WO2024128345A1 - Novel chimeric antigen receptor for targeting b-cell maturation antigen, and use thereof - Google Patents

Novel chimeric antigen receptor for targeting b-cell maturation antigen, and use thereof Download PDF

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WO2024128345A1
WO2024128345A1 PCT/KR2022/020365 KR2022020365W WO2024128345A1 WO 2024128345 A1 WO2024128345 A1 WO 2024128345A1 KR 2022020365 W KR2022020365 W KR 2022020365W WO 2024128345 A1 WO2024128345 A1 WO 2024128345A1
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bcma
car
seq
amino acid
chimeric antigen
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PCT/KR2022/020365
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French (fr)
Korean (ko)
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성백진
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진셀비티 주식회사
성백진
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Priority claimed from KR1020220174221A external-priority patent/KR20240094169A/en
Application filed by 진셀비티 주식회사, 성백진 filed Critical 진셀비티 주식회사
Publication of WO2024128345A1 publication Critical patent/WO2024128345A1/en

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  • the present invention relates to novel chimeric antigen receptors targeting B-cell maturation antigens and uses thereof.
  • CAR-T cells cancer cell-targeting Chimeric Antigen Receptor
  • a chimeric antigen receptor consists of a tumor-specific antigen binding domain (extracellular antigen binding domain), a transmembrane domain (transmembrane domain), a costimulatory domain, and an intracellular signaling domain (intracellular signaling domain).
  • CAR immune cell therapy generally uses gene transduction technology to express, for example, a fusion protein of a minimal antibody binding fragment (single chain fragment variable, scFv) that identifies a tumor-related antigen and a T cell activation sequence on the surface of T cells; T cells expressing the CAR molecules bind tumor antigens in an antigen-dependent, but non-MHC restricted manner and specifically kill tumor cells.
  • CAR immune cell therapies depends on the specificity of the antibodies that identify tumor-related antigens and the affinity with which they bind to the antigens. Therefore, the design of the antigen binding region is an important key to developing new CAR technology.
  • the single domain antibody fragment that is the smallest and can completely bind to the antigen that is, the variable region (VHH) of the heavy chain antibody without the light chain
  • VHH variable region
  • the nanobody structure has high thermal stability, making it easy to use in the production of diagnostic kits, and provides high convenience in storage and use of the finished product when manufactured as an antibody product.
  • CAR modification of a single domain antibody as an antigen-binding region of CAR is one of the development trends of CAR immune cell therapy.
  • BCMA B-cell maturation antigen
  • BAFF B-cell activating factor receptor
  • APRIL B-cell proliferation-inducing ligand
  • Multiple myeloma is a type of blood cancer that occurs when plasma cells abnormally differentiate and proliferate, creating tumors and melting bones, causing pain.
  • multiple myeloma invades the bone marrow and reduces the levels of white blood cells, red blood cells, and platelets, thereby increasing the risk of anemia, infection, and bleeding.
  • myeloma cells produce M protein, an abnormal immune protein, which increases blood concentration, resulting in blood hyperviscosity syndrome or kidney damage.
  • Some of these treatments for multiple myeloma are similar to treatments for other cancers, such as chemotherapy or radiation therapy, stem cell transplantation or bone marrow transplantation, targeted therapy or biologic therapy.
  • Antibody-based cellular immunotherapy has been shown to have substantial clinical benefit for patients suffering from hematological malignancies, especially B-cell non-Hodgkin's lymphoma, but most patients have the problem of relapse or developing secondary resistance. There is a need for effective immunotherapeutic agents to treat myeloma.
  • the present inventors have made diligent efforts to develop a treatment for diseases related to B cells, such as multiple myeloma, and have selected a single domain antibody (sdAb) targeting BCMA, and the selected antibody -Based on the BCMA sdAb, a chimeric antigen receptor (CAR) targeting BCMA and CAR-immune cells expressing it on the surface were finally prepared.
  • sdAb single domain antibody
  • CAR chimeric antigen receptor
  • one object of the present invention is to provide a single domain antibody (sdAb) or antigen-binding fragment thereof that specifically binds to BCMA (B-cell maturation antigen).
  • sdAb single domain antibody
  • BCMA B-cell maturation antigen
  • Another object of the present invention is a chimeric antigen receptor comprising a BCMA-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signal transduction domain.
  • receptor: CAR CAR
  • Another object of the present invention is to provide a polynucleotide encoding the chimeric antigen receptor (CAR).
  • Another object of the present invention is to provide a vector containing the above polynucleotide.
  • Another object of the present invention is to provide isolated immune cells that express the chimeric antigen receptor (CAR) on their surface.
  • CAR chimeric antigen receptor
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases related to BCMA expression.
  • Another object of the present invention is to provide a food composition for preventing or improving diseases related to BCMA expression.
  • Another object of the present invention is to provide a method for producing immune cells, comprising introducing a polynucleotide encoding a chimeric antigen receptor (CAR) or a vector containing the same into isolated immune cells. .
  • CAR chimeric antigen receptor
  • another object of the present invention is a polynucleotide encoding a chimeric antigen receptor (CAR);
  • the present invention provides a method for preventing or treating diseases associated with BCMA expression, comprising administering to a subject a pharmaceutically effective amount of isolated immune cells expressing a chimeric antigen receptor (CAR) on their surface.
  • the present invention provides a CDR1 (complementary determining region 1) represented by any one of the amino acid sequences of SEQ ID NOs: 7 to 9; CDR2 (complementary determining region 2) represented by any one of the amino acid sequences of SEQ ID NOs: 10 to 12; and CDR3 (complementary determining region 3) represented by any one of the amino acid sequences of SEQ ID NOs: 13 to 15, and specifically binds to BCMA (B-cell maturation antigen). sdAb) or antigen-binding fragment thereof.
  • single domain antibodies that specifically bind to BCMA were produced and screened to establish three new single domain antibodies as follows, respectively (i) anti-BCMA sdAb No.7, (ii) anti-BCMA sdAb No.11 and (iii) anti-BCMA sdAb No.19.
  • the single domain antibody is,
  • VHH heavy chain variable domain
  • VHH heavy chain variable domain
  • VHH heavy chain variable domain
  • VHH heavy chain variable domain
  • VHH heavy chain variable domain
  • VHH heavy chain variable domain
  • single-domain antibody generally refers to an antibody fragment consisting of the variable region of an antibody heavy chain (VH region) or the variable region of an antibody light chain (VL region), and refers to a nano antibody. Also referred to as (Nanobody).
  • VH region variable region of an antibody heavy chain
  • VL region variable region of an antibody light chain
  • nanobody nano antibody
  • These single domain antibodies are antibody fragments composed of a single monomeric variable domain, so not only can they selectively bind to a specific antigen like a full antibody, but also have only a heavy chain, so the sequence combination of the CDR region is not as complex as that of a general antibody, so in silico It has the advantage of being easy to build based synthetic libraries.
  • the single domain antibody of the present invention is produced through artificial engineering from the heavy chain antibody of a camelid animal, and is used interchangeably with “VHH (variable heavy chain domains of heavy chain antibody).”
  • CDR generally refers to complementarity determining region, wherein the CDR is primarily associated with the antigenic site.
  • the CDRs of the heavy chain are commonly called CDR1, CDR2 and CDR3, and are numbered sequentially from the N-terminus.
  • CDRs can be defined or identified by conventional methods.
  • the anti-BCMA sdAb of the present invention specifically bound to multiple myeloma H929 cells expressing BCMA.
  • the anti-BCMA sdAb of the present invention can be advantageously applied to the production of a chimeric antigen receptor (CAR) targeting BCMA.
  • CAR chimeric antigen receptor
  • protein, polypeptide and/or amino acid sequence included in the present invention should be understood to include at least functional variants or homologs having the same or similar function as the protein or polypeptide.
  • functional variants may be proteins or polypeptides obtained by substituting, deleting, or adding one or more amino acids in the amino acid sequence of the protein and/or polypeptide.
  • functional variants are amino acid sequences that differ due to substitution, deletion and/or insertion of one or more amino acids, such as 1 to 30, 1 to 20 or 1 to 10 or 1, 2, 3, 4 or 5. It may include a protein or polypeptide having.
  • Functional variants can substantially retain the biological properties of the unmodified protein or polypeptide (substitutions, deletions or additions).
  • functional variants may retain at least 60%, 70%, 80%, 90% or 100% of the biological activity (such as antigen binding ability) of the original protein or polypeptide.
  • a homolog has about 85% or more amino acid sequence homology with the protein and/or polypeptide (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%). %, 97%, 98%, 99% or more) or a polypeptide (e.g., an antibody capable of specifically binding BCMA or a fragment thereof).
  • homology generally refers to similarity, similarity, or correlation between two or more sequences.
  • the present invention provides a chimeric antigen comprising a BCMA-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signal transduction domain.
  • a receptor chimeric antigen receptor: CAR
  • the BCMA-binding domain includes a single domain antibody (sdAb) or an antigen-binding fragment thereof capable of specifically binding to BCMA
  • the single domain antibody (sdAb) includes CDR1 (complementary determining region 1) represented by the amino acid sequence of any one of SEQ ID NOs: 7 to 9; CDR2 (complementary determining region 2) represented by any one of the amino acid sequences of SEQ ID NOs: 10 to 12; and CDR3 (complementary determining region 3) represented by the amino acid sequence of any one of SEQ ID NOs: 13 to 15.
  • CAR chimeric antigen receptor
  • CAR-T chimeric antigen receptor T cells
  • an antigen e.g., tumor-associated antigen (BCMA)
  • BCMA tumor-associated antigen
  • T cell receptor-activating intracellular domains based on the antigenic (e.g. BCMA) specificity of the antibody.
  • Genetically modified CAR-expressing T cells can specifically identify and eliminate target antigen-expressing malignant cells.
  • BCMA B-cell maturation antigen
  • BCMA also known as TNFRSF17, BCM or CD269
  • TNFRSF17, BCM or CD269 is a member of the tumor necrosis receptor (TNFR) family and is predominantly expressed on terminally differentiated B cells, such as memory B cells and plasma cells.
  • BCMA is expressed on tumor cells (e.g., multiple myeloma cells) or is located on the tumor cell surface.
  • “BCMA” of the present invention may include proteins containing mutations of full-length wild-type BCMA, such as point mutations, fragments, insertions, deletions, and splice variants.
  • the term “BCMA-binding domain” generally refers to a domain capable of specifically binding to BCMA protein.
  • the BCMA-binding domain may contain an anti-BCMA antibody or fragment thereof capable of specifically binding to a human BCMA polypeptide or fragment thereof expressed in B cells.
  • binding domain refers to "extracellular domain”, “extracellular binding domain”, “antigen-specific binding domain”, and “Extracellular antigen-specific bidding domain” may be used interchangeably and refers to a CAR domain or fragment that has the ability to specifically bind to a target antigen (e.g. BCMA) do.
  • target antigen e.g. BCMA
  • the anti-BCMA antibody or fragment thereof is used interchangeably with the above-described anti-BCMA sdAb.
  • transmembrane domain generally refers to a domain of CAR that passes through the cell membrane and is connected to an intracellular signaling domain to play a role in signaling.
  • the transmembrane domains include CD8, Fc ⁇ R, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD28, IL-2R ⁇ , CD40, DAP10, MHC class I molecule, TNF receptor protein, and immunoglobulin-like.
  • NKp80 Protein, cytokine receptor, integrin, SLAM protein, activated NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2 , SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 ⁇ , CD8 ⁇ , ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4)
  • costimulatory domain generally refers to an intracellular domain capable of providing immunostimulatory molecules, cell surface molecules required for an effective response of lymphocytes to antigens.
  • the costimulatory domains described above include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, It may be one or more selected from the group consisting of CD7, LIGHT, NKG2C, B7-H3, and CD83, and is preferably 4-1BB represented by the amino acid sequence of SEQ ID NO: 26.
  • intracellular signal transduction domain refers to a domain that is generally located inside a cell and is capable of transmitting signals.
  • the intracellular signaling domain is the intracellular signaling domain of a chimeric antigen receptor.
  • intracellular signaling domains include CD3 ⁇ , Fc ⁇ R, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD28, IL-2R ⁇ , IL-15R- ⁇ , MyD88, DAP10, and DAP12.
  • MHC class I molecule TNF receptor protein, Immunoglobulin-like protein, cytokine receptor, integrin, SLAM protein, activated NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CD40, CDS, ICAM-1, B7 -H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, IA4, CD49D , ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C
  • the present invention may further include a hinge region located between the C terminus of the BCMA-binding domain and the N terminus of the transmembrane domain, wherein the hinge consists of a CD8 hinge, an IgG1 hinge, and an Fc ⁇ RIII ⁇ hinge. It may be one or more types selected from the group, and preferably may be the CD8 hinge represented by the amino acid sequence of SEQ ID NO: 24.
  • the “hinge region” generally refers to the connecting region between the antigen-binding region and the immune cell Fc receptor (FcR)-binding region.
  • a signal peptide may be additionally included at the N terminus of the BCMA-binding domain, and the “signal peptide” generally refers to a peptide chain for guiding protein delivery.
  • the signal peptide may be a short peptide having a length of 5 to 30 amino acids, and in the present invention, the amino acid sequence of SEQ ID NO: 23 was preferably used.
  • the present invention provides a polynucleotide encoding the above-described chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the polynucleotide encoding the chimeric antigen receptor (CAR) is a polynucleotide encoding a BCMA-binding domain; A polynucleotide encoding a transmembrane domain; a polynucleotide coating the costimulatory domain; And it may include a polynucleotide encoding an intracellular signaling domain.
  • the polynucleotide encoding the BCMA-binding domain may preferably be a polynucleotide encoding anti-BCMA sdAb No.7, anti-BCMA sdAb No.11, and anti-BCMA sdAb No.19, and the specific base sequences are described above. It is the same as what was said.
  • the polynucleotide encoding the chimeric antigen receptor (CAR) of the present invention preferably includes a signal peptide represented by the base sequence of SEQ ID NO: 17; Anti-BCMA sdAb represented by the base sequences of SEQ ID NOs: 4 to 6, respectively; CD8 hinge represented by the base sequence of SEQ ID NO: 18; A transmembrane domain represented by the base sequence of SEQ ID NO: 19; 4-1BB (co-stimulatory domain) represented by the base sequence of SEQ ID NO: 20; and CD3 ⁇ (intracellular signaling domain) represented by the base sequence of SEQ ID NO: 21.
  • a signal peptide represented by the base sequence of SEQ ID NO: 17
  • Anti-BCMA sdAb represented by the base sequences of SEQ ID NOs: 4 to 6, respectively
  • CD8 hinge represented by the base sequence of SEQ ID NO: 18
  • a transmembrane domain represented by the base sequence of SEQ ID NO: 19
  • 4-1BB co-stimulatory
  • polynucleotide generally refers to nucleic acid molecules, deoxyribonucleotides or ribonucleotides, or analogs thereof, separated of any length.
  • the polynucleotides of the invention may undergo (1) in-vitro amplification, such as polymerase chain reaction (PCR) amplification; (2) cloning and recombination; (3) purification such as digestion and gel electrophoresis separation; (4) It can be manufactured through synthesis such as chemical synthesis, and preferably the isolated polynucleotide is manufactured by recombinant DNA technology.
  • PCR polymerase chain reaction
  • nucleic acids for encoding antibodies or antigen-binding fragments thereof can be prepared by various methods known in the art, including but not limited to restriction fragment operation of synthetic oligonucleotides or application of SOE PCR. can be manufactured.
  • the present invention provides a vector containing a polynucleotide encoding the chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the term “expression vector” is a gene product containing essential regulatory elements such as a promoter to enable expression of a target gene in an appropriate host cell.
  • the vector may be selected from one or more of plasmids, retroviral vectors, and lentiviral vectors. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself.
  • vectors may contain expression control elements that allow the coding region to be expressed correctly in a suitable host.
  • These regulatory elements are well known to those skilled in the art and include, for example, promoters, ribosome-binding sites, enhancers and other regulatory elements for regulating gene transcription or mRNA translation. can do.
  • the specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally it is a 5' non-specific sequence that participates in transcription initiation and translation initiation, respectively, such as the TATA box, capped sequence, CAAT sequence, etc. -contains a transcribed sequence and a 5' or 3' non-translated sequence.
  • a 5' non-transcriptional expression control sequence may include a promoter region, which may include a promoter sequence for transcribing and regulating a functionally linked nucleic acid.
  • the vector is a recombinant viral vector, preferably a lentiviral vector, and has an operably linked EF1 ⁇ promoter; A polynucleotide encoding a signal peptide; A polynucleotide encoding a BCMA-binding domain; A polynucleotide encoding a transmembrane domain; It contains a polynucleotide encoding an intracellular signaling domain, and may additionally contain a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to increase protein expression.
  • WPRE woodchuck hepatitis virus post-transcriptional regulatory element
  • the EF1 ⁇ promoter may be represented by the nucleotide sequence of SEQ ID NO: 16, and if necessary, the nucleotide sequence of SEQ ID NO: 16 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more , or may contain sequences that are at least 99% identical.
  • the promoter is operably linked to induce expression of an anti-BCMA antibody (sdAb) that is a BCMA-binding domain
  • sdAb anti-BCMA antibody
  • operably linked refers to a nucleic acid expression control sequence to perform a general function. refers to the functional connection between the nucleic acid sequence encoding the protein of interest and the nucleic acid sequence encoding the protein of interest.
  • Operational linkage with a recombinant vector can be made using genetic recombination techniques well known in the art, and site-specific DNA cutting and ligation can be done using enzymes generally known in the art.
  • vectors can be easily introduced into host cells by any method in the art.
  • expression vectors can be transferred into host cells by physical, chemical, or biological means.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and/or exogenous nucleic acids are well known in the related art and can be used with reference to them.
  • a preferred method for introduction of polynucleotides into host cells is calcium phosphate transfection.
  • Biological methods for introducing polynucleotides into host cells include the use of DNA and RNA vectors.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, eg human cells.
  • Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses and adeno-associated viruses, etc.
  • Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Includes.
  • Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).
  • Other methods are available for the state-of-the-art targeted delivery of nucleic acids, such as delivery of polynucleotides using targeted nanoparticles or other suitable submicrometer-sized delivery systems.
  • exemplary delivery vehicles are liposomes.
  • lipid preparations is contemplated for introduction of nucleic acids into host cells (in vitro, ex vivo or in vivo).
  • nucleic acids can be associated with lipids.
  • Nucleic acids associated with lipids may be encapsulated within the aqueous interior of the liposome, dotted within the lipid bilayer of the liposome, attached to the liposome via linkage molecules associated with both the liposome and the oligonucleotide, trapped within the liposome, complexed with the liposome, or , may be dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension within a lipid, contained or complexed with micelles, or otherwise associated with a lipid.
  • the lipid, lipid/DNA or lipid/expression vector association composition is not limited to any particular structure in solution.
  • the present invention includes a polynucleotide encoding the chimeric antigen receptor (CAR) or a vector containing a polynucleotide encoding the chimeric antigen receptor (CAR), and the chimeric antigen receptor Isolated immune cells expressing (CAR) are provided.
  • CAR chimeric antigen receptor
  • CAR chimeric antigen receptor Isolated immune cells expressing
  • the immune cells may be mammalian-derived cells, and may be one or more types selected from the group consisting of T cells, NK cells, NKT cells, B cells, dendritic cells, monocytes, macrophages, eosinophils, basophils, and neutrophils. It may be a T cell, an NK cell, or an NKT cell.
  • the immune cell expressing the chimeric antigen receptor is a polynucleotide encoding the chimeric antigen receptor (CAR) of the present invention or a CAR vector containing the same as an immune cell, such as a T cell or NK. It can be manufactured by introducing it into cells or NKT cells.
  • CAR vectors can be introduced into cells by methods known in the art, such as electroporation and lipofectamine (lipofectamine 2000, Invitrogen).
  • immune effector cells can be transfected by lentiviral vectors to integrate the viral genome carrying CAR molecules into the host genome, ensuring long-term and stable expression of target genes.
  • transposons can be used to introduce CAR transport plasmids (transposons) and transposase transport plasmids into target cells.
  • CAR molecules may be added to the genome by gene editing methods (e.g., CRISPR/Cas9).
  • a lentiviral vector into which a polynucleotide coating BCMA-CAR was inserted was prepared, and the prepared vector was transformed into T cells to prepare BCMA-CAR-T cells.
  • the prepared hBCMA-CAR-T cells express the chimeric antigen receptor targeting BCMA of the present invention.
  • Immune effector cells for producing immune effector cells expressing a chimeric antigen receptor (CAR) can be obtained from a subject, wherein a “subject” is a living organism (e.g., a mammal) against which an immune response can be elicited. Includes. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumor.
