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WO2025084880A1 - Cellule car-t se liant à l'antigène ct83 et son utilisation - Google Patents

Cellule car-t se liant à l'antigène ct83 et son utilisation Download PDF

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WO2025084880A1
WO2025084880A1 PCT/KR2024/016016 KR2024016016W WO2025084880A1 WO 2025084880 A1 WO2025084880 A1 WO 2025084880A1 KR 2024016016 W KR2024016016 W KR 2024016016W WO 2025084880 A1 WO2025084880 A1 WO 2025084880A1
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cancer
antigen
seq
antibody
cells
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Korean (ko)
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김형수
박성택
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Eioncell Inc
Industry Academic Cooperation Foundation Hallym Unviersity
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Eioncell Inc
Industry Academic Cooperation Foundation Hallym Unviersity
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to CAR-T cells binding to CT83 antigen and uses thereof.
  • the complementarity determining region (CDR) of an antibody is a part of the antibody molecule that forms the antigen binding site and allows the antibody to recognize and bind to a specific antigen.
  • CDR is an important factor that determines antibody diversity, and CDR is an important factor in antibody engineering and antibody-based therapeutic development.
  • HeLa cells are a cervical cancer cell line that was collected from a woman named Henrietta Lacks in 1951 for the treatment of malaria. This cell line has a very strong growth ability and can be artificially propagated over a long period of time, so it is widely used in cell biology and cancer research, and is also widely utilized for purposes such as studying the characteristics of tumor cells and evaluating the effectiveness of anticancer treatment.
  • Cervical cancer is a cancer that occurs in the cervix of a woman's uterus (the organ that releases the placenta). This type of cancer is mainly related to infection with the human papillomavirus (HPV). HPV is transmitted through sexual intercourse, and some HPV infections can cause abnormalities in cervical cells, which can lead to cancer.
  • HPV human papillomavirus
  • HT-29 cells are a widely used artificial cell line as one of the colon cancer cell lines. This cell line is derived from human colon cancer tissue and is widely used in colon cancer research and drug testing.
  • Colon cancer is a cancer that occurs in the colon or rectum, and is one of the digestive system cancers. Cancer usually occurs in the lining of the colon, and as the tumor grows, it can invade and spread to surrounding tissues. Colon cancer mainly occurs when intestinal polyps evolve into cancer, and can present various symptoms and signs, which can vary depending on the location, size, and progression of the tumor. Common symptoms of colon cancer include constipation or diarrhea, changes in the shape or color of stool, abdominal pain, weight loss, and fatigue.
  • the inventors of the present invention confirmed that overexpression of CT83 antigen is strongly associated with poor prognosis, and that expression of CT83 antigen promotes proliferation, metastasis, and EMT of cancer cells. Based on the above confirmed results, a recombinant antibody that specifically binds to CT83 antigen and a fusion protein comprising a CT83 protein binding domain, a transmembrane domain, an intracellular co-stimulatory domain, and a signal transduction domain were developed through phage display and bio-panning, and an expression vector comprising the fusion protein and a T cell transformed with the expression vector were developed, thereby completing the present invention.
  • An object of the present invention is to provide a fusion protein comprising a CT83 protein binding domain, a transmembrane domain, an intracellular co-stimulatory domain and a signaling domain.
  • another object of the present invention is to provide a fusion protein, wherein the CT83 protein binding domain is an antibody or an antigen-binding fragment thereof that specifically binds to the CT83 protein.
  • Another object of the present invention is to provide a fusion protein, characterized in that the antibody or antigen-binding fragment thereof is scFv.
  • another object of the present invention is to provide a fusion protein, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 8, and a heavy chain CDR3 of SEQ ID NO: 9; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 10, a light chain CDR2 of SEQ ID NO: 11, and a light chain CDR3 of SEQ ID NO: 12.
  • another object of the present invention is to provide a fusion protein, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 14, and a heavy chain CDR3 of SEQ ID NO: 15; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 16, a light chain CDR2 of SEQ ID NO: 17, and a light chain CDR3 of SEQ ID NO: 18.
  • Another object of the present invention is to provide a fusion protein characterized in that the antibody or antigen-binding fragment thereof specifically binds to an epitope of SEQ ID NO: 23 or 24.
  • Another object of the present invention is to provide a fusion protein characterized in that the antibody or antigen-binding fragment thereof specifically binds to an epitope of SEQ ID NO: 25.
  • Another object of the present invention is to provide an expression vector comprising the fusion protein.
  • Another object of the present invention is to provide a T cell transformed with the expression vector.
  • Another object of the present invention is to provide a composition for preventing or treating cancer, comprising the transformed T cell as an effective ingredient.
  • another object of the present invention is to provide a method for treating cancer, the method comprising the steps of: treating cervical cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, liver cancer, stomach cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, colon cancer, endometrial cancer, ovarian cancer, testis cancer, bladder cancer, renal cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma,
  • a composition for preventing or treating cancer is provided, characterized in that it is selected from the group consisting of multiple myeloid blood cancer and solid cancer.
