WO2018128486A1 - Anti-ceacam6 chimeric antigen receptor specifically binding to ceacam6 - Google Patents
Anti-ceacam6 chimeric antigen receptor specifically binding to ceacam6 Download PDFInfo
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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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- antigen receptor
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- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4613—Natural-killer cells [NK or NK-T]
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- A61K39/4631—Chimeric Antigen Receptors [CAR]
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- 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]
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- 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
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- 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
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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.
Abstract
The present invention relates to an anti-CEACAM6 chimeric antigen receptor (CAR) specifically binding to CEACAM6, and a cell therapeutic agent comprising same. The chimeric antigen receptor according to the present invention is characterized by specifically binding to CEACAM6 and can be a useful target for a targeted anticancer agent. Accordingly, the chimeric antigen receptor according to the present invention can induce immunocytes to move effectively to tumor tissue and be usefully employed in a gene and cell therapy exhibiting highly efficient anticancer effects.
Description
본 발명은 CEACAM6에 특이적으로 결합하는 항-CEACAM6 키메릭 항원 수용체(CAR) 및 이를 포함하는 세포 치료제에 관한 것이다.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.
수십 년 동안 암을 치료하는 방법들은 꾸준히 변화하고 발전해왔다. 1800년대에서부터 1900년대까지는 외과적인 수술 (Surgery), 화학요법 (Chemotherapy), 그리고 방사선 요법 (Radiation therapy)과 같은 방법들이 주로 이뤄졌지만, 이들에 대한 한계점들이 드러나기 시작했다. 가장 대표적으로 기존 치료 방법들은 암이 전이되지 않는 초기의 경우에만 효과가 있으며, 이미 전이가 진행된 상태라면 외과적인 수술 후에도 재발의 가능성이 높게 나타났다. 또한, 화학요법은 고형암 (Solid tumor)에서는 치료 효과가 낮고, 암세포 이외의 정상세포의 성장도 함께 억제시키는 부작용 (Side effect)을 야기시키는 것이 보고되고 있다. 이와 같은 문제점을 해소하고자 최근 항암 면역 치료에 대한 연구가 활발히 진행 중이며, 이는 환자 스스로가 암세포와 싸울 수 있도록 면역 반응을 증가시키는 것을 말한다.For decades, methods of treating cancer have steadily changed and developed. From the 1800s to the 1900s, surgeries, chemotherapy, and radiation therapy were mainly performed, but limitations began to emerge. Most typically, existing treatments are effective only in the early stages when cancer does not metastasize, and if the metastasis has already progressed, there is a high possibility of recurrence after surgical operation. In addition, chemotherapy has been reported to cause side effects that have a low therapeutic effect in solid tumors and also inhibit the growth of normal cells other than cancer cells. In order to solve this problem, researches on anti-cancer immunotherapy have been actively conducted in recent years, which means to increase the immune response so that patients themselves can fight cancer cells.
최근에는 면역세포 요법으로 체내의 면역세포를 꺼내서, 강화시키거나 유전공학적으로 변형시켜 다시 넣어주는 세포치료 방식에 대한 관심이 높아지고 있다. 이의 대표적인 예로는 종양 침윤 림프구 (Tumor Infiltrating Lymphocytes, TIL), 키메릭 항원 수용체 (Chimeric Antigen Receptor, CAR), T세포 수용체 (T-Cell Receptor, TCR) 기술 등이 있으며, 특히 유전자 재조합 변형을 이용한 인공 수용체인 CAR를 이용한 연구가 활발하게 이루어지고 있다.Recently, there has been a growing interest in cell therapy methods that take out immune cells in the body with immune cell therapies, enhance them, or genetically modify them. Typical examples thereof include Tumor Infiltrating Lymphocytes (TIL), Chimeric Antigen Receptors (CAR), and T-Cell Receptors (TCR). Research using CAR as a receptor is being actively conducted.
키메라 항원 수용체 (CAR: chimeric antigen receptr)는 T 세포에 항원 특이성을 전달하도록 설계된 인공 수용체이다. 이들은 T 세포를 활성화하고 특이적 면역성을 제공하도록 선택된 항원 특이적 구성요소, 막관통 구성요소, 및 세포 내 구성요소를 포함한다. 키메라 항원 수용체 발현 T 세포는 암 요법을 포함한 다양한 요법에 사용될 수 있다.Chimeric antigen receptors (CARs) 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.
그러나 CAR-T와 같은 치료제는 종양에 대해 효과적이지만, 일부 경우에 이들 치료는 건강한 조직에 부분적으로 비특이적인 공격으로 인한 부작용을 일으켜 왔다. 이를 극복하기 위하여 현재는 3세대 CAR-T에 대한 연구가 진행 중이며, 이는 보조자극신호 역할을 하는 신호도메인 2개와 인공수용체 (Additional engineered receptor)가 추가되어 '암세포 항원 인식 능력'이 높아져 정상세포를 공격하는 부작용을 최소화하려는 것을 특징으로 한다. 그럼에도 불구하고 현재의 CAR-T 기술은 암세포에서 발현하는 오직 하나의 단백질만을 인지하도록 제조되어 개별적 치료제 개발에 너무 많은 비용이 소모된다는 한계점과 더불어, CAR-T가 한번 주입되면 독성을 가진 T 세포가 암세포들이 제거된 후에도 그 기능이 지속되어 독성을 초래한다는 점, 표적 단백질을 나타내는 정상 세포가 있는 경우 이에 대해서도 비 특이적 공격을 유발하여 치명적인 부작용을 야기하는데 이를 되돌릴 수가 없다는 점의 문제점이 CAR-T 세포 치료제의 개발을 저해하고 있다.However, while therapeutics such as CAR-T are effective against tumors, in some cases these therapies have caused side effects due in part to nonspecific attacks on healthy tissues. In order to overcome this problem, the research on the third generation CAR-T is currently underway, and two signal domains and additional engineered receptors, which act as auxiliary stimulation signals, are added to increase the 'cancer cell antigen recognition ability' to increase normal cells. It is characterized by minimizing attack side effects. Nevertheless, current CAR-T technology is designed to recognize only one protein expressed in cancer cells, which is too costly to develop individual therapeutics. 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.
따라서, 상기와 같은 문제점을 해결할 수 있는 새로운 개량 세포 치료제에 대한 연구가 절실히 필요하다.Therefore, there is an urgent need for research on new and improved cell therapies that can solve the above problems.
종래 치료제의 문제점을 해결하기 위하여 연구한 결과, 본 발명자는 CEACAM6에 특이적으로 결합하는 항원 결합 도메인을 이용한 키메릭 항원 수용체를 제조함으로써, CEACAM6을 특이적으로 표적하고 이를 통해 암세포에서 과발현됨으로써 표적 항암제에 유용한 타깃이 될 수 있음을 확인하였다.As a result of studies to solve the problems of the conventional therapeutic agent, 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.
따라서 본 발명의 목적은 CEACAM6에 특이적으로 결합하는 항원 결합 도메인을 이용한 키메릭 항원 수용체, 이를 이용한 세포 치료제 및 이를 이용한 암 치료 방법을 제공하는 것이다.Accordingly, 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.
상기한 과제를 해결하기 위하여 본 발명은 1) 항원 결합 도메인, 2) 막통과 도메인, 및 3) 세포내 신호 전달 도메인을 포함하는 키메릭 항원 수용체 (CAR)로, 상기 항원 결합 도메인은 CEACAM6에 특이적으로 결합하는 도메인인 것인, 키메릭 항원 수용체 (CAR)를 제공한다.In order to solve the above problems, 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.
또한 본 발명은 상기 키메릭 항원 수용체 (CAR)를 코딩하는 폴리뉴클레오티드를 제공한다.The present invention also provides a polynucleotide encoding the chimeric antigen receptor (CAR).
또한 본 발명은 상기 폴리뉴클레오티드를 포함하는 벡터를 제공한다.The present invention also provides a vector comprising the polynucleotide.
또한 본 발명은 상기 벡터로 형질전환된 세포를 제공한다.The present invention also provides a cell transformed with the vector.
또한 본 발명은 상기 세포를 포함하는 세포치료제를 제공한다.In another aspect, the present invention provides a cell therapy agent comprising the cells.