  • a subject is a living organism (e.g., a mammal) against which an immune response can be elicited. Includes. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, th
  • the T cells can be obtained from blood units collected from the subject using any of many techniques known to those skilled in the art, such as FicollTM separation.
  • Cells from blood are obtained by apheresis, and the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation over a PERCOLLTM gradient or by countercurrent centrifugation.
  • activated T cells are isolated from peripheral blood mononuclear cells (PBMC), and then BCMA-CAR lentivirus is transduced into the T cells to generate BCMA-CAR-T cells.
  • PBMC peripheral blood mononuclear cells
  • BCMA-CAR lentivirus is transduced into the T cells to generate BCMA-CAR-T cells.
  • Manufactured As a result of confirming the BCMA peptide binding ability of the prepared BCMA-CAR-T cells, it was confirmed that as the BCMA peptide increased, the number of BCMA-CAR-T cells expressing CD4 or CD8 that binds to BCMA increased. This means that the BCMA-CAR-T cells prepared in the present invention effectively bind to BCMA.
  • BCMA-CAR-T cells showed a cell-specific killing effect expressing BCMA.
  • the chimeric antigen receptor and CAR-T cells targeting BCMA of the present invention can be usefully used as a composition for preventing or treating diseases related to B cells or BCMA expression.
  • the present invention provides a polynucleotide encoding a chimeric antigen receptor (CAR) targeting the above-described BCMA;
  • a pharmaceutical composition for preventing or treating diseases related to BCMA expression comprising isolated immune cells expressing a chimeric antigen receptor (CAR) targeting BCMA on their surface.
  • the present invention provides a pharmaceutical composition for preventing or treating diseases related to BCMA expression, including an antibody targeting BCMA.
  • BCMA wild-type or mutant BCMA
  • diseases associated with BCMA may be cancer, malignancy, or autoimmune disease.
  • the pharmaceutical composition may further include a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier for oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. can be used.
  • buffers, preservatives, analgesics, solubilizers, and isotonic agents can be used.
  • stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used.
  • the formulation of the pharmaceutical composition can be prepared in various ways by mixing it with the pharmaceutically acceptable carrier described above.
  • it can be manufactured in the form of tablets, troches, capsules, elylsir, suspension, syrup, wafers, etc., and in the case of injections, it can be manufactured in the form of unit dosage ampoules or multiple dosage forms.
  • the pharmaceutical composition may contain a surfactant that can improve membrane permeability.
  • surfactants are derived from steroids, cationic lipids such as N-[1-(2,3-dioleoyl)propyl-N,N,N-trimethylammonium chloride (DOTMA), or cholesterol hemisuccinate. , phosphatidyl glycerol, etc., but are not limited thereto.
  • the present invention provides a method for preventing or treating diseases related to BCMA expression, comprising administering the pharmaceutical composition according to the present invention to an individual.
  • a pharmaceutical composition containing an antibody targeting BCMA can be administered in a pharmaceutically effective amount to prevent or treat diseases related to BCMA expression. It may vary depending on various factors such as the type of disease, the patient's age, weight, nature and severity of symptoms, type of current treatment, number of treatments, form of administration, and route, and can be easily determined by experts in the field.
  • the pharmaceutical composition may be administered together with or sequentially with the pharmacological or physiological components described above, and may also be administered in combination with additional conventional therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. Such administration may be single or multiple administrations. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
  • the term “subject” refers to a mammal suffering from or at risk of a condition or disease that can be alleviated, suppressed, or treated by administering the pharmaceutical composition, and preferably refers to a human.
  • the term 'administration' as used in the present invention means providing the pharmaceutical composition of the present invention to an individual by any suitable method.
  • the present invention provides a pharmaceutical composition that provides an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human as considered by a researcher, veterinarian, doctor or other clinician, that is, alleviation of symptoms of the disease or disorder being treated. It can be administered in a therapeutically effective amount that induces . It is obvious to those skilled in the art that the therapeutically effective dosage and frequency of administration of the pharmaceutical composition of the present invention will vary depending on the desired effect.
  • the optimal dosage to be administered can be easily determined by a person skilled in the art, depending on the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of dosage form, the patient's age, weight, and general health condition. , gender and diet, administration time, administration route and secretion rate of the composition, treatment period, and various factors including concurrently used drugs.
  • the pharmaceutical composition of the present invention can be administered in an amount of 1 to 10,000 mg/kg/day, and may be administered once a day or in several divided doses.
  • the present invention provides a polynucleotide encoding a chimeric antigen receptor (CAR) targeting the above-described BCMA; Alternatively, it provides a food composition for preventing or improving diseases related to BCMA expression, including isolated immune cells expressing a chimeric antigen receptor (CAR) targeting BCMA on the surface.
  • CAR chimeric antigen receptor
  • the food composition of the present invention can be used as a health functional food, food additive, or dietary supplement.
  • a food additive When used as a food additive, it can be appropriately used according to conventional methods, such as adding the active ingredient of the present invention as is or mixing it with other foods or food ingredients.
  • the health functional food refers to a food manufactured by adding the active ingredient of the present invention to food materials such as beverages, teas, spices, gum, and confectionery, or by encapsulating, powdering, or suspending it, and when ingested, it has specific health effects.
  • food materials such as beverages, teas, spices, gum, and confectionery
  • it has the advantage of not having any side effects that may occur when taking the drug for a long time since it is made from food.
  • the health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis.
  • the amount of active ingredients added in such health foods cannot be uniformly specified as it varies depending on the type of health food being targeted, but it can be added within a range that does not damage the original taste of the food, and is usually 0.01 to 50% for the target food.
  • the health functional food of the present invention may be in the form of a beverage.
  • the mixing amount of the active ingredient can be appropriately changed depending on the purpose of use (prevention, health, or therapeutic treatment), and is preferably included in 0.01 to 95% by weight based on the total weight of the food composition. Preferably it is contained in 1 to 80% by weight. If the content is less than 0.01% by weight, the efficiency of taking it may be reduced, and if it exceeds 95% by weight, the rate of increase in effectiveness relative to the amount used is low, making it uneconomical.
  • the active ingredient of the present invention when manufacturing a food or beverage, is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw materials. However, when consumed for a long period of time for health and hygiene purposes or health control, it can be added in an amount below the above range. Since there is no problem in terms of safety, the active ingredient can be used in an amount above the above range. there is.
  • food to which the active ingredient of the present invention can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream. It includes dairy products, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and includes all health foods in the conventional sense.
  • the food composition of the present invention When the food composition of the present invention is manufactured into a beverage, it may contain additional ingredients such as various flavoring agents or natural carbohydrates, like conventional beverages.
  • the natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin; Synthetic sweeteners such as saccharin and aspartame may be used.
  • the natural carbohydrate is contained in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight, based on the total weight of the food composition of the present invention.
  • the food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid. It may include carbonating agents used in beverages, and may include pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages, but is not limited thereto. These ingredients can be used independently or in combination.
  • the ratio of the above additives is not greatly limited, but is preferably contained within the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.
  • the food composition of the present invention can be taken for a long period of time because it has little cytotoxicity in terms of safety.
  • food refers to a natural product or processed product containing one or more nutrients, preferably in a state that can be eaten directly after a certain degree of processing, and is referred to as food in the conventional sense.
  • food additives health functional foods, beverages and beverage additives, etc.
  • the present invention provides a polynucleotide encoding the above-described chimeric antigen receptor (CAR);
  • a method for preventing or treating diseases associated with BCMA expression comprising administering to a subject a pharmaceutically effective amount of isolated immune cells expressing a chimeric antigen receptor (CAR) on their surface.
  • the treatment of the present invention can be used to treat diseases related to BCMA expression, which are the same as the diseases described above, for example, patients diagnosed with cancer.
  • Types of cancer to be treated with CAR and CAR-T cells of the invention include, but are not limited to, multiple myeloma.
  • the treatment method of the present invention may be combined with one or more therapies for cancer selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy, and radiation therapy.
  • the chimeric antigen receptor and CAR-immune cells targeting BCMA produced in the present invention not only effectively bound to BCMA, but also activated CAR-immune cells bound to BCMA.
  • the CAR-immune cells of the present invention effectively kill cells expressing BCMA
  • the chimeric antigen receptor and CAR-immune cells targeting BCMA of the present invention are used to prevent or treat diseases related to B cells or BCMA expression. It can be usefully used as a therapeutic composition.
  • Figure 1 shows data confirming the BCMA binding ability of anti-BCMA sdAb No.7, No.11, and No.19 of the present invention.
  • FIG. 2 is a schematic diagram showing the chimeric antigen receptor (CAR) targeting BCMA of the present invention.
  • Figure 3 is a schematic diagram showing a lentiviral vector expressing a chimeric antigen receptor (BCMA-CAR) targeting BCMA of the present invention and a chimeric antigen receptor expressed in T cells.
  • BCMA-CAR chimeric antigen receptor
  • Figure 4 is a schematic diagram showing a method for producing BCMA-CAR-T cells using a lentivirus expressing BCMA-CAR of the present invention.
  • Figure 5 is data confirming the BCMA binding ability of the BCMA-CAR-T cells of the present invention.
  • Figure 6 is data confirming the effect of killing target cells by the BCMA-CAR-T cells of the present invention.
  • the present inventors screened a library of single-domain antibodies derived from camel to produce a single domain antibody specific for the BCMA peptide.
  • E. coli cells containing a camel antibody library were infected with VCSM13 helper phage and cultured, and then phage particles with antibody proteins expressed on the surface were recovered from the culture medium.
  • Phage panning technology was used to screen for antibodies that bind to BCMA. Panning is an iterative process to enrich phages in the population that possess higher affinity binding to the target of interest.
  • the library was exposed to the selected antigen, and then only the antigen with the highest binding affinity was eluted and amplified to accumulate a population of phages.
  • the bacteriophage selected in the previous step was cloned and selected.
  • phage clones displaying proteins with higher binding affinity were selected compared to phage clones displaying proteins with lower binding affinity.
  • VHH heavy chain variable domain
  • CDR complementarity determining region
  • anti-BCMA sdAb No.7 includes CDR1 represented by the amino acid sequence of SEQ ID NO: 7, CDR2 represented by the amino acid sequence of SEQ ID NO: 10, and CDR3 represented by the amino acid sequence of SEQ ID NO: 13.
  • VHH heavy chain variable domain
  • Anti-BCMA sdAb No.11 is a heavy chain variable domain (VHH) comprising CDR1 represented by the amino acid sequence of SEQ ID NO: 8, CDR2 represented by the amino acid sequence of SEQ ID NO: 11, and CDR3 represented by the amino acid sequence of SEQ ID NO: 14;
  • anti-BCMA sdAb No.19 has a heavy chain variable domain (VHH) comprising CDR1 represented by the amino acid sequence of SEQ ID NO: 9, CDR2 represented by the amino acid sequence of SEQ ID NO: 12, and CDR3 represented by the amino acid sequence of SEQ ID NO: 15. It was confirmed that it consists of.
  • the anti-BCMA sdAb No.7, No.11 and No.19 are composed of heavy chain variable domains represented by the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the anti-BCMA sdAbs are It was confirmed that they were encoded by the base sequences of SEQ ID NOs: 4, 5, and 6, respectively.
  • sequence name sequence number order Anti-BCMA sdAb No.7 amino acid sequence SEQ ID NO: 1 KLEESGGGSVQTGGSLRLTCAASG SIFSSGFMA WFRQAPGKERE FVSGISWRGDSTG YADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYC AARGTDTA YWGQGTQVTVSS base sequence SEQ ID NO: 4 AAGCTGGAGGAGAGCGGTGGTGGCTCCGTGCAGACAGGAGGAAGCTTGCGCCTGACCTGCGCCGCTTCCGGCTCCATTTTTTCATCTGGCCTTCATGGCGTGGTTCCGCCAGGCCCCCCGGGAAGGAGGGAGTTCGTCTCGGGCATCAGTTGGCGTGGTGATTCTACTGGCTACGCGGACAGCGTGAAGGGCCGCTTCACCATCTCTCGGGACAACGCCAAGAACACGGTGGACCTGCAGATGAACTCCCTCAA ACCTGAAGATACTGCTATCTACTGTGCTGCACGCGGGACCGAACACGGTGGACCTGCAGAT
  • the present inventors performed flow cytometry to confirm the specificity for BCMA of the anti-BCMA sdAbs of the present invention established in Example 1 above, whether they could specifically target BCMA.
  • PE-conjugated anti-BCMA antibody Biolegend Inc., cat# 357503, USA
  • PE-conjugated anti-BCMA antibody was used as a secondary antibody.
  • mouse IgG antibody PE-conjugated goat anti-mouse IgG; Biolegend Inc., cat# 405307, USA
  • the antibodies selected in the present invention specifically recognize cells expressing BCMA, they can be useful in various fields such as diagnosis as well as treatment of diseases mediated by cells expressing BCMA.
  • a chimeric antigen receptor (CAR) targeting BCMA As shown in the schematic diagram of FIG. 2, the present inventors used anti-BCMA sdAb No. 7, which was selected as having high specificity for BCMA in Example 1, Using No.11 and No.19, lentiviral vectors (BCMA-CAR lentivirus) expressing a chimeric antigen receptor (CAR) targeting BCMA were produced, respectively.
  • EF1 ⁇ promoter SEQ ID NO: 16
  • a polynucleotide encoding a signal peptide SEQ ID NO: 17
  • a polynucleotide encoding a BCMA-binding domain SEQ ID NO: 4, 5 or 6
  • a polynucleotide encoding the CD8 hinge region SEQ ID NO: 18
  • a polynucleotide encoding a transmembrane domain SEQ ID NO: 19
  • Polynucleotide encoding 4-1BB (co-stimulatory domain) SEQ ID NO: 20
  • Polynucleotide encoding CD3 ⁇ intracellular signaling domain
  • CAR DNA consisting of a polynucleotide (SEQ ID NO: 22) encoding WPRE was synthesized in vitro and inserted into a third-generation lentiviral vector.
  • the sequence information was arranged in Table 2 below.
  • Lentiviral vectors were co-transfected into Lenti-X 293T cells with three vectors: pMDLg/pRRE (Addgene, cat##12251), pMD2.G (Addgene, cat##12259), and pRSV-Rev (Addgene, cat##12253). After infection (co-transfection), BCMA-CAR lentivirus was produced. For co-infection, the three vectors and Lenti-X 293T cells were cultured for 6 hours using Lipofectamine 3000 transfection kit (Invitrogen, cat# L3000-015) and Opti-MEM+GlutaMAX (gibco, cat# 51985-034) medium. .
  • sequence name sequence number order EF1 promoter base sequence SEQ ID NO: 16 GTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA signal peptide base sequence SEQ ID NO: 17 ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG amino acid sequence SEQ ID NO: 23 MALPVTALLLPLALLLHAARP CD8 hinge base sequence SEQ ID NO: 18 ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT amino acid sequence SEQ ID NO: 24 TTTPAPRPPTPAPTIASQPL
  • the present inventors transformed the three types of BCMA-CAR lentiviral vectors produced in Example 3 into T cells to produce BCMA-CAR-T cells (No. 7 CAR-T, No. 11 CAR-T, and No. 11 CAR-T, respectively). 19 CAR-T) was prepared.
  • T cell activation beads T cell activation bead; MiltenylBiotec, cat# 130-091-441
  • BCMA-CAR-T cells were prepared by transducing activated T cells with the BCMA-CAR lentivirus prepared in Example 3, and transduction efficiency was increased using Lenti-boost-p.
  • the present inventors performed flow cytometry to confirm the BCMA peptide binding ability of the BCMA-CAR-T cells.
  • the BCMA-CAR-T cells prepared above were reacted with FITC-BCMA protein (Acrobiosystems, cat#BCA-HF2H3) and anti-CD3, anti-CD4, and anti-CD8 antibodies, followed by fluorescence using a FACS machine. The intensity was measured. During the analysis process, cells expressing CD3 were considered T cells, and the level of FITC expression in T cells was confirmed.
  • FITC-BCMA protein Acrobiosystems, cat#BCA-HF2H3
  • anti-CD3, anti-CD4, and anti-CD8 antibodies followed by fluorescence using a FACS machine. The intensity was measured.
  • cells expressing CD3 were considered T cells, and the level of FITC expression in T cells was confirmed.
  • the present inventors sought to confirm the killing effect of target cells by the BCMA-CAR-T cells of the present invention.
  • %Cytotoxicity [(Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)]
  • chimeric antigen receptor and CAR-T cells targeting BCMA of the present invention can be usefully used as a composition for preventing or treating diseases related to B cells or BCMA expression.

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Abstract

The present invention relates to a novel chimeric antigen receptor for targeting a B-cell maturation antigen, and use thereof. It has been identified that the chimeric antigen receptor for targeting a BCMA and CAR-immune cells, prepared in the present invention, effectively bind to the BCMA and the CAR-immune cells bound to BCMA are activated. In addition, the CAR-immune cells of the present invention have been identified to effectively kill cells in which the BCMA is expressed, and thus the chimeric antigen receptor for targeting BCMA and the CAR-immune cells, of the present invention, can be effectively used as a composition for preventing or treating diseases associated with B cell or BCMA expression.

Description

B-세포 성숙 항원을 표적화하는 신규한 키메라 항원 수용체 및 이의 용도Novel chimeric antigen receptor targeting B-cell maturation antigen and uses thereof
본 발명은 B-세포 성숙 항원을 표적화하는 신규한 키메라 항원 수용체 및 이의 용도에 관한 것이다.The present invention relates to novel chimeric antigen receptors targeting B-cell maturation antigens and uses thereof.
최근 암세포 표적 키메라 항원 수용체(Chimeric Antigen Receptor; CAR) 유전자를 도입하여 생산된 T 세포(CAR-T 세포)의 개선된 항암 효과가 입증되면서, 타겟 암세포에 대한 특이성과 활성화를 촉진하여 항암 치료 효용성을 증진하기 위한 방법으로 CAR-면역세포 치료제 개발에 대한 관심이 대두되고 있다.Recently, the improved anticancer effect of T cells (CAR-T cells) produced by introducing the cancer cell-targeting Chimeric Antigen Receptor (CAR) gene has been demonstrated, increasing the effectiveness of anticancer treatment by promoting specificity and activation for target cancer cells. There is growing interest in developing CAR-immune cell therapy as a way to improve the immune system.
통상적으로 키메라 항원 수용체는 하나의 종양 관련 항원 결합 영역(세포외 항원 결합 도메인), 막관통 영역(막관통 도메인), 공동자극 영역 및 세포내 신호 전달 영역(세포내 신호전달 도메인)으로 구성된다. CAR 면역세포 치료법은 일반적으로 유전자 형질 도입 기술을 통하여, 예컨대, 종양 관련 항원을 식별하는 최소 항체 결합단편(Single chain fragment variable, scFv)과 T 세포 활성화 서열의 융합 단백질을 T 세포 표면에 발현시키고, 상기 CAR 분자를 발현하는 T 세포는 항원의존적이나, 비 MHC 제한된 방식으로 종양 항원과 결합하여, 종양세포를 특이적으로 살해한다.Typically, a chimeric antigen receptor consists of a tumor-specific antigen binding domain (extracellular antigen binding domain), a transmembrane domain (transmembrane domain), a costimulatory domain, and an intracellular signaling domain (intracellular signaling domain). CAR immune cell therapy generally uses gene transduction technology to express, for example, a fusion protein of a minimal antibody binding fragment (single chain fragment variable, scFv) that identifies a tumor-related antigen and a T cell activation sequence on the surface of T cells; T cells expressing the CAR molecules bind tumor antigens in an antigen-dependent, but non-MHC restricted manner and specifically kill tumor cells.
이러한 CAR 면역세포 치료법의 유효성은 종양 관련 항원을 식별하는 항체의 특이성 및 항원과 결합되는 친화력에 의존한다. 따라서, 항원 결합 영역의 설계는 새로운 CAR 기술이 개발되는 데 있어 중요한 관건이다. The effectiveness of these CAR immune cell therapies depends on the specificity of the antibodies that identify tumor-related antigens and the affinity with which they bind to the antigens. Therefore, the design of the antigen binding region is an important key to developing new CAR technology.
