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WO2025234450A1 - Cellule cancéreuse modifiée et composition vaccinale contre le cancer la contenant - Google Patents

Cellule cancéreuse modifiée et composition vaccinale contre le cancer la contenant

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Publication number
WO2025234450A1
WO2025234450A1 PCT/JP2025/016891 JP2025016891W WO2025234450A1 WO 2025234450 A1 WO2025234450 A1 WO 2025234450A1 JP 2025016891 W JP2025016891 W JP 2025016891W WO 2025234450 A1 WO2025234450 A1 WO 2025234450A1
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Prior art keywords
cancer
cancer cells
cells
antigen
expressing
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Pending
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English (en)
Japanese (ja)
Inventor
卓也 建部
功 石田
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TEIKYO HEISEI UNIVERSITY
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TEIKYO HEISEI UNIVERSITY
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Publication of WO2025234450A1 publication Critical patent/WO2025234450A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to modified cancer cells and cancer vaccine compositions containing the same.
  • Anti-PD-1 antibodies have been developed that inhibit immune checkpoints, one of the immune evasion mechanisms of cancer, and are expected to be effective against more than 20 types of cancer. However, only a little over 20% of patients respond to anti-PD-1 antibodies, and complete remission is achieved in only a few percent of cases (Non-Patent Document 1).
  • Non-Patent Document 2 Non-Patent Document 2
  • Non-Patent Document 2 Non-Patent Document 2
  • no recombinant cancer cell vaccine formulations have been known that express antigens derived from foreign pathogenic microorganisms that induce sensitized CTLs against peptides derived from unidentified neoantigens and attack cancer cells.
  • the present invention aims to provide modified cancer cells that are useful for treating or preventing cancer.
  • the present inventors have now discovered that when modified mouse cancer cells expressing a CTL-inducing antigen are administered to mice, the administered modified cancer cells disappear; that when cancer cells are transplanted into mice that have already lost the administered modified cancer cells, the cancer cells also disappear; and that similar results can be obtained when modified cancer cells treated with an antitumor agent are administered.
  • the present inventors have also discovered that similar results can be obtained when HLA-expressing cancer cells are used as the cancer cells and HLA-expressing knock-in mice are used as the recipient mice.
  • the present invention is based on these findings.
  • the following inventions are provided.
  • Genetically modified cancer cells comprising a nucleic acid encoding a cytotoxic T cell-inducing antigen and a nucleic acid encoding IL-2.
  • [4] The genetically modified cancer cell according to any one of [1] to [3] above, wherein the nucleic acid encoding the cytotoxic T cell-inducing antigen and the nucleic acid encoding IL-2 are operably linked to a promoter having transcriptional activity in cancer cells.
  • [5] The genetically modified cancer cell according to any one of [1] to [4] above, which is derived from a subject suffering from cancer.
  • [6] The genetically modified cancer cells according to any one of [1] to [5] above, whose proliferation rate after 24 hours is suppressed to 95% or less.
  • a method for producing genetically modified cancer cells comprising the steps of: (1) introducing a nucleic acid encoding a cytotoxic T cell (CTL)-inducing antigen and a nucleic acid encoding IL-2 into cancer cells isolated from a subject suffering from cancer, thereby obtaining modified cancer cells that express the CTL-inducing antigen and IL-2.
  • CTL cytotoxic T cell
  • IL-2 IL-2-inducing antigen
  • a cancer vaccine composition comprising the genetically modified cancer cells described in any one of [1] to [6] above, or the genetically modified cancer cells produced by the production method described in [7] or [8] above.
  • a combination medicine comprising the cancer vaccine composition according to any one of [9] to [12] above and another drug.
  • the combination drug according to the above-mentioned [13], wherein the other drug is an immunostimulant (preferably an anti-CTLA-4 antibody preparation).
  • a method for treating or preventing cancer comprising administering to a subject suffering from cancer in need thereof the genetically modified cancer cells described in any of [1] to [6] above, the genetically modified cancer cells produced by the production method described in [7] or [8] above, or the cancer vaccine composition described in any of [9] to [12] above.
  • the administration is subcutaneous, intradermal, or topical administration to a subject.
  • the present invention provides modified cancer cells that induce sensitized cytotoxic T cells against neoantigen-derived peptides, triggering attacks on cancer cells.
  • the modified cancer cells of the present invention and cancer vaccine compositions containing them are advantageous in that they can induce tumor immunity even in subjects in whom sufficient cytotoxic immunity against cancer is not induced.