  • another object of the present invention is to provide a method for producing T cells, including a step of transforming the expression vector.
  • Another object of the present invention is to provide a method for preventing or treating cancer using transformed T cells.
  • another object of the present invention is to provide a method for treating cervical cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, liver cancer, stomach cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, colon cancer, endometrial cancer, ovarian cancer, testis cancer, bladder cancer, renal cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, multiple
  • a method for preventing or treating cancer is provided, characterized in that it includes at least one selected from the group consisting of multiple myeloid blood cancer and solid cancer.
  • the present invention provides a fusion protein comprising a CT83 protein binding domain, a transmembrane domain, an intracellular co-stimulatory domain and a signaling domain.
  • the present invention provides a fusion protein, wherein the CT83 protein binding domain is an antibody or an antigen-binding fragment thereof that specifically binds to the CT83 protein.
  • the present invention provides a fusion protein, characterized in that the antibody or antigen-binding fragment thereof is scFv.
  • the present invention provides a fusion protein, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 8, and a heavy chain CDR3 of SEQ ID NO: 9; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 10, a light chain CDR2 of SEQ ID NO: 11, and a light chain CDR3 of SEQ ID NO: 12.
  • the present invention provides a fusion protein, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 14, and a heavy chain CDR3 of SEQ ID NO: 15; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 16, a light chain CDR2 of SEQ ID NO: 17, and a light chain CDR3 of SEQ ID NO: 18.
  • the present invention provides a fusion protein, characterized in that the antibody or antigen-binding fragment thereof specifically binds to an epitope of SEQ ID NO: 23 or 24.
  • the present invention provides a fusion protein, characterized in that the antibody or antigen-binding fragment thereof specifically binds to an epitope of SEQ ID NO: 25.
  • the present invention provides an expression vector comprising the fusion protein.
  • the present invention provides a T cell transformed with the expression vector.
  • the present invention provides a composition for preventing or treating cancer, comprising the transformed T cell as an effective ingredient.
  • the present invention relates to a method for treating cancer, the method comprising treating cancer selected from the group consisting of cervical cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, liver cancer, stomach cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, colon cancer, endometrial cancer, ovarian cancer, testis cancer, bladder cancer, renal cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, and multiple
  • a composition for preventing or treating cancer is provided, characterized in that it is selected from the group consisting of multiple myeloid blood cancer and solid cancer.
  • the present invention provides a method for preventing or treating cancer using the transformed T cells.
  • the present invention is directed to cervical cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, liver cancer, stomach cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, colon cancer, endometrial cancer, ovarian cancer, testis cancer, bladder cancer, renal cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, multiple A method for preventing or treating cancer is provided, characterized in that it includes at least one selected from the group consisting of multiple myeloid blood cancer and solid cancer.
  • a fusion protein comprising the CT83 protein binding domain, the transmembrane domain, the intracellular co-stimulatory domain and the signal transduction domain of the present invention can specifically bind to the CT83 antigen, and can be usefully used in fields related to the diagnosis and treatment of tumors or cancers related to the CT83 antigen by using such fusion protein.
  • Figure 1 shows the ELISA results of 400 anti-CT83 single antibody candidates.
  • Figure 2a shows the ELISA results of SKAI-5, which was selected as an antibody candidate of the present invention.
  • Figure 2b shows the ELISA results of SKAI-33, which was selected as an antibody candidate of the present invention.
  • Figure 3 shows the microarray results confirming binding to the antigen for epitope mapping. It is indicated in dark green in the second row from the top.
  • Figure 4 shows the expression level of KK-LC-1 protein in HeLa cells or HT-29 cells, as measured by Invitrogen's CT83 monoclonal antibody (CL4762).
  • the X-axis corresponds to the PE-H (Phycoerythrin-Height) value
  • the Y-axis corresponds to the cell number (Count).
  • the graph shown on the left is a graph showing the amount of protein expression in HeLa cells.
  • the blue graph on the left in the graph indicates the amount of protein expression when only HeLa is present, and the green graph on the right indicates the amount of protein expression in CT83 PC-hela.
  • the M4 value (median value of fluorescence intensity) corresponds to 1.07%.
  • the graph shown on the right is a graph showing the amount of protein expression in HT-29 cells.
  • the green graph showing a larger value on the graph indicates the amount of protein expression in CT83 PC-hela, and the smaller blue graph indicates the amount of protein expression when only HT-29 is present.
  • the M4 value corresponds to 0.05%.
  • the M4 value is high, indicating that CT83 protein is expressed in large quantities, and for HT-29 cells, the M4 value is low, indicating that CT83 protein is hardly expressed.
  • Figure 5a shows whether SKAI-5, selected as an antibody candidate of the present invention, specifically binds to HeLa cells or HT-29 cells.
  • the x-axis represents the PE-Texas Red-H value, which represents the height of the fluorescence signal reacting with the tandem fluorescent dye in which PE (Phycoerythrin) and Texas Red are combined.
  • the y-axis corresponds to the cell number (Count).
  • the graph shown on the left is a graph showing the degree of binding to HeLa cells.
