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WO2024128345A1 - Nouveau récepteur antigénique chimérique pour cibler un antigène de maturation des lymphocytes b et son utilisation - Google Patents

Nouveau récepteur antigénique chimérique pour cibler un antigène de maturation des lymphocytes b et son utilisation 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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성백진
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Genecell Bt Co Ltd
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C07K2319/00—Fusion polypeptide
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    • C07K2319/00—Fusion polypeptide
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    • C12N2510/00—Genetically modified cells

Definitions

  • 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

La présente invention concerne un nouveau récepteur antigénique chimérique pour cibler un antigène de maturation des lymphocytes B, et son utilisation. Il a été identifié que le récepteur antigénique chimérique pour cibler un BCMA et des cellules immunitaires CAR, préparés selon la présente invention, se lient efficacement au BCMA et les cellules immunitaires CAR liées à BCMA sont activées. De plus, les cellules immunitaires CAR selon la présente invention ont été identifiées pour détruire de manière efficace des cellules exprimant le BCMA, et ainsi le récepteur antigénique chimérique pour cibler BCMA et les cellules immunitaires CAR, selon la présente invention, peut être utilisé de manière efficace en tant que composition pour prévenir ou traiter des maladies associées à l'expression de lymphocytes B ou BCMA.
PCT/KR2022/020365 2022-12-13 2022-12-14 Nouveau récepteur antigénique chimérique pour cibler un antigène de maturation des lymphocytes b et son utilisation Ceased WO2024128345A1 (fr)

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