  • FIG. 1 shows the timing of transplantation of etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing Colon26-Luc cells) and antigen-non-expressing cancer cells (Colon26-Luc cells) into experimental animals and the timing of IVIS imaging (see Example 1 (1-3)).
  • Figure 2 shows the sequences targeted for disruption by the CRISPR-Cas9 method in the production of LLC-Luc cells in which the ⁇ 2M gene has been disrupted ( ⁇ m ⁇ 2M LLC-Luc cells) (see Example 2 (2-1) B).
  • the boxed sequences are the sequences targeted for disruption.
  • the underlined sequences are protospacer adjacent motifs (PAM).
  • FIG. 3 is a diagram showing the enrichment of ⁇ 2M-negative cells together with the results of flow cytometry analysis (see Example 2 (2-1) B).
  • the negative fraction was enriched by H2Db-PE staining.
  • FIG. 4 shows the results of flow cytometry analysis of mouse MHC class I expression in wild-type LLC-Luc cells and ⁇ 2M gene knockout ( ⁇ m ⁇ 2M) LLC-Luc cells (see Example 2 (2-1) c).
  • Figure 5(A) shows the procedure for constructing the human HLA-A2 expression unit DNA used to generate HLA-expressing ⁇ m ⁇ 2M LLC-Luc cells (see (2-2) of Example 2).
  • FIG. 5(B) is a schematic diagram showing a chimeric MHC class I molecule in which the mouse ⁇ 1 and ⁇ 2 domains and mouse ⁇ 2M in the mouse MHC class I molecule are replaced with human ⁇ 1 and ⁇ 2 domains and human ⁇ 2M, respectively, and its behavior within the cell.
  • FIG. 6 shows the results of flow cytometry analysis of HLA expression on the cell surface of the HLA-expressing cancer cells prepared in Example 2.
  • FIG. 7 shows the results of Western blotting analysis of HLA expression in the HLA-expressing cancer cells prepared in Example 2.
  • the genetically modified cancer cells of the present invention are cancer cells modified by gene transfer manipulation, which comprise a nucleic acid encoding a cytotoxic T cell-inducing antigen and a nucleic acid encoding IL-2 in an expressible manner. That is, the genetically modified cancer cells of the present invention are cancer cells into which a nucleic acid encoding a cytotoxic T cell-inducing antigen and a nucleic acid encoding IL-2 have been introduced.
  • Cancer cells to be subjected to gene transfer manipulation can be, for example, cancer cells derived from a subject suffering from cancer. Further, non-limiting examples of cancer cells include cancer cells derived from cancer tissue surgically removed from a subject suffering from cancer.
  • the genetically modified cancer cells of the present invention may be isolated or purified cells.
  • Non-limiting examples of cancer include solid cancers (e.g., colon cancer, lung cancer, mesothelioma, pancreatic cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer, duodenal cancer, small intestine cancer, breast cancer, ovarian cancer, testicular tumors, prostate cancer, liver cancer, thyroid cancer, kidney cancer, uterine cancer, brain tumors, retinoblastoma, skin cancer, sarcoma, malignant bone tumors, bladder cancer), and blood cancers (e.g., leukemias such as acute myeloid leukemia and acute lymphocytic leukemia, and multiple myeloma).
  • solid cancers e.g., colon cancer, lung cancer, mesothelioma, pancreatic cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer, duodenal cancer, small intestine cancer, breast cancer,
  • the cytotoxic T cell-inducing antigen to be expressed in the modified cancer cells is not particularly limited as long as it is an antigen capable of inducing cytotoxic T cells in a mammal, and can be selected, for example, from antigens derived from pathogenic bacteria or viruses (preferably pathogenic viruses).
  • Antigens capable of inducing cytotoxic T cells in mammals are well known to those skilled in the art, and include, for example, antigens that can be presented by MHC class I or HLA class I.
  • Non-limiting examples of antigens capable of inducing cytotoxic T cells include antigens derived from Mycobacterium tuberculosis (e.g., 85A antigen, 85B antigen, MPB51 antigen, Mtb72f antigen) and antigens derived from the novel coronavirus (e.g., spike antigen, nucleocapsid protein antigen, membrane protein antigen).
  • antigens derived from Mycobacterium tuberculosis e.g., 85A antigen, 85B antigen, MPB51 antigen, Mtb72f antigen
  • antigens derived from the novel coronavirus e.g., spike antigen, nucleocapsid protein antigen, membrane protein antigen.