또한 본 발명은 세포를 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for the prevention or treatment of cancer, including the cells as an active ingredient.
또한 본 발명은 상기 세포를 개체에 투여하는 단계를 포함하는 암을 예방 또는 치료하는 방법을 제공한다.The present invention also provides a method for preventing or treating cancer, comprising administering the cells to a subject.
본 발명에 따른 키메릭 항원 수용체는 CEACAM6에 특이적으로 결합하는 것을 특징으로 하며, 표적 항암제에 유용한 타깃이 될 수 있다. 따라서 본 발명에 따른 키메릭 항원 수용체는 종양 조직에 효과적으로 면역세포, 특히 자연살해세포가 이동하도록 유도할 수 있고, 효율 높은 항암 효과를 나타내는 유전자 세포 치료법에 유용하게 이용될 수 있다.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.
도 1은 본 발명에서 사용된 pLVX-AcGFP-C1 벡터를 나타낸 도이다.1 is a diagram showing a pLVX-AcGFP-C1 vector used in the present invention.
도 2는 본 발명에서 사용된 구체적인 관련 서열을 나타낸 도이다.2 is a diagram showing specific related sequences used in the present invention.
도 3은 본 발명의 항-CEACAM6 키메릭 항원 수용체의 예시적 모식도 및 이의 발현 시스템을 나타낸 도이다.Figure 3 is an exemplary schematic diagram of the anti-CEACAM6 chimeric antigen receptor of the present invention and its expression system.
도 4는 CEACAM6-CAR가 도입된 NK92 세포에서 CEACAM6-CAR의 발현유무를 웨스턴블롯을 통해 확인한 결과를 나타낸 도이다.Figure 4 is a diagram showing the results confirmed by Western blot the expression of CEACAM6-CAR in NK92 cells introduced CEACAM6-CAR.
도 5는 CEACAM6-CAR가 도입된 NK92 세포에서 CEACAM6-CAR의 발현유무를 유동세포계수법을 통해 확인한 결과를 나타낸 도이다.5 is a diagram showing the results of confirming the expression of CEACAM6-CAR in the NK92 cells introduced CEACAM6-CAR through flow cytometry.
도 6은 CEACAM6-CAR가 도입된 NK92 세포에서 CEACAM6-CAR의 발현유무를 면역형광법을 통해 확인한 결과를 나타낸 도이다.Figure 6 shows the results of confirming the expression of CEACAM6-CAR in the NK92 cells introduced CEACAM6-CAR through immunofluorescence.
도 7은 A549-CEACAM6 저발현 및 고발현 세포주의 CEACAM6 발현정도를 유동세포분석법을 이용하여 확인한 결과를 나타낸 도이다.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.
도 8은 A549-CEACAM6 저발현 및 고발현 세포주의 CEACAM6 발현정도를 PCR을 이용하여 확인한 결과를 나타낸 도이다.8 is a diagram showing the results of confirming the expression level of CEACAM6 low and high expression cell lines A549-CEACAM6 using PCR.
도 9는 NK92 세포 (NK92) 및 CEACAM6-CAR를 발현하는 NK92 세포 (18B8-CAR)의 A549 세포주에 대한 세포사멸 효과를 칼세인-AM법을 통하여 확인한 결과를 나타낸 도이다.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.
도 10은 NK92 세포 (NK92) 및 CEACAM6-CAR를 발현하는 NK92 세포 (18B8-CAR)의 K562 세포주에 대한 세포 사멸효과를 칼세인-AM법을 통하여 확인한 결과를 나타낸 도이다.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.
도 11은 NK 세포의 사이토카인 분비 변화를 ELISA를 통하여 확인한 결과를 나타낸 도이다.11 is a diagram showing the results confirmed by the ELISA cytokine secretion changes of NK cells.
도 12는 A549 세포주의 CEACAM6 발현정도를 PCR을 통하여 확인한 결과를 나타낸 도이다.12 is a diagram showing the results of confirming the expression level of CEACAM6 by A549 cell line through PCR.
도 13은 CEACAM6에 대한 siRNA를 처리함에 따른 CEACAM6-CAR NK92의 세포사멸 활성을 칼세인-AM법을 통하여 확인한 결과를 나타낸 도이다.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.
도 14는 CEACAM6-CAR가 도입된 NK92 세포의 단일 클론에서 CEACAM6-CAR의 발현 유무를 유동세포분석법을 통해 확인한 결과를 나타낸 도이다.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.
도 15는 유동세포분석법을 이용하여 다양한 암세포에서 CEACAM6의 발현정도를 확인한 결과를 나타낸 도이다.15 is a diagram showing the results of confirming the expression level of CEACAM6 in various cancer cells using flow cytometry.
도 16 및 도 17은 상기 다양한 암세포에 대한 단클론 CEACAM6-CAR NK 세포의 세포사멸 효과를 확인한 결과를 나타낸 도이다.16 and 17 are diagrams showing the results of confirming the apoptosis effect of monoclonal CEACAM6-CAR NK cells to the various cancer cells.
이하, 본 발명에 대하여 상세히 설명한다.EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.
본 발명의 하나의 양태는 1) 항원 결합 도메인, 2) 막통과 도메인, 및 3) 세포내 신호 전달 도메인을 포함하는 키메릭 항원 수용체 (CAR)로, 상기 항원 결합 도메인은 CEACAM6 (Carcinoembryonic antigen-related cell adhesion molecule 6)에 특이적으로 결합하는 도메인인 것인, 키메릭 항원 수용체 (Chimeric Antigen Receptor; CAR)이다.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).
본 발명에 따른 키메릭 항원 수용체는 CEACAM6에 특이적으로 결합하는 것을 특징으로 하므로, CEACAM6에 특이적으로 결합하는 항원 결합 도메인을 포함한다.Since 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.
본 발명의 키메릭 항원 수용체는 표적 항암제에 유용한 타깃이 될 수 있고, 종양 조직에 효과적으로 면역세포가 이동하도록 유도할 수 있으며, 효율 높은 항암 효과를 나타내는 유전자 세포 치료법에 유용하게 이용될 수 있다. 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.
본 발명에서 "항체"는, 면역계 내에서 항원의 자극에 의하여 만들어지는 물질을 의미하는 것으로서 그 종류는 특별히 제한되지 않는다. 또한 본 명세서에서 항체란 항원 결합능을 보유한 항체의 단편, 예컨대, Fab, Fab', F(ab')2 및 Fv 등을 포함하며, 이에 제한되지 않는다.In the present invention, "antibody" means a substance produced by stimulation of an antigen in the immune system, and the kind thereof is not particularly limited. In addition, 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.
본 발명에서 "키메릭 항체" 는, 항체 가변영역 (variable region) 또는 이의 상보성 결정 영역 (complementarity determining region, CDR)이 항체의 나머지 부분과 상이한 동물에서 기원된 항체를 말한다. 이러한 항체는, 예를 들어, 항체 가변 영역은 인간 이외의 동물 (예를 들면, 마우스, 토끼, 가금류 등)에서 유래하고, 항체 불변영역 (constant region)은 인간에서 유래한 항체일 수 있다. 이러한 키메릭 항체는 당업계에 공지된 유전자 재조합 등의 방법으로 제조될 수 있다.As used herein, 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.
본원에서 "중쇄"는, 항원에 대한 특이성을 부여하기 위해 충분한 가변영역의 아미노산 서열을 포함하는 가변영역 도메인 VH 및 3 개의 불변영역 도메인인 CH1, CH2 및 CH3을 포함하는 전체 길이 중쇄 및 이의 단편을 모두 일컫는다.As used herein, “heavy 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.
본원에서 "경쇄"는, 항원에 특이성을 부여하기 위해 충분한 가변영역의 아미노산 서열을 포함하는 가변영역 도메인 VL 및 불변영역 도메인 CL을 포함하는 전체 길이 경쇄 및 이의 단편을 모두 일컫는다.As used herein, “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.
본 발명의 키메릭 항원 수용체를 구성하는 항원 결합 도메인은 주신호가 전달되는 부위로 세포막 외부에 있으며 특정 항원이 있는 타겟 세포의 세포막 리간드(수용체에 결합하여 활성화 하는 물질)를 인지하는 부위를 말한다.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.
