Chimeric antigen receptors targeting bcma and uses thereof

Abstract

The present application relates to a chimeric antigen receptor targeting BCMA and use thereof, in more detail, to an antibody targeting BCMA, a chimeric antigen receptor comprising the above antibody, a chimeric antigen receptor-T cell (CAR-T) expressing the above chimeric antigen receptor. It is confirmed that the chimeric antigen receptor targeting BCMA and the chimeric antigen receptor-T cell prepared in the present application specifically bind to BCMA as an antigen, and the chimeric antigen receptor-T cell binding to BCMA is activated. Also, it is confirmed that the chimeric antigen receptor-T cell of the present application effectively kills cells expressing BCMA, and thus the chimeric antigen receptor targeting BCMA and the chimeric antigen receptor-T cell of the present application can be usefully used as a composition for preventing or treating a disease associated with B cells or expression of BCMA.

Description

Technical Field

[0001] This invention relates to chimeric antigen receptors targeting BCMA and their uses, and more specifically, to antibodies targeting BCMA, chimeric antigen receptors comprising the antibodies, and chimeric antigen receptor-T cells (CAR-T) expressing the chimeric antigen receptors. Background Technology

[0002] Chimeric antigen receptor (CAR) T cells are funded by the generation of chimeric proteins that exhibit specific anti-cancer activity. These proteins combine antibody-based specificity for target antigens with intracellular domains that activate T cell receptors. Typically, chimeric antigen receptors comprise extracellular antigen-binding domains, transmembrane domains, and intracellular signal transduction domains. The extracellular antigen-binding domain may contain a single-chain variable fragment (scFv) targeting a known tumor antigen.

[0003] Chimeric antigen receptors can be expressed on the surface of T cells, which are immune effector cells, using gene transfection technology. When the chimeric antigen receptor expressed on the surface of T cells binds to targeted tumor antigens, the chimeric antigen receptor can activate T cells to initiate a specific anti-tumor response in an antigen-dependent manner.

[0004] On the other hand, B-cell maturation antigens (BCMA), known to be CD269 or TNFRSF17, are members of the tumor necrosis factor receptor family. BCMA has been reported to bind to B-cell activating factor receptor (BAFF) and B-cell proliferation-inducing ligand (APRIL) to promote B-cell survival at different developmental stages. Abnormal signal transduction leads to abnormal B-cell proliferation, which may cause autoimmune diseases such as multiple myeloma and tumorigenesis (Rickert, et al., Immunological Reviews, 2011, Vol. 244: 115-133).

[0005] Multiple myeloma is a type of blood cancer caused by the abnormal differentiation and proliferation of plasma cells. This disease generates tumors and induces pain by dissolving bone. Furthermore, multiple myeloma invades the bone marrow, reducing the levels of white blood cells, red blood cells, and platelets, thereby increasing the risk of anemia, infection, and bleeding. Additionally, myeloma cells produce M protein, an abnormal immune protein, which increases its concentration in the blood, causing hyperviscosity syndrome or kidney damage.

[0006] Some treatments for multiple myeloma are similar to those for other cancers, such as chemotherapy or radiation therapy, stem cell or bone marrow transplantation, targeted therapy, or biological therapy. The practical clinical benefit of antibody-based cell immunotherapy for hematologic malignancies, particularly for patients with B-cell non-Hodgkin lymphoma, has been confirmed, but most patients experience relapse or secondary rejection. Therefore, there is a need for immunotherapy agonists for the treatment of multiple myeloma. To this end, the use of chimeric antigen receptor-T cells is being investigated (Ellebrecht et al., Science 353:179-184, 2016; Carpenter et al., Clin Cancer Res, 19(8):2048-2060, 2013; WO2016-014789; WO 2016 / 014565; WO 2013 / 154760). Summary of the Invention

[0007] Technical issues

[0008] In this invention, as a result of extensive efforts to develop therapeutic agents for B-cell-related diseases such as multiple myeloma, antibodies targeting BCMA were screened. By humanizing the screened anti-BCMA antibodies, chimeric antigen receptors (CARs) and CAR-T cells targeting BCMA were ultimately prepared. It was confirmed that the prepared BCMA-targeting CAR-T cells bind to BCMA and effectively kill BCMA-expressing tumor cells.

[0009] Therefore, the object of the present invention is to provide an antibody targeting BCMA and a chimeric antigen receptor comprising the antibody.

[0010] Another object of the present invention is to provide a polynucleotide encoding a chimeric antigen receptor targeting BCMA, a vector comprising the polynucleotide, and an immune effector cell expressing a chimeric antigen receptor comprising the polynucleotide or the vector.

[0011] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of diseases related to BCMA expression, comprising immune effector cells expressing the chimeric antigen receptor targeting BCMA.

[0012] Technical solution

[0013] To achieve the above objectives, the present invention provides an antibody or fragment thereof that specifically binds to BCMA (B-cell maturation antigen), comprising: a heavy chain variable region comprising a CDR1 region represented by the amino acid sequence of sequence 1, a CDR2 region represented by the amino acid sequence of sequence 2, and a CDR3 region represented by the amino acid sequence of sequence 3; and a light chain variable region comprising a CDR1 region represented by the amino acid sequence of sequence 4, a CDR2 region represented by the amino acid sequence of sequence 5, and a CDR3 region represented by the amino acid sequence of sequence 6.

[0014] In a preferred embodiment of the present invention, the antibody may be an antibody composed of the heavy chain variable region represented by the amino acid sequence of sequence 7 and the light chain variable region represented by the amino acid sequence of sequence 8, or an antibody composed of the heavy chain variable region represented by the amino acid sequence of sequence 13 and the light chain variable region represented by the amino acid sequence of sequence 14.

[0015] Furthermore, the present invention provides a chimeric antigen receptor (CAR), which includes a BCMA-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signal transduction domain. The BCMA-binding domain includes an antibody or fragment thereof capable of specifically binding to BCMA. The antibody includes: a heavy chain variable region comprising a CDR1 region represented by the amino acid sequence of sequence 1, a CDR2 region represented by the amino acid sequence of sequence 2, and a CDR3 region represented by the amino acid sequence of sequence 3; and a light chain variable region comprising a CDR1 region represented by the amino acid sequence of sequence 4, a CDR2 region represented by the amino acid sequence of sequence 5, and a CDR3 region represented by the amino acid sequence of sequence 6.

[0016] In a preferred embodiment of the present invention, the transmembrane domain may be derived from a protein selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.

[0017] In another preferred embodiment of the present invention, the co-stimulatory domain may be derived from a protein selected from the group consisting of CD28, 4-1BB, OX-40 and ICOS, and the signal transduction domain may be derived from CD3ζ.

[0018] In another preferred embodiment of the invention, a hinge region may also be included between the C-terminus of the BCMA-binding domain and the N-terminus of the transmembrane domain, the hinge region being derived from CD8α.

[0019] To achieve another objective, the present invention provides a polynucleotide encoding the above-mentioned chimeric antigen receptor.

[0020] Furthermore, the present invention provides a vector comprising a polynucleotide encoding a chimeric antigen receptor.

[0021] In a preferred embodiment of the present invention, the vector can be a plasmid, a retroviral vector, or a lentiviral vector.

[0022] Furthermore, the present invention provides immune effector cells that contain a polynucleotide encoding the chimeric antigen receptor or a vector containing a polynucleotide encoding the chimeric antigen receptor and express the chimeric antigen receptor.

[0023] In a preferred embodiment of the present invention, the aforementioned immune effector cells may be T cells.

[0024] To achieve another objective, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases associated with BCMA expression, comprising immune effector cells expressing a chimeric antigen receptor targeting BCMA or an antibody or fragment thereof that specifically binds to BCMA.

[0025] In a preferred embodiment of the present invention, the diseases associated with BCMA expression may be multiple myeloma, hematological malignancies, non-Hodgkin's lymphoma, autoantibody-dependent autoimmune disease, systemic lupus erythematosus (SLE), or rheumatoid arthritis.

[0026] The effects of the invention

[0027] It was confirmed that the chimeric antigen receptor and chimeric antigen receptor-T cells prepared in this invention specifically bind to BCMA as an antigen, thereby activating the chimeric antigen receptor-T cells that bind to BCMA.