  • the blue graph on the left in the graph represents the distribution of control cells that were not treated with the antibody, and the red graph on the right represents the distribution of cells treated with SKAI-5.
  • the M3 value (median fluorescence intensity) corresponds to 14.05%.
  • the graph shown on the right is a graph showing the degree of binding to HT-29 cells.
  • the blue graph with a larger value on the graph indicates the distribution of control cells that were not treated with the antibody, and the red graph with a smaller value indicates the distribution of cells treated with SKAI-5.
  • the M3 value (median value of fluorescence intensity) corresponds to 2.42%.
  • the M3 value is high, indicating high binding affinity, and for HT-29 cells, the M3 value is low, indicating almost no binding.
  • Figure 5b shows whether SKAI-33, selected as an antibody candidate of the present invention, specifically binds to HeLa cells or HT-29 cells.
  • the graph shown on the left is a graph showing the degree of binding to HeLa cells.
  • the blue graph on the left in the graph indicates the distribution of control cells that were not treated with the antibody, and the red graph on the right indicates the distribution of cells treated with SKAI-33.
  • the M3 value (median value of fluorescence intensity) corresponds to 22.65%.
  • the graph shown on the right is a graph showing the degree of binding to HT-29 cells.
  • the blue graph with a larger value on the graph indicates the distribution of control cells that were not treated with the antibody, and the red graph with a smaller value indicates the distribution of cells treated with SKAI-33.
  • the M3 value (median value of fluorescence intensity) corresponds to 2.98%.
  • the M3 value is high, indicating high binding affinity, and for HT-29 cells, the M3 value is low, indicating almost no binding.
  • Figure 6a shows key information of one of the epitopes of the antibody SKAI-5 of the present invention.
  • the upper part of Figure 6a shows information on the sequence, length, mass, isoelectric point, charge, hydrophobicity, and extinction coefficient of the epitope
  • the middle part of Figure 6a shows the structure of the epitope
  • the lower part of Figure 6a shows the sequence, sequence number, length, and hydrophobicity index (hydropathy_index) for amino acids constituting the epitope sequence.
  • Figure 6b shows key information of another epitope possessed by the antibody of the present invention, SKAI-5.
  • Figure 6c shows key information on the front sequence of the epitope of the antibody SKAI-33 of the present invention.
  • Figure 6d shows key information on the latter sequence of the epitope of the antibody SKAI-33 of the present invention.
  • Figure 7a shows the position (71-79) of the epitope sequence of the antibody SKAI-5 of the present invention.
  • Figure 7b shows the position (103-113) of another epitope sequence possessed by the antibody SKAI-5 of the present invention.
  • Figure 7c shows the position (55-79) of the epitope sequence of the antibody SKAI-33 of the present invention.
  • Figure 8 shows the structure of the CT-83 CAR lentiviral vector plasmid manufactured using SKAI-5 or 33.
  • Figure 9 shows the virus production process of the CAR expression vector.
  • Figure 10 shows the results of transformation efficiency verification through FACS.
  • the X-axis represents the expression level of green fluorescent protein (GFP), the Y-axis represents the number of cells, and the GFP-expressing cell ratio of the control group (Un) is 1.85%.
  • the GFP-expressing cell ratio of SKAI 5 is 97.7%, and the GFP-expressing cell ratio of SKAI 33 is 97.8%, indicating that the transformation efficiency of 293T cells for lentiviral vector production of SKAI 5 and 3 CARs is high.
  • FIG. 11 shows the results of CAR expression confirmation through FACS.
  • the X-axis represents the expression level of green fluorescent protein (GFP), and the Y-axis represents the number of cells.
  • GFP green fluorescent protein
  • Figure 12 shows the results of an expression experiment on the T cell membrane of CAR-T antibodies.
  • the left side corresponds to the green dot
  • the right side corresponds to the red dot.
  • FIG. 13 shows the results of CAR-T production efficiency experiments.
  • the X-axis represents the forward scatter-height (FSC-H) of the cell size
  • the Y-axis represents the side scatter-height (SCC-H) of the cell, which represents the degree of intracellular granules. Gating was performed to distinguish living cells.
  • the ratio of living cells in the control group (Un) was 73.6%
  • SKAI-33 was 78.4%
  • the ratios of living cells in SKAI-5, 48, and 15 were 89.1%, 79.8, and 77.1%, respectively. It can be seen that the viability of SKAI-5, 15, 33, and 48 of the present invention are higher than the typical reference value.
  • T cell proportions of SKAI-5, 15, 33, and 48 of the present invention are higher than the typical reference value.
  • the expression of GFP was measured, and the X-axis represents the degree of GFP expression and the Y-axis represents the number of cells.
  • the ratio of GFP expressing cells in the control group (Un) was 0.55%
  • SKAI-48 was 69.4%
  • the ratios of GFP expressing cells in SKAI-33, 5, and 15 were 89.0%, 32.9%, and 27.2%, respectively. It can be seen that the transduced T cell ratios of SKAI-5, 15, 33, and 48 of the present invention are higher than the typical reference value.