낙타과 동물 유래 중쇄항체(Heavy chain antibody, HCAb)에서 가장 작고, 항원과 완전히 결합될 수 있는 단일 도메인 항체 단편, 즉, 경쇄가 없는 중쇄항체의 가변 영역(VHH)은, 구조가 간단하고, 분자량은 보통 항체 분자량의 1/10로서, 체외 발현 시스템에서 고효율적으로 발현되고 정제될 수 있으며, 아울러 인간 항체와의 유사성(homology)이 높아 면역원성 위험성도 낮다는 장점이 있다. 고특이성, 고친화력, 면역원성은 낮고, 삼투성이 좋으며, 종양 치료할 때, 통상적인 항체와 접촉될 수 없는 비교적 은폐된 타겟과 접촉될 수 있는 가능성을 갖고 있다. 산업적 활용에 있어서도 나노바디 구조는 높은 열 안정성을 가지고 있어서, 진단키트 제작에의 활용에 용이하고, 항체 제품으로 제작시 완제품에 대한 보관 및 사용에서 높은 편의성을 제공한다.Among the camelid animal-derived heavy chain antibodies (HCAb), the single domain antibody fragment that is the smallest and can completely bind to the antigen, that is, the variable region (VHH) of the heavy chain antibody without the light chain, has a simple structure and a molecular weight of It has the advantage of being 1/10 of the molecular weight of a normal antibody, so that it can be expressed and purified with high efficiency in an in vitro expression system, and also has a low risk of immunogenicity due to its high homology to human antibodies. It has high specificity, high affinity, low immunogenicity, good permeability, and has the potential to contact relatively hidden targets that cannot be contacted by conventional antibodies when treating tumors. In industrial applications, the nanobody structure has high thermal stability, making it easy to use in the production of diagnostic kits, and provides high convenience in storage and use of the finished product when manufactured as an antibody product.
이러한 특성에 기반하여, 단일 도메인 항체를 CAR의 항원 결합 영역으로서 CAR 수식하는 것은 CAR 면역 세포 치료법의 발전 추세 중의 하나이다.Based on these characteristics, CAR modification of a single domain antibody as an antigen-binding region of CAR is one of the development trends of CAR immune cell therapy.
한편, CD269 또는 TNFRSF17로도 알려진 B-세포 성숙 항원(B-cell maturation antigen: BCMA)은 종양괴사인자(tumor necrosis factor) 수용체 패밀리의 구성원이다. BCMA가 B-세포 활성화 인자 수용체(B-cell activating factor receptor: BAFF) 및 B-세포 증식 유도 리간드(B-cell proliferation-inducing ligand: APRIL)와 결합하여 상이한 발달 단계에서 B 세포의 생존을 촉진할 수 있음이 보고된 바 있다. 비정상적인 신호전달은 B 세포의 비정상적인 증식을 초래하여 다발성 골수종 등과 같은 자가 면역 질환(autoimmune diseases) 및 종양형성(tumorigenesis)을 초래할 수 있다.Meanwhile, B-cell maturation antigen (BCMA), also known as CD269 or TNFRSF17, is a member of the tumor necrosis factor receptor family. BCMA binds to B-cell activating factor receptor (BAFF) and B-cell proliferation-inducing ligand (APRIL) to promote B cell survival at different developmental stages. It has been reported that this can be done. Abnormal signaling can lead to abnormal proliferation of B cells, resulting in autoimmune diseases such as multiple myeloma and tumorigenesis.
다발성 골수종은 형질세포가 비정상적으로 분화 및 증식하여 나타나는 혈액암의 일종으로, 이는 종양을 생성하고 뼈를 녹여 통증을 유발한다. 이뿐만 아니라, 다발성 골수종은 골수를 침범하여 백혈구, 적혈구, 혈소판 수치를 감소시킴으로써 빈혈, 감염, 출혈의 위험성도 증가시킨다. 나아가, 골수종 세포는 비정상 면역단백인 M 단백(M protein)을 생성하기도 하며, 이는 혈액의 농도를 증가시켜 혈액과점도증후군을 초래하거나 신장에 손상을 주기도 한다.Multiple myeloma is a type of blood cancer that occurs when plasma cells abnormally differentiate and proliferate, creating tumors and melting bones, causing pain. In addition, multiple myeloma invades the bone marrow and reduces the levels of white blood cells, red blood cells, and platelets, thereby increasing the risk of anemia, infection, and bleeding. Furthermore, myeloma cells produce M protein, an abnormal immune protein, which increases blood concentration, resulting in blood hyperviscosity syndrome or kidney damage.
이러한 다발성 골수종에 대한 일부 치료법은 다른 암에 대한 치료법, 예를 들면, 화학요법 또는 방사선 요법, 줄기세포 이식 또는 골수 이식, 표적화된 요법 또는 생물학적 요법과 유사하다. 항체-기초된 세포 면역요법은 혈액학적 악성, 특히 B 세포 비호지킨 림프종을 앓는 환자에 대한 실제적인 임상적 유익성이 확인되었으나, 대부분의 환자가 재발하거나 2차 저항이 발생하는 문제점이 있으므로, 다발성 골수종을 치료하기 위한 효과적인 면역치료적 작용제가 요구되고 있다.Some of these treatments for multiple myeloma are similar to treatments for other cancers, such as chemotherapy or radiation therapy, stem cell transplantation or bone marrow transplantation, targeted therapy or biologic therapy. Antibody-based cellular immunotherapy has been shown to have substantial clinical benefit for patients suffering from hematological malignancies, especially B-cell non-Hodgkin's lymphoma, but most patients have the problem of relapse or developing secondary resistance. There is a need for effective immunotherapeutic agents to treat myeloma.
상술한 상황 하에서, 본 발명자들은 다발성 골수종 등과 같은 B 세포와 관련된 질환의 치료제를 개발하기 위해 예의 노력한 결과, BCMA를 표적으로 하는 단일 도메인 항체(single domain antibody, sdAb)를 선별하였으며, 상기 선별된 항-BCMA sdAb를 기반으로 하여, 최종적으로 BCMA를 표적으로 하는 키메라 항원 수용체(CAR)및 이를 표면에 발현하는 CAR-면역 세포를 제조하였다. 상기 제조된 BCMA 표적 CAR-면역 세포가 BCMA와 특이적으로 결합하여, BCMA 발현 종양세포를 효과적으로 현저하게 사멸시키는 것을 규명함으로써, 본 발명을 완성하였다.Under the above-described circumstances, the present inventors have made diligent efforts to develop a treatment for diseases related to B cells, such as multiple myeloma, and have selected a single domain antibody (sdAb) targeting BCMA, and the selected antibody -Based on the BCMA sdAb, a chimeric antigen receptor (CAR) targeting BCMA and CAR-immune cells expressing it on the surface were finally prepared. The present invention was completed by confirming that the prepared BCMA-targeted CAR-immune cells specifically bind to BCMA and effectively and significantly kill BCMA-expressing tumor cells.
따라서, 본 발명의 일 목적은, BCMA(B-cell maturation antigen)에 특이적으로 결합하는, 단일 도메인 항체(single domain antibody, sdAb) 또는 이의 항원-결합 단편을 제공하는 데 있다.Accordingly, one object of the present invention is to provide a single domain antibody (sdAb) or antigen-binding fragment thereof that specifically binds to BCMA (B-cell maturation antigen).
또한, 본 발명의 다른 목적은, BCMA-결합 도메인, 막관통 도메인(transmembrane domain), 공동자극 도메인(costimulatory domain) 및 세포 내 신호전달 도메인(intracellular signal transduction domain)을 포함하는 키메라 항원 수용체(chimeric antigen receptor: CAR)를 제공하는 데 있다.In addition, another object of the present invention is a chimeric antigen receptor comprising a BCMA-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signal transduction domain. receptor: CAR).
또한, 본 발명의 또 다른 목적은, 상기 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드를 제공하는 데 있다.In addition, another object of the present invention is to provide a polynucleotide encoding the chimeric antigen receptor (CAR).
또한, 본 발명의 또 다른 목적은, 상기 폴리뉴클레오타이드를 포함하는 벡터를 제공하는 데 있다.Additionally, another object of the present invention is to provide a vector containing the above polynucleotide.
또한, 본 발명의 또 다른 목적은, 상기 키메라 항원 수용체(CAR)를 표면에 발현하는, 단리된 면역 세포를 제공하는 데 있다.Additionally, another object of the present invention is to provide isolated immune cells that express the chimeric antigen receptor (CAR) on their surface.
또한, 본 발명의 또 다른 목적은, BCMA 발현과 관련된 질환 예방 또는 치료용 약학적 조성물을 제공하는 데 있다.In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases related to BCMA expression.
또한, 본 발명의 또 다른 목적은, BCMA 발현과 관련된 질환 예방 또는 개선용 식품 조성물을 제공하는 데 있다.In addition, another object of the present invention is to provide a food composition for preventing or improving diseases related to BCMA expression.
또한, 본 발명의 또 다른 목적은, 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드 또는 이를 포함하는 벡터를, 단리된 면역 세포에 도입시키는 단계를 포함하는, 면역 세포의 제조 방법을 제공하는 데 있다.In addition, another object of the present invention is to provide a method for producing immune cells, comprising introducing a polynucleotide encoding a chimeric antigen receptor (CAR) or a vector containing the same into isolated immune cells. .
또한, 본 발명의 또 다른 목적은, 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드; 또는 키메라 항원 수용체(CAR)를 표면에 발현하는 단리된 면역 세포를, 약학적 유효량으로 대상(subject)에게 투여하는 단계를 포함하는, BCMA 발현과 관련된 질환 예방 또는 치료 방법을 제공하는 데 있다.In addition, another object of the present invention is a polynucleotide encoding a chimeric antigen receptor (CAR); Alternatively, the present invention provides a method for preventing or treating diseases associated with BCMA expression, comprising administering to a subject a pharmaceutically effective amount of isolated immune cells expressing a chimeric antigen receptor (CAR) on their surface.
본 명세서에서 사용한 용어는 단지 설명을 목적으로 사용된 것으로, 한정하려는 의도로 해석되어서는 안된다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서 상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.The terms used herein are for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 실시예가 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥 상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the embodiments belong. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless explicitly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태에 따르면, 본 발명은 서열번호 7 내지 9 중 어느 하나의 아미노산 서열로 표시되는 CDR1(complementary determining region 1); 서열번호 10 내지 12 중 어느 하나의 아미노산 서열로 표시되는 CDR2(complementary determining region 2); 및 서열번호 13 내지 15 중 어느 하나의 아미노산 서열로 표시되는 CDR3(complementary determining region 3);를 포함하며, BCMA(B-cell maturation antigen)에 특이적으로 결합하는, 단일 도메인 항체(single domain antibody, sdAb) 또는 이의 항원-결합 단편을 제공한다.According to one aspect of the present invention, the present invention provides a CDR1 (complementary determining region 1) represented by any one of the amino acid sequences of SEQ ID NOs: 7 to 9; CDR2 (complementary determining region 2) represented by any one of the amino acid sequences of SEQ ID NOs: 10 to 12; and CDR3 (complementary determining region 3) represented by any one of the amino acid sequences of SEQ ID NOs: 13 to 15, and specifically binds to BCMA (B-cell maturation antigen). sdAb) or antigen-binding fragment thereof.
본 발명에서는 BCMA와 특이적으로 결합하는 단일 도메인 항체를 제작하고 스크리닝하여 하기와 같은 신규한 단일 도메인 항체 3 종을 확립하였으며, 각각 (i) 항-BCMA sdAb No.7, (ii) 항-BCMA sdAb No.11 및 (iii) 항-BCMA sdAb No.19로 명명하였다.In the present invention, single domain antibodies that specifically bind to BCMA were produced and screened to establish three new single domain antibodies as follows, respectively (i) anti-BCMA sdAb No.7, (ii) anti-BCMA sdAb No.11 and (iii) anti-BCMA sdAb No.19.
본 발명의 바람직한 구현예에 따르면, 상기 단일 도메인 항체는, According to a preferred embodiment of the present invention, the single domain antibody is,
(i) 서열번호 7의 아미노산 서열로 표시되는 CDR1, 서열번호 10의 아미노산 서열로 표시되는 CDR2 및 서열번호 13의 아미노산 서열로 표시되는 CDR3를 포함하는 중쇄 가변 도메인(VHH)(항-BCMA sdAb No.7); (i) a heavy chain variable domain (VHH) comprising CDR1 represented by the amino acid sequence of SEQ ID NO: 7, CDR2 represented by the amino acid sequence of SEQ ID NO: 10, and CDR3 represented by the amino acid sequence of SEQ ID NO: 13 (anti-BCMA sdAb No .7);
(ii) 서열번호 8의 아미노산 서열로 표시되는 CDR1, 서열번호 11의 아미노산 서열로 표시되는 CDR2 및 서열번호 14의 아미노산 서열로 표시되는 CDR3를 포함하는 중쇄 가변 도메인(VHH)(항-BCMA sdAb No.11); 또는 (ii) a heavy chain variable domain (VHH) comprising CDR1 represented by the amino acid sequence of SEQ ID NO: 8, CDR2 represented by the amino acid sequence of SEQ ID NO: 11, and CDR3 represented by the amino acid sequence of SEQ ID NO: 14 (anti-BCMA sdAb No .11); or
(iii) 서열번호 9의 아미노산 서열로 표시되는 CDR1, 서열번호 12의 아미노산 서열로 표시되는 CDR2 및 서열번호 15의 아미노산 서열로 표시되는 CDR3를 포함하는 중쇄 가변 도메인(VHH)(항-BCMA sdAb No.19);을 포함한다.(iii) a heavy chain variable domain (VHH) comprising CDR1 represented by the amino acid sequence of SEQ ID NO: 9, CDR2 represented by the amino acid sequence of SEQ ID NO: 12, and CDR3 represented by the amino acid sequence of SEQ ID NO: 15 (anti-BCMA sdAb No .19); includes.
상기 (i) 중쇄 가변 도메인(VHH)은 서열번호 1로 표시되는 아미노산 서열로 이루어지고, 서열번호 4로 표시되는 염기서열에 의해 코딩된다.The (i) heavy chain variable domain (VHH) consists of the amino acid sequence shown in SEQ ID NO: 1 and is encoded by the base sequence shown in SEQ ID NO: 4.
상기 (ii) 중쇄 가변 도메인(VHH)은 서열번호 2로 표시되는 아미노산 서열로 이루어지고, 서열번호 5로 표시되는 염기서열에 의해 코딩된다.The (ii) heavy chain variable domain (VHH) consists of the amino acid sequence shown in SEQ ID NO: 2 and is encoded by the base sequence shown in SEQ ID NO: 5.
상기 (iii) 중쇄 가변 도메인(VHH)은 서열번호 3로 표시되는 아미노산 서열로 이루어지고 서열번호 6로 표시되는 염기서열에 의해 코딩된다.The (iii) heavy chain variable domain (VHH) consists of the amino acid sequence shown in SEQ ID NO: 3 and is encoded by the base sequence shown in SEQ ID NO: 6.
본 명세서에서, 용어 "단일 도메인 항체(single-domain antibody, sdAb)"는 통상적으로 항체 중쇄의 가변 영역(VH 영역) 또는 항체 경쇄의 가변 영역(VL 영역)으로 구성되는 항체 단편을 의미하며 나노 항체(Nanobody)로도 지칭된다. 이러한 단일 도메인 항체는 단일 단량체 가변 도메인으로 구성된 항체 단편이므로, 전체 항체와 같이 특정 항원에 선택적으로 결합이 가능할 뿐만 아니라, 중쇄만 가지고 있어 CDR 영역의 서열 조합이 일반적인 항체에 비해 복잡도가 높지 않아 인실리코 기반의 합성 라이브러리 구축이 용이하다는 장점이 있다.As used herein, the term "single-domain antibody (sdAb)" generally refers to an antibody fragment consisting of the variable region of an antibody heavy chain (VH region) or the variable region of an antibody light chain (VL region), and refers to a nano antibody. Also referred to as (Nanobody). These single domain antibodies are antibody fragments composed of a single monomeric variable domain, so not only can they selectively bind to a specific antigen like a full antibody, but also have only a heavy chain, so the sequence combination of the CDR region is not as complex as that of a general antibody, so in silico It has the advantage of being easy to build based synthetic libraries.
본 발명의 단일 도메인 항체는 낙타과 동물의 중쇄항체로부터 인조 공학에 따라 제조되는 것으로, "VHH(variable heavy chain domains of heavy chain antibody)"와 혼용된다. The single domain antibody of the present invention is produced through artificial engineering from the heavy chain antibody of a camelid animal, and is used interchangeably with “VHH (variable heavy chain domains of heavy chain antibody).”
본 출원에서, 용어 "CDR"는 통상적으로 상보성 결정 영역을 의미하는 것으로, CDR는 주로 항원 부위와 결합된다. 중쇄의 CDR는 통상적으로 CDR1, CDR2와 CDR3으로 불리우고, N-단으로부터 순서대로 번호를 매긴다. 본 출원에서, 통상적인 방법으로 CDR를 한정하거나 동정할 수 있다. In this application, the term “CDR” generally refers to complementarity determining region, wherein the CDR is primarily associated with the antigenic site. The CDRs of the heavy chain are commonly called CDR1, CDR2 and CDR3, and are numbered sequentially from the N-terminus. In this application, CDRs can be defined or identified by conventional methods.
본 발명의 일 실시예에서, 도 1에 나타난 바와 같이,본 발명의 항-BCMA sdAb가 BCMA를 발현하는 다발성골수종 H929세포에 특이적으로 결합한 것을 확인하였다.In one example of the present invention, as shown in Figure 1, it was confirmed that the anti-BCMA sdAb of the present invention specifically bound to multiple myeloma H929 cells expressing BCMA.
따라서, 본 발명의 항-BCMA sdAb를 이용하여 BCMA를 표적으로 하는 키메라 항원 수용체(CAR, chimeric antigen receptor) 제조에 유리하게 적용할 수 있다.Therefore, the anti-BCMA sdAb of the present invention can be advantageously applied to the production of a chimeric antigen receptor (CAR) targeting BCMA.
또한, 본 발명에 포함된 단백질, 폴리펩타이드 및/또는 아미노산 서열은 적어도 단백질 또는 폴리펩타이드와 동일하거나 유사한 기능을 갖는 기능성 변이체(functional variants) 또는 상동체를 포함하는 것으로 이해되어야 한다.In addition, the protein, polypeptide and/or amino acid sequence included in the present invention should be understood to include at least functional variants or homologs having the same or similar function as the protein or polypeptide.
본 발명에서, 기능적 변이체(functional variants)는 상기 단백질 및/또는 폴리펩타이드의 아미노산 서열에서 하나 이상의 아미노산을 치환, 결실 또는 첨가함으로써 수득되는 단백질 또는 폴리펩타이드일 수 있다. 예를 들어, 기능적 변이체(functional variants)는 1에서 30, 1에서 20 또는 1에서 10 또는 1, 2, 3, 4 또는 5와 같은 하나 이상의 아미노산의 치환, 결실 및/또는 삽입으로 인해 상이한 아미노산 서열을 갖는 단백질 또는 폴리펩타이드를 포함할 수 있다. 기능적 변이체(functional variants)는 변형되지 않은 단백질 또는 폴리펩타이드 (치환, 결실 또는 첨가)의 생물학적 특성을 실질적으로 유지시킬 수 있다. 예를 들어, 기능적 변이체(functional variants)는 원래 단백질 또는 폴리펩타이드의 생물학적 활성 (항원 결합 능력과 같은)의 적어도 60%, 70%, 80%, 90% 또는 100%로 유지될 수 있다.In the present invention, functional variants may be proteins or polypeptides obtained by substituting, deleting, or adding one or more amino acids in the amino acid sequence of the protein and/or polypeptide. For example, functional variants are amino acid sequences that differ due to substitution, deletion and/or insertion of one or more amino acids, such as 1 to 30, 1 to 20 or 1 to 10 or 1, 2, 3, 4 or 5. It may include a protein or polypeptide having. Functional variants can substantially retain the biological properties of the unmodified protein or polypeptide (substitutions, deletions or additions). For example, functional variants may retain at least 60%, 70%, 80%, 90% or 100% of the biological activity (such as antigen binding ability) of the original protein or polypeptide.
본 발명에서, 상동체는 상기 단백질 및/또는 폴리펩타이드와 아미노산 서열 상동성이 약 85 % 이상(예컨대, 약 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 또는 그 이상)인 단백질 또는 폴리펩타이드(예를 들어 BCMA 또는 이의 단편과 특이적으로 결합할 수 있는 항체)일 수 있다. In the present invention, a homolog has about 85% or more amino acid sequence homology with the protein and/or polypeptide (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%). %, 97%, 98%, 99% or more) or a polypeptide (e.g., an antibody capable of specifically binding BCMA or a fragment thereof).
본 발명에서, 상동성은 일반적으로 둘 이상의 서열 사이의 유사성, 유사성 또는 상관성을 지칭한다.In the present invention, homology generally refers to similarity, similarity, or correlation between two or more sequences.