[0028] Furthermore, it has been confirmed that the chimeric antigen receptor-T cells of the present invention effectively kill cells expressing BCMA. Therefore, the BCMA-targeting chimeric antigen receptor and chimeric antigen receptor-T cells of the present invention can be usefully used as a composition for the prevention or treatment of diseases related to B cell or BCMA expression. Attached Figure Description

[0029] Figure 1 Data to confirm the BCMA binding ability of the humanized anti-BCMA monoclonal antibody (3G4V2).

[0030] Figure 2 This diagram illustrates a lentiviral vector expressing a chimeric antigen receptor targeting BCMA (hBCMA-CAR) and the chimeric antigen receptor expressed in T cells.

[0031] Figure 3 This is a schematic diagram illustrating a method for preparing hBCMA-chimeric antigen receptor-T cells using lentiviruses expressing hBCMA-CAR.

[0032] Figure 4 Data to confirm the BCMA binding capacity of hBCMA-chimeric antigen receptor-T cells.

[0033] Figure 5 The purpose was to confirm the activation of hBCMA-chimeric antigen receptor-T cells and to obtain data on the extent to which IFNγ and CD107a were expressed by 3G4V2-chimeric antigen receptor T cells in the presence of target cells.

[0034] Figure 6 To confirm the effectiveness of hBCMA-chimeric antigen receptor-T cells in killing target cells. Detailed Implementation

[0035] The present invention will now be described in detail.

[0036] Antibodies targeting BCMA

[0037] One embodiment of the present invention relates to an antibody or fragment thereof that specifically binds to BCMA, comprising: a heavy chain variable region comprising a CDR1 region represented by an amino acid sequence of sequence 1, a CDR2 region represented by an amino acid sequence of sequence 2, and a CDR3 region represented by an amino acid sequence of sequence 3; and a light chain variable region comprising a CDR1 region represented by an amino acid sequence of sequence 4, a CDR2 region represented by an amino acid sequence of sequence 5, and a CDR3 region represented by an amino acid sequence of sequence 6.

[0038] In this invention, the aforementioned antibody can be a monoclonal antibody. In this invention, the term "monoclonal antibody" is used, referring to an antibody generated by monoclonal antibody-forming cells that has a uniform primary structure (amino acid arrangement). It recognizes only one antigenic determinant and is typically produced by culturing hybridoma cells that fuse cancer cells and antibody-generating cells. However, it can also be produced by using the obtained antibody gene sequence to express other recombinant proteins in host cells.

[0039] In this invention, the term "antibody" can be used not only in its complete form, having two full-length light chains and two full-length heavy chains, but also as a fragment of an antibody molecule. A fragment of an antibody molecule refers to a fragment that at least retains peptide tag (epitope) binding function, including scFv, Fab, F(ab'), F(ab')2, single domains, etc.

[0040] In antibody fragments, Fab has a structure containing a light chain, a variable region of the heavy chain, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, and has an antigen-binding site. Fab' differs from Fab in that it has a hinge region containing more than one cysteine ​​residue at the C-terminus of the CH1 domain of the heavy chain. The F(ab')2 antibody is generated by forming disulfide bonds through the cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment containing only the variable regions of the heavy chain and the light chain. Techniques for producing Fv fragments are disclosed in international patents WO 88 / 10649, WO 88 / 106630, WO 88 / 07085, WO 88 / 07086, and WO 88 / 09344. Double-chain Fv (dsFv) connects the variable regions of the heavy chain and light chain via disulfide bonds, while single-chain Fv (scFv) typically connects the variable regions of the heavy chain and light chain via peptide linkers via covalent bonds. Such antibody fragments can be obtained using proteases (e.g., papain can be used to partially cleave the entire antibody to obtain Fab, and pepsin can be used to cleave the F(ab')2 fragment), and preferably, they can be prepared using recombinant genetic techniques.

[0041] The monoclonal antibody of the present invention that specifically binds to BCMA can be prepared using all or part of the peptide of the BCMA protein as an immunogen (or antigen). More specifically, as needed, the BCMA, a fusion protein containing the BCMA protein, or a carrier containing the BCMA protein, preferably as an immunogen, together with an adjuvant as an immunostimulant (e.g., Freund's adjuvant), is injected subcutaneously, intramuscularly, intravenously, into the pedisole, or intraperitoneally once or more to sensitize the mammal other than humans. Preferably, the mammal other than humans is a mouse, rat, hamster, guinea pig, chicken, rabbit, cat, dog, pig, goat, sheep, donkey, horse, or cattle (including transgenic mice used to produce human antibodies and other transgenic animals prepared for the purpose of producing antibodies of other animal origin), more preferably a mouse, rat, hamster, guinea pig, chicken, or rabbit. Starting with the first immunization, 1 to 4 immunizations are administered approximately every 1 to 21 days. Antibody-producing cells can be obtained from the sensitized mammal approximately 1 to 10 days after the final immunization. The number of sensitizations and the time intervals can be adjusted according to the characteristics of the immunogen used.

[0042] Hybridoma cells secreting monoclonal antibodies can be prepared according to the methods of Kayla and Milstein et al. (Nature, 1975, Vol. 256, pp. 495-497) and methods based thereon. Hybridoma cells can be prepared by fusing antibody-producing cells from the spleen, lymph nodes, bone marrow, or tonsils taken from the aforementioned immunosensitized animals other than humans, preferably by fusing myeloma cells derived from the spleen with myeloma cells derived from mammals that do not have the ability to produce autoantibodies.

[0043] Cell fusion can be achieved using fusion promoters such as polyethylene glycol or Sendai virus, or by electroporation. For example, antibody-producing cells and mammalian-derived cells capable of unlimited proliferation can be suspended in a medium containing a fusion promoter at a ratio of approximately 1:1 to 1:10 and cultured at approximately 30°C to 40°C for approximately 1 to 5 minutes. Common fusion media such as MEM, RPMI 1640, and Iscove's Modified Dulbecco's Medium are suitable, but serums such as bovine serum are preferably excluded.

[0044] The method for screening hybridoma cell clones that produce the aforementioned monoclonal antibodies involves first transferring the fusion cells obtained as described above to a screening medium such as HAT medium and culturing them at approximately 30°C to 40°C for 3 days to 3 weeks to kill cells other than hybridoma cells. Next, after culturing the hybridoma cells in a microtiter plate or similar medium, immunoassay methods such as radioactive substance-marked immunoantibody (RIA) or enzyme-linked immunosorbent assay (ELISA) can be used to identify fractions that increase reactivity with the immunogen and supernatant used in the aforementioned immune responses in animals other than humans. Then, the clones found above that produce monoclonal antibodies exhibit specific binding affinity to the aforementioned immunogen.

[0045] The monoclonal antibodies of the present invention can be obtained by culturing the aforementioned hybridoma cells in vivo and in vitro. The culturing utilizes conventional methods for culturing mammalian-derived cells, and to extract the monoclonal antibodies from the culture, methods commonly used in the art to which this invention pertains are employed for antibody purification. Various methods can be, for example, salting out, dialysis, filtration, concentration, centrifugation, separate precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography, gel electrophoresis, and isoelectric point electrophoresis, which can be combined as needed. The purified monoclonal antibodies are then concentrated, dried, and prepared as liquids or solids according to their intended use.

[0046] Furthermore, the monoclonal antibody of the present invention can be obtained by the following method: after synthesizing deoxyribonucleic acid (DNA) encoding the variable regions of the heavy chain and the light chain, and genes respectively linked to the basic deoxyribonucleic acid encoding the normal regions of the heavy chain and the light chain (for example, refer to Japanese Publication No. 2007-252372) by polymerase chain reaction (PCR) or chemical synthesis, transformants are prepared by transplanting them into a known vector or the like capable of expressing the above-mentioned genes. Antibodies are then produced by expressing them in hosts such as CHO cells or Escherichia coli. The antibodies are then purified from these culture media using a protein A or G column or the like.

[0047] In one specific embodiment of the present invention, a novel antibody was established by preparing and screening mouse antibodies that specifically bind to BCMA, and named 3G4.

[0048] The 3G4 antibody was confirmed to contain: a heavy chain variable region comprising the CDR1 region represented by the amino acid sequence of sequence 1 (GYTFTSYV), the CDR2 region represented by the amino acid sequence of sequence 2 (IIPYNDGT), and the CDR3 region represented by the amino acid sequence of sequence 3 (ARWNWDGYFDV); and a light chain variable region comprising the CDR1 region represented by the amino acid sequence of sequence 4 (KSLLHSNGITY), the CDR2 region represented by the amino acid sequence of sequence 5 (QMS), and the CDR3 region represented by the amino acid sequence of sequence 6 (TQNLELPFT).

