g., truncated form, extracellular / transmembrane domain) or modified forms thereof (e.g., mutant form, glycosylated / PEGylated, His-tagged / immunofluorescence fusion form) of a BCMA subunit produced by recombinant methods.

[0044] In this invention, the term "targeting BCMA" refers to the ability to specifically bind to BCMA (e.g., human BCMA). For example, a BCMA-targeting chimeric antigen receptor refers to a chimeric antigen receptor that can specifically bind to BCMA; for example, a BCMA-targeting scFv refers to a single-chain antibody that can specifically bind to BCMA (e.g., human BCMA).

[0045] As used in this disclosure, the terms “specific binding” or “specifically binding” refer to a non-random binding reaction between two molecules, such as a binding reaction between an antibody and an antigen. In certain embodiments, the antibody polypeptide provided by this disclosure has a binding affinity (K) to human BCMA. D )≦10 -6 M (for example, ≤ 5 × 10) -7 M, ≤ 2 × 10 -7 M, ≤10 -7 M, ≤ 5 × 10 -8 M, ≤ 2 × 10 -8 M, ≤10 -8 M, ≤ 5 × 10 -9 M, ≤ 4 × 10 -9 M, ≤ 3 × 10 -9 M, ≤ 2 × 10 -9 M, or ≤10-9 M) specifically binds. K used in this disclosure D The ratio of the dissociation rate to the binding rate (K) off / K on ) refers to a method which may be determined by any conventional method known in the art, and includes, but is not limited to, surface plasmon resonance, microscale thermophoresis, high-performance liquid chromatography-mass spectrometry, and flow cytometry (e.g., FACS). In certain embodiments, K D This can preferably be determined by a flow cytometer.

[0046] As used herein, the term “vector” refers to a molecular tool for the transport, transduction, and expression of an exogenous gene (e.g., the polynucleotide of the present invention) containing a desired gene in a target cell, which provides a suitable nucleotide sequence, i.e., a promoter, for initiating transcription in the target cell. A vector may contain an isolated nucleic acid and may be used to deliver the isolated nucleic acid into the cell. Numerous vectors are known in the art and include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmids and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentiviral vectors, and the like.

[0047] As used herein, “expression vector” means a vector containing recombinant polynucleotides that include an expression control sequence operably ligated to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by host cells or in an in vitro expression system. Expression vectors include all known in the art that incorporate recombinant polynucleotides, such as plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0048] As used herein, “cloning vector” refers to a DNA molecule capable of autonomous replication within a host cell, such as a plasmid, cosmid, or phage. Cloning vectors typically contain one or more restriction endonuclease recognition sites and marker genes, in which exogenous DNA sequences are inserted in a defined manner at the restriction endonuclease recognition sites without loss of the vector’s essential biological function, and the marker genes are suitable for the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.

[0049] The host cell may be any prokaryotic or eukaryotic cell containing a cloning vector or expression vector, and includes prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene on the host cell's chromosome or genome. Suitable mammalian host cells include myeloma cells, such as SP2 / 0 cells and NSO cells, as well as Chinese hamster ovary (CHO) cells, hybridoma cell lines, and other mammalian host cells that can be used to express antibodies. Special transgenic animals with modified immune systems can also be used to produce antibodies.

[0050] As used herein, “identity” refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the positions on each sequence aligned for comparison purposes. The molecules are identical at a given position when the positions of the sequences being compared are occupied by the same base. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Identity between two sequences can be calculated by various alignment algorithms and / or programs, including those available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wisconsin), and FASTA or BLAST, which can be used, for example, with default settings. For example, a person skilled in the art can expect polypeptides having at least 70%, 85%, 90%, 95%, 98%, or 99% identity with respect to the particular polypeptides described herein, and preferably polypeptides exhibiting substantially the same function, as well as polynucleotides encoding the aforementioned polypeptides.

