WO2018128486A1 - Récepteur antigénique chimérique anti-ceacam6 se liant de manière spécifique à ceacam6 - Google Patents

Récepteur antigénique chimérique anti-ceacam6 se liant de manière spécifique à ceacam6 Download PDF

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WO2018128486A1
WO2018128486A1 PCT/KR2018/000311 KR2018000311W WO2018128486A1 WO 2018128486 A1 WO2018128486 A1 WO 2018128486A1 KR 2018000311 W KR2018000311 W KR 2018000311W WO 2018128486 A1 WO2018128486 A1 WO 2018128486A1
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car
ceacam6
antigen receptor
chimeric antigen
cells
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Korean (ko)
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최인표
김태돈
서희윤
이수연
홍권표
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한국생명공학연구원
다이노나(주)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46448—Cancer antigens from embryonic or fetal origin
    • A61K39/464482—Carcinoembryonic antigen [CEA]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to an anti-CEACAM6 chimeric antigen receptor (CAR) that specifically binds to CEACAM6 and a cell therapeutic agent comprising the same.
  • CAR chimeric antigen receptor
  • TIL Tumor Infiltrating Lymphocytes
  • CAR Chimeric Antigen Receptors
  • TCR T-Cell Receptors
  • Chimeric antigen receptors are artificial receptors designed to deliver antigen specificity to T cells. These include antigen specific components, transmembrane components, and intracellular components selected to activate T cells and provide specific immunity. Chimeric antigen receptor expressing T cells can be used in a variety of therapies including cancer therapy.
  • CAR-T has the problem that its function continues to be toxic after cancer cells are removed, and if there is a normal cell showing the target protein, it causes a non-specific attack and causes fatal side effects, which cannot be reversed. It is inhibiting the development of cell therapies.
  • the present inventors produced a chimeric antigen receptor using an antigen binding domain that specifically binds to CEACAM6, thereby specifically targeting CEACAM6 and overexpressing it in cancer cells, thereby making it a target anticancer agent. It was confirmed that it can be a useful target for.
  • an object of the present invention is to provide a chimeric antigen receptor using an antigen binding domain that specifically binds to CEACAM6, a cell therapeutic agent using the same, and a method for treating cancer using the same.
  • the present invention provides a chimeric antigen receptor (CAR) comprising 1) an antigen binding domain, 2) a transmembrane domain, and 3) an intracellular signal transduction domain, wherein the antigen binding domain is specific for CEACAM6.
  • Chimeric antigen receptor (CAR) which is a domain that binds to an enemy.
  • the present invention also provides a polynucleotide encoding the chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the present invention also provides a vector comprising the polynucleotide.
  • the present invention also provides a cell transformed with the vector.
  • the present invention provides a cell therapy agent comprising the cells.
  • the present invention also provides a pharmaceutical composition for the prevention or treatment of cancer, including the cells as an active ingredient.
  • the present invention also provides a method for preventing or treating cancer, comprising administering the cells to a subject.
  • the chimeric antigen receptor according to the present invention is characterized by specifically binding to CEACAM6, and may be a useful target for a target anticancer agent. Therefore, the chimeric antigen receptor according to the present invention can effectively induce the migration of immune cells, particularly natural killer cells, to tumor tissues, and can be usefully used for gene cell therapy that exhibits an effective anticancer effect.
  • FIG. 1 is a diagram showing a pLVX-AcGFP-C1 vector used in the present invention.
  • FIG. 2 is a diagram showing specific related sequences used in the present invention.
  • Figure 3 is an exemplary schematic diagram of the anti-CEACAM6 chimeric antigen receptor of the present invention and its expression system.
  • Figure 4 is a diagram showing the results confirmed by Western blot the expression of CEACAM6-CAR in NK92 cells introduced CEACAM6-CAR.
  • FIG. 5 is a diagram showing the results of confirming the expression of CEACAM6-CAR in the NK92 cells introduced CEACAM6-CAR through flow cytometry.
  • Figure 6 shows the results of confirming the expression of CEACAM6-CAR in the NK92 cells introduced CEACAM6-CAR through immunofluorescence.