또한, 본 발명의 다른 양태에 따르면, 본 발명은 BCMA-결합 도메인, 막관통 도메인(transmembrane domain), 공동자극 도메인(costimulatory domain) 및 세포 내 신호전달 도메인(intracellular signal transduction domain)을 포함하는 키메라 항원 수용체(chimeric antigen receptor: CAR)를 제공한다.In addition, according to another aspect of the present invention, the present invention provides a chimeric antigen comprising a BCMA-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signal transduction domain. Provides a receptor (chimeric antigen receptor: CAR).
본 발명의 상기 키메라 항원 수용체(chimeric antigen receptors, CAR)에서, 상기 BCMA-결합 도메인은 BCMA와 특이적으로 결합할 수 있는 단일 도메인 항체(single domain antibody, sdAb) 또는 이의 항원-결합 단편을 포함하며, 상기 단일 도메인 항체(single domain antibody, sdAb)는, 서열번호 7 내지 9 중 어느 하나의 아미노산 서열로 표시되는 CDR1(complementary determining region 1); 서열번호 10 내지 12 중 어느 하나의 아미노산 서열로 표시되는 CDR2(complementary determining region 2); 및 서열번호 13 내지 15 중 어느 하나의 아미노산 서열로 표시되는 CDR3(complementary determining region 3);를 포함하는 것을 특징으로 한다.In the chimeric antigen receptors (CAR) of the present invention, the BCMA-binding domain includes a single domain antibody (sdAb) or an antigen-binding fragment thereof capable of specifically binding to BCMA, , the single domain antibody (sdAb) includes CDR1 (complementary determining region 1) represented by the amino acid sequence of any one of SEQ ID NOs: 7 to 9; CDR2 (complementary determining region 2) represented by any one of the amino acid sequences of SEQ ID NOs: 10 to 12; and CDR3 (complementary determining region 3) represented by the amino acid sequence of any one of SEQ ID NOs: 13 to 15.
본 발명에서, 용어 "키메라 항원 수용체 (CAR)"는 일반적으로 항원 및 하나 이상의 세포 내 도메인과 결합하는 능력을 갖는 세포 외 도메인을 함유하는 융합 단백질을 지칭한다. CAR는 키메라 항원 수용체 T 세포(CAR-T)의 핵심 부분이며, 항원(예를 들어, 종양 관련 항원(BCMA)) 결합 도메인, 막 관통 도메인, 공동 자극 도메인 및 세포 내 신호전달 도메인을 포함할 수 있다. CAR는 항체의 항원(예를 들어 BCMA) 특이성에 기초하여 T 세포 수용체-활성화 세포 내 도메인과 조합될 수 있다. 유전자가 변형된 CAR-발현 T 세포는 표적 항원-발현 악성 세포를 특이적으로 식별하고 제거할 수 있다. As used herein, the term “chimeric antigen receptor (CAR)” generally refers to a fusion protein containing an extracellular domain that has the ability to bind an antigen and one or more intracellular domains. CAR is a core part of chimeric antigen receptor T cells (CAR-T) and may include an antigen (e.g., tumor-associated antigen (BCMA)) binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. there is. CARs can be combined with T cell receptor-activating intracellular domains based on the antigenic (e.g. BCMA) specificity of the antibody. Genetically modified CAR-expressing T cells can specifically identify and eliminate target antigen-expressing malignant cells.
본 발명에서, 용어 "BCMA(B-cell maturation antigen)"는 B-세포 성숙 항원을 지칭한다. BCMA(TNFRSF17, BCM 또는 CD269로도 공지됨)는 종양 괴사 수용체 (TNFR) 패밀리의 구성원이고, 최종 분화 B 세포, 예를 들어 기억 B 세포 및 형질 세포 상에서 우세하게 발현된다. BCMA는 종양 세포(예를 들어, 다발성 골수종 세포)에서 발현되거나 또는 종양 세포 표면에 위치된다. 본 발명의 "BCMA"는 전장 야생형 BCMA의 돌연변이, 예를 들어 점 돌연변이, 단편, 삽입, 결실 및 스플라이스 변이체를 포함하는 단백질을 포함할 수 있다.In the present invention, the term “BCMA (B-cell maturation antigen)” refers to B-cell maturation antigen. BCMA (also known as TNFRSF17, BCM or CD269) is a member of the tumor necrosis receptor (TNFR) family and is predominantly expressed on terminally differentiated B cells, such as memory B cells and plasma cells. BCMA is expressed on tumor cells (e.g., multiple myeloma cells) or is located on the tumor cell surface. “BCMA” of the present invention may include proteins containing mutations of full-length wild-type BCMA, such as point mutations, fragments, insertions, deletions, and splice variants.
본 발명에서, 용어 "BCMA-결합 도메인(BCMA-binding domain)"은 일반적으로 BCMA 단백질에 특이적으로 결합할 수 있는 도메인을 지칭한다. 예를 들어, BCMA-결합 도메인은 B 세포에서 발현된 인간 BCMA 폴리펩타이드 또는 이의 단편에 특이적으로 결합할 수 있는 항-BCMA 항체 또는 이의 단편을 함유할 수 있다. In the present invention, the term “BCMA-binding domain” generally refers to a domain capable of specifically binding to BCMA protein. For example, the BCMA-binding domain may contain an anti-BCMA antibody or fragment thereof capable of specifically binding to a human BCMA polypeptide or fragment thereof expressed in B cells.
본 발명에서, 용어 "결합 도메인(binding domain)"은 "세포 외 도메인(extracellular domain)", "세포 외 결합 도메인(extracellular binding domain)", "항원-특이적 결합 도메인(antigenspecific binding domain)" 및 "세포 외 항원-특이적 결합 도메인(extracellular antigen-specific biding domain)"은 상호 교환적으로 사용될 수 있으며, 표적 항원(예를 들어 BCMA)에 특이적으로 결합하는 능력을 갖는 CAR 도메인 또는 단편을 지칭한다. In the present invention, the term "binding domain" refers to "extracellular domain", "extracellular binding domain", "antigen-specific binding domain", and “Extracellular antigen-specific bidding domain” may be used interchangeably and refers to a CAR domain or fragment that has the ability to specifically bind to a target antigen (e.g. BCMA) do.
본 발명에 있어서, 상기 항-BCMA 항체 또는 이의 단편은 상술한 항-BCMA sdAb와 혼용된다.In the present invention, the anti-BCMA antibody or fragment thereof is used interchangeably with the above-described anti-BCMA sdAb.
본 발명에서, 용어 "막관통 도메인(transmembrane domain)"은 일반적으로 세포막을 통과하고 세포 내 신호전달 도메인에 연결되어 신호전달의 역할을 하는 CAR의 도메인을 지칭한다. 상기 막관통 도메인은 CD8, FcγR, ICOS(CD278), 4-1BB(CD137), OX40(CD134), CD27, CD28, IL-2Rβ, CD40, DAP10, MHC class I 분자, TNF 수용체 단백질, Immunoglobulin-유사 단백질, 사이토카인 수용체, 인테그린, SLAM 단백질, 활성화 NK 세포 수용체, BTLA, Toll 리간드 수용체, CD2, CD7, CD30, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD83 특이적 리간드, CD247, CD3δ, CD3ε, CD3γ, CD3ζ, Ig alpha(CD79a), IL-2Rγ, IL-7Rα, PD-1, TNFSF14, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD154, T 세포 수용체의 알파 쇄, T 세포 수용체의 베타 쇄 및 T 세포 수용체의 제타 쇄로 이루어진 군으로부터 선택된 1 종 이상일 수 있으며, 바람직하게는 서열번호 25의 아미노산 서열로 표시되는 CD8일 수 있다. In the present invention, the term “transmembrane domain” generally refers to a domain of CAR that passes through the cell membrane and is connected to an intracellular signaling domain to play a role in signaling. The transmembrane domains include CD8, FcγR, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD28, IL-2Rβ, CD40, DAP10, MHC class I molecule, TNF receptor protein, and immunoglobulin-like. Protein, cytokine receptor, integrin, SLAM protein, activated NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2 , SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 ( Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8) , SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD83 specific ligand, CD247, CD3δ, CD3ε, CD3γ, CD3ζ, Ig alpha (CD79a), IL-2Rγ, IL-7Rα , PD-1, TNFSF14, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD154, consisting of the alpha chain of the T cell receptor, the beta chain of the T cell receptor, and the zeta chain of the T cell receptor. It may be one or more species selected from the group, preferably CD8 represented by the amino acid sequence of SEQ ID NO: 25.
본 발명에서, 용어 "공동 자극 도메인(costimulatory domain)"은 일반적으로 림프구의 항원에 대한 효과적인 반응에 필요한 세포 표면 분자인 면역 자극 분자를 제공할 수 있는 세포 내 도메인을 지칭한다. 상기 기재된 공동자극 도메인(costimulatory domain)은 CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, 림프구 기능-연관 항원-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 및 CD83로 이루어진 군으로부터 선택된 1종 이상일 수 있으며,바람직하게는 서열번호 26의 아미노산 서열로 표시되는 4-1BB일 수 있다.In the present invention, the term “costimulatory domain” generally refers to an intracellular domain capable of providing immunostimulatory molecules, cell surface molecules required for an effective response of lymphocytes to antigens. The costimulatory domains described above include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, It may be one or more selected from the group consisting of CD7, LIGHT, NKG2C, B7-H3, and CD83, and is preferably 4-1BB represented by the amino acid sequence of SEQ ID NO: 26.
본 발명에서, 용어 "세포 내 신호전달 도메인(intracellular signal transduction domain)"은 일반적으로 세포 내부에 위치하고 신호를 전달할 수 있는 도메인을 지칭한다. 본 발명에서, 세포 내 신호전달 도메인은 키메라 항원 수용체의 세포 내 신호전달 도메인이다. 예를 들어, 세포 내 신호전달 도메인은 CD3ζ, FcγR, ICOS(CD278), 4-1BB(CD137), OX40(CD134), CD27, CD28, IL-2Rβ, IL-15R-α, MyD88, DAP10, DAP12, MHC class I 분자, TNF 수용체 단백질, Immunoglobulin-유사 단백질, 사이토카인 수용체, 인테그린, SLAM 단백질, 활성화 NK 세포 수용체, BTLA, Toll 리간드 수용체, CD2, CD7, CD30, CD40, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD83 특이적 리간드, CD247, CD3δ, CD3ε, CD3γ, CD8, Ig alpha(CD79a), IL-2Rγ, IL-7Rα, PD-1 및 TNFSF14로 이루어진 군으로부터 선택된 1 종 이상일 수 있으며, 바람직하게는 서열번호 27의 아미노산 서열로 표시되는 CD3ζ일 수 있다.In the present invention, the term “intracellular signal transduction domain” refers to a domain that is generally located inside a cell and is capable of transmitting signals. In the present invention, the intracellular signaling domain is the intracellular signaling domain of a chimeric antigen receptor. For example, intracellular signaling domains include CD3ζ, FcγR, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD28, IL-2Rβ, IL-15R-α, MyD88, DAP10, and DAP12. , MHC class I molecule, TNF receptor protein, Immunoglobulin-like protein, cytokine receptor, integrin, SLAM protein, activated NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CD40, CDS, ICAM-1, B7 -H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D , ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE /RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A) , Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD83 specific ligand, CD247, CD3δ, It may be one or more selected from the group consisting of CD3ε, CD3γ, CD8, Ig alpha (CD79a), IL-2Rγ, IL-7Rα, PD-1 and TNFSF14, preferably CD3ζ represented by the amino acid sequence of SEQ ID NO: 27. You can.
본 발명에 있어서, BCMA-결합 도메인의 C 말단 및 막관통 도메인의 N 말단 사이에 위치된 힌지 부위(hinge region)를 추가로 포함할 수 있으며, 상기 힌지는 CD8 힌지, IgG1 힌지 및 FcγRIIIα 힌지로 이루어진 군으로부터 선택된 1 종 이상일 수 있으며, 바람직하게는 서열번호 24의 아미노산 서열로 표시되는 CD8 힌지일 수 있다. 상기 "힌지 부위(hinge region)"는 일반적으로 항원-결합 영역과 면역 세포 Fc 수용체 (FcR)-결합영역 사이의 연결 영역을 지칭한다.In the present invention, it may further include a hinge region located between the C terminus of the BCMA-binding domain and the N terminus of the transmembrane domain, wherein the hinge consists of a CD8 hinge, an IgG1 hinge, and an FcγRIIIα hinge. It may be one or more types selected from the group, and preferably may be the CD8 hinge represented by the amino acid sequence of SEQ ID NO: 24. The “hinge region” generally refers to the connecting region between the antigen-binding region and the immune cell Fc receptor (FcR)-binding region.
본 발명에 있어서, BCMA-결합 도메인의 N 말단에 시그널 펩타이드(signal peptide)를 추가로 포함할 수 있으며, 상기 "시그널 펩타이드(signal peptide)"는 일반적으로 단백질 전달을 안내하기 위한 펩타이드 사슬을 지칭한다. 시그널 펩타이드는 5 내지 30 개의 아미노산 길이를 갖는 짧은 펩타이드일 수 있으며, 본 발명에서는 바람직하게 서열번호 23의 아미노산 서열을 이용하였다.In the present invention, a signal peptide may be additionally included at the N terminus of the BCMA-binding domain, and the “signal peptide” generally refers to a peptide chain for guiding protein delivery. . The signal peptide may be a short peptide having a length of 5 to 30 amino acids, and in the present invention, the amino acid sequence of SEQ ID NO: 23 was preferably used.
또한, 본 발명의 또 다른 양태에 따르면,본 발명은 상술한 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드를 제공한다.In addition, according to another aspect of the present invention, the present invention provides a polynucleotide encoding the above-described chimeric antigen receptor (CAR).
본 발명에 있어서, 상기 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드는 BCMA-결합 도메인를 코딩하는 폴리뉴클레오타이드; 막관통 도메인을 코딩하는 폴리뉴클레오타이드; 공동 자극 도메인을 코팅하는 폴리뉴클레오타이드; 및 세포 내 신호전달 도메인을 코딩하는 폴리뉴클레오타이드를 포함할 수 있다. In the present invention, the polynucleotide encoding the chimeric antigen receptor (CAR) is a polynucleotide encoding a BCMA-binding domain; A polynucleotide encoding a transmembrane domain; a polynucleotide coating the costimulatory domain; And it may include a polynucleotide encoding an intracellular signaling domain.
상기 BCMA-결합 도메인을 코딩하는 폴리뉴클레오타이드는 바람직하게 항-BCMA sdAb No.7, 항-BCMA sdAb No.11 및 항-BCMA sdAb No.19를 코딩하는 폴리뉴클레오타이드일 수 있으며, 구체적인 염기서열은 상술한 바와 같다. The polynucleotide encoding the BCMA-binding domain may preferably be a polynucleotide encoding anti-BCMA sdAb No.7, anti-BCMA sdAb No.11, and anti-BCMA sdAb No.19, and the specific base sequences are described above. It is the same as what was said.
본 발명의 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드는 바람직하게, 서열번호 17의 염기서열로 표시되는 시그널 펩타이드; 각각 서열번호 4 내지 6의 염기서열로 표시되는 항-BCMA sdAb; 서열번호 18의 염기서열로 표시되는 CD8 힌지; 서열번호 19의 염기서열로 표시되는 막관통 도메인; 서열번호 20의 염기서열로 표시되는 4-1BB(공동자극 도메인); 및 서열번호 21의 염기서열로 표시되는 CD3ζ(세포 내 신호전달 도메인)로 구성될 수 있다.The polynucleotide encoding the chimeric antigen receptor (CAR) of the present invention preferably includes a signal peptide represented by the base sequence of SEQ ID NO: 17; Anti-BCMA sdAb represented by the base sequences of SEQ ID NOs: 4 to 6, respectively; CD8 hinge represented by the base sequence of SEQ ID NO: 18; A transmembrane domain represented by the base sequence of SEQ ID NO: 19; 4-1BB (co-stimulatory domain) represented by the base sequence of SEQ ID NO: 20; and CD3ζ (intracellular signaling domain) represented by the base sequence of SEQ ID NO: 21.
본 발명에서, 용어 "폴리뉴클레오타이드"는 일반적으로 임의의 길이로 분리된 핵산 분자(nucleic acid molecule), 데옥시리보뉴클레오티드 또는 리보뉴클레오티드, 또는 그의 유사체를 지칭한다. 일부 구현예에서, 본 발명의 폴리뉴클레오타이드는 (1) 중합효소 연쇄반응(PCR) 증폭과 같은 in-vitro 증폭; (2) 클로닝 및 재조합; (3) 절단(digestion) 및 겔 전기영동 분리와 같은 정제; (4) 화학 합성과 같은 합성을 통해 제조될 수 있으며, 바람직하게 분리된 폴리뉴클레오타이드는 재조합 DNA 기술에 의해 제조된다. 본 발명에서, 항체 또는 이의 항원 결합 단편을 코딩하기 위한 핵산은 합성 올리고뉴클레오티드의 제한 단편 조작(restriction fragment operation) 또는 SOE PCR의 적용을 포함하지만 이에 제한하지 않고, 당업계에 공지된 다양한 방법에 의해 제조될 수 있다.In the present invention, the term “polynucleotide” generally refers to nucleic acid molecules, deoxyribonucleotides or ribonucleotides, or analogs thereof, separated of any length. In some embodiments, the polynucleotides of the invention may undergo (1) in-vitro amplification, such as polymerase chain reaction (PCR) amplification; (2) cloning and recombination; (3) purification such as digestion and gel electrophoresis separation; (4) It can be manufactured through synthesis such as chemical synthesis, and preferably the isolated polynucleotide is manufactured by recombinant DNA technology. In the present invention, nucleic acids for encoding antibodies or antigen-binding fragments thereof can be prepared by various methods known in the art, including but not limited to restriction fragment operation of synthetic oligonucleotides or application of SOE PCR. can be manufactured.
또한, 본 발명의 또 다른 양태에 따르면,본 발명은 상기 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드를 포함하는 벡터를 제공한다. In addition, according to another aspect of the present invention, the present invention provides a vector containing a polynucleotide encoding the chimeric antigen receptor (CAR).
본 발명에서, 용어 "벡터(expression vector)"는 적당한 숙주세포 내에서 목적 유전자가 발현할 수 있도록 프로모터 등의 필수적인 조절 요소를 포함하는 유전자 제조물이다. 벡터는 플라스미드, 레트로바이러스(retroviral) 벡터 및 렌티바이러스(lentiviral) 벡터 중 하나 이상으로부터 선택될 수 있다. 적당한 숙주로 형질전환되면, 벡터는 숙주 게놈과 무관하게 복제하고 기능할 수 있거나, 또는 일부 경우에 게놈 그 자체에 통합될 수 있다. In the present invention, the term “expression vector” is a gene product containing essential regulatory elements such as a promoter to enable expression of a target gene in an appropriate host cell. The vector may be selected from one or more of plasmids, retroviral vectors, and lentiviral vectors. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself.
또한, 벡터는 코딩 영역이 적합한 숙주에서 정확하게 발현될 수 있게 하는 발현 제어 요소를 포함할 수 있다. 이러한 조절 요소는 당업자에게 잘 알려져 있으며, 예를 들어 프로모터, 리보솜 결합 부위(ribosome-binding site), 인핸서(enhancer) 및 유전자 전사(transcription) 또는 mRNA 번역(translation)을 조절하기 위한 다른 조절 요소를 포함할 수 있다. 발현 조절 서열의 특정 구조는 종 또는 세포 유형의 기능에 따라 달라질 수 있으나, 일반적으로 TATA 박스(box), 캡핑된(capped) 서열, CAAT 서열 등과 같은 전사 개시 및 번역 개시에 각각 참여하는 5' 비-전사 서열, 및 5' 또는 3' 비-번역 서열을 함유한다. 예를 들어, 5' 비-전사 발현 조절 서열은 기능적으로 연결된 핵산을 전사 및 조절하기 위한 프로모터 서열을 포함할 수 있는 프로모터 영역을 포함할 수 있다. In addition, vectors may contain expression control elements that allow the coding region to be expressed correctly in a suitable host. These regulatory elements are well known to those skilled in the art and include, for example, promoters, ribosome-binding sites, enhancers and other regulatory elements for regulating gene transcription or mRNA translation. can do. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally it is a 5' non-specific sequence that participates in transcription initiation and translation initiation, respectively, such as the TATA box, capped sequence, CAAT sequence, etc. -contains a transcribed sequence and a 5' or 3' non-translated sequence. For example, a 5' non-transcriptional expression control sequence may include a promoter region, which may include a promoter sequence for transcribing and regulating a functionally linked nucleic acid.