[0051] As used herein, “isolated” means modified or removed from its natural state. For example, nucleic acids or peptides that are naturally present in living animals are not “isolated,” but the same nucleic acids or peptides that have been partially or completely separated from the material that coexists with them in their natural state are “isolated.” Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment, for example, within a host cell.

[0052] Unless otherwise specified, the nucleic acid molecules of proteins or nucleotide sequences that “encode” the amino acid sequences of proteins as used herein encompass all nucleotide sequences that encode the same amino acid sequence, as well as their degenerate counterparts. A nucleotide sequence may also contain one or more introns.

[0053] As used herein, the term "mutant" refers to a polypeptide variant obtained by substituting at least one residue or by adding at least one amino acid residue to the N-terminus or C-terminus of the amino acid sequence of the parent molecule.

[0054] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, horses, cattle, chickens, rats, mice, amphibians, reptiles, and the like. Unless otherwise indicated, the terms "patient" and "subject" are used interchangeably. In this invention, the preferred subject is human.

[0055] As used herein, the term “treat” means administering to a subject an effective amount of cells having a polynucleotide sequence of a target gene modified ex vivo in accordance with the method described herein, such that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, for example, a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, a reduction in one or more symptoms, a reduction in the severity of the disease, stabilization of the disease state (i.e., no exacerbation), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete remission), whether detectable or undetectable. Treatment may mean extending survival compared to the expected survival without treatment. Therefore, it will be understood by those skilled in the art that treatment may improve the state of the disease but may not result in a complete cure of the disease. As used herein, the term “treat” includes prevention. Alternatively, treatment is “effective” if the progression of the disease is reduced or halted. "Treatment" can also mean extending survival compared to the expected survival rate without treatment. Patients requiring treatment include those diagnosed with disorders related to the expression of polynucleotide sequences, and those who may develop such disorders due to genetic predisposition or other factors.

[0056] As used herein, the term “autoimmune disease” is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to autoantigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, diabetes mellitus (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthritis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, and similar conditions.

[0057] Where a numerical limit or range is claimed herein, the endpoints are inclusive. In addition, all values ​​and subranges within a numerical limit or range are specifically inclusive. [Examples]

[0058] Example 1: Synthesis of the scFv gene In specific embodiments of the present invention, the nucleic acids encoding the scFv shown in Table 2 can be synthesized by genetic engineering. The nucleic acid fragments encoding the scFv of the present invention were synthesized at Beijing Biomed Gene Technology. The scFv fragment contains VH, VL, and a linker region that links the VH domain and the VL domain together to form an antigen-binding site that specifically recognizes BCMA. A single-chain antibody is generally a continuous chain of amino acids encoded by a single nucleotide chain, and the scFv can be further modified by amino acid deletions, insertions, substitutions, additions, and other modifications known in the art. In addition, methods for introducing the above modifications onto nucleic acid sequences according to some amino acid sequence, such that the modifications of scFv are preferably carried out at the nucleic acid level, are also known to those skilled in the art.

[0059] Example 2. Construction of CAR molecules and lentiviral packaging. As shown in Figure 1, the single-chain antibody scFv, hinge region, transmembrane domain, CD28 or 4-1BB costimulatory domain, and CD3ζ domain combine to form the CAR structure of Example 1. The hinge region and transmembrane domain are selected from CD8α.