  • the amino acid sequences of these antigens and the nucleotide sequences encoding them are available in public databases.
  • the nucleotide sequence and amino acid sequence of the 85A antigen can also be the nucleotide sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 6, and the nucleotide sequence and amino acid sequence of the Spike antigen can also be the nucleotide sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 8, respectively.
  • the IL-2 expressed in the modified cancer cells is mammalian IL-2, and when the modified cancer cells are human, human IL-2 can be preferably used.
  • the amino acid sequence of wild-type IL-2 and the nucleotide sequence encoding it are publicly known.
  • the nucleotide sequence and amino acid sequence of human wild-type IL-2 registered in the National Library of Medicine under ACCESSION: BC066257 can be used, and the nucleotide sequence and amino acid sequence of mouse wild-type IL-2 registered in the National Library of Medicine under ACCESSION: NM_008366 can be used.
  • the nucleotide sequence and amino acid sequence of human wild-type IL-2 can also be the nucleotide sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4.
  • the nucleotide sequence and amino acid sequence of mouse wild-type IL-2 can also be the nucleotide sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.
  • IL-2 expressed in modified cancer cells may also be wild-type IL-2, or a modified IL-2 in which several (e.g., 1 to 10, 1 to 6, 1 to 4, 1 to 3, or 1 or 2) amino acids have been substituted, inserted, added, and/or deleted, and which retains the immune response-modulating function of wild-type IL-2, and a nucleic acid encoding the modified IL-2.
  • the modified IL-2 may also consist of an amino acid sequence that is 80% or more identical (preferably, 85% or more, 90% or more, 92% or more, 94% or more, 96% or more, 98% or more, or 99% or more) to the amino acid sequence of wild-type IL-2.
  • identity refers to the degree of identity when the sequences being compared are appropriately aligned, and refers to the percentage of exact amino acid matches between the sequences. Identity is determined, for example, by taking into account the presence of gaps in the sequence and the nature of the amino acids (Wilbur, Natl. Acad. Sci. U.S.A. 80:726-730 (1983)).
  • the alignment can be performed, for example, by using any algorithm, and specifically, homology search software such as BLAST (Basic local alignment search tool) (Altschul et al., J. Mol. Biol.
  • FASTA Phrase et al., Methods in Enzymology 183:63-69 (1990)
  • Smith-Waterman Method. Enzym., 164, 765 (1988)
  • Identity can be calculated using, for example, a known homology search program such as those described above, for example, the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using default parameters.
  • NCBI National Center for Biotechnology Information
  • the base sequences encoding the antigens and IL-2 to be expressed in modified cancer cells may be codon-optimized to enable expression in cancer cells and/or to increase the amount of expression in cancer cells. Codon optimization is well known to those skilled in the art and can be performed using known methods commonly used in this field.
  • nucleic acids encoding cytotoxic T cell-inducing antigens and nucleic acids encoding IL-2 into cancer cells can be achieved by gene transfer manipulation.
  • Gene transfer manipulation of cancer cells can be achieved by chemical, physical, or biological methods. Chemical methods include transfection using cationic polymers, cationic lipids, calcium phosphate, etc. Physical methods include electroporation and microinjection. Biological methods include methods using viral vectors.
  • the nucleic acids to be introduced into cancer cells can be in the form of recombinant nucleic acid constructs to ensure stable expression in cancer cells, and expression vectors such as plasmids or viral vectors can be used.
  • gene transfer into cancer cells can be achieved by chemical or physical methods such as electroporation.
  • gene transfer into cancer cells can be achieved by infecting the cancer cells with viral particles.
  • Viral vectors that can be used include, but are not limited to, retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses.
  • Expression vectors, reagents, and equipment used for gene transfer are well known to those skilled in the art, and commercially available products can be used. Gene transfer into cancer cells can be performed in vitro or ex vivo.
  • the nucleic acid molecules encoding the cytotoxic T cell-inducing antigen and IL-2 can be operably linked to a promoter with transcriptional activity in cancer cells so that they are expressed in the cancer cells into which they are introduced.
  • Promoters with transcriptional activity in cancer cells are well known to those of skill in the art, and an appropriate one can be selected for the present invention.
  • a promoter with cancer cell-specific transcriptional activity can be used.
  • Non-limiting examples of promoters with cancer cell-specific transcriptional activity include the TERT promoter, the AFP promoter (liver cancer-specific), the CEA promoter (pancreatic cancer and colorectal cancer-specific), and the PSA promoter (prostate cancer-specific).