  • the X-axis represents the expression level of APC, and the Y-axis represents the number of cells.
  • the ratio of APC expressing cells in the control group (Un) was 0.97%
  • SKAI-48 was 20.1%
  • the ratios of APC expressing cells in SKAI-33, 5, and 15 were 27.0%, 27.0%, and 26.2%, respectively. It can be seen that the CAR expressing cell ratios of SKAI-5, 15, 33, and 48 of the present invention meet the typical standard.
  • Figure 14 shows the results of the CAR-T safety confirmation experiment.
  • Figure 15 shows the results of an experiment to confirm the expression of CT83 on cancer cell lines.
  • the X-axis represents the expression level of Alexa-488 fluorescence
  • the Y-axis represents the number of cells
  • the graphs of the isotype control and Alexa-488 fluorescence almost overlap.
  • the red graph on the left in MB-231 represents the isotype control
  • the blue graph on the right represents the expression level of CT83. It can be confirmed that CT83 antigen is expressed in 75.1% of cells. It can be confirmed that the ratios of CT83 antigen expressing cells are 80.1% and 75.1% in H358 and KATO-III, respectively.
  • Figure 16 shows the results of an experiment to confirm whether CAR-T specifically recognizes CT83 through a cytotoxicity experiment.
  • Figure 17 shows the results of an experiment to confirm whether CAR-T specifically recognizes CT83 by checking the amount of IFN- ⁇ secretion.
  • Figure 18 shows the results of changes in tumor volume when the CAR-T treatment of the present invention is administered.
  • Figure 19 shows the results of changes in body weight of mice when administered the CAR-T treatment of the present invention.
  • the present invention may include T cells transformed with an expression vector of a CT-83 CAR lentiviral vector plasmid manufactured using SKAI-5 or 33.
  • “Phage display” is an experimental method for detecting interactions between proteins or between proteins and other target substances. By cloning a gene into a bacteriophage and displaying the expressed protein on the surface of the phage, the presence or degree of interaction can be measured by binding strength with other proteins or other target substances.
  • Panning is a method of selecting antibodies or antigen-binding fragments that bind to a specific antigen. Using a phage library, various antibody fragments are expressed on phages, and then specific binding to the antigen is confirmed. By confirming the genetic information encoded by the phages that have specific binding, antibodies can be produced.
  • the "bio-panning" of the present invention is a technology for selecting peptides that bind to a specific target substance based on affinity, and through this, it is known that the sequence of a peptide that binds to a specific target can be identified and utilized for antibody development and pharmaceutical development.
  • the amino acid sequence of the antibody of the present invention is shown in Table 2.
  • the CT83 antigen is a protein expressed by the CT83 gene and is also called CXorf61, KKLC1, Kita-kyushu lung cancer antigen 1, KK-LC-1, or Cancer/testis antigen 83. It has a molecular weight of approximately 26.8 kDa and is known to be one of the carcinogenic factors in the human body.
  • the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CT83, comprising a heavy chain variable region comprising CDR 1, CDR 2 and CDR 3; and a light chain variable region comprising CDR 1, CDR 2 and CDR 3.
  • the above antibody or antigen-binding fragment thereof may be characterized by specifically binding to an epitope of SEQ ID NO: 23 or 24.
  • the above antibody or antigen-binding fragment thereof may be characterized by specifically binding to an epitope of SEQ ID NO: 25.
  • the above antibody or antigen-binding fragment thereof may be a scFv that specifically binds to CT83.
  • the scFv may mean a single-chain fragment antibody and may mean a linkage of variable regions of light and heavy chains of immunoglobulin.
  • the present invention provides a gene encoding an antibody or an antigen-binding fragment thereof that specifically binds to CT83.
  • the antibody or an antigen-binding fragment thereof can be produced.
  • the present invention provides a vector containing the gene.
  • the above vector may refer to, but is not limited to, a plasmid, a cosmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a retroviral vector, a lentiviral vector, an adenoviral vector, a liposome, or an exosome.
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • retroviral vector a retroviral vector
  • a lentiviral vector an adenoviral vector
  • a liposome or an exosome.
  • the present invention provides a host cell comprising the vector.
  • the method for injecting the vector into the host cell may be a lentiviral vector, but is not limited thereto.
  • the present invention provides a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to CT83 protein, comprising a step of culturing a host cell containing the vector.
  • the present invention provides a composition for preventing or treating cancer, comprising the antibody or an antigen-binding fragment thereof as an effective ingredient.
  • the present invention provides a method for treating cancer using the composition for preventing or treating cancer.
  • the method for treating cancer may target mammals.
  • the mammal may refer to an animal that has developed cancer, including dogs, cats, giraffes, tigers, whales, sheep, goats, monkeys, camels, cows, pigs, elephants, and horses.
  • the present invention provides an antibody-drug conjugate in which a pharmaceutical composition for preventing or treating cancer is conjugated to an antibody or an antigen-binding fragment thereof that binds to CT83.
  • the above antibody-drug conjugate for preventing or treating cancer may mean an antibody or an antigen-binding fragment thereof conjugated with a linker or a secondary antibody.