[0060] 1) The scFv gene targeting BCMA was synthesized by gene synthesis (Beijing Biomed Gene Technology). 2) Using an existing CAR plasmid as a template, nucleic acid fragments containing the CD8α hinge region, CD8α transmembrane region, 4-1BB intracellular region (corresponding to NP_001552.2), and CD3ζ intracellular region (corresponding to NP_000725.1) of the CAR molecule were cloned by PCR. 3) Using the gene obtained in step 1) and the nucleic acid fragment obtained in step 2) as templates, a complete nucleic acid fragment of the BCMA-targeting CAR was cloned by PCR. 4) The complete nucleic acid fragment obtained in step 3) was inserted into the lentiviral vector pLenti6.3 / V5 (Thermo Fisher, Waltham, MA, USA) by restriction digestion and ligation to obtain a lentiviral transfer plasmid containing the gene for the BCMA-targeting CAR (Figure 2). 5) Lentiviral packaging plasmids pLP / VSVG, pLP1 / MDK, pLP2 / RSK (Thermo Fisher, Waltham, MA, USA) and the transfer plasmid obtained in step 4) were transfected into HEK293T cells with Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA), and the culture medium was collected after 48 hours. After removing cell debris by centrifugation at 300 g, the cells were centrifuged at 25,000 rpm for 3 hours using an ultracentrifuge. The precipitate was dissolved in 1 mL of physiological saline. This was the desired lentiviral vector.

[0061] Example 3. Preparation of CAR-T cells T cells were isolated from peripheral blood mononuclear cells of healthy volunteers (Miaotong (Shanghai) Biological Technology Co., Ltd., China) using CD3 / CD28 dynabeads (Thermo Fisher). The isolated and purified T cells (at this point, the T cells were attached to CD3 / CD28 dynabeads) were measured in 1.0 × 10⁶ units. 6 Cells were cultured at a concentration of 1 / mL in X-VIVO15 medium (Lonza, Switzerland) containing IL-2 (Shandong Jintai Biological Engineering, China) (500 IU / mL). After 48 hours of culture, T cells were infected with the above lentiviral vector. 24 hours after viral infection, the cells were centrifuged, the medium was changed, and fresh X-VIVO15 containing IL-2 (500 IU / mL) was added to continue the culture. After 8 days of cell culture, all cells in the culture system were collected, the DynaBeads were removed from the culture system using a magnetic rack, and the T cells were centrifuged and counted.

[0062] Example 4. Analysis of lentiviral transduction efficiency 1.0 × 10 6 Cells were isolated from prepared CAR-T cells, resuspended in 200 μL of DPBS, and the supernatant was removed after centrifugation at 1000 g. The cell precipitate was resuspended in 100 μL of DPBS, 3 μL of antibody was added, and the mixture was incubated at room temperature for 20 minutes. The cells were then resuspended in 200 μL of DPBS, and the supernatant was removed after centrifugation at 1000 g. The cells were resuspended in 100 μL of DPBS, and the CAR content in each cell group was detected by flow cytometry (NovoCyte2060R, ACEA Biosciences, San Diego, CA, USA). As shown in Figure 3, the transduction efficiency of each CAR gene ranged from 35 to 65% at lentivirus infection MOI = 0.5.

[0063] Example 5. Detection of BCMA expression on the surface of target cells In this example, the expression of BCMA on the surface of human myeloma cell line NCI-H929 and human leukemia cell line K562 was cited as an example.

[0064] Both NCI-H929 and K562 cells were purchased from ATCC. NCI-H929 and K562 cells were cultured to the active proliferation stage, and 1.0 × 10⁶ cells were obtained. 6 Cells were isolated from each cell type. The cells were resuspended in 200 μL of DPBS, centrifuged at 1000 g, and the supernatant was removed. The cell precipitate was resuspended in 100 μL of DPBS, 5 μL of fluorescently labeled BCMA antibody (Milteny Biotech, USA) was added, and the mixture was incubated at room temperature for 20 minutes. The cells were then resuspended in 200 μL of DPBS, and the supernatant was removed after centrifuging at 1000 g. The cells were resuspended in 100 μL of DPBS, and the ratio of BCMA expression in the two cell types was detected using a flow cytometer (NovoCyte2060R, ACEA Biosciences, San Diego, CA, USA). As shown in Figure 4, the surface expression level of BCMA was high in NCI-H929 cells, while K562 cells expressed almost no BCMA.

[0065] Example 6. Efficiency of BCMACAR-T during target cell death In this example, we used the detection of the cell death efficiency of CAR-T cells containing a BCMA-specific scFv, CD8α hinge region, CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ domain against BCMA-positive target cells as an example.