  • expression vectors containing a nucleic acid encoding a cytotoxic T cell-inducing antigen and a nucleic acid encoding IL-2 can be carried out according to methods well known to those of skill in the art.
  • nucleic acids include DNA and RNA, as well as modified forms of these and artificial nucleic acids, but DNA is preferred.
  • DNA also includes cDNA, genomic DNA, and chemically synthesized DNA. Note that nucleic acid (molecule) and polynucleotide can be used interchangeably.
  • modified cancer cells When the modified cancer cells are used as a cancer treatment vaccine, as described below, viable modified cancer cells are preferred from the perspective of efficacy, but from the perspective of safety, modified cancer cells whose proliferation ability has been inhibited are desirable.
  • Methods for inhibiting the proliferation ability of modified cancer cells include, for example, contact with an anticancer drug and irradiation.
  • anticancer drugs include those that inhibit cell proliferation and exhibit antitumor effects, such as etoposide, mitoxantrone, docetaxel, and mitomycin C.
  • the type and concentration of the anticancer agent to be contacted with the modified cancer cells can be determined based on the proliferation rate of the cancer cells.
  • the modified cancer cells can be contacted with a certain concentration of anticancer agent, and the anticancer agent can be contacted with the modified cancer cells at a concentration such that the proliferation rate (%) after 24 hours is 60-95%, preferably 70-90% (see Example 1(1-1)K).
  • the proliferation rate (%) after 24 hours indicates the ratio of the number of surviving cancer cells 24 hours after treatment to the number of surviving cancer cells before treatment (i.e., the number of surviving untreated cancer cells).
  • a method for producing modified cancer cells comprising the steps of: (1) introducing a nucleic acid encoding a cytotoxic T-cell-inducing antigen and a nucleic acid encoding IL-2 into cancer cells isolated from a subject suffering from cancer, thereby obtaining modified cancer cells expressing the cytotoxic T-cell-inducing antigen and IL-2.
  • the production method of the present invention may further comprise, after step (1), (2) a step of immersing the modified cancer cells in an anticancer agent.
  • the production method of the present invention may also further comprise, prior to step (1), a step of isolating cancer cells from a subject suffering from cancer.
  • the production method of the present invention can be carried out according to the description of the modified cancer cells of the present invention.
  • modified cancer cells When modified cancer cells are produced using cancer cells isolated from a subject suffering from cancer, the modified cancer cells can be referred to as autologous gene-modified cancer cells.
  • the production method of the present invention may further comprise administering the modified cancer cells obtained by step (1) or (2) to a subject suffering from cancer in need thereof (preferably the subject from which the cancer cells were isolated).
  • the production method of the present invention can be referred to as a method for treating or preventing cancer.
  • the present invention provides a cancer vaccine composition comprising the modified cancer cells of the present invention.
  • the present invention also provides a pharmaceutical composition for treating or preventing cancer comprising the modified cancer cells of the present invention.
  • the modified cancer cells of the present invention also include modified cancer cells produced by the production method of the present invention (the same applies hereinafter). Because the cancer vaccine composition of the present invention contains the modified cancer cells as an active ingredient, in the present invention, the term "cancer vaccine composition" is used interchangeably with "cancer vaccine cell preparation.”
  • cancer vaccine composition refers to a method for treating or preventing cancer by using the immune system to identify cancer cells present in the body as foreign substances and activating or proliferating immune cells involved in their elimination.
  • cancer vaccine composition of the present invention can be used for the treatment and/or prevention of cancer.
  • the cancer vaccine composition of the present invention is advantageous in that it can remove cancer cells that could not be removed in such cases.
  • Surgical cancer therapy also has a certain probability of causing cancer metastasis and/or recurrence
  • the cancer vaccine composition of the present invention is advantageous in that it can prevent such post-operative cancer metastasis and/or recurrence. That is, the cancer vaccine composition of the present invention can preferably be used to treat metastatic and/or recurrent cancer or to prevent cancer metastasis and/or recurrence, and more preferably can be used to prevent cancer metastasis and/or recurrence after surgical therapy.
  • compositions for preventing post-operative cancer metastasis and/or recurrence in cancer patients which comprise the modified cancer cells of the present invention or the cancer vaccine composition of the present invention as an active ingredient.
  • These compositions can be provided as pharmaceuticals.
  • treatment includes, but is not limited to, therapeutic benefit, and is used to mean measures to achieve beneficial or desired results.
  • therapeutic benefit means a complete cure or improvement of the disease being treated.