본 발명의 구체적인 구현예에서, 상기 벡터는 재조합 바이러스 벡터로, 바람직하게는 렌티바이러스 벡터이며, 작동가능하게 연결된 EF1α 프로모터; 시그널 펩타이드를 코딩하는 폴리뉴클레오타이드; BCMA-결합 도메인을 코딩하는 폴리뉴클레오타이드; 막관통 도메인을 코딩하는 폴리뉴클레오타이드; 세포 내 신호전달 도메인을 코딩하는 폴리뉴클레오타이드를 포함하며, 단백질 발현을 증가시키기 위해 WPRE(woodchuck hepatitis virus post-transcriptional regulatory element)를 추가로 포함할 수 있다. In a specific embodiment of the invention, the vector is a recombinant viral vector, preferably a lentiviral vector, and has an operably linked EF1α promoter; A polynucleotide encoding a signal peptide; A polynucleotide encoding a BCMA-binding domain; A polynucleotide encoding a transmembrane domain; It contains a polynucleotide encoding an intracellular signaling domain, and may additionally contain a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to increase protein expression.
상기 EF1α 프로모터는 서열번호 16의 염기서열로 표시될 수 있으며, 필요에 따라 상기 서열번호 16의 염기서열과 90% 이상, 93% 이상, 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상 동일한 서열을 포함할 수 있다.The EF1α promoter may be represented by the nucleotide sequence of SEQ ID NO: 16, and if necessary, the nucleotide sequence of SEQ ID NO: 16 and 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more , or may contain sequences that are at least 99% identical.
또한, 상기 프로모터는 BCMA-결합 도메인인 항-BCMA 항체(sdAb)의 발현을 유도하도록 작동 가능하게 연결되어 있으며 여기서, "작동 가능하게 연결된(operably linked)"은 일반적 기능을 수행하도록 핵산 발현조절 서열과 목적하는 단백질을 코딩하는 핵산 서열이 기능적으로 연결되어 있는 것을 말한다. 재조합 벡터와의 작동적 연결은 당해 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술분야에서 일반적으로 알려진 효소 등을 사용한다. Additionally, the promoter is operably linked to induce expression of an anti-BCMA antibody (sdAb) that is a BCMA-binding domain, where "operably linked" refers to a nucleic acid expression control sequence to perform a general function. refers to the functional connection between the nucleic acid sequence encoding the protein of interest and the nucleic acid sequence encoding the protein of interest. Operational linkage with a recombinant vector can be made using genetic recombination techniques well known in the art, and site-specific DNA cutting and ligation can be done using enzymes generally known in the art.
유전자를 세포 내로 도입하고 발현시키는 방법은 관련 기술분야에 공지되어 있다. 발현 벡터와 관련하여, 벡터는 관련 기술분야의 임의의 방법에 의해 숙주 세포 내로 용이하게 도입될 수 있다. 예를 들어, 발현 벡터는 물리적, 화학적, 또는 생물학적 수단에 의해 숙주 세포 내로 옮겨질 수 있다.Methods for introducing and expressing genes into cells are known in the art. With regard to expression vectors, the vectors can be easily introduced into host cells by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.
폴리뉴클레오티드를 숙주 세포 내로 도입하기 위한 물리적 방법은 인산칼슘 침전, 리포펙션, 입자 충격, 미세주사, 전기천공 등을 포함한다. 벡터 및/또는 외인성 핵산을 포함하는 세포를 생산하는 방법은 관련 기술분야에 널리 공지되어 있으며 이를 참조하여 이용할 수 있다. 숙주 세포 내로 폴리뉴클레오티드의 도입을 위한 바람직한 방법은 인산칼슘 형질감염이다.Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and/or exogenous nucleic acids are well known in the related art and can be used with reference to them. A preferred method for introduction of polynucleotides into host cells is calcium phosphate transfection.
숙주 세포 내로 폴리뉴클레오티드를 도입하기 위한 생물학적 방법은 DNA 및 RNA 벡터의 사용을 포함한다. 바이러스 벡터, 및 특히 레트로바이러스 벡터는 유전자를 포유동물, 예를 들어 인간 세포 내로 삽입하기 위해 가장 널리 사용되는 방법이 되었다. 다른 바이러스 벡터는 렌티바이러스, 폭스바이러스, 단순 포진 바이러스, 아데노바이러스 및 아데노-연관 바이러스 등으로부터 유래될 수 있다. Biological methods for introducing polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, eg human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses and adeno-associated viruses, etc.
숙주 세포 내로 폴리뉴클레오티드를 도입하기 위한 화학적 수단은 콜로이드성 분산액 시스템, 예컨대 거대분자 복합체, 나노캡슐, 마이크로구체, 비드, 및 수중유 에멀젼, 미셀, 혼합된 미셀, 및 리포솜을 비롯한 지질-기반 시스템을 포함한다. 시험관 내 및 생체 내에서 전달 비히클로서 사용하기 위한 예시적인 콜로이드성 시스템은 리포솜 (예를 들어, 인공 막 소포)이다. 핵산의 최신 기술의 표적화된 전달, 예컨대 표적화된 나노입자 또는 다른 적합한 마이크로미터-미만 크기의 전달 시스템을 사용한 폴리뉴클레오티드의 전달을 위한 다른 방법이 이용 가능하다.Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Includes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles). Other methods are available for the state-of-the-art targeted delivery of nucleic acids, such as delivery of polynucleotides using targeted nanoparticles or other suitable submicrometer-sized delivery systems.
비-바이러스 전달 시스템이 이용되는 경우에, 예시적인 전달 비히클은 리포솜이다. 지질 제제의 사용은 숙주세포 내로의 핵산의 도입(시험관 내, 생체 외 또는 생체 내)을 위해 고려된다. 또 다른 측면에서, 핵산은 지질과 회합될 수 있다. 지질과 회합된 핵산은 리포솜의 수성 내부에 캡슐화되거나, 리포솜의 지질 이중층 내에 점재되거나, 리포솜 및 올리고뉴클레오티드 둘 다와 회합된 연결 분자를 통해 리포솜에 부착되거나, 리포솜 내에 포획되거나, 리포솜과 복합체화되거나, 지질 함유 용액 중에 분산되거나, 지질과 혼합되거나, 지질과 조합되거나, 지질 내에 현탁액으로서 함유되거나, 미셀과 함께 함유 또는 복합체화되거나, 또는 지질과 달리 회합될 수 있다. 지질, 지질/DNA 또는 지질/발현 벡터 회합 조성물은 용액 중의 임의의 특정한 구조로 제한되지 않는다.When non-viral delivery systems are used, exemplary delivery vehicles are liposomes. The use of lipid preparations is contemplated for introduction of nucleic acids into host cells (in vitro, ex vivo or in vivo). In another aspect, nucleic acids can be associated with lipids. Nucleic acids associated with lipids may be encapsulated within the aqueous interior of the liposome, dotted within the lipid bilayer of the liposome, attached to the liposome via linkage molecules associated with both the liposome and the oligonucleotide, trapped within the liposome, complexed with the liposome, or , may be dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension within a lipid, contained or complexed with micelles, or otherwise associated with a lipid. The lipid, lipid/DNA or lipid/expression vector association composition is not limited to any particular structure in solution.
또한,본 발명의 또 다른 양태에 따르면, 본 발명은 상기 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드 또는 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드를 포함하는 벡터를 포함하고, 상기 키메라 항원 수용체(CAR)를 발현하는, 단리된 면역 세포를 제공한다.In addition, according to another aspect of the present invention, the present invention includes a polynucleotide encoding the chimeric antigen receptor (CAR) or a vector containing a polynucleotide encoding the chimeric antigen receptor (CAR), and the chimeric antigen receptor Isolated immune cells expressing (CAR) are provided.
본 발명에 있어서, 상기 면역 세포는 포유동물 유래 세포일 수 있고, T 세포, NK 세포, NKT 세포, B 세포, 수지상 세포, 단핵구, 마크로파지, 호산구, 호염기구 및 호중구로 이루어진 군으로부터 선택된 1 종 이상일 수 있으며, 바람직하게는 T 세포 또는 NK 세포 또는 NKT 세포일 수 있다.In the present invention, the immune cells may be mammalian-derived cells, and may be one or more types selected from the group consisting of T cells, NK cells, NKT cells, B cells, dendritic cells, monocytes, macrophages, eosinophils, basophils, and neutrophils. It may be a T cell, an NK cell, or an NKT cell.
본 발명에 있어서, 상기 키메라 항원 수용체(CAR)를 발현하는 면역 세포는, 본 발명의 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드 또는 이를 포함하는 CAR 벡터를 면역 세포, 예를 들어 T 세포 또는 NK 세포 또는 NKT 세포내로 도입시켜 제조할 수 있다. In the present invention, the immune cell expressing the chimeric antigen receptor (CAR) is a polynucleotide encoding the chimeric antigen receptor (CAR) of the present invention or a CAR vector containing the same as an immune cell, such as a T cell or NK. It can be manufactured by introducing it into cells or NKT cells.
구체적으로, CAR 벡터는 전기천공법, 리포펙타민(lipofectamine 2000, Invitrogen) 등과 같은 당업계에 공지된 방법에 의해 세포 내로 도입될 수 있다. 예를 들어, 면역 이펙터 세포는 렌티바이러스 벡터에 의해 형질 감염되어 CAR 분자를 운반하는 바이러스 게놈을 숙주 게놈에 통합시켜 표적 유전자의 장기적이고 안정적인 발현을 보장할 수 있다. 다른 예를 들어, 전이인자(transposon)는 CAR 운반 플라스미드(transposon) 및 전이효소 운반 플라스미드를 표적 세포 내로 도입하는데 이용될 수 있다. 다른 예를 들어, CAR 분자는 유전자 편집방법 (예컨대 CRISPR / Cas9)에 의해 게놈에 첨가될 수 있다.Specifically, CAR vectors can be introduced into cells by methods known in the art, such as electroporation and lipofectamine (lipofectamine 2000, Invitrogen). For example, immune effector cells can be transfected by lentiviral vectors to integrate the viral genome carrying CAR molecules into the host genome, ensuring long-term and stable expression of target genes. As another example, transposons can be used to introduce CAR transport plasmids (transposons) and transposase transport plasmids into target cells. As another example, CAR molecules may be added to the genome by gene editing methods (e.g., CRISPR/Cas9).
본 발명의 일 실시예에서는, BCMA-CAR를 코팅하는 폴리뉴클레오타이드가 삽입된 렌티바이러스 벡터를 제조하였으며, 제조된 벡터를 T 세포에 형질전환 시켜 BCMA-CAR-T 세포를 제조하였다. 제조된 hBCMA-CAR-T 세포에서는 본 발명의 BCMA를 표적으로 하는 키메라 항원 수용체를 발현하게 된다. In one embodiment of the present invention, a lentiviral vector into which a polynucleotide coating BCMA-CAR was inserted was prepared, and the prepared vector was transformed into T cells to prepare BCMA-CAR-T cells. The prepared hBCMA-CAR-T cells express the chimeric antigen receptor targeting BCMA of the present invention.
키메라 항원 수용체(CAR)를 발현하는 면역 이펙터 세포 제조를 위한 면역 이펙터 세포는 대상체로 부터 수득할 수 있으며, 상기 "대상체"는 면역 반응이 도출될 수 있는 살아있는 유기체 (예를 들어, 포유동물)를 포함한다. 대상체의 예는 인간, 개, 고양이, 마우스, 래트, 및 그의 트랜스제닉 종을 포함한다. T 세포는 말초 혈액 단핵 세포, 골수, 림프절 조직, 제대혈, 흉선 조직, 감염 부위로부터의 조직, 복수, 흉막 삼출, 비장 조직, 및 종양을 비롯한 수많은 공급원으로부터 수득될 수 있다.Immune effector cells for producing immune effector cells expressing a chimeric antigen receptor (CAR) can be obtained from a subject, wherein a “subject” is a living organism (e.g., a mammal) against which an immune response can be elicited. Includes. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumor.
상기 T 세포는 통상의 기술자에게 공지된 임의의 많은 기술, 예를 들면, 피콜(Ficoll)™ 분리를 사용하여 대상체로부터 수집된 혈액 단위로부터 수득될 수 있다. 혈액으로부터 세포는 분리반출술에 의해 수득되며, 분리반출술 생성물은 전형적으로 T 세포, 단핵구, 과립구, B 세포를 비롯한 림프구, 다른 유핵 백혈구, 적혈구, 및 혈소판을 함유한다. The T cells can be obtained from blood units collected from the subject using any of many techniques known to those skilled in the art, such as Ficoll™ separation. Cells from blood are obtained by apheresis, and the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
분리반출술에 의해 수집된 세포는 혈장 분획을 제거하고 세포를 후속 프로세싱 단계를 위해 적절한 완충제 또는 배지에 두기 위해 세척될 수 있다. T 세포는 적혈구를 용해시키고, 예를 들어 퍼콜(PERCOLL)™ 구배를 통한 원심분리에 의해 또는 역류 원심 분리에 의해 단핵구를 고갈시킴으로써 말초 혈액 림프구로부터 단리된다.Cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation over a PERCOLL™ gradient or by countercurrent centrifugation.
본 발명의 일 실시예에서, 말초 혈액 단핵세포(peripheral blood mononuclear cell, PBMC)로 부터 활성화된 T 세포를 분리한 다음, BCMA-CAR 렌티바이러스를 T 세포에 형질도입시켜 BCMA-CAR-T 세포를 제조하였다. 제조된 BCMA-CAR-T 세포의 BCMA 펩타이드 결합능을 확인한 결과, BCMA 펩타이드 증가에 따라, BCMA와 결합하는 CD4 또는 CD8를 발현하는 BCMA-CAR-T 세포가 증가하는 것을 확인하였다. 이는 본 발명에서 제조한 BCMA-CAR-T 세포가 효과적으로 BCMA와 결합하는 것을 의미한다.In one embodiment of the present invention, activated T cells are isolated from peripheral blood mononuclear cells (PBMC), and then BCMA-CAR lentivirus is transduced into the T cells to generate BCMA-CAR-T cells. Manufactured. As a result of confirming the BCMA peptide binding ability of the prepared BCMA-CAR-T cells, it was confirmed that as the BCMA peptide increased, the number of BCMA-CAR-T cells expressing CD4 or CD8 that binds to BCMA increased. This means that the BCMA-CAR-T cells prepared in the present invention effectively bind to BCMA.
본 발명의 일 실시예에서, hBCMA-CAR-T 세포에 의한 표적 세포의 사멸효과를 확인한 결과, BCMA-CAR-T 세포는 BCMA를 발현하는 세포-특이적으로 사멸 효과를 보이는 것을 확인하였다. In one embodiment of the present invention, as a result of confirming the killing effect of target cells by hBCMA-CAR-T cells, it was confirmed that BCMA-CAR-T cells showed a cell-specific killing effect expressing BCMA.
즉, 본 발명의 BCMA를 표적으로 하는 키메라 항원 수용체 및 CAR-T 세포는 B 세포 또는 BCMA 발현과 관련된 질환 예방 또는 치료용 조성물로 유용하게 활용할 수 있다. That is, the chimeric antigen receptor and CAR-T cells targeting BCMA of the present invention can be usefully used as a composition for preventing or treating diseases related to B cells or BCMA expression.
또한, 본 발명의 또 다른 양태에 따르면, 본 발명은 상술한 BCMA를 표적하는 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드; 또는 BCMA를 표적하는 키메라 항원 수용체(CAR)를 표면에 발현하는 단리된 면역 세포를 포함하는 BCMA 발현과 관련된 질환 예방 또는 치료용 약학적 조성물을 제공한다. In addition, according to another aspect of the present invention, the present invention provides a polynucleotide encoding a chimeric antigen receptor (CAR) targeting the above-described BCMA; Alternatively, it provides a pharmaceutical composition for preventing or treating diseases related to BCMA expression, comprising isolated immune cells expressing a chimeric antigen receptor (CAR) targeting BCMA on their surface.
또한, 본 발명은 BCMA를 표적으로 하는 항체를 포함하는 BCMA 발현과 관련된 질환 예방 또는 치료용 약학적 조성물을 제공한다. Additionally, the present invention provides a pharmaceutical composition for preventing or treating diseases related to BCMA expression, including an antibody targeting BCMA.
BCMA(야생형 또는 돌연변이체 BCMA) 발현과 관련된 질환은 암, 악성종양 또는 자가면역 질환일 수 있다. 바람직하게 다발성골수종(multiple myeloma), 혈액암, 비-호지킨 림프종(non-Hodgkin's lymphoma), 자가항체-의존성 자가면역 질환(autoantibody-dependent autoimmune disease), 전신홍반루프스(systemic lupus erythematosus, SLE) 및 류마티스 관절염(rheumatoid arthritis)으로 이루어진 군으로부터 선택된 1 종 이상일 수 있다.Diseases associated with BCMA (wild-type or mutant BCMA) expression may be cancer, malignancy, or autoimmune disease. Preferably multiple myeloma, blood cancer, non-Hodgkin's lymphoma, autoantibody-dependent autoimmune disease, systemic lupus erythematosus (SLE) and It may be one or more types selected from the group consisting of rheumatoid arthritis.
상기 약학적 조성물은 약학적으로 허용되는 담체를 추가로 포함할 수 있다. 경구 투여 시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등을 사용할 수 있고, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등을 혼합하여 사용할 수 있으며, 국소 투여용의 경우 에는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다.The pharmaceutical composition may further include a pharmaceutically acceptable carrier. For oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. can be used. For injections, buffers, preservatives, analgesics, solubilizers, and isotonic agents can be used. , stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used.
상기 약학적 조성물의 제형은 상술한 약제학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를들어, 경구 투여 시에는 정제, 트로키, 캡슐, 엘릴시르, 서스펜션, 시럽, 웨이퍼 등의 형태로 제조될 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조될 수 있다.The formulation of the pharmaceutical composition can be prepared in various ways by mixing it with the pharmaceutically acceptable carrier described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elylsir, suspension, syrup, wafers, etc., and in the case of injections, it can be manufactured in the form of unit dosage ampoules or multiple dosage forms.
또한, 상기 약학적 조성물은 막 투과성을 향상시킬 수 있는 계면활성제를 포함할 수 있다. 이러한 계면활성제는 스테로이드에서 유도된 것이거나 N-[1-(2,3-디올레오일)프로필-N,N,N-트리메틸암모늄클로라이드(DOTMA) 등의 양이온성 지질, 또는 콜레스테롤 헤미숙시네이트, 포스파티딜 글리세롤 등의 각종 화합물일 수 있으나, 이에 한정되는 것은 아니다.Additionally, the pharmaceutical composition may contain a surfactant that can improve membrane permeability. These surfactants are derived from steroids, cationic lipids such as N-[1-(2,3-dioleoyl)propyl-N,N,N-trimethylammonium chloride (DOTMA), or cholesterol hemisuccinate. , phosphatidyl glycerol, etc., but are not limited thereto.
또한, 본 발명은 본 발명에 따른 약학적 조성물을 개체에 투여하는 단계를 포함하는 BCMA 발현과 관련된 질환을 예방하거나 치료하는 방법을 제공한다. 상기 BCMA를 표적하는 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드; 또는 BCMA를 표적하는 키메라 항원 수용체(CAR)를 표면에 발현하는 단리된 면역 세포; 또는 BCMA를 표적으로 하는 항체를 포함하는 약학적 조성물은 BCMA 발현과 관련된 질환을 예방 또는 치료하기 위해 약학적으로 효과적인 양으로 투여될 수 있다. 질환 종류, 환자의 연령, 체중, 증상의 특성 및 정도, 현재 치료법의 종류, 치료 회수, 투여 형태 및 경로 등 다양한 요인에 따라 달라질 수 있으며, 해당분야의 전문가들에 의해 용이하게 결정될 수 있다.Additionally, the present invention provides a method for preventing or treating diseases related to BCMA expression, comprising administering the pharmaceutical composition according to the present invention to an individual. A polynucleotide encoding a chimeric antigen receptor (CAR) targeting the BCMA; or isolated immune cells expressing on their surface a chimeric antigen receptor (CAR) targeting BCMA; Alternatively, a pharmaceutical composition containing an antibody targeting BCMA can be administered in a pharmaceutically effective amount to prevent or treat diseases related to BCMA expression. It may vary depending on various factors such as the type of disease, the patient's age, weight, nature and severity of symptoms, type of current treatment, number of treatments, form of administration, and route, and can be easily determined by experts in the field.