[0049] Preferably, the 3G4 antibody is composed of a heavy chain variable region represented by the amino acid sequence of sequence 7 and a light chain variable region represented by the amino acid sequence of sequence 8. Furthermore, the heavy chain variable region of the 3G4 antibody can be encoded by the base sequence of sequence 9, and the light chain variable region of the 3G4 antibody can be encoded by the base sequence of sequence 10. When the 3G4 antibody exists in scFv form, it can be linked in a light chain variable region-linker-heavy chain variable region manner, preferably represented by the amino acid sequence of sequence 11 or encoded by the base sequence of sequence 12.

[0050] In another specific embodiment of the present invention, 3G4, which is an anti-BCMA antibody, is prepared as a humanized antibody with a structure corresponding to that of humans, and is named 3G4V2.

[0051] The heavy chain variable region CDR and light chain variable region CDR of the above-mentioned 3G4V2 are the same as those of 3G4, making the remaining part except for the CDR portion humanized. Preferably, 3G4V2 is composed of the heavy chain variable region represented by the amino acid sequence of sequence 13 and the light chain variable region represented by the amino acid sequence of sequence 14, and the heavy chain variable region of the 3G4V2 antibody can be encoded by the base sequence of sequence 15, and the light chain variable region of the 3G4V2 antibody can be encoded by the base sequence of sequence 16. When the above-mentioned 3G4 antibody exists in the form of scFv, it can be linked in the manner of light chain variable region-linker-heavy chain variable region, preferably represented by the amino acid sequence of sequence 17 or encoded by the base sequence of sequence 18.

[0052] In another specific embodiment of the present invention, the results of confirming whether 3G4V2, as a humanized anti-BCMA antibody, specifically binds to BCMA are as follows: Figure 1 As shown, no binding to 3G4V2 was confirmed in A549 cells that do not express hBCMA, but an increased binding capacity of 3G4V2 to A549 cells that express hBCMA was confirmed.

[0053] Therefore, in this invention, a chimeric antigen receptor targeting BCMA is prepared using 3G4V2 as a humanized anti-BCMA antibody.

[0054] In this invention, the term "humanized antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies produced by humans and / or prepared using one of the technical centers for preparing human antibodies disclosed in this application. The definition of a humanized antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0055] Furthermore, it should be understood that the proteins, peptides, and / or amino acid sequences included in this invention include at least functional variants or homologs having the same or similar functions as the proteins or peptides.

[0056] In this invention, the functional variant can be a protein or polypeptide obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence of the aforementioned protein and / or polypeptide. The functional variant can substantially retain the biological characteristics of the unmodified protein or polypeptide (substitution, deletion, addition). For example, the functional variant can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (such as antigen-binding ability) of the original protein or polypeptide.

[0057] In this invention, the homolog can be a protein or polypeptide (e.g., an antibody capable of specifically binding to BCMA or its fragments) that has amino acid sequence homology of 85% or more (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the aforementioned protein and / or polypeptide.

[0058] In this invention, homology generally refers to the similarity or correlation between two or more sequences.

[0059] Chimeric antigen receptor targeting BCMA

[0060] Another embodiment of the present invention relates to a chimeric antigen receptor comprising a BCMA-binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signal transduction domain, characterized in that the BCMA-binding domain comprises an antibody or fragment thereof capable of specifically binding to BCMA, wherein the antibody comprises: a heavy chain variable region comprising a CDR1 region represented by the amino acid sequence of sequence 1, a CDR2 region represented by the amino acid sequence of sequence 2, and a CDR3 region represented by the amino acid sequence of sequence 3; and a light chain variable region comprising a CDR1 region represented by the amino acid sequence of sequence 4, a CDR2 region represented by the amino acid sequence of sequence 5, and a CDR3 region represented by the amino acid sequence of sequence 6.

[0061] In this invention, the term "chimeric antigen receptor" generally refers to a fusion protein containing an antigen and an extracellular domain capable of binding to one or more intracellular domains. The chimeric antigen receptor is a core component of chimeric antigen receptor T cells (CAR-T cells) and may include an antigen-binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signal transduction domain. The chimeric antigen receptor can combine with the T cell receptor-activating intracellular domain based on the antigen specificity of the antibody (e.g., BCMA). Genetically modified CAR-expressing T cells can recognize and eliminate target antigen-expressing malignant cells.

[0062] In this invention, the term "BCMA (B-cell maturation antigen)" refers to the B-cell maturation antigen. BCMA (also known as TNFRSF17, BCM, or CD269) is a member of the tumor necrosis receptor (TNFR) family and is preferentially expressed in terminally differentiated B cells, such as memory B cells and plasma cells. BCMA is expressed in tumor cells (e.g., multiple myeloma cells) or located on the surface of tumor cells. The "BCMA" of this invention can include proteins containing mutations, such as point mutations, fragments, insertions, deletions, and splice variants of the full-length wild-type BCMA.

[0063] In this invention, the term "BCMA-binding domain" generally refers to a domain capable of specifically binding to the BCMA protein. For example, the BCMA-binding domain may contain an anti-BCMA antibody or a fragment thereof that specifically binds to a human BCMA polypeptide or fragment thereof expressed in B cells.

[0064] In this invention, the term "binding domain" can be used interchangeably with "extracellular domain," "extracellular binding domain," "antigen-specific binding domain," and "extracellular antigen-specific bidding domain," referring to a chimeric antigen receptor domain or fragment that has the ability to specifically bind to a target antigen (e.g., BCMA).

[0065] In this invention, the anti-BCMA antibody or its fragment is used as the anti-BCMA antibody, which is a monoclonal antibody, preferably scFv (single chain variable fragment). In this invention, 3G4V2 is used as a humanized anti-BCMA antibody.

[0066] In this invention, the term "transmembrane domain" generally refers to a domain of a chimeric antigen receptor that functions as a signal transduction device by connecting to an intracellular signal transduction domain via the cell membrane. The aforementioned transmembrane domain may originate from proteins selected from the group consisting of CD8α, CD4, CD2, CD137, CD80, CD86, CD152, and PD1, and preferably may be represented by the amino acid sequence of sequence 29.

[0067] In this invention, a "co-stimulatory domain" generally refers to an intracellular domain of an immunostimulatory molecule that provides the cell surface molecule required for an effective antigenic response against lymphocytes. The co-stimulatory domains described above may include the co-stimulatory domain of CD28, the co-stimulatory domains of the TNF receptor family such as OX40 and 4-1BB, and preferably, 4-1BB as represented by the amino acid sequence of sequence 30.

[0068] In this invention, "intracellular signal transduction domain" generally refers to a domain located within the cell capable of transmitting signals. In this invention, the intracellular signal transduction domain is the intracellular signal transduction domain of a chimeric antigen receptor. For example, the intracellular signal transduction domain can be selected from the CD3ζ intracellular domain, CD28 intracellular domain, 4-1BB intracellular domain, and OX40 intracellular domain; preferably, it can be CD3ζ represented by the amino acid sequence of sequence 31.

[0069] In this invention, a hinge region may also be included between the C-terminus of the BCMA-binding domain and the N-terminus of the transmembrane domain. This hinge region is derived from CD8α and, preferably, can be represented by the amino acid sequence of sequence 28. The aforementioned "hinge region" generally refers to the connection region between the antigen-binding region and the immune cell Fc receptor (FcR)-binding region.

[0070] In this invention, a signal peptide may also be included at the N-terminus of the BCMA-binding domain. The term "signal peptide" generally refers to a peptide chain used to guide protein delivery. The signal peptide can be a short peptide with a length of 5 to 30 amino acids; preferably, the amino acid sequence at sequence 27 is used in this invention.

[0071] Polynucleotides encoding chimeric antigen receptors and vectors expressing chimeric antigen receptors Another embodiment of the present invention relates to a polynucleotide encoding the above-described chimeric antigen receptor.

[0072] In this invention, the polynucleotide encoding the chimeric antigen receptor may include polynucleotides encoding the BCMA-binding domain, polynucleotides encoding the transmembrane domain, polynucleotides encoding the co-stimulatory domain, and polynucleotides encoding the intracellular signal transduction domain.

[0073] Preferably, the polynucleotide encoding the BCMA-binding domain can be a polynucleotide encoding a 3G4 antibody or a 3G4V2 antibody, and the specific base sequence is as described above.