  • prevention is used to mean reducing the probability of contracting a disease or the probability of a disease recurring.
  • treatment and prevention are not limited to complete treatment and prevention, but may also be treatment and prevention to the extent that a person skilled in the art would recognize as having potential therapeutic and preventive effects (e.g., slowing the progression of a disease, inhibiting the worsening of a disease, delaying the onset of a disease).
  • the term "subject” refers to mammals, including humans, and examples of non-human mammals include mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, and monkeys.
  • the cancer vaccine composition of the present invention can be used in combination with other drugs.
  • “combination” means administering multiple active ingredients simultaneously or separately to the same subject for treatment or prevention.
  • the multiple active ingredients may be contained in the same composition, or may be contained separately in different compositions.
  • other drugs that can be used in combination with the cancer vaccine composition of the present invention include immunostimulants.
  • immunostimulants include antibody preparations such as anti-CTLA-4 antibodies, anti-PD-1 antibodies, or anti-PD-L1 antibodies.
  • the two When the cancer vaccine composition of the present invention is used in combination with another drug, the two may be administered simultaneously or sequentially. When the cancer vaccine composition of the present invention and the other drug are administered sequentially, they may be administered continuously or with an interval between administrations. When administered sequentially, the order of administration is not particularly limited, but when an immunostimulant is used in combination, it is preferable to administer the cancer vaccine composition of the present invention first from the perspective of inducing killer T cells against neoantigens.
  • a combination drug comprising the cancer vaccine composition of the present invention and another drug.
  • the term "combination drug” refers to a combination of different compositions each containing multiple active ingredients separately.
  • the combination drug of the present invention can be used for the treatment and/or prevention of cancer.
  • the combination drug of the present invention can also be administered in accordance with the description of the cancer vaccine composition.
  • the administration route for the cancer vaccine composition of the present invention can be selected from routes suitable for administering the cell preparation, and examples include subcutaneous administration, intradermal administration, topical administration, intraperitoneal administration, intravenous administration, and intramuscular administration.
  • administration sites for subcutaneous or intradermal administration include the back, arm, and thigh.
  • the compositions may be administered at the same site or at different sites.
  • the administration site may be the site where the cancer has developed or nearby, or the site where the cancer is expected to develop, recur, or metastasize or nearby.
  • Dosage forms suitable for administering the cancer vaccine composition of the present invention include, for example, injections. These preparations can be formulated using pharmaceutically acceptable carriers by methods commonly used in the art (for example, known methods described in the General Provisions for Preparations of the 18th Edition of the Japanese Pharmacopoeia, etc.). Pharmaceutically acceptable carriers include, for example, excipients, binders, diluents, stabilizers, buffers, colorants, emulsifiers, dispersants, suspending agents, preservatives, soothing agents, etc.
  • the cancer vaccine composition of the present invention may be in a form that is dissolved or suspended in water or another suitable solvent at the time of use.
  • the dosage can be determined depending on the sex, age, and weight of the recipient, symptoms, dosage form, and route of administration.
  • the single dose of the cancer vaccine composition of the present invention for an adult can be, for example, in the range of 1.0 x 10 to 1.0 x 10 (preferably 1.0 x 10 to 1.0 x 10 ) in terms of the number of modified cancer cells of the present invention, but is not limited thereto.
  • the active ingredient in the above-mentioned dose can also be administered in multiple divided doses.
  • the cancer vaccine composition of the present invention can be administered multiple times (e.g., 2 to 4 times, preferably 2 to 3 times) to enhance the therapeutic or preventive effect.
  • the interval between administrations can be, for example, 1, 2, 3, 4, 5, or 6 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or 1, 2, 3, 4, 5, or 6 months, but is not limited to these.
  • the modified cancer cells of the present invention or the cancer vaccine composition of the present invention can be administered to a subject at risk of developing cancer, thereby reducing the risk of developing the disease.
  • subject at risk of developing cancer refers to a subject who has no subjective symptoms of cancer or has not been diagnosed with cancer, but who is at risk of developing cancer in the future, such as a person who has not been diagnosed with cancer or a cancer patient who has undergone surgery to remove cancer tissue.
  • reducing the risk of developing cancer means reducing the probability of developing cancer, and reducing the probability of developing cancer improves the prognosis of cancer.
  • another aspect of the present invention provides a composition for reducing the risk of developing cancer and a composition for improving prognosis in cancer treatment, which contain the modified cancer cells of the present invention or the cancer vaccine composition of the present invention as an active ingredient.