상기 약학적 조성물은 상기한 약리학적 또는 생리학적 성분과 함께 투여되거나 순차적으로 투여될 수 있으며, 또한 추가의 종래의 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 이러한 투여는 단일 또는 다중 투여일 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition may be administered together with or sequentially with the pharmacological or physiological components described above, and may also be administered in combination with additional conventional therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. Such administration may be single or multiple administrations. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
본 명세서에서 사용된 용어 "개체"는 상기 약학적 조성물을 투여하여 경감, 억제 또는 치료될 수 있는 상태 또는 질환을 앓고 있거나 그러한 위험이 있는 포유동물을 의미하며, 바람직하게 사람을 의미한다.As used herein, the term “subject” refers to a mammal suffering from or at risk of a condition or disease that can be alleviated, suppressed, or treated by administering the pharmaceutical composition, and preferably refers to a human.
본 발명에서 사용되는 용어 '투여'는 임의의 적절한 방법으로 개체에게 본 발명의 약학 조성물을 제공하는 것을 의미한다. 본 발명은 약학 조성물은 연구자, 수의사, 의사 또는 기타 임상에 의해 생각되는 조직계, 동물 또는 인간에서 생물학적 또는 의학적 반응을 유도하는 유효 성분 또는 약학적 조성물의 양, 즉 치료되는 질환 또는 장애의 증상의 완화를 유도하는 양인 치료상 유효량으로 투여할 수 있다. 본 발명의 약학 조성물에 대한 치료상 유효 투여량 및 투여횟수는 원하는 효과에 따라 변화될 것임은 당업자에게 자명하다. 그러므로, 투여될 최적의 투여량은 당업자에 의해 쉽게 결정될 수 있으며, 질환의 종류, 질환의 중증도, 조성물에 함유된 유효성분 및 다른 성분의 함량, 제형의 종류, 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여시간, 투여 경로 및 조성물의 분비율, 치료기간, 동시 사용되는 약물을 비롯한 다양한 인자 등에 따라 조절될 수 있다. 본 발명의 약학 조성물은 1~10,000㎎/㎏/일의 양으로 투여할 수 있으며, 하루에 한번 투여할 수도 있고, 수 회에 나누어 투여할 수도 있다.The term 'administration' as used in the present invention means providing the pharmaceutical composition of the present invention to an individual by any suitable method. The present invention provides a pharmaceutical composition that provides an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human as considered by a researcher, veterinarian, doctor or other clinician, that is, alleviation of symptoms of the disease or disorder being treated. It can be administered in a therapeutically effective amount that induces . It is obvious to those skilled in the art that the therapeutically effective dosage and frequency of administration of the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by a person skilled in the art, depending on the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of dosage form, the patient's age, weight, and general health condition. , gender and diet, administration time, administration route and secretion rate of the composition, treatment period, and various factors including concurrently used drugs. The pharmaceutical composition of the present invention can be administered in an amount of 1 to 10,000 mg/kg/day, and may be administered once a day or in several divided doses.
또한, 본 발명의 또 다른 양태에 따르면, 본 발명은 상술한 BCMA를 표적하는 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드; 또는 BCMA를 표적하는 키메라 항원 수용체(CAR)를 표면에 발현하는 단리된 면역 세포를 포함하는 BCMA 발현과 관련된 질환 예방 또는 개선용 식품 조성물을 제공한다.In addition, according to another aspect of the present invention, the present invention provides a polynucleotide encoding a chimeric antigen receptor (CAR) targeting the above-described BCMA; Alternatively, it provides a food composition for preventing or improving diseases related to BCMA expression, including isolated immune cells expressing a chimeric antigen receptor (CAR) targeting BCMA on the surface.
본 발명의 식품 조성물은 건강기능식품, 식품 첨가제 또는 식이보조제로 사용될 수 있다.The food composition of the present invention can be used as a health functional food, food additive, or dietary supplement.
식품 첨가제로 사용할 경우, 본 발명의 유효성분을 그대로 첨가하거나, 다른 식품 또는 식품 성분과 함께 혼합하여 사용되는 등 통상적인 방법에 따라 적절하게 사용될 수 있다. When used as a food additive, it can be appropriately used according to conventional methods, such as adding the active ingredient of the present invention as is or mixing it with other foods or food ingredients.
상기 건강기능식품이란, 본 발명의 유효 성분을 음료, 차류, 향신료, 껌, 과자류 등의 식품소재에 첨가하거나, 캡슐화, 분말화, 현탁액 등으로 제조한 식품으로, 이를 섭취할 경우 건강상 특정한 효과를 가져오는 것을 의미하나, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용시 발생할 수 있는 부작용 등이 없는 장점이 있다. 이와 같이 하여 얻어지는 본 발명의 건강기능식품은, 일상적으로 섭취하는 것이 가능하기 때문에 매우 유용하다. 이와 같은 건강식품에 있어서의 유효 성분의 첨가량은, 대상인 건강식품의 종류에 따라 달라 일률적으로 규정할 수 없지만, 식품 본래의 맛을 손상시키지 않는 범위에서 첨가하면 되며, 대상 식품에 대하여 통상 0.01 내지 50 중량%, 바람직하기로는 0.1 내지 20 중량%의 범위이다. 또한, 과립, 정제 또는 캡슐형태의 식품의 경우에는 통상 0.1 내지 100 중량% 바람직하기로는 0.5 내지 80 중량%의 범위에서 첨가하면 된다. 한 구체예에서, 본 발명의 건강기능식품은 음료의 형태일 수 있다.The health functional food refers to a food manufactured by adding the active ingredient of the present invention to food materials such as beverages, teas, spices, gum, and confectionery, or by encapsulating, powdering, or suspending it, and when ingested, it has specific health effects. However, unlike regular drugs, it has the advantage of not having any side effects that may occur when taking the drug for a long time since it is made from food. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The amount of active ingredients added in such health foods cannot be uniformly specified as it varies depending on the type of health food being targeted, but it can be added within a range that does not damage the original taste of the food, and is usually 0.01 to 50% for the target food. % by weight, preferably in the range of 0.1 to 20% by weight. In addition, in the case of foods in the form of granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a beverage.
또한, 상기 유효 성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 변경될 수 있음은 물론이며, 식품 조성물 총 중량에 대하여 0.01~95중량%로 포함되는 것이 바람직하며, 더욱 바람직하게는 1~80중량%로 포함되는 것이다. 그 함량이 0.01중량% 미만일 경우에는 복용의 효율성이 떨어질 수 있으며, 95중량%를 초과할 경우에는 사용량 대비 효과 상승률이 낮아 비경제적일 수 있다.In addition, the mixing amount of the active ingredient can be appropriately changed depending on the purpose of use (prevention, health, or therapeutic treatment), and is preferably included in 0.01 to 95% by weight based on the total weight of the food composition. Preferably it is contained in 1 to 80% by weight. If the content is less than 0.01% by weight, the efficiency of taking it may be reduced, and if it exceeds 95% by weight, the rate of increase in effectiveness relative to the amount used is low, making it uneconomical.
구체적인 예로, 식품 또는 음료의 제조 시에는 본 발명의 유효 성분은 원료에 대하여 15중량% 이하, 바람직하게는 10중량% 이하의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하여 장기간 섭취할 경우에는 상기 범위 이하의 양으로 첨가될 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다. As a specific example, when manufacturing a food or beverage, the active ingredient of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw materials. However, when consumed for a long period of time for health and hygiene purposes or health control, it can be added in an amount below the above range. Since there is no problem in terms of safety, the active ingredient can be used in an amount above the above range. there is.
상기 식품의 종류에는 특별한 제한은 없으나, 본 발명의 유효 성분을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 수프, 음료수, 차, 드링크제, 알코올 음료, 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no particular limitation on the type of food, but examples of food to which the active ingredient of the present invention can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream. It includes dairy products, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and includes all health foods in the conventional sense.
본 발명의 식품 조성물이 음료로 제조될 경우 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등의 추가 성분을 포함할 수 있다. 상기 천연 탄수화물로는 포도당, 과당 등의 모노사카라이드; 말토오스, 수크로오스 등의 디사카라이드; 덱스트린, 사이클로덱스트린 등의 천연 감미제; 사카린, 아스파르탐 등의 합성 감미제 등이 사용될 수 있다. 상기 천연 탄수화물은 본 발명의 식품 조성물 총 중량에 대하여 0.01~10중량%, 바람직하게는 0.01~0.1중량%로 포함된다.When the food composition of the present invention is manufactured into a beverage, it may contain additional ingredients such as various flavoring agents or natural carbohydrates, like conventional beverages. The natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin; Synthetic sweeteners such as saccharin and aspartame may be used. The natural carbohydrate is contained in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight, based on the total weight of the food composition of the present invention.
본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 포함할 수 있으며, 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 포함할 수 있으나 이에 제한되지 않는다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 상기의 첨가제 비율은 크게 제한되지는 않으나, 본 발명의 식품 조성물 총 중량에 대하여 0.01~0.1중량% 범위내로 포함되는 것이 바람직하다.The food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid. It may include carbonating agents used in beverages, and may include pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages, but is not limited thereto. These ingredients can be used independently or in combination. The ratio of the above additives is not greatly limited, but is preferably contained within the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.
건강 및 위생을 목적으로 하거나 건강 조절을 목적으로 하는 장기간의 섭취인 경우, 본 발명의 식품 조성물은 안전성 면에서 세포 독성이 거의 없기 때문에 장기간 복용이 가능하다.In the case of long-term intake for the purpose of health and hygiene or health control, the food composition of the present invention can be taken for a long period of time because it has little cytotoxicity in terms of safety.
본 명세서에서 식품이란 함은 영양소를 한 가지 또는 그 이상 함유하고 있는 천연물 또는 가공품을 의미하며, 바람직하게는 어느 정도의 가공 공정을 거쳐 직접 먹을 수 있는 상태가 된 것을 의미하며, 통상적인 의미로서의 식품, 식품 첨가제, 건강 기능성 식품, 음료 및 음료 첨가제 등을 모두 포함하는 의도이다.In this specification, food refers to a natural product or processed product containing one or more nutrients, preferably in a state that can be eaten directly after a certain degree of processing, and is referred to as food in the conventional sense. , food additives, health functional foods, beverages and beverage additives, etc.
또한, 본 발명의 또 다른 양태에 따르면, 본 발명은 상술한 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드; 키메라 항원 수용체(CAR)를 표면에 발현하는 단리된 면역 세포를, 약학적 유효량으로 대상(subject)에게 투여하는 단계를 포함하는, BCMA 발현과 관련된 질환 예방 또는 치료 방법을 제공한다.In addition, according to another aspect of the present invention, the present invention provides a polynucleotide encoding the above-described chimeric antigen receptor (CAR); Provided is a method for preventing or treating diseases associated with BCMA expression, comprising administering to a subject a pharmaceutically effective amount of isolated immune cells expressing a chimeric antigen receptor (CAR) on their surface.
본 발명의 치료는 BCMA 발현과 관련된 질환은 앞서 설명한 질환과 동일하며, 예컨대, 암으로 진단받은 환자를 치료하는데 사용될 수 있다. 본 발명의 CAR 및 CAR-T 세포로 치료될 암의 유형은 다발성골수종을 포함하나 이에 제한되지는 않는다. The treatment of the present invention can be used to treat diseases related to BCMA expression, which are the same as the diseases described above, for example, patients diagnosed with cancer. Types of cancer to be treated with CAR and CAR-T cells of the invention include, but are not limited to, multiple myeloma.
본 발명의 치료 방법은 항체 요법, 화학요법, 사이토카인 요법, 수지상 세포 요법, 유전자 요법, 호르몬 요법, 레이저 광 요법 및 방사선 요법의 군으로부터 선택된 암에 대한 1종 이상의 요법과 조합될 수 있다.The treatment method of the present invention may be combined with one or more therapies for cancer selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy, and radiation therapy.
본 발명의 방법은 상술한 바를 이용하므로, 이와 중복된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다.Since the method of the present invention uses the above-described information, duplicate content is omitted to avoid excessive complexity of the specification.
본 발명에서 제조된 BCMA를 표적으로 하는 키메라 항원 수용체 및 CAR-면역 세포는 BCMA와 효과적으로 결합할 뿐만 아니라, BCMA와 결합한 CAR-면역 세포의 활성화가 이루어진 것을 확인하였다. 또한, 본 발명의 CAR-면역 세포는 BCMA를 발현하는 세포를 효과적으로 사멸시키는 것을 확인하였으므로, 본 발명의 BCMA를 표적으로 하는 키메라 항원 수용체 및 CAR-면역 세포는 B 세포 또는 BCMA 발현과 관련된 질환 예방 또는 치료용 조성물로 유용하게 활용할 수 있다.It was confirmed that the chimeric antigen receptor and CAR-immune cells targeting BCMA produced in the present invention not only effectively bound to BCMA, but also activated CAR-immune cells bound to BCMA. In addition, since it was confirmed that the CAR-immune cells of the present invention effectively kill cells expressing BCMA, the chimeric antigen receptor and CAR-immune cells targeting BCMA of the present invention are used to prevent or treat diseases related to B cells or BCMA expression. It can be usefully used as a therapeutic composition.
도 1은 본 발명의 항-BCMA sdAb No.7, No.11 및 No.19의 BCMA 결합능을 확인한 데이터이다.Figure 1 shows data confirming the BCMA binding ability of anti-BCMA sdAb No.7, No.11, and No.19 of the present invention.
도 2는 본 발명의 BCMA를 표적으로 하는 키메라 항원 수용체(CAR)를 나타낸 모식도이다.Figure 2 is a schematic diagram showing the chimeric antigen receptor (CAR) targeting BCMA of the present invention.
도 3은 본 발명의 BCMA를 표적으로 하는 키메라 항원 수용체(BCMA-CAR)를 발현하는 렌티바이러스 벡터 및 T 세포에서 발현된 키메라 항원 수용체를 나타낸 모식도이다. Figure 3 is a schematic diagram showing a lentiviral vector expressing a chimeric antigen receptor (BCMA-CAR) targeting BCMA of the present invention and a chimeric antigen receptor expressed in T cells.
도 4는 본 발명의 BCMA-CAR를 발현하는 렌티바이러스를 이용한 BCMA-CAR-T 세포 제조방법을 나타낸 모식도이다. Figure 4 is a schematic diagram showing a method for producing BCMA-CAR-T cells using a lentivirus expressing BCMA-CAR of the present invention.
도 5는 본 발명의 BCMA-CAR-T 세포의 BCMA 결합능을 확인한 데이터이다.Figure 5 is data confirming the BCMA binding ability of the BCMA-CAR-T cells of the present invention.
도 6은 본 발명의 BCMA-CAR-T 세포에 의한 표적 세포의 사멸 효과를 확인한 데이터이다.Figure 6 is data confirming the effect of killing target cells by the BCMA-CAR-T cells of the present invention.
이하, 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the examples are only for illustrating the present invention in more detail, and it is understood by those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. It will be self-evident.
실시예 1. BCMA를 표적으로 하는 단일 도메인 항체 제조 및 선별Example 1. Preparation and selection of single domain antibodies targeting BCMA
본 발명자들은 BCMA 펩타이드 특이적인 단일 도메인 항체를 제조하기 위해, 낙타에서 유래된 단일-도메인 항체(single-domain antibody)의 라이브러리로부터 스크리닝하였다. The present inventors screened a library of single-domain antibodies derived from camel to produce a single domain antibody specific for the BCMA peptide.
보다 구체적으로, 낙타의 항체 라이브러리를 갖는 대장균 세포를 VCSM13 헬퍼파아지 (helper phage)로 감염시켜 배양한 후 배양액으로부터 항체 단백질이 표면에 발현된 (surface displayed) 파아지 파티클을 회수하였다. BCMA와 결합하는 항체를 스크리닝하기 위하여 파아지 패닝 기술을 이용하였다. 패닝은 대상 표적에 더 높은 친화성 결합을 보유한 모집단 내의 파지를 농축하기 위한 반복 공정이다.More specifically, E. coli cells containing a camel antibody library were infected with VCSM13 helper phage and cultured, and then phage particles with antibody proteins expressed on the surface were recovered from the culture medium. Phage panning technology was used to screen for antibodies that bind to BCMA. Panning is an iterative process to enrich phages in the population that possess higher affinity binding to the target of interest.
먼저, 라이브러리를 선택한 항원에 노출시킨 뒤 가장 높은 결합 친화력(binding affinity)을 가진 항원만 용리 및 증폭시켜 파지의 집단(population)을 축적하였다. 다음으로, 콜로니 피킹 단계로 각 고유 단백질 결합체를 분리하기 위해 이전 단계에서 선택한 박테리오 파지를 클로닝하여 선택하였다. 마지막으로 패닝 과정 동안 결합 친화력(binding affinity)이 더 낮았던 단백질을 표시하는 파지 클론에 비해 결합 친화력이 더 높은 단백질을 표시하는 파지 클론들을 선택하였다. First, the library was exposed to the selected antigen, and then only the antigen with the highest binding affinity was eluted and amplified to accumulate a population of phages. Next, in order to isolate each unique protein complex in the colony picking step, the bacteriophage selected in the previous step was cloned and selected. Lastly, during the panning process, phage clones displaying proteins with higher binding affinity were selected compared to phage clones displaying proteins with lower binding affinity.
이 선택 과정은 정성적이므로 항체-항원 상호작용을 평가하는 보다 정량적인 ELISA 검출을 진행하여 BCMA에 결합하는 단일-도메인(single-domain) 항체를 선별하였고, 상기 선별된 BCMA 단일 도메인 항체 3 종을 각각 항-BCMA sdAb No.7, 항-BCMA sdAb No.11 및 항-BCMA sdAb No.19로 명명하였다. 또한, 이들의 VHH(variable heavy chain domains of heavy chain antibody)를 시퀀싱하여 프레임 영역 FR과 상보성 결정 영역 CDR을 확인하였다.Since this selection process is qualitative, more quantitative ELISA detection to evaluate antibody-antigen interaction was performed to select single-domain antibodies that bind to BCMA, and each of the three selected BCMA single domain antibodies was selected. They were named anti-BCMA sdAb No.7, anti-BCMA sdAb No.11 and anti-BCMA sdAb No.19. In addition, their VHH (variable heavy chain domains of heavy chain antibody) were sequenced to confirm the frame region FR and complementarity-determining region CDR.
상술한 서열분석 결과에 따른 항체의 중쇄 가변 도메인(VHH)에 대한 서열정보는 하기 표 1에 정렬하였으며, 하기 표 1에서 밑줄친 부분은 상보성 결정 영역(complementarity determining region; CDR)를 의미한다.Sequence information for the heavy chain variable domain (VHH) of the antibody according to the above-described sequence analysis results is arranged in Table 1 below, and the underlined portion in Table 1 refers to the complementarity determining region (CDR).
표 1에 나타낸 바와 같이, 항-BCMA sdAb No.7은 서열번호 7의 아미노산 서열로 표시되는 CDR1, 서열번호 10의 아미노산 서열로 표시되는 CDR2 및 서열번호 13의 아미노산 서열로 표시되는 CDR3를 포함하는 중쇄 가변 도메인(VHH); 항-BCMA sdAb No.11은 서열번호 8의 아미노산 서열로 표시되는 CDR1, 서열번호 11의 아미노산 서열로 표시되는 CDR2 및 서열번호 14의 아미노산 서열로 표시되는 CDR3를 포함하는 중쇄 가변 도메인(VHH); 및 항-BCMA sdAb No.19는 서열번호 9의 아미노산 서열로 표시되는 CDR1, 서열번호 12의 아미노산 서열로 표시되는 CDR2 및 서열번호 15의 아미노산 서열로 표시되는 CDR3를 포함하는 중쇄 가변 도메인(VHH)으로 구성되는 것을 확인하였다.As shown in Table 1, anti-BCMA sdAb No.7 includes CDR1 represented by the amino acid sequence of SEQ ID NO: 7, CDR2 represented by the amino acid sequence of SEQ ID NO: 10, and CDR3 represented by the amino acid sequence of SEQ ID NO: 13. heavy chain variable domain (VHH); Anti-BCMA sdAb No.11 is a heavy chain variable domain (VHH) comprising CDR1 represented by the amino acid sequence of SEQ ID NO: 8, CDR2 represented by the amino acid sequence of SEQ ID NO: 11, and CDR3 represented by the amino acid sequence of SEQ ID NO: 14; And anti-BCMA sdAb No.19 has a heavy chain variable domain (VHH) comprising CDR1 represented by the amino acid sequence of SEQ ID NO: 9, CDR2 represented by the amino acid sequence of SEQ ID NO: 12, and CDR3 represented by the amino acid sequence of SEQ ID NO: 15. It was confirmed that it consists of.