[0074] Preferably, the polynucleotide encoding the chimeric antigen receptor of the present invention comprises: a signal peptide represented by the base sequence of sequence 21; 3G4V2 as an anti-BCMA antibody represented by the base sequence of sequence 18; a transmembrane domain represented by the base sequence of sequence 23; a 4-1BB (co-stimulatory domain) represented by the base sequence of sequence 24; and a CD3ζ (intracellular signal transduction domain) represented by the base sequence of sequence 25.

[0075] Furthermore, a polynucleotide encoding a hinge region may be included between the polynucleotide encoding the BCMA-binding domain and the transmembrane domain, preferably the CD8 hinge region represented by the base sequence of sequence 22.

[0076] In this invention, the term "polynucleotide" generally refers to a nucleic acid molecule, deoxyribonucleotide, ribonucleotide, or similar substance thereof isolated into an arbitrary length. In some examples, the polynucleotides of this invention can be prepared by: (1) in vitro amplification such as polymerase chain reaction (PCR); (2) cloning and recombination; (3) purification such as digestion and gel electrophoresis; and (4) synthesis such as chemical synthesis. Preferably, the isolated polynucleotides can be prepared by recombinant deoxyribonucleic acid (DRCA) technology. In this invention, nucleic acids encoding antibodies or their antigen-binding fragments can be prepared using methods including restriction fragment operation or overlap extension polymerase chain reaction (SOE PCR), but are not limited thereto. Various methods known in the art to which this invention pertains can be used for preparation.

[0077] Another embodiment of the present invention relates to a vector comprising the above-described polynucleotide encoding a chimeric antigen receptor.

[0078] In this invention, the term "expression vector" refers to a gene preparation that contains essential regulatory elements such as promoters in a manner that enables the expression of a target gene within a suitable host cell. The vector may be selected from one or more of plasmids, retroviral vectors, and lentiviral vectors. When transformed into a suitable host, the vector can replicate independently of the host genome and perform its corresponding function, or, in some cases, can integrate with the genome itself.

[0079] Furthermore, the vector can contain expression control elements that enable the coding region to be correctly expressed in the appropriate host. Such regulatory elements are well known to those skilled in the art and may include, for example, promoters, ribosome-binding sites, enhancers, and other regulatory elements that regulate gene transcription or messenger RNA (mRNA) translation. The specific structure of the expression regulatory sequence can vary depending on the species or cell type and function, typically including 5' non-transcriptional sequences involved in transcription and translation initiation, such as TATA boxes, capped sequences, and CAAT sequences, as well as 5' or 3' non-translational sequences. For example, the 5' non-transcriptional expression regulatory sequence may contain a promoter region, which may contain promoter sequences for transcription and regulation of functionally linked nucleic acids.

[0080] In a specific embodiment of the present invention, the vector is a recombinant viral vector, preferably a lentiviral vector, comprising: an operably linked EF1α promoter; a polynucleotide encoding a signal peptide; a polynucleotide encoding a BCMA-binding domain; a polynucleotide encoding a transmembrane domain; and a polynucleotide encoding an intracellular signal transduction domain. To increase protein expression, it may also contain WPRE (woodchuck hepatitis virus post-transcriptional regulatory element). Figure 2 ).

[0081] The aforementioned EF1α promoter can be represented by the base sequence of sequence 19, and can have a sequence that is 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more or more identical to the base sequence of sequence 19 as needed.

[0082] Furthermore, the aforementioned promoter can be operably linked to induce the expression of anti-hBCMA antibody (scFv), which serves as a BCMA-binding domain. "Operably linked" means performing a general function by functionally linking the nucleic acid expression regulatory sequence to the nucleic acid sequence encoding the target protein. Operable linking to the recombinant vector can be prepared using gene recombination techniques well-known in the art to which this invention pertains, and epitope-specific deoxyribonucleic acid (DNA) cleavage and ligation can be performed using enzymes commonly used in the art to which this invention pertains.

[0083] Methods for introducing and expressing genes into cells are well known in the relevant technical fields. Relatedly, expression vectors can be easily introduced into host cells using any method within the relevant technical field. For example, expression vectors can be transferred into host cells using physical, chemical, or biological methods.

[0084] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, liposome transfection, particle impaction, microinjection, and electroporation. Methods for producing vectors and / or cells containing exogenous nucleic acids are well known in the relevant technical fields. For example, see [Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY]. Preferably, calcium phosphate transfection is used for introducing polynucleotides into host cells.

[0085] Biological methods for introducing polynucleotides into host cells include the use of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) vectors. Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses, and adeno-associated viruses.

[0086] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, magnetic beads, and lipid-based systems like oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery carrier in test tubes and in vivo is the liposome (e.g., artificial membrane vesicles). Targeted delivery of nucleic acids can be achieved using state-of-the-art technologies; for example, other methods for delivering polynucleotides can utilize targeted nanoparticles or other suitable sub-micrometer-sized delivery systems.

[0087] In the case of using non-viral delivery systems, liposomes are an illustrative delivery vector. The use of lipid formulations can be considered for the introduction of nucleic acids into host cells (in vitro, in vivo, or intracellularly). On the other hand, nucleic acids can be associated with lipids. Lipid-associated nucleic acids may be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linkers associated with both liposomes and oligonucleotides, captured within liposomes, forming complexes with liposomes, dispersed in solutions containing lipids, mixed with lipids, combined with lipids, contained within lipids in suspension form, contained or complexed with micelles, or otherwise associated with lipids. Lipids, lipid / DNA, or lipid / expression vector associated compositions are not limited to any specific structure in solution.

[0088] Chimeric antigen receptor expressing immune effector cells

[0089] Another embodiment of the present invention relates to an immune effector cell that contains a polynucleotide encoding the chimeric antigen receptor or a vector containing a polynucleotide encoding the chimeric antigen receptor and expressing the chimeric antigen receptor.

[0090] In this invention, the aforementioned immune effector cells can be mammalian-derived cells, preferably T cells or natural killer (NK) cells.

[0091] In this invention, the aforementioned immune effector cells expressing chimeric antigen receptors can be prepared by introducing the chimeric antigen receptor vector of this invention into immune effector cells, for example, into T cells or natural killer cells.

[0092] Specifically, chimeric antigen receptor vectors can be introduced into cells using methods known in the art, such as electroporation and lipofectamine (lipofectamine 2000, Invitrogen). For example, immune effector cells can be transfected with lentiviral vectors to integrate the viral genome transporting the chimeric antigen receptor molecule with the host genome, ensuring long-term and stable expression of the target gene. As another example, transposons can be used during the introduction of chimeric antigen receptor transport plasmids and transferase transport plasmids into target cells. Furthermore, chimeric antigen receptor molecules can be added to the genome using gene editing methods (e.g., CRISPR / Cas9).

[0093] In a specific embodiment of the present invention, such as Figure 2As shown, a lentiviral vector encoding a polynucleotide of hBCMA-CAR was prepared, and hBCMA-chimeric antigen receptor-T cells were prepared by transforming the prepared vector into T cells. The chimeric antigen receptor targeting BCMA of the present invention was expressed in the prepared hBCMA-chimeric antigen receptor-T cells.

[0094] Immune effector cells for preparing chimeric antigen receptor-expressing immune effector cells can be obtained from subjects, including living organisms (e.g., mammals) capable of eliciting an immune response. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic strains. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.

[0095] The aforementioned T cells can be produced using any of the various techniques known to those skilled in the art, such as using Ficoll. TM Apheresis is the process of obtaining cells from blood units collected from a patient. Cells are obtained from blood through apheresis, and typical apheresis products contain T cells, monocytes, granulocytes, lymphocytes such as B cells, other nucleated blood cells, red blood cells, and platelets.

[0096] Cells collected via apheresis, after removal of plasma fractions, can be washed for subsequent processing steps and for preservation in appropriate buffers or culture media. After lysing red blood cells, for example by PERCOLL... TM T cells are isolated from peripheral blood lymphocytes after monocytes are depleted by gradient centrifugation or countercurrent centrifugation.

[0097] In a specific embodiment of the present invention, such as Figure 3 As shown, activated T cells were isolated from peripheral blood mononuclear cells (PBMCs), and hBCMA-CAR lentivirus was transduced into these T cells to prepare hBCMA-chimeric antigen receptor-T cells. The results confirming the hBCMA peptide binding capacity of the prepared hBCMA-chimeric antigen receptor-T cells are as follows: Figure 4 As shown, it was confirmed that with the increase of hBCMA peptide, the number of hBCMA-chimeric antigen receptor-T cells expressing CD4 or CD8 that bind to hBCMA also increased. This indicates that the hBCMA-chimeric antigen receptor-T cells prepared in this invention effectively bind to BCMA.