  • FIG. 7 is a diagram showing the results of confirming the expression level of CEACAM6 low and high expression cell lines A549-CEACAM6 using flow cytometry.
  • FIG. 8 is a diagram showing the results of confirming the expression level of CEACAM6 low and high expression cell lines A549-CEACAM6 using PCR.
  • FIG. 9 is a diagram showing the results of confirming the apoptosis effect on the A549 cell line of NK92 cells (NK92) and NK92 cells (18B8-CAR) expressing CEACAM6-CAR through the calcein-AM method.
  • FIG. 10 is a diagram showing the results of confirming the cell killing effect on the K562 cell line of NK92 cells (NK92) and NK92 cells (18B8-CAR) expressing CEACAM6-CAR through the calcein-AM method.
  • FIG. 11 is a diagram showing the results confirmed by the ELISA cytokine secretion changes of NK cells.
  • FIG. 12 is a diagram showing the results of confirming the expression level of CEACAM6 by A549 cell line through PCR.
  • Figure 13 shows the results of confirming the apoptosis activity of CEACAM6-CAR NK92 by treatment with siRNA for CEACAM6 through the calcein-AM method.
  • Figure 14 shows the results of confirming the expression of CEACAM6-CAR in a single clone of NK92 cells introduced CEACAM6-CAR through flow cytometry.
  • 15 is a diagram showing the results of confirming the expression level of CEACAM6 in various cancer cells using flow cytometry.
  • 16 and 17 are diagrams showing the results of confirming the apoptosis effect of monoclonal CEACAM6-CAR NK cells to the various cancer cells.
  • One aspect of the invention is a chimeric antigen receptor (CAR) comprising 1) an antigen binding domain, 2) a transmembrane domain, and 3) an intracellular signal transduction domain, wherein the antigen binding domain is CEACAM6 (Carcinoembryonic antigen-related Chimeric Antigen Receptor (CAR), which is a domain that specifically binds to cell adhesion molecule 6).
  • CAR Carcinoembryonic antigen-related Chimeric Antigen Receptor
  • the chimeric antigen receptor according to the present invention is characterized in that it specifically binds to CEACAM6, it comprises an antigen binding domain that specifically binds to CEACAM6.
  • the chimeric antigen receptor of the present invention can be a useful target for a target anticancer agent, can induce immune cells to migrate effectively to tumor tissues, and can be usefully used for gene cell therapy that exhibits an effective anticancer effect.
  • antibody means a substance produced by stimulation of an antigen in the immune system, and the kind thereof is not particularly limited.
  • the antibody herein includes fragments of antibodies having antigen-binding ability, such as, but not limited to, Fab, Fab ', F (ab') 2, Fv, and the like.
  • a "chimeric antibody” refers to an antibody that originates in an animal whose antibody variable region or complementarity determining region (CDR) thereof is different from the rest of the antibody.
  • Such antibodies can be, for example, antibody variable regions derived from animals other than humans (eg, mice, rabbits, poultry, etc.) and antibody constant regions can be antibodies derived from humans.
  • Such chimeric antibodies can be prepared by methods such as genetic recombination known in the art.
  • variable chain refers to a full length heavy chain and fragment thereof comprising a variable region domain VH comprising three amino acid sequences of variable regions sufficient to confer specificity for an antigen and three constant region domains CH1, CH2 and CH3 All are called.
  • light chain refers to both the full length light chain and fragments thereof including the variable region domain VL and the constant region domain CL, which comprise an amino acid sequence of sufficient variable region to confer specificity to the antigen.
  • the antigen binding domain constituting the chimeric antigen receptor of the present invention refers to a site through which the main signal is transmitted and is located outside the cell membrane and recognizes a cell membrane ligand (a substance that binds to and activates a receptor) of a target cell having a specific antigen.
  • the transmembrane domain of the present invention is a site that connects the antigen binding domain and the co-stimulatory, essential signaling domain between the cell membranes, and the intracellular signal transduction domain activates the immune response of immune cells by binding the antigen binding domain. It means the site to make.