  • the linker may be characterized by being selected from the group consisting of lysine conjugation, cysteine conjugation, and position-specific conjugation, but is not limited thereto.
  • the purpose of the present invention is to provide a fusion protein comprising a CT83 protein binding domain, a transmembrane domain, an intracellular co-stimulatory domain, and a signal transduction domain.
  • the fusion protein can be expressed and function as a chimeric antigen receptor (CAR) on an immune cell.
  • CAR chimeric antigen receptor
  • the chimeric antigen receptor may include a CT83 antigen binding domain, a transmembrane domain, and an intracellular co-stimulatory domain and a signal transduction domain, and the provision of the present invention can effectively treat cancer by inducing effective death of cancer cells related to solid cancer and T cell activation.
  • the pharmaceutical composition for preventing or treating cancer may mean at least one selected from the group consisting of a toxin, a chemotherapeutic agent, a cytotoxic anticancer agent, a targeted anticancer agent, an immunotherapy agent, an immune checkpoint inhibitor, an antibiotic, an ADP-ribosyl transferase, a radioisotope, and a nucleolytic enzyme.
  • the above cancers are cervical cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, liver cancer, stomach cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, colon cancer, endometrial cancer, ovarian cancer, testis cancer, bladder cancer, renal cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, multiple It may mean multiple myeloid blood cancer, and/or solid tumor.
  • the present invention provides a kit for detecting a tumor or cancer, comprising an antibody or an antigen-binding fragment thereof that binds to CT83.
  • the above CT83 antigen can act as a biomarker in the kit for detecting the tumor or cancer, and an antibody or an antigen-binding fragment thereof that binds to the CT83 antigen can be used as a reactant.
  • the present invention provides a method for detecting a tumor or cancer, comprising the steps of treating a cancer-related sample with an antibody or an antigen-binding fragment thereof that binds to CT83 to form a CT83 antigen-antibody complex; and the step of detecting the CT83 antigen-antibody complex.
  • the present invention provides a method for providing tumor or cancer diagnosis information, comprising the steps of treating a cancer-related sample with an antibody or an antigen-binding fragment thereof that binds to CT83 to form a CT83 antigen-antibody complex; and the step of detecting the CT83 antigen-antibody complex.
  • SKAI-5 and 33 proteins are antibodies or antigen-binding fragments that specifically bind to the CT83 antigen, and that SKAI-5 and 33 proteins can be combined with drugs for preventing or treating cancer to utilize them as ADCs, or chimeric antigen receptor T-cells (CAR-T) can be produced to utilize them for anticancer treatment.
  • an antibody library was constructed and expressed as follows.
  • OPAL library oriented peptide array library, Construction of a Large synthetic human scFv library with six diversified CDRs and high functional diversity.
  • the antibody expressed by the OPAL library comprises a human variable heavy chain encoded by the gene Vh3-23 located at V3-23 and a human variable light chain encoded by the gene V ⁇ 1g located at 1g, and CDR-H1, H2, L1, L2, and L3 comprise some CDR loops and CDR sequences combined or combined with CDR sequences derived from humans.
  • CDR-H3 consists of four sublibraries, including AE, BE, CF, and DF (Table 1), and forms an intraloop disulfide bond (C-(Xaa)4-C) between cysteines in the DF sublibrary.
  • n means that the corresponding amino acid is repeated n times.
  • ER2738 E. coli, AMID BIOSCIENCES, cat. No. ER-201 was transformed with the OPAL library, and 20 MOI of helper phage (VCSM13) was added to 2xYT medium (BD Difco TM, 2xYT powder) containing 50 ug/ml carbenicillin and 70 ug/ml kanamycin, and cultured at 30°C for 14 h. Afterwards, the bacteriophage was activated (rescued) using 4% Polyethylene glycol 8000 (PEG 8000).
  • PEG 8000 Polyethylene glycol 8000
  • biopanning was performed as follows.
  • CT83 Amino acid sequence Expression cell Tags KK-LC-1 (CT83) (NM_001017978) 5 TP309391 (N-ter)-MNFYLLLASSILCALIVFWKYRRFQRNTGEMSSNSTALALVRPSSSGLINSNTDNNLAVYDLSRDILNNFPHSIARQKRILVNLSMVENKLVELEHTLLSKGFRGASPHRKST-(C-ter) HEK293T (C-ter)-Myc/DDK recombinant human Lung cancer antigen 1 (CT83)-VLPs (Active) 6 CSB-MP711093HU (N-ter)-MNFYLLLASSILCALIVFWKYRRFQRNTGEMSSNSTALALVRPSSSGLINSNTDNNLAVYDLSRDILNNFPHSIARQKRILVNLSMVENKLVELEHTLLSKGFRGASPHRKST-(C-ter) Mammalian cells (N-ter)- 6xHis-tag
  • CT83 antigen was bound to epoxy magnetic beads (M-270 Epoxy) overnight at 4°C, and then incubated with scFv-expressing phage at 37°C for 2 hours. Afterwards, the CT83-bound phage was eluted using 100 mM Triethylamine (TEA) and infected with new ER2738 cells. The bio-panning process of specifically binding the scFv-expressing bacteriophage to the CT83 antigen and then eluted using TEA was repeated three times. By repeating the bio-panning process, substances that nonspecifically bind to the antigen could be removed.