[0098] In another specific example of the invention, to confirm the activation of hBCMA-chimeric antigen receptor-T cells, the expression levels of IFNγ and CD107a by 3G4V2-chimeric antigen receptor-T cells were confirmed in the presence of target cells. The results are as follows: Figure 5 As shown, T cells were not activated in K562 cells that did not express BCMA, while in the presence of H929 cells that expressed BCMA, T cells were activated and IFNγ expression was increased.

[0099] In another specific embodiment of the present invention, the results confirming the killing effect of hBCMA-chimeric antigen receptor-T cells on target cells are as follows: Figure 6 As shown, hBCMA-chimeric antigen receptor-T cells were confirmed to have a specific killing effect on RPMI8226 cells and H929 cells expressing BCMA.

[0100] That is, the chimeric antigen receptor and chimeric antigen receptor-T cell targeting BCMA of the present invention can be usefully used as a composition for the prevention or treatment of diseases related to B cells or BCMA expression.

[0101] Compositions for the prevention or treatment of diseases associated with BCMA expression.

[0102] Another embodiment of the present invention relates to a pharmaceutical composition for the prevention or treatment of diseases associated with BCMA expression, comprising immune effector cells expressing a chimeric antigen receptor targeting BCMA.

[0103] Another embodiment of the present invention relates to a pharmaceutical composition for the prevention or treatment of B-cell-related diseases, comprising an antibody targeting BCMA.

[0104] Diseases associated with B cells or with the expression of BCMA (wild-type or mutant BCMA) can be cancer, malignant tumors, or autoimmune diseases. Preferably, they can be multiple myeloma, hematologic malignancies, non-Hodgkin's lymphoma, autoantibody-dependent autoimmune diseases, systemic lupus erythematosus, or rheumatoid arthritis.

[0105] The above-mentioned pharmaceutical compositions may also contain pharmaceutically acceptable carriers. For oral administration, binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc., may be used. In the case of injectable formulations, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., may be mixed in. For topical administration, bases, excipients, lubricants, preservatives, etc., may be used.

[0106] The dosage forms of the above-mentioned pharmaceutical compositions can be prepared in a variety of ways by mixing them with the above-mentioned pharmaceutically acceptable carriers. For example, when administered orally, they can be prepared as tablets, sugar-coated tablets, capsules, liquids, sustained-release formulations, syrups, round tablets, etc. In the case of injections, they can be prepared as single-dose ampoules or multiple-dose formulations.

[0107] Furthermore, the above-mentioned pharmaceutical composition may contain a surfactant capable of improving membrane permeability. The surfactant may be a cationic lipid induced from steroids or such as N-[1-(2,3-diol oil)propyl-N,N,N-trimethylammonium chloride (DOTMA), or various mixtures such as cholesterol monosuccinate and phosphatidylglycerol, but is not limited thereto.

[0108] Furthermore, the present invention provides a method for preventing or treating cancer, comprising the step of administering the pharmaceutical composition of the present invention to an individual. For the prevention or treatment of B-cell-related diseases, a pharmaceutically effective amount of the above-described pharmaceutical composition comprising hBCMA-chimeric antigen receptor-T cells or anti-hBCMA antibodies can be administered. The dosage can vary depending on various factors such as the type of disease, the patient's age, weight, the characteristics and severity of symptoms, the type of current treatment, the frequency of treatment, the form of administration, and the route of administration, and can be easily determined by experts in the relevant field.

[0109] The above-described pharmaceutical composition can be administered co-administered or sequentially with the aforementioned pharmacological or physiological components, and can be administered in combination with additional existing therapeutic agents, or sequentially or simultaneously with prior therapeutic agents. Such administration can be a single dose or multiple doses. Importantly, the dosage should be determined to achieve the maximum effect with the minimum amount without side effects, taking all the above factors into account; this can be easily determined by someone skilled in the art.

[0110] As used in this specification, the term "individual" refers to a mammal that is in a state that can be alleviated, suppressed, or treated by administration of the above-described pharmaceutical composition or that is at risk of developing a disease, preferably a human.

[0111] The term "administration" as used in this invention refers to the delivery of the pharmaceutical composition of this invention to an individual by any suitable method. The pharmaceutical composition of this invention can be administered by researchers, veterinarians, physicians, or other clinical means at an amount of the active ingredient or pharmaceutical composition that induces a biological or medical response in an imaginable tissue system, animal, or human, i.e., at a therapeutically effective amount that induces relief of symptoms of the disease or disorder to be treated. It will be apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration of the pharmaceutical composition of this invention vary depending on the desired effect. Therefore, the optimal dosage can be easily determined by a practitioner and can be adjusted based on various factors such as the type and severity of the disease, the content of the active ingredient and other components in the composition, the type of dosage form, the patient's age, weight, general health status, sex, and diet, the time of administration, the route of administration, the secretion rate of the composition, the duration of treatment, and concurrent medications. The pharmaceutical composition of this invention can be administered at doses ranging from 1 mg / kg / day to 10,000 mg / kg / day, either once a day or divided into several doses.

[0112] The following preferred embodiments are provided to aid in understanding the present invention. However, these embodiments are provided merely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited to these embodiments.

[0113] Example 1: Preparation and screening of antibodies targeting BCMA

[0114] To screen for antibodies specific to the BCMA peptide, hybridoma cells that produce antibodies that bind to BCMA are prepared for antibody screening.

[0115] First, using a method known in the technical field to which this invention pertains, mice were immunized with BCMA peptide (sequence 19; Acrobiosystems Inc., cat#BCA-H522y), spleen cells were extracted and then fused with mouse myeloma cells to prepare hybridoma cells.

[0116] Because the mouse myeloma cells used for cell fusion lack hypoxanthine-guanidine-phosphoribosyl-transferase (HGPRT), they cannot survive in HAT medium. However, hybridoma cells can survive in HAT medium by fusing with spleen cells. Using this method, only hybridoma cells can be proliferated; therefore, they can be continuously proliferated in HAT medium until conventional hybridoma cells are established.

[0117] To produce BCMA-binding antibodies in hybridoma cells, a limiting dilution method was used. First, clones proliferating from a single cell were screened by dividing the cells into sub-cells of 96 wells. This process was repeated three times to screen hybridoma cells that would produce BCMA-binding antibodies.

[0118] The antibody screened in hybridoma cells was named 3G4 antibody (anti-hBCMA monoclonal antibody), and its amino acid sequence was analyzed. Sequence information of the heavy chain and light chain variable regions of the antibody based on the sequence analysis results is shown in Table 1 below. The underlined portions in Table 1 indicate the complementarity determining region (CDR).

[0119] Table 1. Sequence information of 3G4 antibody

[0120]

[0121]

[0122] As shown in Table 1, the 3G4 antibody was confirmed to contain: a heavy chain variable region, comprising the CDR1 region represented by the amino acid sequence of sequence 1 (GYTFTSYV), the CDR2 region represented by the amino acid sequence of sequence 2 (IIPYNDGT), and the CDR3 region represented by the amino acid sequence of sequence 3 (ARWNWDGYFDV); and a light chain variable region, comprising the CDR1 region represented by the amino acid sequence of sequence 4 (KSLLHSNGITY), the CDR2 region represented by the amino acid sequence of sequence 5 (QMS), and the CDR3 region represented by the amino acid sequence of sequence 6 (TQNLELPFT).

[0123] Specifically, the aforementioned 3G4 antibody is composed of a heavy chain variable region represented by the amino acid sequence of sequence 7 and a light chain variable region represented by the amino acid sequence of sequence 8. Furthermore, the heavy chain variable region of the 3G4 antibody can be encoded by the base sequence of sequence 9, and the light chain variable region of the 3G4 antibody can be encoded by the base sequence of sequence 10.

[0124] Example 2: Preparation of humanized antibodies based on 3G4 antibody

[0125] The 3G4 antibody screened in Example 1 above was prepared into a humanized antibody with a structure corresponding to that of humans.