  • the chimeric antigen receptor of the present invention is characterized in that the antigen binding domain specifically binds to CEACAM6, which CEACAM6 is an abbreviation of Carcinoembryonic antigenrelated cell adhesion molecule 6, and can be specified as NM_002483 and NM_002483 in humans.
  • the antigen binding domain of the present invention is a substance that specifically binds to CEACAM6, and may be an antibody or an antibody fragment, and the fragment of the antibody may be an scFv.
  • the antigen binding domain may consist of a heavy chain variable region, a linker sequence, a light chain variable region.
  • the heavy chain variable region may include an amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence represented by SEQ ID NO: 1 capable of encoding a functional equivalent to an amino acid expressed by the nucleotide sequence represented by SEQ ID NO: 1; At least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and may consist of an amino acid sequence encoded by a base sequence having sequence homology.
  • the light chain variable region may include an amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 2 or a nucleotide sequence represented by SEQ ID NO: 2 capable of encoding a functional equivalent to an amino acid expressed by the nucleotide sequence represented by SEQ ID NO: 2; At least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and may consist of an amino acid sequence encoded by a base sequence having sequence homology.
  • amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence having 95% or more sequence homology with the nucleotide sequence of SEQ ID NO: 2, respectively, is substantially identical to the amino acid sequence encoded by the nucleotide sequences of SEQ ID NOs: 1 and 2. It can show the physiological activity of.
  • the antibody or fragment of the antibody of the present invention may further include a linker, through which the heavy chain variable region and the light chain variable region may be interconnected.
  • the linker may be used without limitation as long as it is a component capable of forming the VH-linker-VL domain by connecting the heavy chain variable region and the light chain variable region, and preferably encoded by SEQ ID NO: 3 or a nucleotide sequence showing 95% or more homology thereto. Consisting of an amino acid sequence.
  • the antigen binding domain of the anti-CEACAM6 chimeric antigen receptor of the invention may be linked to the transmembrane domain by a hinge region, a spacer region or a combination thereof.
  • the hinge region or spacer region of the present invention may comprise a Myc epitope, and the Myc epitope region, which is a hinge region or spacer region of the present invention, is an amino acid sequence encoded by SEQ ID NO: 4 or a nucleotide sequence exhibiting at least 95% homology therewith. It may be made of.
  • One membrane transmembrane domain of the chimeric antigen receptor of the invention is CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. It may include a transmembrane domain of a protein selected from the group consisting of. In one example, the transmembrane domain may be a transmembrane domain of CD28, which may be composed of SEQ ID NO: 9 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.
  • the intracellular signal transduction domain which is a component of the chimeric antigen receptor of the present invention, can use any intracellular signaling domain known in the art without limitation.
  • the intracellular signal transduction domain may be a DAP10, CD3 zeta or a combination thereof, but is not limited thereto.
  • the chimeric antigen receptor of the present invention can exhibit a killing effect on cancer cells, particularly cells expressing CEACAM6, with high activity by using DAP10 and CD3 zeta as intracellular signaling domains.
  • DAP10 functions as a co-stimulatory domain and has at least SEQ ID NO: 10 or at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence Having homology, it may be composed of an amino acid sequence exhibiting a function substantially equivalent to that of the amino acid sequence represented by SEQ ID NO: 10; CD3 zeta functions as an NK cell activating domain and has at least SEQ ID NO: 11 or at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence homology. It may be composed of an amino acid sequence showing a function substantially equivalent to the amino acid sequence represented by SEQ ID NO: 11.
  • the intracellular signaling domains of the present invention comprise SEQ ID NO: 10 or an amino acid sequence that exhibits at least 95% homology thereto; Or SEQ ID NO: 11 or an amino acid sequence showing 95% or more homology thereto.
  • the antigen binding domain of the present invention may comprise a signal peptide for domain exposure.
  • the signal peptide may be CD8 alpha, and in the case of CD8 alpha, the signal peptide of the present invention may be encoded by a nucleotide sequence of at least 95% homology with an amino acid encoded by the nucleotide sequence of SEQ ID NO. It may consist of an amino acid sequence.
  • Another aspect of the invention is a polynucleotide capable of encoding (encoding) the anti-CEACAM6 chimeric antigen receptor described above.