본 발명의 막통과 도메인 (Transmembrane domain)은 항원 결합 도메인과 보조자극, 필수 신호전달 도메인을 세포막 사이로 연결하는 부위이며, 세포내 신호 전달 도메인은 항원 결합 도메인의 결합에 의해 면역 세포의 면역반응을 활성화 시키는 부위를 의미한다.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.
본 발명의 키메릭 항원 수용체는 항원 결합 도메인이 CEACAM6에 특이적으로 결합하는 것을 특징으로 하며, 상기 CEACAM6은 Carcinoembryonic antigenrelated cell adhesion molecule 6의 약어이며, 인간에서는 NM_002483 및 NM_002483로 특정될 수 있다.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.
본 발명의 항원 결합 도메인은 CEACAM6에 특이적으로 결합하는 물질로, 항체 또는 항체 단편일 수 있으며, 항체의 단편은 scFv일 수 있다.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.
CEACAM6과 특이적으로 결합할 수 있는 항-CEACAM6 키메릭 항원 수용체에서, 항원 결합 도메인은 중쇄 가변영역, 링커 서열, 경쇄 가변영역으로 이루어질 수 있다. 상기 중쇄 가변영역은 서열번호 1로 표시되는 염기서열에 의하여 암호화되는 아미노산 서열 또는 서열번호 1의 염기서열에 의해 발현되는 아미노산과 기능적 동등물을 암호화할 수 있는 상기 서열번호 1로 표시되는 염기서열과 적어도 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 염기서열에 의하여 암호화되는 아미노산 서열로 이루어질 수 있다.In an anti-CEACAM6 chimeric antigen receptor capable of specifically binding to CEACAM6, 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.
상기 경쇄 가변영역은 서열번호 2로 표시되는 염기서열에 의하여 암호화되는 아미노산 서열 또는 서열번호 2의 염기서열에 의해 발현되는 아미노산과 기능적 동등물을 암호화할 수 있는 상기 서열번호 2로 표시되는 염기서열과 적어도 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 염기서열에 의하여 암호화되는 아미노산 서열로 이루어질 수 있다.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.
서열번호 1의 염기서열 또는 서열번호 2의 염기서열과 각각 95% 이상의 서열 상동성을 갖는 염기서열에 의해 암호화되는 아미노산 서열은 서열번호 1 및 2의 염기서열에 의하여 암호화되는 아미노산 서열과 실질적으로 동질의 생리활성을 나타낼 수 있다.The 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.
본 발명의 항체 또는 항체의 단편은 링커를 추가로 포함할 수 있으며 링커를 통해 중쇄 가변영역 및 경쇄 가변영역이 상호 연결될 수 있다. 링커는 중쇄 가변 영역 및 경쇄 가변영역을 연결하여 VH-링커-VL 도메인을 이룰 수 있는 성분이라면 제한없이 사용할 수 있으며, 바람직하게는 서열번호 3 또는 이와 95% 이상의 상동성을 나타내는 염기서열에 의해 암호화되는 아미노산 서열로 이루어질 수 있다.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.
본 발명의 항-CEACAM6 키메라 항원 수용체의 항원 결합 도메인은 힌지 영역, 스페이서 영역 또는 이들의 조합에 의하여 막통과 도메인에 연결될 수 있다. 본 발명의 힌지 영역 또는 스페이서 영역은 Myc 에피토프를 포함할 수 있으며, 본 발명의 힌지 영역 또는 스페이서 영역인 Myc 에피토프 영역은 서열번호 4 또는 이와 95% 이상의 상동성을 나타내는 염기 서열에 의해 코딩된 아미노산 서열로 이루어지는 것일 수 있다.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.
본 발명의 키메릭 항원 수용체의 일 구성요소인 막 통과 도메인은 CD28, CD3 엡실론, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 및 CD154로 이루어진 군으로부터 선택되는 단백질의 막통과 도메인을 포함하는 것일 수 있다. 일례로, 상기 막 통과 도메인은 CD28의 막통과 도메인일 수 있고, 이는 서열번호 9 또는 이와 95% 이상의 상동성을 나타내는 아미노산 서열로 이루어진 것일 수 있으나, 이에 제한되는 것은 아니다.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.
또한 본 발명의 키메릭 항원 수용체의 일 구성요소인 세포 내 신호 전달 도메인은 당분야에 알려진 세포 내 신호전달 도메인을 제한없이 사용할 수 있다. 본 발명의 일 구현예로서, 상기 세포 내 신호 전달 도메인은 DAP10, CD3 제타 (zeta) 또는 이들의 조합일 수 있으며, 이에 한정되는 것은 아니다.In addition, 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. In one embodiment of the present invention, the intracellular signal transduction domain may be a DAP10, CD3 zeta or a combination thereof, but is not limited thereto.
본 발명의 키메릭 항원 수용체는 세포 내 신호전달 도메인로 DAP10 및 CD3 zeta를 이용함으로써 높은 활성으로 암 세포, 특히 CEACAM6를 발현하는 세포에 대한 사멸효과를 나타낼 수 있다. 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.
이 경우, DAP10은 공동-자극성 (Co-stimulatory) 도메인으로 기능하며, 서열번호 10 또는 이와 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 10으로 표시되는 아미노산 서열과 실질적으로 동등한 기능을 나타내는 아미노산 서열로 이루어질 수 있고; CD3 제타 (zeta)는 NK 세포 활성화 도메인으로 기능하며, 서열번호 11 또는 이와 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 11로 표시되는 아미노산 서열과 실질적으로 동등한 기능을 나타내는 아미노산 서열로 이루어 질 수 있다. 따라서 본 발명의 세포내 신호전달 도메인은 서열번호 10 또는 이와 95% 이상의 상동성을 나타내는 아미노산 서열; 또는 서열번호 11 또는 이와 95% 이상의 상동성을 나타내는 아미노산 서열로 이루어지는 것일 수 있다.In this case, 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. Thus, 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.
또한 본 발명의 항원 결합 도메인은 도메인 노출을 위한 신호 펩타이드를 포함할 수 있다. 상기 신호 펩타이드는 CD8 알파일 수 있으며, CD8 알파인 경우, 본 발명의 신호 펩타이드는 서열번호 6의 염기서열로 암호화되는 아미노산 또는 서열번호 6의 염기서열과 95% 이상의 상동성을 염기서열에 의해 암호화되는 아미노산 서열로 이루어진 것일 수 있다.In addition, 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.
또한 본 발명의 다른 하나의 양태는 상기 기술한 항-CEACAM6 키메릭 항원 수용체를 코딩 (암호화)할 수 있는 폴리뉴클레오티드이다. 본 발명의 항원 수용체를 암호화하는 폴리뉴클레오티드는 코돈의 축퇴성 (degeneracy)으로 인하여 또는 상기 항원 수용체를 발현시키고자 하는 생물에서 선호되는 코돈을 고려하여, 코딩영역으로부터 발현되는 항원 수용체의 아미노산 서열을 변화시키지 않는 범위 내에서 코딩영역에 다양한 변형이 이루어질 수 있고, 코딩영역을 제외한 부분에서도 유전자의 발현에 영향을 미치지 않는 범위 내에서 다양한 변형 또는 수식이 이루어질 수 있으며, 그러한 변형 유전자 역시 본 발명의 범위에 포함됨을 당업자는 잘 이해할 수 있을 것이다. 즉, 본 발명의 폴리뉴클레오티드는 이와 동등한 활성을 갖는 단백질을 코딩하는 한, 하나 이상의 핵산 염기가 치환, 결실, 삽입 또는 이들의 조합에 의해 변이될 수 있으며, 이들 또한 본 발명의 범위에 포함된다.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. That is, as long as the polynucleotide of the present invention encodes a protein having equivalent activity, one or more 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.
또한 본 발명의 다른 하나의 양태는 상기 폴리뉴클레오티드를 포함하는 벡터, 상기 벡터로 형질전환된 세포이다.Another embodiment of the present invention is a vector comprising the polynucleotide, a cell transformed with the vector.