[0126] Specifically, using the germline sequence of the human antibody as a frame, the 3G4 antibody was prepared as a humanized antibody through CDR grafting, which involved replacing the CDR of the mouse antibody that binds to BCMA with the CDR of the human antibody. The humanized antibody was named 3G4V2, and its amino acid sequence was analyzed. Sequence information regarding the variable regions of the antibody heavy chain and light chain based on the sequence analysis results is shown in Table 2 below. The underlined portions in Table 2 indicate complementarity-determining regions.

[0127] Table 2. Sequence information of the 3G4V2 antibody

[0128]

[0129]

[0130]

[0131] As shown in Table 2, it was confirmed that the 3G4V2 antibody is composed of the heavy chain variable region represented by the amino acid sequence of sequence 13 and the light chain variable region represented by the amino acid sequence of sequence 14. Furthermore, the heavy chain variable region of the 3G4V2 antibody is encoded by the base sequence of sequence 15, and the light chain variable region of the 3G4V2 antibody is encoded by the base sequence of sequence 16.

[0132] Example 3: Confirmation of the BCMA binding ability of the anti-BCMA antibody

[0133] In this invention, it was confirmed that the 3G4V2 prepared as a humanized anti-BCMA antibody in Example 2 above can specifically target BCMA.

[0134] First, to produce the 3G4V2 antibody, the heavy chain variable region represented by the base sequence of sequence 15 was cloned into the heavy chain antibody expression vector for human antibody IgG1, and the light chain variable region represented by the base sequence of sequence 16 was cloned into the light chain antibody expression vector for human antibody Kappa. The expression vectors were then co-transfected into CHO cells to produce the antibody. The produced antibody was purified using a Protein A column (Thermo Fisher Scientific, cat#20356).

[0135] Then, A549 cells and hBCMA-expressing A549 cells were treated with 1 mg of the purified antibody described above, followed by treatment with a secondary antibody (Biolegend, CAT#409304) specific for the human IgG Fc moiety. After reaction with the antibody, the PE fluorescence of the secondary antibody was measured by flow cytometry.

[0136] The results are as follows Figure 1 As shown, no binding to 3G4V2 was confirmed in A549 cells that do not express hBCMA, but the binding ability of A549 cells expressing hBCMA to 3G4V2 was confirmed to be increased to 60.1%.

[0137] Example 4: Preparation of a chimeric antigen receptor expression vector targeting BCMA

[0138] In this invention, a lentiviral vector (hBCMA-CAR lentivirus) expressing a chimeric antigen receptor targeting BCMA, comprising 3G4V2, a humanized anti-BCMA antibody prepared in Example 2 above, was prepared.

[0139] like Figure 3 As shown in the schematic diagram, a chimeric antigen receptor deoxyribonucleic acid (CAR DNA) (Sequence 32) composed of an EF1α promoter (Sequence 20), a polynucleotide encoding a signal peptide (Sequence 21), a polynucleotide encoding a BCMA-binding domain (Sequence 18), a polynucleotide encoding a CD8 hinge region (Sequence 22), a polynucleotide encoding a transmembrane domain (Sequence 23), a polynucleotide encoding a 4-1BB (co-stimulatory domain) (Sequence 24), a polynucleotide encoding a CD3ζ (intracellular signal transduction domain) (Sequence 25), and a polynucleotide encoding WPRE (Sequence 26) is synthesized in vitro and inserted into a third-generation lentiviral vector.

[0140] Lentiviral vectors were produced by co-transfecting Lenti-X 293T cells with three vectors: pMDLg / pRRE (Addgene, cat##12251), pMD2.G (Addgene, cat##12259), and pRSV-Rev (Addgene, cat##12253). For co-transfection, the three vectors were cultured with Lenti-X 293T cells for 6 hours using a Lipofectamine 3000 transfection kit (Invitrogen, cat#L3000-015) and Opti-MEM+GlutaMAX (gibco, cat#51985-034) medium.

[0141] Example 5: Preparation of hBCMA-chimeric antigen receptor-T cells

[0142] In this invention, hBCMA-chimeric antigen receptor-T cells (or 3G4V2-chimeric antigen receptor-T cells) are prepared by transforming the hBCMA-CAR lentiviral vector prepared in Example 4 above into T cells.

[0143] Specifically, such as Figure 3 As shown, after isolating peripheral blood mononuclear cells (PBMCs) from the blood, T cells were activated using a T cell activation bead (Miltenyl Biotec, CAT#130-091-441). The activated T cells were then transduced with the hBCMA-CAR lentivirus prepared in Example 4 above to prepare hBCMA-chimeric antigen receptor-T cells, using Lenti-boost-p to increase transduction efficiency.

[0144] The hBCMA peptide binding capacity of hBCMA-chimeric antigen receptor-T cells was confirmed using flow cytometry. The prepared hBCMA-chimeric antigen receptor-T cells were reacted with FITC-hbCMA protein, anti-CD3, anti-CD4, and anti-CD8 antibodies, and the fluorescence intensity was measured using flow cytometry fluorescence sorting (FACS). During the analysis, cells expressing CD3 were used as T cells to confirm the expression level of FITC in T cells.

[0145] The results are as follows Figure 4 As shown, it was confirmed that with the increase of hBCMA peptide, the number of hBCMA-chimeric antigen receptor-T cells expressing CD4 or CD8 that bind to hBCMA also increased. This indicates that the hBCMA-chimeric antigen receptor-T cells prepared in this invention effectively bind to BCMA.

[0146] Example 6: Confirmation of hBCMA-chimeric antigen receptor-T cell activation via hBCMA peptide

[0147] In this invention, in order to confirm whether the hBCMA-chimeric antigen receptor-T cells prepared in Example 5 above are activated by the BCMA peptide, the expression levels of IFNγ and CD107a of the hBCMA-chimeric antigen receptor-T cells are confirmed in the presence of target cells.

[0148] Targeted cells included K562 cells (ATCC, cat#CCL-243) that do not express BCMA and H929 cells (ATCC, cat#CRL-9068) that express BCMA. hBCMA-chimeric antigen receptor-T cells were reacted with target cells at ratios of 2:1, 1:1, 0.5:1, and 0:1 for specified time periods. Surface and intracellular antibodies were used for staining, and flow cytometry was used to measure (BCMA protein, INF-γ, CD107a, CD3, CD4, and CD8 staining). The expression levels of IFNγ and CD107a in hBCMA-CAR-T cells reacting with target cells were confirmed using a 0:1 ratio (chimeric antigen receptor T cells only).

[0149] The results are as follows Figure 5 As shown, it was confirmed that T cells were not activated in K562 cells that do not express BCMA, but in the presence of H929 cells that express BCMA, T cells were activated and IFNγ expression was increased.

[0150] Example 7: Confirmation of the killing effect of hBCMA-chimeric antigen receptor-T cells on BCMA-expressing cells

[0151] In this invention, the killing effect of hBCMA-chimeric antigen receptor-T cells on target cells was confirmed.

[0152] K562 cells (non-BCMA-expressing), RPMI8226 cells (BCMA-expressing), and H929 cells (hBCMA-chimeric antigen receptor-T cells) were used as target cells and mixed with hBCMA-chimeric antigen receptor-T cells at ratios of 1:4, 1:2, 1:1, 1:0.5, and 1:0.25, respectively. The chemiluminescence was measured using the CytoTox-Glo Cytotoxicity Assay (Promega, CAT#G9291). The cell-killing effect was calculated using the measured values ​​according to the following mathematical formula 1.

[0153] Mathematical Formula 1

[0154] % Cytotoxicity = [(Experimental-Effector Spontaneous-Target Spontaneous) / (Target Maximum-Target Spontaneous)]×100

[0155] Experimental: Luminescence values ​​derived from the culture medium containing co-cultured target cells and chimeric antigen receptor-T cells.

[0156] Effector Spontaneous: Luminescence values ​​derived from a culture medium containing only chimeric antigen receptor-T cells.

[0157] Target Spontaneous: The luminescence value derived from a culture medium containing only target cells.

[0158] Target Maximum: The luminescence value at 100% lysis of target cells (using lysis reagent (Lysis Reagent)).

[0159] The results are as follows Figure 6 As shown, hBCMA-chimeric antigen receptor-T cells were confirmed to specifically kill RPMI8226 and H929 cells expressing BCMA.

[0160] That is, the chimeric antigen receptor and chimeric antigen receptor-T cell targeting BCMA of the present invention can be usefully used for the prevention or treatment of diseases related to B cells or BCMA expression.