  • the polynucleotide encoding the antigen receptor of the present invention changes the amino acid sequence of the antigen receptor expressed from the coding region, due to the degeneracy of the codon or in view of the codons preferred in the organism to express the antigen receptor.
  • Various modifications may be made to the coding region within a range not to be made, and various modifications or modifications may be made within the range not affecting the expression of genes in parts other than the coding region, and such modified genes may also be included in the scope of the present invention. Those skilled in the art will appreciate that included.
  • nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, which are also included in the scope of the present invention.
  • Another embodiment of the present invention is a vector comprising the polynucleotide, a cell transformed with the vector.
  • vectors of the invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like.
  • Suitable vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers and can be prepared in various ways depending on the purpose.
  • a lentiviral vector (Clontech, 632155) may be used.
  • pLVX-AcGFP-C1 which is a vector used in an embodiment of the present invention, is shown in FIG. 1.
  • the antigen receptor of the present invention may be introduced into cells through the vector to transform the cells, wherein the cells are preferably T cells, tumor infiltrating lymphocytes, B cells, NK cells, or NK-T cells, and some In an embodiment, the cells can be T cells or NK cells, or regulatory T cells. In some embodiments, the cells are obtained or prepared from bone marrow, peripheral blood, peripheral blood mononuclear cells, or umbilical cord blood. In some embodiments, the cell is a human cell.
  • chimeric antigen receptors can be transformed into NK cells using a vector comprising a polynucleotide capable of encoding the anti-CEACAM6 chimeric antigen receptor described above.
  • cells transformed by introducing an antigen receptor may recognize CEACAM6 as antigen and bind specifically to CEACAM6, express a CEACAM6-specific chimeric antigen receptor on the cell surface, and CAR-T cells or CAR-NK Intracellular signal transduction domains, such as upon contact with and ligation of tumor antigens, such as cells, can be used to induce activation of immune cells and to induce tumor specific death.
  • CAR-NK cells refer to cells in which the chimeric antigen receptor of the present invention is introduced into NK (Natural killer) cells.
  • the cells have the advantages of anti-cancer specific target therapy, which is an existing advantage of CAR-T therapeutic agents, including the chimeric antigen receptor according to the present invention.
  • it may also have the advantage that it can be used as a general-purpose therapeutic agent through the terminal modification of aptamer fused with CEACAM6, which can bind to the chimeric antigen receptor.
  • another aspect of the present invention provides a method for preventing or treating a cell therapeutic agent comprising the cell and a pharmaceutical composition for preventing or treating cancer comprising the same as an active ingredient or administering the cell to a subject. Way.
  • the cells are, for example, T cells, tumor infiltrating lymphocytes, natural killer cells, natural killer T cells, or progenitor cells, such as hematopoietic stem cells, mesenchymal stromal cells, stems.
  • progenitor cells such as hematopoietic stem cells, mesenchymal stromal cells, stems.
  • Cells, pluripotent stem cells, and embryonic stem cells which can be used in cell therapies such as chemotherapy.
  • the cells may come from a donor or may be cells obtained from a patient.
  • the cells can be used, for example, for regeneration to replace the function of diseased cells.
  • the cells can also be modified to express heterologous genes so that the biological agent can be delivered to a specific microenvironment such as, for example, diseased bone marrow or metastatic deposits.
  • the pharmaceutical composition for preventing or treating cancer of the present invention may further include a conjugate in which a conjugate material is fused to CEACAM6, and the method for preventing or treating the cancer may include administering a conjugate in which the conjugate material is fused to CEACAM6. It may further comprise the step. According to the conjugated material conjugated to CEACAM6, it can specifically bind to a target cell and exhibit excellent anticancer effect.
  • Cancer in the present invention may include any carcinoma known in the art without limitation, specifically may be lung cancer.
  • the cells provided in the present invention are cells expressing a chimeric antigen receptor having an antigen binding domain capable of specifically binding to CEACAM6, wherein the chimeric antigen receptor is for example a response to a target cell of a conventional CAR-T therapeutic agent.