  • epoxy magnetic beads M-270 Epoxy
  • bio-panning was additionally performed once more using KK-LC-1 (CT83) (NM_001017978) human recombinant protein (CAT#: TP309391, Origene) (Table 2) to discover antibody candidates.
  • a set of 400 monoclonal antibody candidates were randomly selected from independently formed colonies cultured after the above four rounds of panning (Fig. 1). ELISA analysis was performed on each candidate expressed in the form of scFv, and 50 antibody candidates that specifically bind to KK-LC-1 (CT-83) compared to BSA and KK-LC-1 VLPs compared to BSA were selected.
  • NGS analysis was performed on the above 50 selected antibody candidates, and 46 antibody candidates were selected after excluding overlapping antibodies and antibodies with frame shifts.
  • the scFv of the 46 antibody candidates was reproduced using the gene sequences, and the recombinant KK-LC-1/BSA and KK-LC-1 VLPs/BSA were analyzed by ELISA to select the final antibody candidates that can specifically bind to the CT83 protein.
  • the final antibody candidates were named SKAI-5, SKAI-15, SKAI-33, and SKAI-48, and the heavy and light chain CDR amino acid sequences of SKAI-5 and SKAI-33 among the antibody candidates are listed in Table 3.
  • Sequence number 7 Sequence number 8 Sequence number 9 (N-ter)-NYSMS-(C-ter) (N-ter)-SLSSGGGST-(C-ter) (N-ter)-ARRKTRFDY-(C-ter) VL CDR1 CDR2 CDR3 Sequence number 10 Sequence number 11 Sequence number 12 (N-ter)-SGSSSNIGSNTVN-(C-ter) (N-ter)-ADSQ-(C-ter) (N-ter)-GSWDSSLNG-(C-ter) SKAI-33 VH CDR1 CDR2 CDR3 Sequence number 13 Sequence number 14 Sequence number 15 (N-ter)-SYSMS-(C-ter) (N-ter)-AISSSGGNK-(C-ter) (N-ter)-ARRKKLFDY-(C-ter) VL CDR1 CDR2 CDR3 Sequence number 16 Sequence number 17 Sequence number 18 (N-ter)-S
  • SKAI-5 The ELISA results of SKAI-5 are as shown in Fig. 2a.
  • TMB (3,3',5,5' - tetramethylbenzidine) was added, and 20 minutes later, the absorbance at 450 nm was measured using a plate reader.
  • Example 3 The final antibody candidates identified in Example 3 were manufactured as IgG antibodies, and the following experiments were performed to clearly identify the amino acid sequence of the site where the antibodies bind to the CT83 protein.
  • the scFv sequence of SKAI-5 or 33 that binds to CT83 was inserted into the human IgG vector system (TGEX-HC, Antibody design labs, MX001, and TGEX-LC, Antibody design labs, MX002) by the following method.
  • a gene cloning vector was constructed so that IgG can be produced using the base sequence encoding scFv by amplifying the antibody gene binding region using forward and reverse primers containing restriction enzyme sites.
  • the base sequences of the primers are listed in Table 4.
  • Sequence number 19 Sequence number 20 (BSSHll) NNGCGCGCACTCCGAGGTGCAGCTGTTG (BsmBl) NNCGTCTCNATGCTGAGCTCACGGTGAC TGEX-VL Sequence number 21 Sequence number 22 (BspEI) NNNNNNTCCGGACAGTCTGTGCTGACT (BsaI) NNGGTCTCNTTCGTAGGACCGTCAGCT
  • a vector was produced using the following method so that the above IgG can be expressed in mammalian cells.
  • the VH primer was designed to have restriction sites for BSSHll at the 5' end and BsmBl at the 3' end, and the VL primer was designed to have restriction sites for BspEI at the 5' end and BsaI at the 3' end.
  • the base sequences of the above VH primers are the base sequences of SEQ ID NOs: 19 and 20, and the base sequences of the VL primers are the base sequences of SEQ ID NOs: 21 and 22.
  • the amplified VH fragment was inserted into the TGEX-HC (Antibody design labs, MX001) vector, and the VL fragment was inserted into the TGEX-LC (Antibody design labs, MX002) vector.
  • the plasmids were amplified using Escherichia coli DH5 ⁇ -competent cells, and cloning was confirmed by DNA sequencing.