본 발명에서 사용되는 벡터는 당 분야에 공지된 벡터를 다양하게 사용할 수 있고, 상기 항원 수용체를 생산하고자 하는 숙주세포의 종류에 따라 프로모터 (promoter), 종결자 (terminator), 인핸서 (enhancer) 등과 같은 발현조절 서열, 막 표적화 또는 분비를 위한 서열 등을 적절히 선택하고 목적에 따라 다양하게 조합할 수 있다. 본 발명의 벡터는 플라스미드 벡터, 코즈미드 벡터, 박테리오 파아지 벡터 및 바이러스 벡터 등을 포함하나 이에 제한되지 않는다. 적합한 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 같은 발현 조절 엘리먼트 외에도 막 표적화 또는 분비를 위한 시그널 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다.As the vector used in the present invention, various vectors known in the art may be used, and a promoter, a terminator, an enhancer, or the like may be used according to the type of the host cell to produce the antigen receptor. Expression control sequences, sequences for membrane targeting or secretion, and the like may be appropriately selected and combined in various ways depending on the purpose. 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.
본 발명에서는 바람직한 일 예로서, 렌티-바이러스용 벡터 (Clontech사, 632155)를 사용할 수 있고, 구체적으로 본 발명의 실시예에서 사용되는 벡터인 pLVX-AcGFP-C1를 도 1에 나타내었다.In the present invention, as a preferred example, a lentiviral vector (Clontech, 632155) may be used. Specifically, pLVX-AcGFP-C1, which is a vector used in an embodiment of the present invention, is shown in FIG. 1.
또한 본 발명의 항원 수용체를 상기 벡터를 통해 세포에 도입하여 세포를 형질전환 시킬 수 있고, 상기 세포는 T 세포, 종양 침윤 림프구, B 세포, NK 세포, 또는 NK-T 세포인 것이 바람직하며, 일부 실시양태에서, 세포는 T 세포 또는 NK 세포, 또는 조절 T 세포일 수 있다. 일부 실시양태에서, 상기 세포는 골수, 말초혈액, 말초혈액단핵세포 또는 제대혈로부터 얻거나 제조된다. 일부 실시양태에서, 세포는 인간 세포이다.In addition, 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.
본 발명의 일 예로서, 상기 기술한 항-CEACAM6 키메릭 항원 수용체를 코딩할 수 있는 폴리뉴클레오티드를 포함하는 벡터를 이용하여 키메릭 항원 수용체를 NK 세포에 형질전환 시킬 수 있다.As an example of the present invention, 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.
상기와 같이 항원 수용체가 도입되어 형질전환된 세포는 CEACAM6을 항원으로 인식하여 CEACAM6 특이적으로 결합할 수 있으며, 세포 표면에 CEACAM6 특이적인 키메릭 항원 수용체를 발현하고, CAR-T 세포 또는 CAR-NK 세포와 같은 활성, 예컨대 종양 항원과 접촉 및 결찰 시 세포내 신호 전달도메인을 통해 면역 세포의 활성화를 유도하고 종양 특이적 사멸을 유도할 수 있도록 할 수 있다.As 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.
본 발명에 있어, 특히 CAR-NK 세포는 NK (Natural killer) 세포에 본 발명의 키메릭 항원 수용체가 도입된 세포를 의미한다. 상기 세포는 CAR-T 치료제의 기존 장점인 항암 특이적 표적 치료의 장점을 가지면서, 본 발명에 따른 키메릭 항원 수용체를 포함하여 치료 반응을 on/off 할 수 있는 스위치 기능을 통해 기존의 문제점인 독성 문제를 해결할 뿐만 아니라, 키메릭 항원 수용체와 결합될 수 있는 CEACAM6과 융합된 압타머의 말단 변형을 통해 범용의 치료제로 사용할 수 있다는 장점 또한 가질 수 있다.In the present invention, in particular, 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. In addition to solving the toxicity problem, 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.
따라서 본 발명의 다른 하나의 양태는 상기 세포를 포함하는 세포 치료제 및 이를 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물 또는 상기 세포를 개체에 투여하는 단계를 포함하는 암을 예방 또는 치료하는 방법이다. Accordingly, 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.
본 발명에 있어 상기 세포는, 예를 들어, T 세포, 종양 침윤 림프구, 자연 살해 세포, 자연 살해 T 세포, 또는 전구 세포 (progenitor cell), 예를 들어, 조혈 줄기세포, 중간엽 간질 세포, 줄기세포, 전분화능 줄기세포, 및 배아 줄기세포일 수 있으며, 이들은 항암치료 등의 세포 요법에 사용될 수 있다. 세포는 공여자로부터 올 수 있거나, 환자로부터 얻어진 세포가 될 수 있다. 세포는 예를 들어, 병에 걸린 세포의 기능을 대체하는 재생에 사용될 수 있다. 세포는 또한 이종 유전자를 발현하도록 변형되어 생물학 제제가, 예를 들어, 병에 걸린 골수 또는 전이성 침착물과 같은 특정 미세 환경으로 전달될 수 있다.In the present invention, 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. 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.
또한, 본 발명의 상기 암의 예방 또는 치료용 약학적 조성물은 CEACAM6에 접합 물질이 융합된 접합체를 추가적으로 포함할 수 있고, 상기 암을 예방 또는 치료하는 방법은 CEACAM6에 접합 물질이 융합된 접합체를 투여하는 단계를 더 포함할 수 있다. 상기 CEACAM6에 접합된 접합 물질에 따라 표적하는 세포에 특이적으로 결합하여 우수한 항암 효과를 나타낼 수 있다.In addition, 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.
본 발명에 있어 암은 당 분야 알려진 모든 암종을 제한없이 포함할 수 있으며, 구체적으로 폐암일 수 있다.Cancer in the present invention may include any carcinoma known in the art without limitation, specifically may be lung cancer.
특히 본 발명에서 제공된 세포는 CEACAM6에 특이적으로 결합할 수 있는 항원 결합 도메인을 가진 키메릭 항원 수용체를 발현하는 세포로, 상기 키메릭 항원 수용체가 예컨대 통상적인 CAR-T 치료제의 표적 세포에 대한 반응 개시(on)/중지(off)를 CEACAM6과 융합된 압타머 또는 중간 매개체와 함께 조절할 수 있으므로, 후속 세포 요법, 치료 세포의 활성이 증가 또는 감소될 필요가 있는 상황에서 매우 유익할 수 있는 안전성 스위치를 포함한다. 예를 들어, 키메라 항원 수용체를 발현하는 T 세포가 환자에게 제공되는 경우, 어떤 상황에서는, 부작용, 예를 들어 탈표적 (off-target) 독성이 있을 수 있다. 또는, 예를 들어, 치료 세포가 종양 세포, 또는 종양 크기를 감소시키는 작용을 할 수 있고, 더 이상 필요하지 않을 수 있다. 이러한 상황에서는, CEACAM6의 조절을 통해 치료 세포가 더 이상 활성화되지 않도록 조절할 수 있다.In particular, 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. For example, where T cells expressing a chimeric antigen receptor are provided to a patient, in some circumstances there may be side effects, eg off-target toxicity. Or, for example, 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.
본 발명에 있어서, 용어 "단위 용량"은 포유동물을 위한 단일 투여량으로서 적합한 물리적으로 개별 단위를 나타내며, 원하는 희석제와 함께 원하는 면역원 자극 효과를 얻도록 계산된 예정된 양의 약학 조성물을 포함한다. 접종물의 단위 용량에 대한 구체적인 내용은 약학 조성물의 고유한 특성 및 달성될 구체적인 면역학적 효과에 의해서 영향을 받으며, 그에 따라 결정된다.In the present invention, the term “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.
구체적인 적용을 위한 유효량은 치료될 질환 또는 병태, 투여될 구체적인 조성물, 피험체의 크기, 및/또는 질환 또는 병태의 중증도와 같은 인자에 따라 달라질 수 있다. 과도한 실험 없이도 본원에 제시된 특정 조성물의 유효량은 경험적으로 결정될 수 있다.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.
본 발명의 다른 하나의 양태는, 상기 키메릭 항원 수용체 (CAR)를 발현하는 세포의 암의 예방 또는 치료 용도이다.Another aspect of the invention is the use of the prophylaxis or treatment of cancer of cells expressing the chimeric antigen receptor (CAR).