[0161] Industrial availability

[0162] In this invention, it was confirmed that the screening antibody specifically recognizes cells expressing BCMA. Using the antibody established above, it was confirmed that the chimeric antigen receptor and chimeric antigen receptor-T cells targeting BCMA not only effectively bind to BCMA, but also activate the chimeric antigen receptor-T cells that bind to BCMA.

[0163] Furthermore, it has been confirmed that the chimeric antigen receptor-T cells of the present invention effectively kill cells expressing BCMA. Therefore, the BCMA-specific antibody, the chimeric antigen receptor targeting BCMA, and the chimeric antigen receptor-T cells of the present invention can be usefully used as a composition for the prevention or treatment of diseases related to BCMA expression. sequence list <110> National Cancer Center Protahn Bio Co., Ltd. <120> Chimeric antigen receptors targeting BCMA and their applications <130> POPC212231PCTCN <150> KR10-2020-0073714 <151> 2020-06-17 <150> PCT / KR2021 / 006671 <151> 2021-05-28 <160> 32 <170> PatentIn version 3.2 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 3G4_VH_CDR1 <400> 1 Gly Tyr Thr Phe Thr Ser Tyr Val 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 3G4_VH_CDR2 <400> 2 Ile Ile Pro Tyr Asn Asp Gly Thr 1 5 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 3G4_VH_CDR3 <400> 3 Ala Arg Trp Asn Trp Asp Gly Tyr Phe Asp Val 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 3G4_VL_CDR1 <400> 4 Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr 1 5 10 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 3G4_VL_CDR2 <400> 5 Gln Met Ser 1 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 3G4_VL_CDR3 <400> 6 Thr Gln Asn Leu Glu Leu Pro Phe Thr 1 5 <210> 7 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> 3G4_VH chain <400> 7 Gln Val Gln Leu Lys Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Trp Asn Trp Asp Gly Tyr Phe Asp Val Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 8 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 3G4_VL chain <400> 8 Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly 1 5 10 15 Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Phe Tyr Cys Thr Gln Asn 85 90 95 Leu Glu Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 9 <211> 354 <212> Deoxyribonucleic acid <213> Artificial Sequence <220> <223> 3G4_VH chain <400> 9 caggtgcagc tgaaggagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60 tcctgcaagg cttctggata cacattcact agctatgtta tgcactgggt gaagcagaag 120 cctgggcagg gccttgagtg gattggatat attattcctt acaatgatgg tactaagtac 180 aatgagaagt tcaaaggcaa ggccacactg acttcagaca aatcctccag tacagcctac 240 atggagctca gcagcctgac ctctgaggac tctgcgctct attactgtgc aagatggaac 300 tgggacgggt acttcgatgt ctggggcgca gggaccacgg tcaccgtctc ctca 354 <210> 10 <211> 336 <212> Deoxyribonucleic acid <213> Artificial Sequence <220> <223> 3G4_VL chain <400> 10 gatattgtga tgacgcaggc tgcattctcc aatccagtca ctcttggaac atcagcttcc 60 atctcctgca ggtctagtaa gagtctccta catagtaatg gcatcactta tttgtattgg 120 tatctgcaga agccaggcca gtctcctcag ctcctgattt atcagatgtc caaccttgcc 180 tcaggagtcc cagacaggtt cagtagcagt gggtcaggaa ctgatttcac actgagaatc 240 agcagagtgg aggctgagga tgtgggtgtt ttttactgta ctcaaaatct agaacttcca 300 ttcacgttcg gctcggggac aaagttggaa ataaaa 336 <210> 11 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> 3G4_scFv <400> 11 Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly 1 5 10 15 Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Phe Tyr Cys Thr Gln Asn 85 90 95 Leu Glu Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu Lys Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser 130 135 140 Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Val 145 150 155 160 Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly 165 170 175 Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys 180 185 190 Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr Met 195 200 205 Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Leu Tyr Tyr Cys Ala 210 215 220 Arg Trp Asn Trp Asp Gly Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr 225 230 235 240 Val Thr Val Ser Ser 245 <210> 12 <211> 735 <212> deoxyribonucleic acid <213> Artificial Sequence <220> <223> 3G4_scFv <400> 12 gatattgtga tgacgcaggc tgcattctcc aatccagtca ctcttggaac atcagcttcc 60 atctcctgca ggtctagtaa gagtctccta catagtaatg gcatcactta tttgtattgg 120 tatctgcaga agccaggcca gtctcctcag ctcctgattt atcagatgtc caaccttgcc 180 tcaggagtcc cagacaggtt cagtagcagt gggtcaggaa ctgatttcac actgagaatc 240 agcagagtgg aggctgagga tgtgggtgtt ttttactgta ctcaaaatct agaacttcca 300 ttcacgttcg gctcggggac aaagttggaa ataaaaggtg gtggtggttc gggtggtggt 360 ggttcgggtg gtggtggttc gcaggtgcag ctgaaggagt ctggacctga gctggtaaag 420 cctggggctt cagtgaagat gtcctgcaag gcttctggat acacattcac tagctatgtt 480 atgcactggg tgaagcagaa gcctgggcag ggccttgagt ggattggata tattattcct 540 tacaatgatg gtactaagta caatgagaag ttcaaaggca aggccacact gacttcagac 600 aaatcctcca gtacagccta catggagctc agcagcctga cctctgagga ctctgcgctc 660 tattactgtg caagatggaa ctgggacggg tacttcgatg tctggggcgc agggaccacg 720 gtcaccgtct cctca 735 <210> 13 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> 3G4V2_VH chain <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Asn Trp Asp Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 14 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 3G4V2_VL chain <400> 14 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Thr Gln Asn 85 90 95 Leu Glu Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 15 <211> 354 <212> Deoxyribonucleic acid <213> Artificial Sequence <220> <223> 3G4V2_VH chain <400> 15 caggtgcagc tggtgcagag cggcgccgag gtgaagaagc cgggtgcttc cgtgaaggtg 60 tcctgtaagg cctctggcta caccttcacc agctacgtga tgcattgggt ccgccaggcc 120 cccggacagc gcctggagtg gatcggttac atcatcccgt acaacgacgg cactaagtac 180 aacgagaaat ttcagggccg agtgaccctg acctccgaca aatccagctc gaccgcctac 240 atggagctgt cttctctgcg ctcggaggac accgcggttt attactgtgc tcgttggaac 300 tgggatggct atttcgacgt gtggggccag ggaacgaccg tcaccgtgtc gtcc 354 <210> 16 <211> 336 <212> Deoxyribonucleic acid <213> Artificial Sequence <220> <223> 3G4V2_VL OPEN <400> 16 60. gcatcgtga tgacccagag ccctttgtct cttcctgtca ctccggggga gccagcttct atctcatgcc gatcttccaa gagcctgctg cactcaaatg gcatcaccta cctctattgg tacctgcaga agcccgggca atcccctcag ttgctcatct atcagatgtc taaccgcgcc tccggtgtcc ccgaccgctt cagctcctct ggctccggca ccgactttac tctgaagata 240 tcccgcgtgg aggccgaga tgtgggcgtg tactactgca ctcagaacct ggaactgccc 300 ttcaccttcg gccagggcac caagctggag atcaag <210> 17 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> 3G4V2_scFv <400> 17 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Thr Gln Asn 85 90 95 Leu Glu Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser 130 135 140 Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Val 145 150 155 160 Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile Gly 165 170 175 Tyr Ile Ile Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Gln 180 185 190 Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr Met 195 200 205 Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala 210 215 220 Arg Trp Asn Trp Asp Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr 225 230 235 240 Val Thr Val Ser Ser 245 <210> 18 <211> 735 <212> Deoxyribonucleic acid <213> Artificial Sequence <220> <223> 3G4V2_scFv <400> 18 gacatcgtga tgacccagag ccctttgtct cttcctgtca ctccggggga gccagcttct 60 atctcatgcc gatcttccaa gagcctgctg cactcaaatg gcatcaccta cctctattgg 120 tacctgcaga agcccgggca atcccctcag ttgctcatct atcagatgtc taaccgcgcc 180 tccggtgtcc ccgaccgctt cagctcctct ggctccggca ccgactttac tctgaagata 240 tcccgcgtgg aggccgaaga tgtgggcgtg tactactgca ctcagaacct ggaactgccc 300 ttcaccttcg gccagggcac caagctggag atcaagggcg gtggtggctc cggcggaggg 360 ggttctggag gcggcggctc ccaggtgcag ctggtgcaga gcggcgccga ggtgaagaag 420 ccgggtgctt ccgtgaaggt gtcctgtaag gcctctggct acaccttcac cagctacgtg 480 atgcattggg tccgccaggc ccccggacag cgcctggagt ggatcggtta catcatcccg 540 tacaacgacg gcactaagta caacgagaaa tttcagggcc gagtgaccct gacctccgac 600 aaatccagct cgaccgccta catggagctg tcttctctgc gctcggagga caccgcggtt 660 tattactgtg ctcgttggaa ctgggatggc tatttcgacg tgtggggcca gggaacgacc 720 gtcaccgtgt cgtcc 735 <210> 19 <211> 54 <212> PRT <213> Artificial Sequence <220> <223> BCMA peptide <400> 19 Met Leu Gln Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser 1 5 10 15 Leu Leu His Ala Cys Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr 20 25 30 Pro Pro Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser 35 40 45 Val Lys Gly Thr Asn Ala 50 <210> 20 <211> 1178 <212> Deoxyribonucleic Acid <213> Artificial Sequence <220> <223> EF1 promoter <400> 20 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 60 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 120 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 180 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 240 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 300 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 360 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 420 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 480 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 540 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 600 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 660 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 720 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 780 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 840 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 900 cctcagccgt cgcttcatgt gactccactg agtaccgggc gccgtccagg cacctcgatt 960 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 1020 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 1080 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 1140 tggttcaaag tttttttctt ccatttcagg tgtcgtga 1178 <210> 21 <211> 63 <212> Deoxyribonucleic acid <213> Artificial Sequence <220> <223> signal peptides <400> twenty one atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccg 63 <210> twenty two <211> 135 <212> DNA <213> Artificial Sequence <220> <223> CD8 hinge <400> twenty two accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> twenty three <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Transmembrane domain <400> twenty three atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60 accctttact gc 72 <210> twenty four <211> 126 <212> DNA <213> Artificial Sequence <220> <223> 4-1BB <400> twenty four aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120 gaactg 126 <210> 25 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> CD3_zeta <400> 25 agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttggggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgc 336 <210> 26 <211> 591 <212> DNA <213> Artificial Sequence <220> <223> Post-transcriptional regulatory elements of marmot hepatitis virus <400> 26 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc 60 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta 120 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt 180 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg 240 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta 300 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt 360 tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg 420 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca 480 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc 540 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct g 591 <210> 27 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide <400> 27 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 28 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> CD8 hinge <400> 28 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 29 <211> twenty four <212> PRT <213> Artificial Sequence <220> <223> Transmembrane domain <400> 29 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 30 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> 4‑1BB <400> 30 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 31 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CD3_zeta <400> 31 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 32 <211> 2594 <212> Deoxyribonucleic acid <213> Artificial Sequence <220> <223> CAR for lentiviral insertion <400> 32 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 60 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 120 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 180 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 240 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 300 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 360 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 420 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 480 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 540 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 600 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 660 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 720 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 780 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 840 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 900 cctcagccgt cgcttcatgt gactccactg agtaccgggc gccgtccagg cacctcgatt 960 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 1020 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 1080 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 1140 tggttcaaag tttttttctt ccatttcagg tgtcgtgatc tagaggatcc gacatcgtga tgacccagag ccctttgtct cttcctgtca ctccgggggga gccagcttct atctcatgcc 1260 gatcttccaa gagcctgctg cactcaaatg gcatcaccta cctctattgg tacctgcaga agcccgggca atcccctcag ttgctcatct atcagatgtc taaccgcgcc tccggtgtcc 1380. ccgaccgctt cagctcctct ggctccggca ccgactttac tctgaagata tccccgcgtgg 1440 aggccgaaga tgtggggcgtg tactactgca ctcagaacct ggaactgccc ttcaccttcg gccagggcac caagctggag atcaagggcg gtggtggctc cggcggaggg ggttctggag 1560. gcggcggctc ccaggtgcag ctggtgcaga gcggcgccga ggtgaagaag ccgggtgctt ccgtgaaggt gtcctgtaag gcctctggct acaccttcac cagctacgtg atgcattggg 1680 tccgccaggc ccccggacag cgcctggagt ggatcggtta catcatcccg tacaacgacg 1740 gcactaagta caacgagaaa tttcagggcc gagtgaccct gacctccgac aaatccagct cgaccgccta catggagctg tcttctctgc gctcggagga caccgcggtt tattactgtg ctcgttggaa ctgggatggc tatttcgacg tgtggggcca gggaacgacc gtcaccgtgt 1920 cgtccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc 1980 cccgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg 2040 ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact tgtggggtcc 2100 ttctcctgtc actggttatc accctttact gcaaacgggg cagaaagaaa ctcctgtata 2160 tattcaaaca accatttatg agaccagtac aaactactca agaggaagat ggctgtagct 2220 gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc agcaggagcg 2280 cagacgcccc cgcgtacaag cagggccaga accagctcta taacgagctc aatctaggac 2340 gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag atggggggaa 2400 agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa gataagatgg 2460 cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag gggcacgatg 2520 gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt cacatgcagg 2580 ccctgcccccc tcgc 2594