구체적으로, 상기 항-BCMA sdAb No.7, No.11 및 No.19는, 각각 서열번호 1, 2 및 3의 아미노산 서열로 표시되는 중쇄가변 도메인으로 구성되며, 또한, 상기 항-BCMA sdAb들은 각각 서열번호 4, 5 및 6의 염기서열로 코딩되는 것을 확인하였다.Specifically, the anti-BCMA sdAb No.7, No.11 and No.19 are composed of heavy chain variable domains represented by the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the anti-BCMA sdAbs are It was confirmed that they were encoded by the base sequences of SEQ ID NOs: 4, 5, and 6, respectively.
서열 명sequence name 서열번호sequence number 서열order
항-BCMA sdAb, No.7Anti-BCMA sdAb, No.7 아미노산 서열
amino acid sequence
 		서열번호 1SEQ ID NO: 1 KLEESGGGSVQTGGSLRLTCAASGSIFSSGFMAWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAARGTDTAYWGQGTQVTVSSKLEESGGGSVQTGGSLRLTCAASG SIFSSGFMA WFRQAPGKERE FVSGISWRGDSTG YADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYC AARGTDTA YWGQGTQVTVSS
염기서열base sequence 서열번호 4SEQ ID NO: 4 AAGCTGGAGGAGAGCGGTGGTGGCTCCGTGCAGACAGGAGGAAGCTTGCGCCTGACCTGCGCCGCTTCCGGCTCCATTTTTTCATCTGGCTTCATGGCGTGGTTCCGCCAGGCCCCCGGGAAGGAGAGGGAGTTCGTCTCGGGCATCAGTTGGCGTGGTGATTCTACTGGCTACGCGGACAGCGTGAAGGGCCGCTTCACCATCTCTCGGGACAACGCCAAGAACACGGTGGACCTGCAGATGAACTCCCTCAAACCTGAAGATACTGCTATCTACTACTGTGCTGCACGCGGGACCGACACCGCCTATTGGGGCCAGGGCACCCAGGTCACCGTGTCGTCCAAGCTGGAGGAGAGCGGTGGTGGCTCCGTGCAGACAGGAGGAAGCTTGCGCCTGACCTGCGCCGCTTCCGGCTCCATTTTTTCATCTGGCCTTCATGGCGTGGTTCCGCCAGGCCCCCCGGGAAGGAGAGGGAGTTCGTCTCGGGCATCAGTTGGCGTGGTGATTCTACTGGCTACGCGGACAGCGTGAAGGGCCGCTTCACCATCTCTCGGGACAACGCCAAGAACACGGTGGACCTGCAGATGAACTCCCTCAA ACCTGAAGATACTGCTATCTACTACTGTGCTGCACGCGGGACCGACACCGCCTATTGGGGCCAGGGCACCCAGGTCACCGTGTCGTCC
CDR1 CDR1 서열번호 7SEQ ID NO: 7 SIFSSGFMASIFSSGFMA
CDR2CDR2 서열번호 10SEQ ID NO: 10 FVSGISWRGDSTGFVSGISWRGDSTG
CDR3CDR3 서열번호 13SEQ ID NO: 13 AARGTDTAAARGTDTA
항-BCMA sdAb, No.11Anti-BCMA sdAb, No.11 아미노산 서열
amino acid sequence
 		서열번호 2SEQ ID NO: 2 KLEESGGGSVQTGGSLRLTCAASGSISRLDNMGWFRQAPGKERELVAAINSKSNKTNYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCANWSFDKHRAYWGQGTQVTVSSKLEESGGGSVQTGGSLRLTCAASG SISRLDNMG WFRQAPGKERE LVAAINSKSNKTN YADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYC ANWSFDKHRA YWGQGTQVTVSS
염기서열
base sequence
 		서열번호 5SEQ ID NO: 5 AAGCTGGAGGAGAGCGGTGGGGGCTCCGTGCAGACAGGAGGCTCCCTCCGCCTGACTTGCGCCGCTTCCGGTTCTATCTCTCGTTTGGATAATATGGGCTGGTTCCGCCAGGCCCCCGGGAAGGAGAGGGAGCTGGTGGCTGCAATTAATTCCAAGAGCAACAAGACCAACTACGCGGACAGTGTGAAGGGCCGCTTCACCATCTCGCGGGACAACGCGAAAAACACGGTTGACCTGCAGATGAACAGCCTTAAACCTGAAGACACCGCCATCTACTACTGTGCCAACTGGTCTTTTGATAAGCACCGCGCTTATTGGGGCCAGGGCACCCAGGTCACCGTGTCCTCCAAGCTGGAGGAGAGCGGTGGGGGCTCCGTGCAGACAGGAGGCTCCCTCTCCGCCTGACTTGCGCCGCTTCCGGTTTCTATCTCTCGTTTGGATAATATGGGCTGGTTCCGCCAGGCCCCCGGGAAGGAGAGGGAGCTGGTGGCTGCAATTAATTCCAAGAGCAACAAGACCAACTACGCGGACAGTGTGAAGGGCCGCTTCACCATCTCTCGCGGGACAACGCGAAAAAACACGGTTGACCTGCAGATGAACAGCCTTAAACC TGAAGACACGCCATCTACTACTGTGCCAACTGGTCTTTTGATAAGCACCGCGCTTATTGGGGCCAGGGCACCCAGGTCACCGTGTCCTCC
CDR1 CDR1 서열번호 8SEQ ID NO: 8 SISRLDNMGSISRLDNMG
CDR2CDR2 서열번호 11SEQ ID NO: 11 LVAAINSKSNKTNLVAINSKSNKTN
CDR3CDR3 서열번호 14SEQ ID NO: 14 ANWSFDKHRAANWSFDKHRA
항-BCMA sdAb, No.19Anti-BCMA sdAb, No.19 아미노산 서열
amino acid sequence
 		서열번호 3SEQ ID NO: 3 KLEESGGGSVQTGGSLRLTCAASGIIFSDDAIGWFRQAPGKERELVSLIDSKSSTYYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCNDHKRRQAMEYWGQGTQVTVSSKLEESGGGSVQTGGSLRLTCAASG IIFSDDAIG WFRQAPGKERE LVSLIDSKSSTY YADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYC NDHKRRQAME YWGQGTQVTVSS
염기서열
base sequence
 		서열번호 6SEQ ID NO: 6 AAGCTGGAGGAGAGCGGAGGAGGCTCCGTGCAGACTGGGGGCTCCCTTCGCCTGACCTGTGCCGCTTCCGGTTCGATTTTTTCAAGCGATGTGATGGCATGGTTCCGCCAGGCCCCCGGGAAGGAGCGCGAGTTCGTCTCTGGCATCAGTTGGCGTGGTGATTCTACCGGCTACGCGGACAGCGTGAAGGGCCGCTTCACCATCTCTCGGGACAACGCGAAGAACACGGTTGACCTGCAGATGAACTCCCTCAAACCTGAAGACACCGCCATCTACTACTGCACTACATTGAGGGAGGCTCAGTCCGCCCCCCACAACCTGCAAGCCGCCTATTGGGGCCAGGGCACCCAGGTCACCGTGTCGTCCAAGCTGGAGGAGAGCGGAGGAGGCTCCGTGCAGACTGGGGGCTCCCTTCGCCTGACCTGTGCCGCTTCCGGTTCGATTTTTTCAAGCGATGTGATGGCATGGTTCCGCCAGGCCCCCCGGGAAGGAGCGCGAGTTCGTCTCTGGCATCAGTTGGCGTGGTGATTCTACCGGCTACGCGGACAGCGTGAAGGGCCGCTTCACCATCTCTCGGGACAACGCGAAGAACACGGTTGACCTGCAGATGAACTCCC TCAAACCTGAAGACACCGCCATCTACTACTGCACTACATTGAGGGAGGCTCAGTCCGCCCCCCACAACCTGCAAGCCGCCTATTGGGGCCAGGGCACCCAGGTCACCGTGTCGTCC
CDR1 CDR1 서열번호 9SEQ ID NO: 9 IIFSDDAIGIIFSDDAIG
CDR2CDR2 서열번호 12SEQ ID NO: 12 LVSLIDSKSSTYLVSLIDSKSSTY
CDR3CDR3 서열번호 15SEQ ID NO: 15 NDHKRRQAMENDHKRRQAME
실시예 2. 항-BCMA sdAb의 결합능 확인 Example 2. Confirmation of binding ability of anti-BCMA sdAb
본 발명자들은 상기 실시예 1에서 확립한 본 발명의 항-BCMA sdAb들이 BCMA를 특이적으로 표적화할 수 있는지 BCMA에 대한 특이성을 확인하기 위해, 유세포분석을 실시하였다. The present inventors performed flow cytometry to confirm the specificity for BCMA of the anti-BCMA sdAbs of the present invention established in Example 1 above, whether they could specifically target BCMA.
간략하게는, BCMA를 발현하는 다발성골수종 H929 세포 5x105 개와 항-BCMA No.7, No.11 및 No.19 1 ㎍ 각각을 30 분간 반응시킨 다음, 2차 항체로 표면(surface)을 염색한 후, 유세포분석기로 측정하였다.Briefly, 5x105 BCMA-expressing multiple myeloma H929 cells were reacted with 1 μg each of anti-BCMA No.7, No.11, and No.19 for 30 minutes, and then the surface was stained with a secondary antibody. Afterwards, it was measured using a flow cytometer.
이때, 양성대조군으로 PE-컨쥬게이션된 항-BCMA 항체(PE-conjugated anti-BCMA antibody; Biolegend Inc., cat# 357503, 미국) 5㎍를 사용하였으며, 2차 항체로는 PE-컨쥬게이션된 항-마우스 IgG 항체(PE-conjugated goat anti-mouse IgG; Biolegend Inc., cat# 405307, 미국) 5㎍를 사용하였다. At this time, 5㎍ of PE-conjugated anti-BCMA antibody (Biolegend Inc., cat# 357503, USA) was used as a positive control, and PE-conjugated anti-BCMA antibody was used as a secondary antibody. -5 μg of mouse IgG antibody (PE-conjugated goat anti-mouse IgG; Biolegend Inc., cat# 405307, USA) was used.
그 결과, 도 1에 나타낸 바와 같이, 본 발명의 항-BCMA sdAb No.7, No.11, No.19 모두 BCMA를 발현하는 세포와 특이적으로 결합하는 것을 확인하였다.As a result, as shown in Figure 1, it was confirmed that the anti-BCMA sdAb No.7, No.11, and No.19 of the present invention all specifically bound to cells expressing BCMA.
이는, 본 발명에서 선별한 항체들이 BCMA를 발현하는 세포를 특이적으로 인식하였으므로, BCMA를 발현하는 세포에 의해 매개되는 질환의 치료뿐만 아니라 진단 등 다양한 분야에 유용하게 활용할 수 있음을 입증한다.This demonstrates that since the antibodies selected in the present invention specifically recognize cells expressing BCMA, they can be useful in various fields such as diagnosis as well as treatment of diseases mediated by cells expressing BCMA.
실시예 3. BCMA를 표적으로 하는 키메라 항원 수용체(CAR) 발현 벡터 제작Example 3. Construction of a chimeric antigen receptor (CAR) expression vector targeting BCMA
본 발명자들은, 도 2의 모식도에 나타낸 것과 같은 BCMA를 표적으로 하는 키메라 항원 수용체(CAR)를 제작하기 위하여, 상기 실시예 1에서 BCMA에 대한 특이성이 높은 것으로 선별된 항-BCMA sdAb No.7, No.11, No.19를 이용하여, BCMA를 표적으로 하는 키메라 항원수용체(CAR)를 발현하는 렌티바이러스 벡터(BCMA-CAR 렌티바이러스)를 각각 제작하였다. In order to construct a chimeric antigen receptor (CAR) targeting BCMA as shown in the schematic diagram of FIG. 2, the present inventors used anti-BCMA sdAb No. 7, which was selected as having high specificity for BCMA in Example 1, Using No.11 and No.19, lentiviral vectors (BCMA-CAR lentivirus) expressing a chimeric antigen receptor (CAR) targeting BCMA were produced, respectively.
도 3의 모식도에 나타낸 바와 같이, EF1α 프로모터(서열번호 16); 시그널 펩타이드를 코딩하는 폴리뉴클레오타이드(서열번호 17); BCMA-결합 도메인을 코딩하는 폴리뉴클레오타이드(서열번호 4, 5 또는 6); CD8 힌지 부위를 코딩하는 폴리뉴클레오타이드(서열번호 18); 막관통 도메인을 코딩하는 폴리뉴클레오타이드(서열번호 19); 4-1BB(공동자극 도메인)을 코딩하는 폴리뉴클레오타이드(서열번호 20); CD3ζ(세포내 신호전달도메인)을 코딩하는 폴리뉴클레오타이드(서열번호 21); 및 WPRE를 코딩하는 폴리뉴클레오타이드(서열번호 22)로 구성된 CAR DNA를 생체외(in vitro)에서 합성하여 3세대 렌티바이러스 벡터에 삽입하였다. 상기 서열정보는 하기 표 2에 정렬하였다.As shown in the schematic diagram of Figure 3, EF1α promoter (SEQ ID NO: 16); A polynucleotide encoding a signal peptide (SEQ ID NO: 17); A polynucleotide encoding a BCMA-binding domain (SEQ ID NO: 4, 5 or 6); A polynucleotide encoding the CD8 hinge region (SEQ ID NO: 18); A polynucleotide encoding a transmembrane domain (SEQ ID NO: 19); Polynucleotide encoding 4-1BB (co-stimulatory domain) (SEQ ID NO: 20); Polynucleotide encoding CD3ζ (intracellular signaling domain) (SEQ ID NO: 21); and CAR DNA consisting of a polynucleotide (SEQ ID NO: 22) encoding WPRE was synthesized in vitro and inserted into a third-generation lentiviral vector. The sequence information was arranged in Table 2 below.
렌티바이러스 벡터는 pMDLg/pRRE(Addgene, cat# #12251) pMD2.G(Addgene, cat##12259), pRSV-Rev(Addgene, cat##12253)의 세 가지 벡터 함께 Lenti-X 293T 세포로 공동 감염(co-transfection)시킨 다음, BCMA-CAR 렌티바이러스를 생산하였다. 공동 감염을 위해 Lipofectamine 3000 transfection kit(Invitrogen, cat# L3000-015)와 Opti-MEM+GlutaMAX(gibco, cat# 51985-034) 배지를 사용하여 세 가지 벡터와 Lenti-X 293T 세포를 6시간 배양하였다.Lentiviral vectors were co-transfected into Lenti-X 293T cells with three vectors: pMDLg/pRRE (Addgene, cat##12251), pMD2.G (Addgene, cat##12259), and pRSV-Rev (Addgene, cat##12253). After infection (co-transfection), BCMA-CAR lentivirus was produced. For co-infection, the three vectors and Lenti-X 293T cells were cultured for 6 hours using Lipofectamine 3000 transfection kit (Invitrogen, cat# L3000-015) and Opti-MEM+GlutaMAX (gibco, cat# 51985-034) medium. .
서열 명sequence name 서열번호sequence number 서열order
EF1 프로모터EF1 promoter 염기서열base sequence 서열번호 16SEQ ID NO: 16 GTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA
시그널 펩타이드signal peptide 염기서열 base sequence 서열번호 17SEQ ID NO: 17 ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG
아미노산서열amino acid sequence 서열번호 23SEQ ID NO: 23 MALPVTALLLPLALLLHAARPMALPVTALLLPLALLLHAARP
CD8 힌지CD8 hinge 염기서열 base sequence 서열번호 18SEQ ID NO: 18 ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT
아미노산서열amino acid sequence 서열번호 24SEQ ID NO: 24 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
막관통 도메인transmembrane domain 염기서열 base sequence 서열번호 19SEQ ID NO: 19 ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC
아미노산서열amino acid sequence 서열번호 25SEQ ID NO: 25 IYIWAPLAGTCGVLLLSLVITLYCIYIWAPLAGTCGVLLLSLVITLYC
4-1BB4-1BB 염기서열 base sequence 서열번호 20SEQ ID NO: 20 AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAAGAAGGAGGATGTGAACTG
아미노산서열amino acid sequence 서열번호 26SEQ ID NO: 26 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
CD3ξCD3ξ 염기서열 base sequence 서열번호 21SEQ ID NO: 21 AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGG CAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC
아미노산서열amino acid sequence 서열번호 27SEQ ID NO: 27 RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
WPREWPRE 염기서열base sequence 서열번호 22SEQ ID NO: 22 ATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCA CCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCC TGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG
실시예 4. hBCMA-CAR-T 세포 제조Example 4. Preparation of hBCMA-CAR-T cells
본 발명자들은 상기 실시예 3에서 제작한 3종의 BCMA-CAR 렌티바이러스 벡터를 T 세포에 형질 전환시켜 BCMA-CAR-T 세포(각각 No.7 CAR-T, No.11 CAR-T, No.19 CAR-T)를 제조하였다.The present inventors transformed the three types of BCMA-CAR lentiviral vectors produced in Example 3 into T cells to produce BCMA-CAR-T cells (No. 7 CAR-T, No. 11 CAR-T, and No. 11 CAR-T, respectively). 19 CAR-T) was prepared.
보다 구체적으로, 도 4에 나타낸 바와 같이, 혈액에서 말초혈액단핵세포(peripheral blood mononuclear cell, PBMC)를 분리한 다음, T 세포 활성화 비드(T cell activation bead; MiltenylBiotec, cat# 130-091-441)를 사용해 T 세포를 활성화시켰다. 활성화된 T 세포에 상기 실시예 3에서 제조한 BCMA-CAR 렌티바이러스를 T 세포에 형질 도입시켜 BCMA-CAR-T 세포를 제조하였으며, Lenti-boost-p를 사용하여 형질 도입 효율을 증가시켰다. More specifically, as shown in Figure 4, peripheral blood mononuclear cells (PBMC) were isolated from the blood, and then T cell activation beads (T cell activation bead; MiltenylBiotec, cat# 130-091-441) was used to activate T cells. BCMA-CAR-T cells were prepared by transducing activated T cells with the BCMA-CAR lentivirus prepared in Example 3, and transduction efficiency was increased using Lenti-boost-p.
또한, 본 발명자들은 상기 BCMA-CAR-T 세포의 BCMA 펩타이드 결합능을 확인하기 위하여, 유세포분석(Flow Cytometry)을 실시하였다.In addition, the present inventors performed flow cytometry to confirm the BCMA peptide binding ability of the BCMA-CAR-T cells.
간략하게는, 상기에서 제조한 BCMA-CAR-T세포를 FITC-BCMA 단백질(Acrobiosystems, cat#BCA-HF2H3)과 anti-CD3, anti-CD4, anti-CD8 항체와 반응시킨 후 FACS 기계를 이용해 형광 세기를 측정하였다. 분석과정에서 CD3를 발현하는 세포를 T 세포로 하여 T 세포안에서 FITC 발현 정도를 확인하였다.Briefly, the BCMA-CAR-T cells prepared above were reacted with FITC-BCMA protein (Acrobiosystems, cat#BCA-HF2H3) and anti-CD3, anti-CD4, and anti-CD8 antibodies, followed by fluorescence using a FACS machine. The intensity was measured. During the analysis process, cells expressing CD3 were considered T cells, and the level of FITC expression in T cells was confirmed.
그 결과, 도 5에 나타낸 바와 같이, BCMA 펩타이드 증가에 따라, BCMA와 결합하는 CD4 또는 CD8를 발현하는 BCMA-CAR-T 세포가 증가하는 것을 확인하였다.As a result, as shown in Figure 5, it was confirmed that as the BCMA peptide increased, the number of BCMA-CAR-T cells expressing CD4 or CD8 that binds to BCMA increased.
이는 본 발명에서 제조한 BCMA-CAR-T 세포가 효과적으로 BCMA와 결합하는 것을 입증한다.This demonstrates that the BCMA-CAR-T cells prepared in the present invention effectively bind to BCMA.
실시예 5. BCMA 발현 세포에 대한 BCMA-CAR-T 세포의 사멸 효과 확인Example 5. Confirmation of the killing effect of BCMA-CAR-T cells on BCMA-expressing cells
본 발명자들은 본 발명의 BCMA-CAR-T 세포에 의한 표적세포의 사멸효과를 확인하고자 하였다.The present inventors sought to confirm the killing effect of target cells by the BCMA-CAR-T cells of the present invention.