키메릭 항원 수용체, 및 이의 암 예방 또는 치료 용도에 대해서는 상기 설명한 바와 같다.Chimeric antigen receptors, and their cancer prophylactic or therapeutic uses, are as described above.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예 1. 벡터 백본Example 1 Vector Backbone
본 발명에 사용될 벡터로는 렌티-바이러스용 벡터 (Clontech사, 632155)를 사용하였으며, 구체적으로 도 1에 나타낸 pLVX-AcGFP-C1을 이용하였다. 실험에 사용하고자 하는 Kozak 서열과 AcGFP1을 삭제한 후, XhoI을 제한효소로 사용하였다. 구체적인 관련 서열을 도 2에 나타내었다.As a vector to be used in the present invention, 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.
실시예 2. 항-CEACAM6 키메릭 항원 수용체 도출Example 2. Derivation of anti-CEACAM6 chimeric antigen receptor
본 발명의 CEACAM6에 특이적으로 결합하는 키메릭 항원 수용체의 각 도메인을 코딩하는 핵산을 포함하는 플라스미드를 다음과 같은 방법으로 제조하였다.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.
먼저, CEACAM6에 특이적으로 결합할 수 있는 항원결합 도메인으로서, 항-CEACAM6 키메릭 항체 또는 이의 항체 단편을 도출하고자 하였으며, ScFv의 서열 도출은 인간 CD8 알파를 이용하여 수행하였다. 항원 결합 도메인을 포함하는 키메릭 항원 수용체에서 항원 결합도메인 (엑토 도메인)은 CEACAM6을 표적으로 하는 것을 특징으로 하며, VH-링커-VL로 구성되는 도메인을 갖고 CD8 알파 영역을 신호 펩타이드로 하여 도메인이 도출된다.First, 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. In a chimeric antigen receptor comprising an antigen binding domain, the antigen binding domain (ecto 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. Derived.
구체적으로, ScFv의 서열은 항 CEACAM6 (18B8)-A549 하이브리도마 (hybridoma)에서 하이브리도마 항체 유전자 클로닝 키트 (TB326-Ig-primer Sets, Novagen)을 사용하여 분석하였다. 먼저 항 CEACAM6 (18B8)-A549 하이브리도마에서 RNA를 분리한 후, 상기 클로닝 키트 제조사의 메뉴얼에 따라 중쇄 (heavy chain)과 경쇄 (light chain) 각각의 3` 말단 프라이머와 함께 First strand cDNA 합성 기법으로 cDNA를 합성하고, 합성된 cDNA의 서열을 ‘IMGTV-QUEST’ 웹사이트에서 분석하여 18B8 항체의 중쇄 및 경쇄 각각의 IgG 서열 및 CDR 부위를 분석하였다. 하이브리도마로부터 얻은 항 18B8 서열 정보를 이용하여 인간 CD8 알파의 신호 펩타이드 (signal peptide), Myc 에피토프 및 CD8 힌지 영역을 포함한 18B8-ScFv 유전자 재조합을 바이오니아 (Bioneer, 대전)에 의뢰하였다. 재조합 시 신호 펩타이드의 5` 말단에 제한효소 XhoI의 서열을 추가하고, 18B8의 VH-링커-VL 앞뒤로 제한효소 SfiI의 서열을 포함시켰으며, CD8 힌지 영역의 3` 말단 쪽으로 HindIII의 제한효소 서열을 추가하여 제작하였다. 상기 재조합된 서열에서 SfiI 제한효소의 서열을 이용하여 기존의 FC가 태그 (tag)되어 있는 다른 CAR 벡터에서 엑토 도메인 (Ecto Domain)을 18B8의 ScFv로 치환하는 방식으로 18B8-CAR를 제작하거나 또는 신호 펩타이드 5` 말단의 XhoI 서열과 CD8 힌지 영역의 3` 말단의 HindIII 서열을 이용하여 기존 CAR의 엑토 도메인을 18B8-Myc 힌지로 치환하는 방식으로 18B8-CAR를 제작하였다. Specifically, the sequence of ScFv was analyzed using the hybridoma antibody gene cloning kit (TB326-Ig-primer Sets, Novagen) in anti CEACAM6 (18B8) -A549 hybridomas. First, 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. When recombination, the 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.
막 투과 영역은 인간 CD28 유전자의 힌지에서 세포질 영역 (cytoplasmic region)을 이용하였다. 구체적으로, 정방향 프라이머 (서열번호 12)에 제한효소 BamH1의 서열을 추가하고, 역방향 프라이머 (서열번호 13)에 제한효소 EcoRI 서열을 추가하여 제작하였으며, Jurkat 세포의 cDNA에 위의 프라이머를 이용하여 PCR을 수행하여 막투과 영역의 DNA를 얻었다.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.
신호 전달 도메인 중 Dap10을 공동-자극성 도메인으로 사용하였다. 구체적으로, 정방향 프라이머 (서열번호 14)에 제한효소 EcoRI의 서열을 추가하고, 역방향 프라이머 (서열번호 15)에 제한효소 NotI서열을 추가하여 제작하였으며, primary mature NK 세포의 cDNA에 상기 프라이머를 이용하여 PCR을 진행하여 공동-자극성 도메인을 제작하였다.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.
NK 세포 활성화 도메인으로 CD3 제타를 두 종류의 프라이머 (정방향 프라이머: 서열번호 16, 역방향 프라이머: 서열번호 17)를 이용하여 제작하였다. 구체적으로, Jurkat 세포의 cDNA에 상기 프라이머를 이용하여 PCR을 진행하여 활성화 도메인을 제작하였다. 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.
상기 각 도메인을 각각의 제한효소를 이용하여 순차적으로 연결하였고, 각 도메인에 대응하는 구체적인 서열 정보는 다음과 같다.Each domain was sequentially linked using respective restriction enzymes, and specific sequence information corresponding to each domain is as follows.
[표 1]TABLE 1
구체적으로, 신호 펩타이드는 인간 CD8 알파 영역에 해당하는 서열번호 6으로 나타내었으며, 표적 특이적 인식 도메인 중 중쇄 가변 영역은 서열번호 1로, 링커는 서열번호 3으로, 경쇄 가변영역은 서열번호 2로 나타내었다. 연결 도메인 (스페이서)은 서열번호 4의 Myc 에피토프 또는 서열번호 18의 인간 Fc로 나타내었고, 막투과 영역으로 CD28을 서열번호 5로 나타내었다. 하나 이상의 세포내 신호 도메인은 공동 자극성 도메인으로 DAP-10을 서열번호 7에, NK 세포 활성화 도메인으로 CD3 제타를 서열번호 8에 나타내었다.Specifically, 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.
실시예 3. 항-CEACAM6 키메릭 항원 수용체가 도입된 세포의 제조Example 3. Preparation of cells into which anti-CEACAM6 chimeric antigen receptor has been introduced
상기 실시예 2에 나타낸 항-CEACAM6 키메릭 항원 수용체 (18B8-CAR)를 코딩하는 폴리뉴클레오티드를 벡터에 도입하고, 이를 이용하여 형질전환된 세포를 제조하였며, CEACAM6 특이적 항원결합 도메인을 포함하는 본 발명의 키메릭 항원 수용체 및 이의 발현시스템의 모식도를 도 3에 나타내었다.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.
먼저, 상기 실시예 1의 pLVX-AcGFP-C1에서 AcGFP가 제거된 벡터를 기본벡터로 하여 상기 벡터 내 MCS의 XhoI과 XbaI의 제한효소를 이용하여 실시예 2의 항-CEACAM6 키메릭 항원 수용체 (CEACAM6-CAR)를 코딩하는 폴리뉴클레오티드를 벡터에 삽입하였다.First, 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.