[0066] 2 x 10 6 NCI-H929 target cells (ATCC) or BCMA expression-negative K562 cells were resuspended in 1 mL of saline, 5 μL of calcein AM (concentration 1 μg / μL, Thermo Fisher, USA) were added, gently mixed, and then incubated in a 37°C water bath for 5 minutes to label the target cells. Excess dye was removed by washing twice with 10 mL of saline, and the cells were resuspended in 1 mL of saline for counting. Each cell was 1 × 10⁶. 5NCI-H929 or K562 cells labeled on were added to each well of a 48-well cell culture plate (Corning, NY, USA), and various CAR-T cells were added according to an E:T ratio of 5:1. The plates were placed in a 5% CO2 cell incubator at 37°C for 6 hours, and the culture supernatant was taken for detection. The fluorescence value of the cell supernatant was detected by a fluorescence microplate reader (Varioscan Lux, Thermo Fisher) (excitation wavelength: 495 nm, emission wavelength: 515 nm).

[0067] Figures 5 to 17 show that CAR-T proteins possessing BCMA-targeting scFv, a co-stimulatory signaling domain, and a CD3ζ signaling domain can all effectively kill NCI-H929 cells by recognizing the BCMA target protein. These CAR-T proteins are more effective against NCI-H929 cells than Bluebird Bio's BCMA-targeting CAR-T (C-CAR-T, sequence reference Seq No. 15 in International Publication No. 2016 / 014789), but they cannot effectively kill BCMA-negative K562 cells. This indicates that these CAR-T proteins specifically recognize BCMA via scFv, activating the CAR-T and enabling the CAR-T to kill tumor cells.

[0068] Figure 18 shows that most CAR-T cells (with the exception of O5-CAR-T) exhibit higher cell death efficiency against NCI-H929 cells compared to C-CAR-T cells, indicating that these CAR-T cells are more reactive against tumor cells.

[0069] Example 7. Cytokine expression of BCMACAR-T stimulated by target cells In this example, we described the detection of cytokine interferon-gamma (IFN-γ) expression in BCMA-positive target cells (NCI-H929) and BCMA-negative cells (K562) after stimulation with a CAR-T receptor containing scFv-01, which specifically recognizes BCMA, as well as the CD8α hinge region, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3ζ domain.

[0070] T cells and CAR-T cells were co-incubated with NCI-H929 cells or K562 cells for 6 hours in a 37°C 5% CO2 incubator (effector cell ratios were 10:1, 5:1, and 1:1, respectively). The supernatant was collected, and cytokine expression was detected using the CBA Human Th1 / Th2 / Th17 Cytokine Kit (BD Biosciences).

[0071] Figure 19 shows that BCMACAR-T cells co-incubated with BCMA-positive target cells (NCI-H929) exhibited significantly enhanced IFN-γ induction compared to T cells. Simultaneously, an increase in the ratio of effector cells to NCI-H929 cells enhanced the cytokine release effect. However, after co-incubation of BCMACAR-T cells with BCMA-negative cells K562, the IFN-γ induction effect did not differ significantly from that of the T cell group. The above experimental results indicate that the CAR-T cells of the present invention can specifically recognize BCMA target antigens and thereby produce cytokines involved in cytotoxicity.