간략하게는, 표적세포로 BCMA를 발현하지 않는 K562 세포와 BCMA를 발현하는 H929 세포를 이용하였으며, BCMA-CAR-T 세포와 1:4, 1:2, 1:1, 1:0.5 및 1:0.25 비율이 되도록 혼합한 다음, 루미네센스(CytoTox-Glo Cytotoxicity Assay, Promega, cat# G9291)를 측정하였다. 측정한 값으로 하기 수학식 1을 이용하여 세포 사멸 정도를 계산하였다:Briefly, K562 cells, which do not express BCMA, and H929 cells, which express BCMA, were used as target cells, and 1:4, 1:2, 1:1, 1:0.5, and 1:4 with BCMA-CAR-T cells. After mixing to a ratio of 0.25, luminescence (CytoTox-Glo Cytotoxicity Assay, Promega, cat# G9291) was measured. The degree of cell death was calculated using the measured values using Equation 1 below:
[수학식 1][Equation 1]
%Cytotoxicity = [(Experimental - Effctor Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)] X 100%Cytotoxicity = [(Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)]
[Experimental: 표적세포 및 T 세포 복합 배양의 배지로 부터 도출된 발광(Luminescence)값, Effctor Spontaneous: T 세포 배지로부터 도출된 발광값, Target Spontaneous: 표적세포 배지로부터 도출된 발광값, Target Maximum: 표적세포의 100% 용해(용해시약(Lysis Reagent) 이용)로부터 도출된 발광값].[Experimental: Luminescence value derived from the medium of target cells and T cell complex culture, Effctor Spontaneous: Luminescence value derived from the T cell medium, Target Spontaneous: Luminescence value derived from the target cell medium, Target Maximum: Target Luminescence value derived from 100% lysis of cells (using Lysis Reagent)].
그 결과, 도 6에 나타낸 바와 같이, BCMA-CAR-T 세포는 BCMA를 발현하는 H929 세포를 보다 특이적으로 사멸시키는 것을 확인하였다.As a result, as shown in Figure 6, it was confirmed that BCMA-CAR-T cells more specifically kill H929 cells expressing BCMA.
이는, 본 발명의 BCMA를 표적으로 하는 키메라 항원 수용체 및 CAR-T 세포가 B세포 또는 BCMA 발현과 관련된 질환 예방 또는 치료용 조성물로 유용하게 활용할 수 있음을 입증한다.This demonstrates that the chimeric antigen receptor and CAR-T cells targeting BCMA of the present invention can be usefully used as a composition for preventing or treating diseases related to B cells or BCMA expression.

Claims (25)

  1. 서열번호 7 내지 9 중 어느 하나의 아미노산 서열로 표시되는 CDR1(complementary determining region 1); 서열번호 10 내지 12 중 어느 하나의 아미노산 서열로 표시되는 CDR2(complementary determining region 2); 및 서열번호 13 내지 15 중 어느 하나의 아미노산 서열로 표시되는 CDR3(complementary determining region 3);를 포함하며,CDR1 (complementary determining region 1) represented by any one of the amino acid sequences of SEQ ID NOs: 7 to 9; CDR2 (complementary determining region 2) represented by any one of the amino acid sequences of SEQ ID NOs: 10 to 12; and CDR3 (complementary determining region 3) represented by the amino acid sequence of any one of SEQ ID NOs: 13 to 15,
    BCMA(B-cell maturation antigen)에 특이적으로 결합하는 단일 도메인 항체(single domain antibody, sdAb) 또는 이의 항원-결합 단편.A single domain antibody (sdAb) or antigen-binding fragment thereof that specifically binds to BCMA (B-cell maturation antigen).
  2. 제1항에 있어서,According to paragraph 1,
    상기 단일 도메인 항체는,The single domain antibody,
    (i) 서열번호 7의 아미노산 서열로 표시되는 CDR1, 서열번호 10의 아미노산 서열로 표시되는 CDR2 및 서열번호 13의 아미노산 서열로 표시되는 CDR3를 포함하는 중쇄 가변 도메인(VHH);(i) a heavy chain variable domain (VHH) comprising CDR1 represented by the amino acid sequence of SEQ ID NO: 7, CDR2 represented by the amino acid sequence of SEQ ID NO: 10, and CDR3 represented by the amino acid sequence of SEQ ID NO: 13;
    (ii) 서열번호 8의 아미노산 서열로 표시되는 CDR1, 서열번호 11의 아미노산 서열로 표시되는 CDR2 및 서열번호 14의 아미노산 서열로 표시되는 CDR3를 포함하는 중쇄 가변 도메인(VHH);또는(ii) a heavy chain variable domain (VHH) comprising CDR1 represented by the amino acid sequence of SEQ ID NO: 8, CDR2 represented by the amino acid sequence of SEQ ID NO: 11, and CDR3 represented by the amino acid sequence of SEQ ID NO: 14; or
    (iii) 서열번호 9의 아미노산 서열로 표시되는 CDR1, 서열번호 12의 아미노산 서열로 표시되는 CDR2 및 서열번호 15의 아미노산 서열로 표시되는 CDR3를 포함하는 중쇄 가변 도메인(VHH);을 포함하는 것을 특징으로 하는,(iii) a heavy chain variable domain (VHH) comprising CDR1 represented by the amino acid sequence of SEQ ID NO: 9, CDR2 represented by the amino acid sequence of SEQ ID NO: 12, and CDR3 represented by the amino acid sequence of SEQ ID NO: 15; to,
    BCMA(B-cell maturation antigen)에 특이적으로 결합하는 단일 도메인 항체(single domain antibody, sdAb) 또는 이의 항원-결합 단편.A single domain antibody (sdAb) or antigen-binding fragment thereof that specifically binds to BCMA (B-cell maturation antigen).
  3. 제2항에 있어서,According to paragraph 2,
    상기 (i) 중쇄 가변 도메인(VHH)은 서열번호 1로 표시되는 아미노산 서열로 이루어진 것을 특징으로 하는,The (i) heavy chain variable domain (VHH) is characterized in that it consists of the amino acid sequence represented by SEQ ID NO: 1,
    BCMA(B-cell maturation antigen)에 특이적으로 결합하는 단일 도메인 항체(single domain antibody, sdAb) 또는 이의 항원-결합 단편.A single domain antibody (sdAb) or antigen-binding fragment thereof that specifically binds to BCMA (B-cell maturation antigen).
  4. 제3항에 있어서,According to paragraph 3,
    상기 (i) 중쇄 가변 도메인(VHH)은 서열번호 4로 표시되는 염기서열에 의해 코딩되는 것을 특징으로 하는,The (i) heavy chain variable domain (VHH) is characterized in that it is encoded by the base sequence represented by SEQ ID NO: 4,
    BCMA(B-cell maturation antigen)에 특이적으로 결합하는 단일 도메인 항체(single domain antibody, sdAb) 또는 이의 항원-결합 단편.A single domain antibody (sdAb) or antigen-binding fragment thereof that specifically binds to BCMA (B-cell maturation antigen).
  5. 제2항에 있어서,According to paragraph 2,
    상기 (ii)중쇄 가변 도메인(VHH)은 서열번호 2로 표시되는 아미노산 서열로 이루어진 것을 특징으로 하는,The (ii) heavy chain variable domain (VHH) is characterized in that it consists of the amino acid sequence represented by SEQ ID NO: 2,
    BCMA(B-cell maturation antigen)에 특이적으로 결합하는 단일 도메인 항체(single domain antibody, sdAb) 또는 이의 항원-결합 단편.A single domain antibody (sdAb) or antigen-binding fragment thereof that specifically binds to BCMA (B-cell maturation antigen).
  6. 제5항에 있어서,According to clause 5,
    상기 (ii) 중쇄 가변 도메인(VHH)은 서열번호 5로 표시되는 염기서열에 의해 코딩되는 것을 특징으로 하는,The (ii) heavy chain variable domain (VHH) is characterized in that it is encoded by the base sequence represented by SEQ ID NO: 5,
    BCMA(B-cell maturation antigen)에 특이적으로 결합하는 단일 도메인 항체(single domain antibody, sdAb) 또는 이의 항원-결합 단편.A single domain antibody (sdAb) or antigen-binding fragment thereof that specifically binds to BCMA (B-cell maturation antigen).
  7. 제2항에 있어서,According to paragraph 2,
    상기 (iii) 중쇄 가변 도메인(VHH)은 서열번호 3으로 표시되는 아미노산 서열로 이루어진 것을 특징으로 하는,The (iii) heavy chain variable domain (VHH) is characterized in that it consists of the amino acid sequence represented by SEQ ID NO: 3,
    BCMA(B-cell maturation antigen)에 특이적으로 결합하는 단일 도메인 항체(single domain antibody, sdAb) 또는 이의 항원-결합 단편.A single domain antibody (sdAb) or antigen-binding fragment thereof that specifically binds to BCMA (B-cell maturation antigen).
  8. 제7항에 있어서,In clause 7,
    상기 (iii) 중쇄 가변 도메인(VHH)은 서열번호 6으로 표시되는 염기서열에 의해 코딩되는 것을 특징으로 하는,The (iii) heavy chain variable domain (VHH) is characterized in that it is encoded by the base sequence represented by SEQ ID NO: 6,
    BCMA(B-cell maturation antigen)에 특이적으로 결합하는 단일 도메인 항체(single domain antibody, sdAb) 또는 이의 항원-결합 단편.A single domain antibody (sdAb) or antigen-binding fragment thereof that specifically binds to BCMA (B-cell maturation antigen).
  9. BCMA-결합 도메인, 막관통 도메인(transmembrane domain), 공동자극 도메인(costimulatory domain) 및 세포 내 신호전달 도메인(intracellular signal transduction domain)을 포함하는 키메라 항원 수용체(chimeric antigen receptor: CAR)로서, A chimeric antigen receptor (CAR) comprising a BCMA-binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signal transduction domain,
    상기 BCMA-결합 도메인은 BCMA와 특이적으로 결합할 수 있는 단일 도메인 항체(single domain antibody, sdAb) 또는 이의 항원-결합 단편을 포함하며,The BCMA-binding domain includes a single domain antibody (sdAb) or an antigen-binding fragment thereof capable of specifically binding to BCMA,
    상기 단일 도메인 항체(single domain antibody, sdAb)는, 서열번호 7 내지 9 중 어느 하나의 아미노산 서열로 표시되는 CDR1(complementary determining region 1); 서열번호 10 내지 12 중 어느 하나의 아미노산 서열로 표시되는 CDR2(complementary determining region 2); 및 서열번호 13 내지 15 중 어느 하나의 아미노산 서열로 표시되는 CDR3(complementary determining region 3);를 포함하는 것을 특징으로 하는, 키메라 항원 수용체(chimeric antigen receptors, CAR).The single domain antibody (sdAb) includes CDR1 (complementary determining region 1) represented by the amino acid sequence of any one of SEQ ID NOs: 7 to 9; CDR2 (complementary determining region 2) represented by any one of the amino acid sequences of SEQ ID NOs: 10 to 12; and CDR3 (complementary determining region 3) represented by the amino acid sequence of any one of SEQ ID NOs: 13 to 15. Chimeric antigen receptors (CAR).
  10. 제9항에 있어서,According to clause 9,
    상기 막관통 도메인은 CD8,FcγR, ICOS(CD278), 4-1BB(CD137), OX40(CD134), CD27, CD28, IL-2Rβ, CD40, DAP10, MHC class I 분자, TNF 수용체 단백질, Immunoglobulin-유사 단백질, 사이토카인 수용체, 인테그린, SLAM 단백질, 활성화 NK 세포 수용체, BTLA, Toll 리간드 수용체, CD2, CD7, CD30, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD83 특이적 리간드, CD247, CD3δ, CD3ε, CD3γ, CD3ζ, Ig alpha(CD79a), IL-2Rγ, IL-7Rα, PD-1, TNFSF14, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD154, T 세포 수용체의 알파 쇄, T 세포 수용체의 베타 쇄 및 T 세포 수용체의 제타 쇄로 이루어진 군으로부터 선택된 1 종 이상인 것을 특징으로 하는,The transmembrane domain is CD8, FcγR, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD28, IL-2Rβ, CD40, DAP10, MHC class I molecule, TNF receptor protein, Immunoglobulin-like Protein, cytokine receptor, integrin, SLAM protein, activated NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2 , SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 ( Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8) , SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD83 specific ligand, CD247, CD3δ, CD3ε, CD3γ, CD3ζ, Ig alpha (CD79a), IL-2Rγ, IL-7Rα , PD-1, TNFSF14, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD154, consisting of the alpha chain of the T cell receptor, the beta chain of the T cell receptor, and the zeta chain of the T cell receptor. Characterized by one or more species selected from the group,
    키메라 항원 수용체(chimeric antigen receptors, CAR).Chimeric antigen receptors (CAR).
  11. 제9항에 있어서,According to clause 9,
    상기 공동자극 도메인은 CD27, CD28,CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, 림프구 기능-연관 항원-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3 및 CD83로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는,The costimulatory domains include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C. Characterized in that it is one or more selected from the group consisting of B7-H3 and CD83,
    키메라 항원 수용체(chimeric antigen receptors, CAR).Chimeric antigen receptors (CAR).
  12. 제9항에 있어서,According to clause 9,
    상기 세포 내 신호전달 도메인은 CD3ζ, FcγR, ICOS(CD278), 4-1BB(CD137), OX40(CD134), CD27, CD28, IL-2Rβ, IL-15R-α, MyD88, DAP10, DAP12, MHC class I 분자, TNF 수용체 단백질, Immunoglobulin-유사 단백질, 사이토카인 수용체, 인테그린, SLAM 단백질, 활성화 NK 세포 수용체, BTLA, Toll 리간드 수용체, CD2, CD7, CD30, CD40, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD83 특이적 리간드, CD247, CD3δ, CD3ε, CD3γ, CD8, Ig alpha(CD79a), IL-2Rγ, IL-7Rα, PD-1 및 TNFSF14로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는,The intracellular signaling domains include CD3ζ, FcγR, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD28, IL-2Rβ, IL-15R-α, MyD88, DAP10, DAP12, MHC class. I molecule, TNF receptor protein, Immunoglobulin-like protein, cytokine receptor, integrin, SLAM protein, activated NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CD40, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108) , SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD83 specific ligand, CD247, CD3δ, CD3ε, CD3γ , CD8, Ig alpha (CD79a), IL-2Rγ, IL-7Rα, PD-1, and at least one selected from the group consisting of TNFSF14,
    키메라 항원 수용체(chimeric antigen receptors, CAR).Chimeric antigen receptors (CAR).
  13. 제9항에 있어서,According to clause 9,
    상기 BCMA-결합 도메인의 C 말단 및 막관통 도메인의 N 말단 사이에 힌지 부위(hinge region)가 추가로 포함되는 것을 특징으로 하는,Characterized in that a hinge region is further included between the C terminus of the BCMA-binding domain and the N terminus of the transmembrane domain,
    키메라 항원 수용체(chimeric antigen receptors, CAR).Chimeric antigen receptors (CAR).
  14. 제13항에 있어서,According to clause 13,
    상기 힌지는 CD8α 힌지, IgG1 힌지 및 FcγRIIIα 힌지로 이루어진 군으로부터 선택된 1 종 이상인 것을 특징으로 하는,The hinge is characterized in that at least one selected from the group consisting of CD8α hinge, IgG1 hinge, and FcγRIIIα hinge,
    키메라 항원 수용체(chimeric antigen receptors, CAR).Chimeric antigen receptors (CAR).
  15. 제9항에 있어서, According to clause 9,
    상기 키메라 항원 수용체는 추가로 시그널(signal) 펩타이드에 연결되는 것을 특징으로 하는,The chimeric antigen receptor is further linked to a signal peptide,
    키메라 항원 수용체(chimeric antigen receptors, CAR).Chimeric antigen receptors (CAR).
  16. 제9항 내지 제15항 중 어느 한 항의 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드.A polynucleotide encoding the chimeric antigen receptor (CAR) of any one of claims 9 to 15.
  17. 제9항 내지 제15항 중 어느 한 항의 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드를 포함하는 벡터.A vector comprising a polynucleotide encoding the chimeric antigen receptor (CAR) of any one of claims 9 to 15.
  18. 제17항에 있어서, According to clause 17,
    상기 벡터는 플라스미드(plasmid), 레트로바이러스(retroviral) 벡터 및 렌티바이러스(lentiviral) 벡터로 이루어진 군으로부터 선택된 1 종 이상인 것을 특징으로 하는, The vector is characterized in that one or more types selected from the group consisting of plasmid, retroviral vector, and lentiviral vector,
    벡터.vector.
  19. 제9항 내지 제15항 중 어느 한 항의 키메라 항원 수용체(CAR)를 표면에 발현하는, 단리된 면역 세포.An isolated immune cell expressing on its surface the chimeric antigen receptor (CAR) of any one of claims 9 to 15.
  20. 제19항에 있어서,According to clause 19,
    상기 단리된 면역 세포는, T 세포, NK 세포, NKT 세포, B 세포, 수지상 세포, 단핵구, 마크로파지, 호산구, 호염기구 및 호중구로 이루어진 군으로부터 선택된 1 종 이상인 것을 특징으로 하는,The isolated immune cells are characterized in that one or more types selected from the group consisting of T cells, NK cells, NKT cells, B cells, dendritic cells, monocytes, macrophages, eosinophils, basophils and neutrophils,
    단리된 면역 세포.Isolated immune cells.
  21. 제9항 내지 제15항 중 어느 한 항의 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드; 또는 제9항 내지 제15항 중 어느 한 항의 키메라 항원 수용체(CAR)를 표면에 발현하는 단리된 면역 세포를 유효성분으로 포함하는, BCMA 발현과 관련된 질환 예방 또는 치료용 약학적 조성물.A polynucleotide encoding the chimeric antigen receptor (CAR) of any one of claims 9 to 15; Or a pharmaceutical composition for preventing or treating diseases related to BCMA expression, comprising as an active ingredient isolated immune cells expressing the chimeric antigen receptor (CAR) of any one of claims 9 to 15 on the surface.
  22. 제21항에 있어서,According to clause 21,
    상기 BCMA 발현과 관련된 질환은 다발성골수종(multiple myeloma), 혈액암, 비-호지킨 림프종(non-Hodgkin'symphoma), 자가항체-의존성 자가면역 질환(autoantibody-dependent autoimmune disease), 전신홍반루프스(systemic lupus erythematosus, SLE) 및 류마티스 관절염(rheumatoid arthritis)으로 이루어진 군으로부터 선택된 1 종 이상인 것을 특징으로 하는,Diseases related to BCMA expression include multiple myeloma, blood cancer, non-Hodgkin's lymphoma, autoantibody-dependent autoimmune disease, and systemic lupus erythematosus. Characterized by at least one selected from the group consisting of lupus erythematosus (SLE) and rheumatoid arthritis,
    BCMA 발현과 관련된 질환 예방 또는 치료용 약학적 조성물.Pharmaceutical composition for preventing or treating diseases related to BCMA expression.
  23. 제9항 내지 제15항 중 어느 한 항의 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드; 또는 제9항 내지 제15항 중 어느 한 항의 키메라 항원 수용체(CAR)를 표면에 발현하는 단리된 면역 세포를 유효성분으로 포함하는, BCMA 발현과 관련된 질환 예방 또는 개선용 식품 조성물.A polynucleotide encoding the chimeric antigen receptor (CAR) of any one of claims 9 to 15; Or a food composition for preventing or improving diseases related to BCMA expression, comprising as an active ingredient isolated immune cells expressing the chimeric antigen receptor (CAR) of any one of claims 9 to 15 on the surface.
  24. 제9항 내지 제15항 중 어느 한 항의 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드 또는 이를 포함하는 벡터를, 단리된 면역 세포에 도입시키는 단계를 포함하는, 면역세포의 제조 방법.A method for producing immune cells, comprising introducing a polynucleotide encoding the chimeric antigen receptor (CAR) of any one of claims 9 to 15 or a vector containing the same into isolated immune cells.
  25. 제9항 내지 제15항 중 어느 한 항의 키메라 항원 수용체(CAR)를 코딩하는 폴리뉴클레오타이드; 또는 제9항 내지 제15항 중 어느 한 항의 키메라 항원 수용체(CAR)를 표면에 발현하는 단리된 면역 세포를, 약학적 유효량으로 대상(subject)에게 투여하는 단계를 포함하는, BCMA 발현과 관련된 질환 예방 또는 치료 방법.A polynucleotide encoding the chimeric antigen receptor (CAR) of any one of claims 9 to 15; or a disease associated with BCMA expression, comprising administering to a subject a pharmaceutically effective amount of isolated immune cells expressing the chimeric antigen receptor (CAR) of any one of claims 9 to 15 on the surface. Prevention or treatment method.
PCT/KR2022/020365 2022-12-13 2022-12-14 Novel chimeric antigen receptor for targeting b-cell maturation antigen, and use thereof WO2024128345A1 (en)

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KR10-2022-0174221 2022-12-13

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