  • On / off can be controlled with aptamers or intermediates fused with CEACAM6, so safety switches that can be very beneficial in subsequent cell therapy, situations where the activity of the treated cells needs to be increased or decreased It includes.
  • T cells expressing a chimeric antigen receptor are provided to a patient, in some circumstances there may be side effects, eg off-target toxicity.
  • the therapeutic cells may act to reduce tumor cells, or tumor size, and may no longer be needed. In such situations, the regulation of CEACAM6 can be used to regulate the treatment cells so that they are no longer active.
  • unit dose refers to physically discrete units suited as a single dosage for a mammal and includes a predetermined amount of pharmaceutical composition calculated to achieve the desired immunogen stimulating effect with the desired diluent.
  • the details of the unit dose of the inoculum are influenced and determined according to the inherent properties of the pharmaceutical composition and the specific immunological effects to be achieved.
  • the effective amount for a specific application may vary depending on factors such as the disease or condition to be treated, the specific composition to be administered, the size of the subject, and / or the severity of the disease or condition. Even without undue experimentation, an effective amount of a particular composition presented herein can be determined empirically.
  • Another aspect of the invention is the use of the prophylaxis or treatment of cancer of cells expressing the chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • a lentiviral vector (Clontech, 632155) was used. Specifically, pLVX-AcGFP-C1 shown in FIG. 1 was used. After removing the Kozak sequence and AcGFP1 to be used in the experiment, XhoI was used as a restriction enzyme. Specific relevant sequences are shown in FIG. 2.
  • Plasmids containing nucleic acids encoding each domain of the chimeric antigen receptor specifically binding to CEACAM6 of the present invention were prepared by the following method.
  • an antigen-binding domain capable of specifically binding to CEACAM6 was intended to derive an anti-CEACAM6 chimeric antibody or antibody fragment thereof, and derivation of ScFv was performed using human CD8 alpha.
  • the antigen binding domain ecto domain
  • the antigen binding domain is characterized by targeting CEACAM6, has a domain consisting of VH-linker-VL, and has a domain consisting of a CD8 alpha region as a signal peptide.
  • the sequence of ScFv was analyzed using the hybridoma antibody gene cloning kit (TB326-Ig-primer Sets, Novagen) in anti CEACAM6 (18B8) -A549 hybridomas.
  • RNA was isolated from anti-CEACAM6 (18B8) -A549 hybridoma, and then first strand cDNA synthesis was performed with the 3 ′ terminal primer of each of heavy and light chains according to the cloning kit manufacturer's manual.
  • CDNA was synthesized, and the sequence of the synthesized cDNA was analyzed on the 'IMGTV-QUEST' website to analyze the IgG sequence and CDR region of each of the heavy and light chains of the 18B8 antibody.
  • Anti 18B8 sequence information from hybridomas was used to request Bioneer (Bioneer), 18B8-ScFv gene recombination, including signal peptide, Myc epitope and CD8 hinge region of human CD8 alpha.
  • Bioneer Bioneer
  • 18B8-ScFv gene recombination including signal peptide, Myc epitope and CD8 hinge region of human CD8 alpha.
  • sequence of restriction enzyme XhoI was added at the 5 ⁇ end of the signal peptide
  • the sequence of restriction enzyme SfiI was included before and after the VH-linker-VL of 18B8, and the restriction enzyme sequence of HindIII toward the 3 ⁇ end of the CD8 hinge region. In addition, it was produced.
  • 18B8-CAR is produced or a signal is generated by replacing an ecto domain with an ScFv of 18B8 in another CAR vector tagged with an existing FC using the sequence of SfiI restriction enzyme in the recombinant sequence.
  • 18B8-CAR was constructed by replacing the ecto domain of the existing CAR with the 18B8-Myc hinge using the XhoI sequence at the 5 'end of the peptide and the HindIII sequence at the 3' end of the CD8 hinge region.
  • the membrane permeation region used a cytoplasmic region at the hinge of the human CD28 gene. Specifically, the restriction enzyme BamH1 sequence was added to the forward primer (SEQ ID NO: 12), and the restriction enzyme EcoRI sequence was added to the reverse primer (SEQ ID NO: 13), and PCR was performed using the above primers on the cDNA of Jurkat cells. Was performed to obtain DNA of the transmembrane region.