Claims

1. An isolated antibody that specifically binds to BCMA, The aforementioned antibody is 1) Heavy chain variable domain VH including VH-CDR1 containing SEQ ID NO: 7, VH-CDR2 containing SEQ ID NO: 10, and VH-CDR3 containing SEQ ID NO: 14, and light chain variable domain VL including VL-CDR1 containing SEQ ID NO: 17, VL-CDR2 containing SEQ ID NO: 20, and VL-CDR3 containing SEQ ID NO: 21, 2) VH including VH-CDR1 containing SEQ ID NO: 8, VH-CDR2 containing SEQ ID NO: 11, and VH-CDR3 containing SEQ ID NO: 15, and VL-CDR1 containing SEQ ID NO: 18, VL-CDR2 containing SEQ ID NO: 20, and VL-CDR3 containing SEQ ID NO: 21, or 3) VH including VH-CDR1 containing SEQ ID NO: 8, VH-CDR2 containing SEQ ID NO: 12, and VH-CDR3 containing SEQ ID NO: 14, and VL including VL-CDR1 containing SEQ ID NO: 17, VL-CDR2 containing SEQ ID NO: 20, and VL-CDR3 containing SEQ ID NO: 21, Antibodies containing antibodies.

2. 1) VH containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 23, and VL containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 33, 2) VH containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 24, and VL containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 33, 3) VH containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 25, and VL containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 35, 4) VH containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 27, and VL containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 37, 5) VH containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 28, and VL containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 38, 6) VH containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 29, and VL containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 33, 7) VH containing an amino acid sequence of SEQ ID NO: 30, or having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto, and VL containing an amino acid sequence of SEQ ID NO: 39, or having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto, 8) VH containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 32, and VL containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 41, 9) VH containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 28, and VL containing an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with respect to SEQ ID NO: 42, or 10) The antibody according to claim 1, comprising VH, which contains an amino acid sequence of SEQ ID NO: 29, or having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto, and VL, which contains an amino acid sequence of SEQ ID NO: 43, or having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

3. The antibody according to claim 1, comprising an amino acid sequence presented in SEQ ID NOs: 44, 45, 47, 49, 50, 51, 52, 54, 55, or 56, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

4. The antibody according to any one of claims 1 to 3, wherein the antibody is scFv.

5. A chimeric antigen receptor that targets BCMA, comprising the scFv described in claim 4, and optionally further comprising at least one of a hinge region, a transmembrane region, a costimulatory domain, and an intracellular signaling domain.

6. The chimeric antigen receptor according to claim 5, wherein the co-stimulatory domain is selected from CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, or any combination thereof.

7. The chimeric antigen receptor according to claim 6, wherein the costimulatory domain is 4-1BB, and 4-1BB comprises the amino acid sequence of SEQ ID NO:

57.

8. The chimeric antigen receptor according to claim 6, wherein the intracellular signaling domain is CD3ζ, and the CD3ζ comprises the amino acid sequence of SEQ ID NO:

58.

9. An isolated nucleic acid molecule encoding an antibody according to any one of claims 1 to 4 or a chimeric antigen receptor according to any one of claims 5 to 8.

10. A vector comprising the nucleic acid molecule described in claim 9.

11. A host cell comprising the nucleic acid molecule described in claim 9 or the vector described in claim 10.

12. A composition or kit comprising an antibody according to any one of claims 1 to 4, a chimeric antigen receptor according to any one of claims 5 to 8, a nucleic acid molecule according to claim 9, a vector according to claim 10, at least one host cell according to claim 11, and a pharmaceutically acceptable excipient, diluent, or carrier.

13. Use of an antibody according to any one of claims 1 to 4, a chimeric antigen receptor according to any one of claims 5 to 8, a nucleic acid molecule according to claim 9, a vector according to claim 10, a host cell according to claim 11, or a composition according to claim 12 in the preparation of a pharmaceutical for the prevention or treatment of B cell-associated neoplasms, autoimmune diseases, or infectious diseases caused by viruses or bacteria.

14. The use according to claim 13, wherein the B-cell-related neoplasm is at least one selected from acute leukemia, chronic leukemia, polycythemia vera, lymphoma, multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

15. The acute leukemia is selected from acute lymphoblastic leukemia and acute myeloid leukemia, The aforementioned chronic leukemia is selected from chronic lymphocytic leukemia and chronic myeloid leukemia. The aforementioned lymphoma is selected from Hodgkin's disease and non-Hodgkin's lymphoma. The use described in claim 14.

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