  • Expi-CHO-s cells 6x10 ⁇ 6 Expi-CHO-s cells were prepared, and 15 ug each of heavy and light chain IgG plasmids were reacted in Opti-pro SFM medium (ThermoFisher scientific, Cat no. 12309019) for 5 minutes. After 5 minutes, they were mixed with ExpifectamineTM CHO to form a complex. The formed complex was transfected into the prepared Expi-CHO-s cells (ThermoFisher scientific, A29127) without exceeding 5 minutes of reaction. The next day, ExpiCHOTM Feed and ExpiFectamineTM CHO Enhancer (ExpiFectamineTM CHO Transfection kit, ThermoFisher scientific, cat no. A29129) that increase nutrient supply and protein production efficiency were added. Cells were harvested when the cell viability was approximately 75% (approximately 7 days after transfection).
  • a monoclonal antibody was purified using Protein A Resin (Amicogen).
  • the produced antibody volume 50 ml
  • 1 ml of protein A resin ⁇ 40 mg human IgG/ml resin
  • the mixture was loaded onto an affinity chromatography column and eluted with 0.1 M glycine HCl buffer (pH 2.7).
  • the purified antibody was concentrated using a 50 kDa cut off amicon (merck millipore), and the antibody concentration was measured using a protein assay kit (Pierce TM, BCA Protein Assay kit, Thermo Scientific).
  • Epitope mapping using microarray was performed to identify the antigen binding site (epitope) of the selected antibodies against the target antigen (CT83).
  • the SKAI-5 or 33 antibody manufactured by Example 4-1 was biotinylated. Thereafter, the biotinylated antibody sample was subjected to microarray chip analysis for approximately 18,000 CT83 antigen peptide positions to confirm binding to the CT83 antigen (Fig. 3).
  • Antigen-antibody binding was confirmed by measuring overlapping biotinylation signals.
  • the sequence, sequence number, length, mass, isoelectric point, charge, hydrophobicity, and extinction coefficient information of the major epitopes binding to SKAI-5 or 33 are shown in Figures 6a to 6d, and the positions of the repeat sequences were confirmed ( Figure 7).
  • the confirmed epitope sequences are listed in (Table 5).
  • SKAI-5 Epitope 1 (SEQ ID NO: 23)
  • Epitope 2 (SEQ ID NO: 24) (N-ter)-PHSIARQKR-(C-ter) (N-ter)-FRGASPHRKST-(C-ter)
  • SKAI-33 Sequence number Epitope 25 (N-ter)-NNLAVYDLLSRDILNNFPHSIARQKR-(C-ter)
  • the HeLa cells used in this experiment are cancer cells that express the CT83 (KK-LC-1) protein, and HT-29 are cancer cells that do not express the CT83 (KK-LC-1) protein.
  • HT-29 are cancer cells that do not express the CT83 (KK-LC-1) protein.
  • HeLa and HT-29 cells were collected by trypsinization, washed with PBS, and centrifuged to precipitate the cells twice.
  • the cells were prepared by diluting 1/100 with mouse monoclonal antibody against CT83 antigen (Invitrogen, Catalog # MA5-24711) and incubating for 1 hour at room temperature. After 1 hour, the secondary antibody was diluted 1/100 and incubated for 1 hour at room temperature, and analyzed by forward scattered light (FSC).
  • FSC forward scattered light
  • CAR-T therapy using chimeric antigen receptors is a next-generation anticancer drug that targets specific antigens and can be expected to have an anticancer effect even with a single administration.
  • a fusion protein including a polynucleotide encoding a chimeric antigen receptor (CAR) using SKAI-5 or 33 was produced, and a CT-83 CAR-T vector including a sequence encoding the fusion protein as follows was designed and produced, and a virus of the vector was produced.
  • CAR chimeric antigen receptor
  • the above vector was prepared using scFv that specifically binds to CT-83.
  • anti-CT83 KK-LC-1
  • scFv anti-CT83 capable of recognizing CT-83 of target cells
  • ZsGreen1 a type of green fluorescent protein (GFP), was inserted into the vector.
  • the vector was constructed to include anti-CT83 scFv, which is a CT83 protein binding domain, CD8 ⁇ hinge and transmembrane domains, which are transmembrane domains, 4-1BB costimulatory domain, which is a type of intracellular costimulatory domain, CD3 ⁇ activation domain, which is a type of signaling domain, and ZsGreen1 fluorescent protein.
  • anti-CT83 scFv which is a CT83 protein binding domain
  • CD8 ⁇ hinge and transmembrane domains which are transmembrane domains
  • 4-1BB costimulatory domain which is a type of intracellular costimulatory domain
  • CD3 ⁇ activation domain which is a type of signaling domain
  • ZsGreen1 fluorescent protein ZsGreen1 fluorescent protein.
  • CT-83 CAR-T vector manufactured to include SKAI-5 or 33 is as shown in Figures 8a and b.
  • the 293T cells were transfected to include a CAR structural coding gene including an scFv that can specifically bind to CT83.
  • T cells expressing CAR were cultured and amplified into millions through the above process.
  • specific growth factors and stimulants were used to promote the proliferation of T cells.
  • four factors consisting of Rev, GAG/POL, VSV-G, and the target gene (SKAI-5 or 33) were mixed in an optimal ratio to HEK-293T cells to perform virus transfection, and more specifically, 18 ⁇ g of Rev, 18 ⁇ g of GAG/POL, 7 ⁇ g of VSV-G, and 30 ⁇ g of the target gene (SKAI-5 or 33) were mixed to perform transfection.