  • Dap10 in the signal transduction domain was used as the co-stimulatory domain. Specifically, a sequence of restriction enzyme EcoRI was added to a forward primer (SEQ ID NO: 14), and a restriction enzyme NotI sequence was added to a reverse primer (SEQ ID NO: 15), and the primer was used for cDNA of primary mature NK cells. PCR was performed to produce co-stimulatory domains.
  • CD3 zeta was prepared using two kinds of primers (forward primer: SEQ ID NO: 16, reverse primer: SEQ ID NO: 17) as the NK cell activation domain. Specifically, PCR was performed on the cDNA of Jurkat cells using the primers to prepare an activation domain.
  • Each domain was sequentially linked using respective restriction enzymes, and specific sequence information corresponding to each domain is as follows.
  • the signal peptide is represented by SEQ ID NO: 6 corresponding to the human CD8 alpha region, the heavy chain variable region of SEQ ID NO: 1, the linker is SEQ ID NO: 3, the light chain variable region is SEQ ID NO: 2 Indicated.
  • the linking domain (spacer) is represented by the Myc epitope of SEQ ID NO: 4 or the human Fc of SEQ ID NO: 18, and CD28 as SEQ ID NO: 5 as the transmembrane region.
  • One or more intracellular signal domains represented DAP-10 as SEQ ID NO: 7 as the co-stimulatory domain and CD3 zeta as SEQ ID NO: 8 as the NK cell activation domain.
  • a polynucleotide encoding the anti-CEACAM6 chimeric antigen receptor (18B8-CAR) shown in Example 2 was introduced into a vector, and transformed cells were prepared using the same, and the cell containing the CEACAM6 specific antigen binding domain.
  • a schematic diagram of the chimeric antigen receptor and its expression system of the present invention is shown in FIG. 3.
  • the anti-CEACAM6 chimeric antigen receptor (CEACAM6) of Example 2 using the restriction enzymes of XhoI and XbaI of MCS in the vector, using the vector from which AcGFP was removed in pLVX-AcGFP-C1 of Example 1 Polynucleotides encoding -CAR) were inserted into the vector.
  • the vector containing CEACAM6 (18B8) -CAR was transformed into HEK293T cells with viral packaging vectors (PMDLg / RRE, RSV / REV, VSVG), from which a lentiviral expressing CEACAM6-CAR was obtained. Got it.
  • the lentiviral was concentrated using an ultrafast centrifuge, the lentiviral expressing the concentrated CEACAM6-CAR was infected with HEK293T or Hela cells, and the amount of Myc epitope of CEACAM6-CAR was confirmed by flow cytometry to determine the infection unit. Calculated.
  • NK cells and the amount of lentiviruses were calculated so that the multiplicity of infection (MOI) was 10, and the lentiviruses expressing CEACAM6-CAR were infected with NK cells by spinoculation method (360g, 90min, RT) .
  • Infected NK cells were incubated at 37 ° C., 5% CO 2 for 5 hours, and then changed to fresh culture medium. After 3 days, the cells were treated with 3 ug / ml of puromycin for the selection of infected NK cells. The cells were also treated with puromycin in uninfected NK cells, and the control cells were treated with puromycin. The culture was performed using a culture medium treated with puromycin until annihilation.
  • infected NK cells were proliferated or expanded by exchange with medium without puromycin.
  • Alpha-MEM containing 12.5% fetal calf serum, 12.5% horse serum, 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM polyacid and 200 U / ml recombinant IL- for the proliferation or expansion of the selected NK cells.
  • the experiment was performed using a medium using 2.
  • 18B8-CAR NK92 tagged with the original NK92 and human FC and 18B8-CAR NK92 cells tagged with the Myc-hinge were obtained to obtain the proteins present in the cells, and CD3 zeta (santa cruz; Expression of CEACAM6 was confirmed using an antibody corresponding to sc-166435) using a Western blot, and the results are shown in FIG. 4.
  • the NK cells of the present invention into which the anti-CEACAM6 chimeric antigen receptor was introduced express the anti-CEACAM6 chimeric antigen receptor.