그 다음, CEACAM6 (18B8)-CAR를 포함하는 벡터를 viral packaging vector (PMDLg/RRE, RSV/REV, VSVG)와 함께 HEK293T 세포에 형질전환시키고, 그로부터 CEACAM6-CAR를 발현하는 렌티바이러스 (Lentivirus)를 얻었다. 렌티바이러스를 초고속원심분리기를 이용하여 농축시키고, 상기 농축된 CEACAM6-CAR를 발현하는 렌티바이러스를 HEK293T 또는 Hela 세포에 감염시킨 뒤, CEACAM6-CAR의 Myc 에피토프 양을 유동세포계수법으로 확인하여 Infection Unit을 계산하였다. 감염다중도 (Multiplicity of infection, MOI)가 10이 되도록 NK 세포 수 및 렌티바이러스의 양을 계산하고, CEACAM6-CAR를 발현하는 렌티바이러스를 NK 세포에 spinoculation 방법 (360g, 90min, RT)으로 감염시켰다. 감염된 NK 세포를 37℃, 5% CO2 조건에서 5시간 동안 배양한 후, 신선한 배양배지로 갈아주었다. 3일 뒤 감염된 NK 세포의 선별을 위하여 3 ug/ml 농도의 퓨로마이신 (puromycin)을 처리하여 배양을 진행하였으며, 대조군으로 감염되지 않은 NK 세포에도 퓨로마이신을 처리하고, 대조군 세포가 퓨로마이신에 의해 전멸할 때까지 퓨로마이신이 처리된 배양배지를 이용하여 배양을 진행하였다. 대조군 세포가 전멸한 시점에서 감염된 NK 세포를 퓨로마이신이 없는 배지로 교환하여 증식 또는 확장시켰다. 상기 선별된 NK 세포의 증식 또는 확장을 위하여 Alpha-MEM 함유 12.5% 우태아 혈청, 12.5% 말혈청, 0.2 mM 이노시톨, 0.1 mM 2-머캅토에탄올, 0.02 mM 폴릭산 및 200 U/ml 재조합 IL-2을 이용한 배지를 이용하여 실험을 수행하였다. Next, 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. The number of 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. At the time point at which control cells were annihilated, 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.
실시예 4. 항-CEACAM6 키메릭 항원 수용체가 도입된 NK 세포에서 CAR의 발현확인Example 4 Confirmation of CAR Expression in NK Cells Incorporated with Anti-CEACAM6 Chimeric Antigen Receptor
본 발명의 항-CEACAM6 키메릭 항원 수용체가 도입된 상기 실시예 3의 NK 세포에서 CAR의 발현 유무를 확인하였다.The expression of CAR was confirmed in the NK cells of Example 3 in which the anti-CEACAM6 chimeric antigen receptor of the present invention was introduced.
구체적으로, 본래의 NK92와 인간 FC가 태그된 18B8-CAR NK92 및 Myc-힌지가 태그된 18B8-CAR NK92 세포를 수득하여 세포 내에 존재하는 단백질을 얻고, CAR의 활성도메인인 CD3 제타 (santa cruz; sc-166435)에 대응하는 항체를 이용하여 CEACAM6의 발현을 웨스턴블롯을 이용하여 확인하였으며, 그 결과를 도 4에 나타내었다. Specifically, 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.
도 4에 나타낸 바와 같이, 항-CEACAM6 키메릭 항원 수용체가 도입된 본 발명의 NK 세포는 항-CEACAM6 키메릭 항원 수용체를 발현하고 있음을 확인하였다. As shown in FIG. 4, it was confirmed that the NK cells of the present invention into which the anti-CEACAM6 chimeric antigen receptor was introduced express the anti-CEACAM6 chimeric antigen receptor.
또한, 본래의 NK92와 18B8-CAR NK92에 각 태그 단백질 (FC 및 Myc)에 해당하는 항체 (FC;eBioscience;12-4988-82, Myc;CTS;9B11)를 1 ug/100 ul의 농도로 4℃, 암소에서 30분 동안 반응시킨 후 유동세포계수법 (BD;FacsCantoII)을 이용하여 CEACAM6-CAR의 발현을 확인하였고, 면역형광법 (Immunofluorescent, IF) 또한 상기 Fc 항체 또는 Myc 항체를 이용하여 세포를 염색하고, DAPI를 이용하여 핵을 염색한 후 confocal을 통하여 CEACAM6-CAR의 발현을 확인하였으며, 그 결과를 각각 도 5 및 도 6에 나타내었다.In addition, antibodies (FC; eBioscience; 12-4988-82, Myc; CTS; 9B11) corresponding to the respective tagged proteins (FC and Myc) in the original NK92 and 18B8-CAR NK92 were added at a concentration of 1 ug / 100 ul. After 30 minutes of reaction in the dark, the expression of CEACAM6-CAR was confirmed using flow cytometry (BD; FacsCantoII), and immunofluorescent (Immunofluorescent, IF) stained cells using the Fc antibody or Myc antibody. After staining the nucleus using DAPI, the expression of CEACAM6-CAR was confirmed through confocal, and the results are shown in FIGS. 5 and 6, respectively.
도 5 및 도 6에 나타낸 바와 같이, 항-CEACAM6 키메릭 항원 수용체가 도입된 본 발명의 NK 세포는 항-CEACAM6 키메릭 항원 수용체를 발현하고 있음을 확인하였다. As shown in FIG. 5 and FIG. 6, it was confirmed that the NK cells of the present invention into which the anti-CEACAM6 chimeric antigen receptor was introduced express the anti-CEACAM6 chimeric antigen receptor.
실시예 5. CEACAM6-CAR NK 세포의 세포사멸 효과 확인Example 5. Confirmation of Apoptosis Effect of CEACAM6-CAR NK Cells
실시예 3에서 제조한 CEACAM6-CAR NK92 세포를 이용하여 암세포에 대한 세포사멸 효과를 확인하였다.CEACAM6-CAR NK92 cells prepared in Example 3 was used to confirm the effect of apoptosis on cancer cells.
먼저, CEACAM6를 낮게 발현하는 A549-CEACAM6 Low (저발현)와 CEACAM6를 높게 발현하는 A549-CEACAM6 High (고발현) (인간 폐 암종; RPMI1640 (10% FBS)) 세포주에 CEACAM6 항체 (santa Cruz Biotechnology; sc-59899)를 1 ug/100 ul의 농도로 처리하고, 4℃, 암소에서 30분 동안 반응시켜 염색한 후 유동세포계수법 (BD; FacsCantoII)를 이용하여 CEACAM6의 발현을 확인하였으며, 그 결과를 도 7에 나타내었다. 또한, CEACAM6의 DNA 서열에 대응하는 프라이머를 이용하여 A549의 CEACAM6 RNA 레벨을 PCR을 통하여 확인하였으며, 그 결과를 도 8에 나타내었다. First, 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. In addition, using a primer corresponding to the DNA sequence of CEACAM6, the CEACAM6 RNA level of A549 was confirmed by PCR, and the results are shown in FIG. 8.
도 7 및 도 8에서 나타낸 바와 같이, A549-CEACAM6 Low 세포는 CEACAM6를 발현하지 않으나, A549-CEACAM6 High 세포는 CEACAM6를 발현하고 있음을 확인하였다.As shown in FIG. 7 and FIG. 8, it was confirmed that A549-CEACAM6 Low cells did not express CEACAM6, but A549-CEACAM6 High cells expressed CEACAM6.
그 다음, CEACAM6의 발현에 차이를 보이는 상기 A549-CEACAM6 Low 및 High 세포를 칼세인 (Calcein)-AM으로 염색하고 (5 ug/ml calcein, 37℃, 5% CO2, 암소에서 1시간 반응), 염색된 A549 세포주에 본래의 NK92 세포 (NK92) 및 CEACAM6-CAR를 발현하는 NK92 세포 (18B8-CAR)를 1:1과 5:1 (NK92, 18B8-NK92: A549)의 비율로 혼합하고, 37℃, 5% CO2 조건에서 4시간 동안 반응시켜 A549 세포주에 대한 세포 사멸효과를 칼세인-AM법을 통하여 확인하였다. 대조군으로는 칼세인이 염색된 AU565에 RPMI1640 (10% FBS)만 처리한 군 (spontaneous value)과 2% triton X-100 (maximum value)을 처리한 군을 사용하였으며, 세포사멸 활성 (killin effect)은 아래와 같은 방법으로 계산하였다. Then, 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). To the stained A549 cell line, 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. As a control group, the group treated with RPMI1640 (10% FBS) and the group treated with 2% triton X-100 (maximum value) were treated with calcein-stained AU565. Was calculated by the following method.
세포사멸 활성 (%) = (조건에 따른 calcein release value-spontaneous value) / (maximum value-spontaneous value) x 100 Apoptotic activity (%) = (calcein release value-spontaneous value) / (maximum value-spontaneous value) x 100
그 결과를 도 9에 나타내었다.The results are shown in FIG.