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to BCMA, characterized in that, Include: The heavy chain variable region includes the CDR1 region represented by the amino acid sequence of sequence 1, the CDR2 region represented by the amino acid sequence of sequence 2, and the CDR3 region represented by the amino acid sequence of sequence 3. as well as The light chain variable region includes the CDR1 region represented by the amino acid sequence of sequence 4, the CDR2 region represented by the amino acid sequence of sequence 5, and the CDR3 region represented by the amino acid sequence of sequence 6.

2. The antibody or antigen-binding fragment thereof that specifically binds to BCMA according to claim 1, characterized in that, The antibody is an antibody composed of the heavy chain variable region represented by the amino acid sequence of sequence 7 and the light chain variable region represented by the amino acid sequence of sequence 8, or an antibody composed of the heavy chain variable region represented by the amino acid sequence of sequence 13 and the light chain variable region represented by the amino acid sequence of sequence 14.

3. A chimeric antigen receptor, comprising: a BCMA-binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signal transduction domain, characterized in that, The BCMA-binding domain contains an antibody or its antigen-binding fragment capable of specifically binding to BCMA. The antibody comprises: The heavy chain variable region includes the CDR1 region represented by the amino acid sequence of sequence 1, the CDR2 region represented by the amino acid sequence of sequence 2, and the CDR3 region represented by the amino acid sequence of sequence 3. as well as The light chain variable region includes the CDR1 region represented by the amino acid sequence of sequence 4, the CDR2 region represented by the amino acid sequence of sequence 5, and the CDR3 region represented by the amino acid sequence of sequence 6.

4. The chimeric antigen receptor according to claim 3, characterized in that, The transmembrane domain is a protein selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.

5. The chimeric antigen receptor according to claim 3, characterized in that, The co-stimulatory domain is a protein selected from the group consisting of CD28, 4-1BB, OX-40 and ICOS.

6. The chimeric antigen receptor according to claim 3, characterized in that, The signal transduction structure domain is CD3ζ.

7. The chimeric antigen receptor according to claim 3, characterized in that, A hinge region is also included between the C-terminus of the BCMA-bonded structural domain and the N-terminus of the transmembrane structural domain.

8. A polynucleotide, characterized in that, The chimeric antigen receptor is encoded according to any one of claims 3 to 7.

9. A carrier, characterized in that, It comprises a polynucleotide encoding the chimeric antigen receptor of any one of claims 3 to 7.

10. The carrier according to claim 9, characterized in that, The vector is a plasmid or a retroviral vector.

11. The carrier according to claim 10, characterized in that, The vector is a lentiviral vector.

12. An immune effector cell, characterized in that, It comprises a polynucleotide encoding the chimeric antigen receptor of any one of claims 3 to 7, or a vector comprising said polynucleotide.

13. The immune effector cell according to claim 12, characterized in that, The immune effector cells are T cells or natural killer cells.

14. A pharmaceutical composition for the prevention or treatment of diseases associated with BCMA expression, characterized in that, It includes the immune effector cells as described in claim 12.

15. The pharmaceutical composition according to claim 14 for the prevention or treatment of diseases associated with BCMA expression, characterized in that, The diseases associated with BCMA expression are hematologic malignancies or autoantibody-dependent autoimmune diseases. The hematologic malignancies mentioned above are multiple myeloma or non-Hodgkin's lymphoma. The autoantibody-dependent autoimmune diseases mentioned above are systemic lupus erythematosus (SLE) or rheumatoid arthritis.

Images