  • the supernatant containing lentivirus particles released into the medium was collected. Ultracentrifugation was performed during the process, and the process was performed at 17,200 g and 4°C for 24 hours.
  • the ratio of GFP (Green Fluorescent Protein)-positive 293T cells was confirmed by Fluorescence-Activated Cell Sorting (FACS) (Beckman Coulter, B53013).
  • the GFP positivity rate was confirmed through FACS 48 hours after transformation, and a high transformation efficiency of more than 97% was confirmed (Fig. 10).
  • the virus titer was calculated and transformation of activated T cells was performed.
  • the reverse calculation formula is as follows, and the reverse calculation results are as shown in Table 6 below.
  • N Number of cells transduced
  • V T Transduction Volume, mL
  • the titer of a typical lentivirus for research purposes is generally in the range of 10 ⁇ 6 to 10 ⁇ 8 TU/mL, and it was confirmed that a virus with a typical level of titer was produced in this experiment.
  • human IgG-APC antibody Human Immunoglobulin G-Allophycocyanin antibody
  • RFP Red Fluorescent Protein
  • the cell viability of CAR-T containing SKAI-5 or 33 was found to be over 80%, which was confirmed to be over 70%, which is the minimum cell viability index generally recognized in the industry as an effective treatment.
  • CD3+ cell ratio analysis was all over 90%, which was an appropriate level, and CAR+CD3+ was also confirmed to be over 50%, exceeding the typical level of 2-30% to be recognized as an effective treatment.
  • the above drawing 14(a) is a standard curve, and as shown in the above drawing, as the virus concentration on the X-axis increases, the Cq value on the Y-axis decreases, confirming that VSV-G is detected faster as the virus concentration increases.
  • the above experiment is an experiment to evaluate the risk of infection through VSV-G targeting CAR-T produced through the virus, and the results of the above experiment showed that the risk of infection may increase as the virus concentration increases.
  • the CAR-T produced through the present invention embodiment is safe from infection by residual viruses.
  • MDA-MB-231 breast cancer
  • H358 lung cancer
  • KATO-III gastric cancer
  • CT83 was expressed in the above cell lines. As shown in Fig. 15, while CT83 was found to be negative in the control group, 293T, it was observed to be positive in MDA-MB-231 (breast cancer), H358 (lung cancer), and KATO-III (gastric cancer), confirming that CT83 was expressed on the cell membrane of the cancer cell-related cell lines.
  • IFN- ⁇ is an important cytokine that enhances anti-tumor immune responses, and an increase in the secretion amount of IFN- ⁇ indicates that the CAR-T cells of the present invention have recognized and activated target cells.
  • the amount of IFN- ⁇ secretion was analyzed using ELISA.
  • the CT83 CAR-T manufactured by the embodiment of the present invention reacted with the CT83 antigen and exhibited a cytotoxic effect on cancer cell lines, and through this, it was found that the CAR-T manufactured by the embodiment of the present invention had excellent anticancer efficacy in vivo.
  • a fusion protein comprising a polynucleotide encoding a chimeric antigen receptor (CAR) of the present invention can specifically bind to a CT83 antigen, and such fusion protein can be usefully used in fields related to diagnosis and treatment of tumors or cancers associated with the CT83 antigen.
  • CAR chimeric antigen receptor

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Abstract

L'objectif de la présente invention est de fournir un CAR-T pour l'antigène CT83, et son utilisation. Une protéine recombinante a été produite à l'aide d'informations concernant l'antigène CT83, et un anticorps anti-CT83 (KK-LC-1) se liant à l'antigène a été produit par criblage biologique. Un CAR-T a été développé à l'aide de scFv de deux anticorps recombinants sélectionnés SKAI-5 ou SKAI-33, et la cytotoxicité contre une lignée cellulaire exprimant l'antigène CT83 a été identifiée, et ainsi la présente invention peut être utilisée de manière efficace dans des applications liées au traitement du cancer.
PCT/KR2024/016016 2023-10-20 2024-10-21 Cellule car-t se liant à l'antigène ct83 et son utilisation Pending WO2025084880A1 (fr)

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Citations (4)

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KR20170138548A (ko) * 2015-04-22 2017-12-15 큐어백 아게 종양 질병 치료용 rna를 포함하는 조성물
KR20200071079A (ko) * 2017-09-27 2020-06-18 유니버시티 오브 써던 캘리포니아 공동-자극을 위한 신규한 플랫폼, 신규한 car 설계 및 입양 세포 치료를 위한 다른 향상
KR20210137427A (ko) * 2019-01-06 2021-11-17 아빈투스 바이오, 인코포레이티드 Car t 세포 방법 및 작제물
JP2023532278A (ja) * 2020-06-25 2023-07-27 ザ・メソジスト・ホスピタル 抗原特異的ｔ細胞受容体及びキメラ抗原受容体、並びにがん免疫療法のための免疫シグナル伝達モジュレーションにおける使用方法

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