도 9에 나타낸 바와 같이, CEACAM6-CAR NK92 세포는 CEACAM6가 과발현되는 A549-High 세포에 대해서만 세포사멸 효과에 차이가 남을 확인하였다. As shown in Figure 9, CEACAM6-CAR NK92 cells were confirmed that the difference in apoptosis effect only for A549-High cells over-express CEACAM6.
또한, CEACAM6 발현하지 않는 세포에 대한 본 발명의 CEACAM6-CAR NK92의 세포사멸 효과를 확인하였다.In addition, the apoptosis effect of CEACAM6-CAR NK92 of the present invention on the cells that do not express CEACAM6 was confirmed.
구체적으로, CEACAM6 발현하지 않는 것으로 알려진 K562 세포에 본래의 NK92 세포 (NK92) 및 CEACAM6-CAR를 발현하는 NK92 세포 (18B8-CAR)를 처리하고 Calcein-AM법을 통하여 세포사멸 효과를 확인하였으며, 그 결과를 도 10에 나타내었다.Specifically, 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. The results are shown in FIG.
도 10에 나타낸 바와 같이, CEACAM6 발현하지 않는 세포에 대한 NK92 세포 및 CEACAM6-CAR NK92 세포의 활성은 동등함을 확인하였다. As shown in Figure 10, it was confirmed that the activity of NK92 cells and CEACAM6-CAR NK92 cells to the cells that do not express CEACAM6 is equivalent.
실시예 6. CEACAM6-CAR NK92의 세포활성 확인Example 6 Confirmation of Cell Activity of CEACAM6-CAR NK92
NK 세포의 사이토카인 분비를 확인함으로써 본 발명의 CEACAM6-CAR NK세포의 활성을 확인하였다. By confirming cytokine secretion of NK cells, the activity of CEACAM6-CAR NK cells of the present invention was confirmed.
구체적으로, 상기 실시예 5의 A549-CEACAM6 Low (저발현) 및 High (고발현) 세포에 본래의 NK92 및 CEACAM6-CAR NK92를 10:1의 비율로 혼합하고, 37℃, 5% CO2 조건에서 4시간 동안 반응시킨 후 상등액에 분비된 각 사이토카인 (Interleukin 6; IL-6, Interferon-gamma; IFN-r, tumor necrosis factor-alpha; TNF-a)의 양을 ELISA (ebioscience ELISA Kit; 각각 88-7066-88, 88-7316-88 및 EB127689)를 통하여 확인하였으며, 그 결과를 도 11에 나타내었다. Specifically, 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 ℃, 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.
도 11에 나타낸 바와 같이, 본 발명의 CEACAM6-CAR NK92 세포는 CEACAM6가 높게 발현되는 A549-CEACAM6 고발현 세포에서 더 많은 양의 사이토카인을 분비하는 것을 확인하였으며, 이를 통해 본 발명의 CEACAM6-CAR NK92 세포는 CEACAM6에 높은 활성을 나타냄을 확인하였다. As shown in Figure 11, 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.
실시예 7. CEACAM6에 대한 CEACAM6-CAR NK 세포의 특이성 (Specificity) 확인Example 7 Identification of Specificity of CEACAM6-CAR NK Cells Against CEACAM6
본 발명의 CEACAM6-CAR NK 세포가 CEACAM6에 특이적으로 결합하는지의 유무를 확인하였다. It was confirmed whether the CEACAM6-CAR NK cells of the present invention specifically bind to CEACAM6.
구체적으로, CEACAM6에 대한 siRNA (BIONEER; 1029726, 1029729 및 1029733)를 A549 세포 주에 lipofectamin (invitrogen; 11514-015, 18324-020)를 이용하여 형질전환 시킨 후, 세포 내 단백질 발현을 항 CEACAM6 항체로 확인하여 단백질 발현 정도를 웨스턴블롯으로 확인하였으며, 그 결과를 도 12에 나타내었다. Specifically, 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.
도 12에 나타낸 바와 같이, A549 세포는 CEACAM6에 대한 siRNA를 처리함에 따라 CEACAM6의 발현이 감소됨을 확인하였다.As shown in Figure 12, A549 cells were confirmed that the expression of CEACAM6 is reduced by treatment with siRNA for CEACAM6.
또한, 상기 A549에 CEACAM6에 대한 siRNA를 처리함에 따른 CEACAM6-CAR NK92의 세포사멸 활성을 칼세인-AM법을 통하여 확인하였으며, 그 결과를 도 13에 나타내었다. In addition, the apoptosis activity of CEACAM6-CAR NK92 by treating the siRNA for CEACAM6 in A549 was confirmed by calcein-AM method, and the results are shown in FIG. 13.
도 13에 나타낸 바와 같이, siRNA 처리에 따라 CEACAM6 발현이 감소된 A549 세포에 대하여 CEACAM6-CAR NK의 세포사멸 활성이 감소되었음을 확인하였으며, 이를 통해 CEACAM6-CAR NK 세포는 표적 항원인 CEACAM6에 특이적으로 활성을 나타냄을 확인하였다.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.
실시예Example
8. 8.
단클론Monoclonal
(Single clone) (Single clone)
CEACAM6CEACAM6
-CAR -CAR
NKNK
세포의 발현 확인 Expression of Cells
단클론의 CEACAM6-CAR NK 세포를 제조하여 이의 CAR 발현 유무를 확인하였다.Monoclonal CEACAM6-CAR NK cells were prepared and their CAR expression was confirmed.
먼저 CEACAM6 (18B8)-CAR를 도입한 상기 실시예 3의 NK92 세포의 수를 측정하여 100 cells/10 ml이 되도록 배양배지에 18B8-CAR NK92 세포를 희석하고, 96 웰 플레이트에 100 ul씩 소분하여 깔아주었다. 2일 또는 3일에 한 번씩 현미경을 통하여 단클론이 형성되는 웰을 확인하고 세포를 계속 배양하여 증식시켜 CEACAM6-CAR NK 세포의 단일 클론을 얻었다.First, 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.
상기 단일 클론으로 얻어진 CEACAM6-CAR NK92 세포의 CAR 발현유무를 Myc 항체를 이용하여 유동세포분석법을 통해 실시예 4와 동일한 방법으로 확인하였으며, 그 결과를 도 14에 나타내었다.CAR expression of CEACAM6-CAR NK92 cells obtained as the monoclonal was confirmed in the same manner as in Example 4 by flow cytometry using Myc antibody, the results are shown in FIG.
도 14에 나타낸 바와 같이, 단일 클론으로 얻어진 CEACAM6-CAR NK92 세포는 94%의 높은 형질 도입율로 CEACAM6-CAR를 발현함을 확인하였다. As shown in Figure 14, it was confirmed that CEACAM6-CAR NK92 cells obtained from a single clone express CEACAM6-CAR with a high transduction rate of 94%.
실시예 9. 다양한 암세포에 대한 단클론 CEACAM6-CAR NK 세포의 세포사멸 효과 변화 확인Example 9 Confirmation of Changes in Apoptotic Effects of Monoclonal CEACAM6-CAR NK Cells on Various Cancer Cells
상기 실시예에서 이용한 A549 세포주 외에 다양한 암세포주에 대하여 단클론 CEACAM6-CAR NK 세포의 세포사멸 효과를 확인하였다.The apoptosis effect of monoclonal CEACAM6-CAR NK cells was confirmed against various cancer cell lines in addition to the A549 cell line used in the above examples.
구체적으로, CEACAM6를 발현하는 암세포를 선별하기 위하여 다양한 암세포를 대상으로 CEACAM6에 대응하는 항체를 반응시켜 상기 실시예 5와 동일한 방법으로 유동세포분석법을 이용하여 각 암세포에서 CEACAM6의 발현을 확인하였으며, 그 결과를 도 15에 나타내었다.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.
상기 CEACAM6의 발현정도를 확인한 다양한 암세포 중 A549(+), AU565, CFPAC-1, SK-BR-3, SKOV-3 및 K562를 선별한 후, 각 세포에 대한 단클론 CEACAM6-CAR NK 세포 (18B8/L1.3-4)의 세포사멸 효과를 실시예 5와 동일한 방법으로 Calcein-AM법 및 LDH 세포독성 키트 (Thermo scientific;88954)의 메뉴얼에 따라 LDH 분석을 이용하여 확인하였으며, 그 결과를 도 16 도 17에 나타내었다.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.