  • the NK cells of the present invention into which the anti-CEACAM6 chimeric antigen receptor was introduced express the anti-CEACAM6 chimeric antigen receptor.
  • CEACAM6-CAR NK92 cells prepared in Example 3 was used to confirm the effect of apoptosis on cancer cells.
  • CEACAM6 antibody (santa Cruz Biotechnology; A549-CEACAM6 Low (low expression) expressing CEACAM6 low and A549-CEACAM6 High (high expression) expressing CEACAM6 high (human lung carcinoma; RPMI1640 (10% FBS)) cell line; sc-59899) was treated at a concentration of 1 ug / 100 ul, and reacted for 30 minutes at 4 ° C. in the dark and stained, and then the expression of CEACAM6 was confirmed using flow cytometry (BD; FacsCantoII). 7 is shown.
  • BD FacsCantoII
  • the A549-CEACAM6 Low and High cells showing a difference in the expression of CEACAM6 were stained with Calcein-AM (5 ug / ml calcein, 37 ° C., 5% CO 2 , 1 hour reaction in the cow).
  • Calcein-AM 5 ug / ml calcein, 37 ° C., 5% CO 2 , 1 hour reaction in the cow.
  • the original NK92 cells (NK92) and NK92 cells expressing CEACAM6-CAR (18B8-CAR) were mixed at a ratio of 1: 1 and 5: 1 (NK92, 18B8-NK92: A549),
  • the cell death effect on the A549 cell line was confirmed by calcein-AM method by reacting at 37 ° C. and 5% CO 2 for 4 hours.
  • the group treated with RPMI1640 (10% FBS) and the group treated with 2% triton X-100 (maximum value) were treated with calcein-stained AU56
  • Apoptotic activity (%) (calcein release value-spontaneous value) / (maximum value-spontaneous value) x 100
  • CEACAM6-CAR NK92 cells were confirmed that the difference in apoptosis effect only for A549-High cells over-express CEACAM6.
  • K562 cells not known to express CEACAM6 were treated with native NK92 cells (NK92) and NK92 cells expressing CEACAM6-CAR (18B8-CAR), and the apoptosis effect was confirmed through the Calcein-AM method.
  • NK92 native NK92 cells
  • NK92 cells expressing CEACAM6-CAR (18B8-CAR) were treated with native NK92 cells (NK92) and NK92 cells expressing CEACAM6-CAR (18B8-CAR)
  • CEACAM6-CAR NK cells of the present invention By confirming cytokine secretion of NK cells, the activity of CEACAM6-CAR NK cells of the present invention was confirmed.
  • Example 5 A549-CEACAM6 Low (low expression) and High (high expression) cells of Example 5 were mixed with the original NK92 and CEACAM6-CAR NK92 in a ratio of 10: 1, 37 °C, 5% CO 2 conditions After 4 hours of reaction in the supernatant secretion of each cytokine (Interleukin 6; IL-6, Interferon-gamma; IFN-r, tumor necrosis factor-alpha; TNF-a) the amount of ELISA (ebioscience ELISA Kit; respectively) 88-7066-88, 88-7316-88 and EB127689) and the results are shown in FIG.
  • cytokine Interleukin 6; IL-6, Interferon-gamma; IFN-r, tumor necrosis factor-alpha; TNF-a
  • ELISA ebioscience ELISA Kit
  • the CEACAM6-CAR NK92 cells of the present invention was confirmed to secrete a greater amount of cytokines in A549-CEACAM6 high expression cells expressing high CEACAM6, through which the CEACAM6-CAR NK92 of the present invention The cells were confirmed to exhibit high activity on CEACAM6.
  • CEACAM6-CAR NK cells of the present invention specifically bind to CEACAM6.
  • siRNA for CEACAM6 (BIONEER; 1029726, 1029729 and 1029733) was transformed into lipofectamin (invitrogen; 11514-015, 18324-020) in an A549 cell line, and then intracellular protein expression was expressed with anti-CEACAM6 antibody. The degree of protein expression was confirmed by Western blot, and the results are shown in FIG. 12.