도 16 및 도 17에 나타낸 바와 같이, 단클론 CEACAM6-CAR NK92 세포는 CEACAM6가 발현하는 다양한 암세포에 대해 세포사멸 효과가 있음을 확인하였다.As shown in FIG. 16 and FIG. 17, it was confirmed that monoclonal CEACAM6-CAR NK92 cells have an apoptosis effect against various cancer cells expressing CEACAM6.
또한, 단클론 CEACAM6-CAR NK92 세포는 CEACAM6를 적게 발현하거나 발현하지 않는 암세포인 SK-BR-3, SKOV-3 및 K562에 대해서는 본래의 NK92 세포와 비교하여 세포사멸 효과가 차이나지 않았으며, 이를 통해 단클론 CEACAM6-CAR NK92 세포의 사멸효과는 암세포의 CEACAM6에 특이적임을 확인하였다.In addition, 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.
Claims (26)
1) 항원 결합 도메인,1) an antigen binding domain,
2) 막통과 도메인, 및2) transmembrane domains, and
3) 세포내 신호 전달 도메인을 포함하는 키메릭 항원 수용체(CAR) 로,3) a chimeric antigen receptor (CAR) comprising an intracellular signal transduction domain,
상기 항원 결합 도메인은 CEACAM6 (Carcinoembryonic antigen-related cell adhesion molecule 6)에 특이적으로 결합하는 도메인인 것인, 키메릭 항원 수용체 (CAR).The antigen binding domain is a domain that specifically binds to Carcinoembryonic antigen-related cell adhesion molecule (CEACAM6), chimeric antigen receptor (CAR).
제1항에 있어서, 상기 항원 결합 도메인은 CEACAM6에 특이적으로 결합하는 항체 또는 항체 단편인 것인, 키메릭 항원 수용체.The chimeric antigen receptor of claim 1, wherein the antigen binding domain is an antibody or antibody fragment that specifically binds to CEACAM6.
제2항에 있어서, 상기 항체의 단편은 scFv인 것인, 키메릭 항원 수용체 (CAR)The chimeric antigen receptor (CAR) of claim 2, wherein the fragment of antibody is a scFv.
제2항에 있어서, 상기 항체 또는 항체의 단편은 서열번호 1의 염기서열에 의하여 암호화되는 아미노산 서열로 이루어진 중쇄 가변영역을 포함하는 것인, 키메릭 항원 수용체 (CAR).The chimeric antigen receptor (CAR) according to claim 2, wherein the antibody or fragment thereof comprises a heavy chain variable region consisting of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1.
제2항에 있어서, 상기 항체 또는 항체의 단편은 서열번호 2의 염기서열에 의하여 암호화되는 아미노산 서열로 이루어진 경쇄 가변영역을 포함하는 것인, 키메릭 항원 수용체 (CAR).The chimeric antigen receptor (CAR) according to claim 2, wherein the antibody or fragment thereof comprises a light chain variable region consisting of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2.
제2항에 있어서, 항체 또는 항체의 단편은 링커를 추가로 포함하는 것인, 키메릭 항원 수용체 (CAR).The chimeric antigen receptor (CAR) of claim 2, wherein the antibody or fragment of the antibody further comprises a linker.
제6항에 있어서, 상기 링커는 서열번호 3의 염기서열에 의하여 암호화되는 아미노산 서열로 이루어진 것인, 키메릭 항원 수용체 (CAR).The chimeric antigen receptor (CAR) according to claim 6, wherein the linker is composed of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO.
제1항에 있어서, 상기 항원 결합 도메인은 힌지 영역, 스페이서 영역 또는 이들의 조합에 의하여 막통과 도메인에 연결되는 것인, 키메릭 항원 수용체 (CAR).The chimeric antigen receptor (CAR) of claim 1, wherein the antigen binding domain is linked to the transmembrane domain by a hinge region, a spacer region or a combination thereof.
제8항에 있어서, 상기 힌지 영역, 스페이서 영역 또는 이들의 조합은 Myc 에피토프를 포함하는 것인, 키메릭 항원 수용체 (CAR).The chimeric antigen receptor (CAR) of claim 8, wherein the hinge region, spacer region or combination thereof comprises a Myc epitope.
제9항에 있어서, 상기 스페이서는 서열번호 4의 염기서열에 의하여 암호화되는 아미노산 서열로 이루어진 것인, 키메릭 항원 수용체 (CAR).10. The chimeric antigen receptor (CAR) according to claim 9, wherein the spacer consists of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO.
제1항에 있어서, 상기 막통과 도메인은 CD28, CD3 엡실론, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 및 CD154로 이루어진 군으로부터 선택되는 단백질의 막통과 도메인을 포함하는 것인, 키메릭 항원 수용체 (CAR)The transmembrane domain of claim 1, wherein the transmembrane domain is selected from the group consisting of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. Chimeric antigen receptor (CAR), comprising the transmembrane domain of the protein being
제11항에 있어서, 상기 막통과 도메인은 CD28의 막통과 도메인을 포함하는 것인, 키메릭 항원 수용체 (CAR).The chimeric antigen receptor (CAR) of claim 11, wherein the transmembrane domain comprises the transmembrane domain of CD28.
제12항에 있어서, 상기 CD28의 막통과 도메인은 서열번호 9의 아미노산 서열로 이루어진 것인, 키메릭 항원 수용체 (CAR).The chimeric antigen receptor (CAR) of claim 12, wherein the transmembrane domain of CD28 consists of the amino acid sequence of SEQ ID NO.
제1항에 있어서, 상기 세포 내 신호전달 도메인은 DAP10, CD3 제타 또는 이들의 조합을 포함하는 것인, 키메릭 항원 수용체 (CAR).The chimeric antigen receptor (CAR) of claim 1, wherein the intracellular signaling domain comprises DAP10, CD3 zeta or a combination thereof.
제14항에 있어서, 상기 세포내 신호전달 도메인은 서열번호 10의 아미노산 서열; 또는 서열번호 11의 아미노산 서열로 이루어진 것인, 키메릭 항원 수용체 (CAR).The method of claim 14, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 10; Or a chimeric antigen receptor (CAR) consisting of the amino acid sequence of SEQ ID NO.
제1항에 있어서, 상기 항원 결합 도메인은 신호 펩타이드를 포함하는 것인, 키메릭 항원 수용체 (CAR).The chimeric antigen receptor (CAR) of claim 1, wherein the antigen binding domain comprises a signal peptide.
제16항에 있어서, 상기 신호 펩타이드는 CD8 알파인, 키메릭 항원 수용체 (CAR).The chimeric antigen receptor (CAR) of claim 16, wherein the signal peptide is CD8 alpine.
제16항에 있어서, 상기 신호 펩타이드는 서열번호 6의 염기서열에 의하여 암호화되는 아미노산 서열로 이루어진 것인, 키메릭 항원 수용체 (CAR).The chimeric antigen receptor (CAR) of claim 16, wherein the signal peptide consists of an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 6. 18.
제1항 내지 제18항 중 어느 한 항의 키메릭 항원 수용체 (CAR)를 코딩하는 폴리뉴클레오티드.A polynucleotide encoding the chimeric antigen receptor (CAR) of any one of claims 1-18.
제19항의 폴리뉴클레오티드를 포함하는 벡터.A vector comprising the polynucleotide of claim 19.
제20항의 벡터로 형질전환된 세포.A cell transformed with the vector of claim 20.
제21항에 있어서, 상기 세포는 NK 세포, T 세포 또는 조절 T 세포인 것인, 세포.The cell of claim 21, wherein the cell is an NK cell, a T cell, or a regulatory T cell.
제21항의 세포를 포함하는 세포치료제.Cell therapy comprising the cell of claim 21.
제21항의 세포를 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer, comprising the cell of claim 21 as an active ingredient.
제21항의 세포를 개체에 투여하는 단계를 포함하는 암을 예방 또는 치료하는 방법.A method of preventing or treating cancer comprising administering to a subject a cell of claim 21.
제21항의 세포를 개체로부터 분리된 표본과 접촉시키는 단계를 포함하는, 암의 진단방법.A method of diagnosing cancer, comprising contacting a cell of claim 21 with a sample isolated from an individual.
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