  • A549 cells were confirmed that the expression of CEACAM6 is reduced by treatment with siRNA for CEACAM6.
  • CEACAM6-CAR NK As shown in FIG. 13, it was confirmed that the apoptosis activity of CEACAM6-CAR NK was reduced on A549 cells in which CEACAM6 expression was reduced by siRNA treatment, through which CEACAM6-CAR NK cells were specifically targeted to CEACAM6, a target antigen. It was confirmed to show activity.
  • Example 3 measure the number of NK92 cells of Example 3 in which CEACAM6 (18B8) -CAR was introduced, dilute 18B8-CAR NK92 cells in culture medium to 100 cells / 10 ml, and subdivide 100 ul into 96 well plates. I laid it. Once every 2 or 3 days, the wells in which monoclonal cells were formed through a microscope were identified, and the cells were continuously cultured and expanded to obtain a single clone of CEACAM6-CAR NK cells.
  • CEACAM6-CAR NK92 cells obtained from a single clone express CEACAM6-CAR with a high transduction rate of 94%.
  • Example 5 Specifically, in order to screen for cancer cells expressing CEACAM6, the antibodies corresponding to CEACAM6 were reacted with various cancer cells to confirm the expression of CEACAM6 in each cancer cell using flow cytometry in the same manner as in Example 5. The results are shown in FIG.
  • A549 (+), AU565, CFPAC-1, SK-BR-3, SKOV-3 and K562 were selected from various cancer cells confirming the expression level of CEACAM6, and then monoclonal CEACAM6-CAR NK cells (18B8 / L1.3-4) apoptosis effect was confirmed using LDH analysis according to the manual of the Calcein-AM method and LDH cytotoxicity kit (Thermo scientific; 88954) in the same manner as in Example 5, the results are shown in Figure 16 It is shown in FIG.
  • the monoclonal CEACAM6-CAR NK92 cells showed no difference in apoptosis effect compared to the original NK92 cells for SK-BR-3, SKOV-3, and K562, which are cancer cells that express little or no CEACAM6.
  • the killing effect of CEACAM6-CAR NK92 cells was confirmed to be specific for CEACAM6 of cancer cells.

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Abstract

La présente invention concerne un récepteur antigénique chimérique (CAR) anti-CEACAM6 se liant de manière spécifique à CEACAM6, et un agent thérapeutique cellulaire le comprenant. Le récepteur antigénique chimérique selon la présente invention est caractérisé en ce qu'il se lie de manière spécifique à CEACAM6 et peut être une cible utile pour un agent anticancéreux ciblé. Le récepteur récepteur antigénique chimérique selon la présente invention peut induire des immunocytes pour se déplacer efficacement vers un tissu tumoral et être utilement employé dans un gène et une thérapie cellulaire présentant des effets anticancéreux hautement efficaces.
PCT/KR2018/000311 2017-01-05 2018-01-05 Récepteur antigénique chimérique anti-ceacam6 se liant de manière spécifique à ceacam6 WO2018128486A1 (fr)

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US20150152181A1 (en) * 2012-05-07 2015-06-04 The Trustees Of Dartmouth College Anti-b7-h6 antibody, fusion proteins, and methods of using the same
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WO2016150899A2 (fr) * 2015-03-23 2016-09-29 Bayer Pharma Aktiengesellschaft Anticorps anti-ceacam6 et leurs utilisations
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US20150152181A1 (en) * 2012-05-07 2015-06-04 The Trustees Of Dartmouth College Anti-b7-h6 antibody, fusion proteins, and methods of using the same
WO2015095895A1 (fr) * 2013-12-20 2015-06-25 Fred Hutchinson Cancer Research Center Molécules effectrices chimériques marquées et leurs récepteurs
WO2016150899A2 (fr) * 2015-03-23 2016-09-29 Bayer Pharma Aktiengesellschaft Anticorps anti-ceacam6 et leurs utilisations
US20160361360A1 (en) * 2015-06-12 2016-12-15 Immunomedics, Inc. Disease therapy with chimeric antigen receptor (car) constructs and t cells (car-t) or nk cells (car-nk) expressing car constructs

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