  • These compositions can be provided as pharmaceuticals.
  • Another aspect of the present invention provides a method for treating or preventing cancer, comprising administering a therapeutically or prophylactically effective amount of the modified cancer cells of the present invention or the cancer vaccine composition of the present invention to a subject suffering from cancer in need thereof.
  • the therapeutic or preventive method of the present invention can be carried out in accordance with the description of the modified cancer cells of the present invention and the cancer vaccine composition of the present invention.
  • a method for reducing the risk of developing cancer or a method for improving prognosis in cancer treatment comprising administering a therapeutically or prophylactically effective amount of modified cancer cells of the present invention or a cancer vaccine composition of the present invention to a subject in need thereof.
  • a method for preventing postoperative cancer metastasis and/or recurrence in a cancer patient or a method for treating metastatic and/or recurrent cancer in a cancer patient comprising administering a therapeutically or prophylactically effective amount of modified cancer cells of the present invention or a cancer vaccine composition of the present invention to a patient in need thereof.
  • the method for reducing the risk of developing cancer, the method for improving prognosis in cancer treatment, the method for preventing postoperative cancer metastasis and/or recurrence in a cancer patient, and the method for treating metastatic and/or recurrent cancer in a cancer patient can be carried out according to the descriptions of the modified cancer cells of the present invention and the cancer vaccine composition of the present invention.
  • Another aspect of the present invention provides use of the modified cancer cells of the present invention for the manufacture of a cancer vaccine composition, a pharmaceutical composition for treating or preventing cancer, a cancer immunotherapy preparation, a composition for reducing the risk of developing cancer, a composition for improving prognosis in cancer treatment, a composition for preventing cancer metastasis and/or recurrence after surgery in cancer patients, or a composition for treating metastatic and/or recurrent cancer in cancer patients.
  • the use of the present invention can be carried out in accordance with the descriptions of the modified cancer cells of the present invention and the cancer vaccine composition of the present invention.
  • Example 1 Analysis of antitumor and cancer vaccine effects using cancer cells expressing a CTL-inducing antigen
  • cancer cells genetically modified to express IL-2 and an antigen that induces cytotoxic T cells against cancer induce antitumor immunity mediated by cytotoxic T cells.
  • Meth-A cells were diluted with PBS and mixed with Matrigel (Corning, #356234, NY, USA) (1:1) on ice for later use.
  • the cell/Matrigel mixture was injected subcutaneously using a 1 mL syringe.
  • the cells were diluted with PBS and injected subcutaneously using a 1 mL syringe.
  • Plasmid construction ⁇ 85A-mIL2 plasmid> A DNA fragment encoding mIL2 (SEQ ID NO: 1) containing BstXI and NotI sites at the 5' and 3' ends, respectively, was amplified by polymerase chain reaction (PCR) using the mIL2 cDNA ORF clone (Sino Biological) as a template. The amplified DNA fragment encoding mIL2 was substituted for the DNA encoding GFP in pIRES-AcGFP1 (Takara Bio) to obtain pIRES-mIL2.
  • PCR polymerase chain reaction
  • a DNA fragment encoding TERTp-85A was inserted into pIRES-mIL2 to obtain TERTp-85A/pIRES-mIL2.
  • the amplified DNA fragment was inserted into pBluescript SK(+) (Stratagene/Agilent) to obtain the plasmid vector (TERTp-85A-IRES-mIL2).
  • telomerase promoter The human telomerase promoter (TERTp) containing a BamHI site at the 5' end and an EcoRI site containing an ATG at the 3' end was amplified by polymerase chain reaction (PCR) as a template.
  • the amplified DNA fragment was inserted into pBluescript SK(+) (Stratagene/Agilent) to obtain the TERTp/pBluescript SK(+) plasmid.
  • a Spike-encoding DNA fragment containing an EcoRI site at the 5' end and a SalI site excluding the ATG at the 3' end was amplified by PCR using the Spike cDNA ORF clone (Sino Biological) as a template.
  • the amplified DNA fragment encoding Spike (SEQ ID NO: 7) was inserted into the TERTp/pBluescript SK(+) plasmid to obtain TERTp-Spike/pBluescript SK(+).
  • a DNA fragment encoding IRES-mIL2-polyA having SalI at the 5' end and XhoI at the 3' end was amplified by PCR using the pIRES-mIL2 plasmid as a template.
  • the amplified DNA fragment was inserted into TERTp-Spike/pBluescript SK(+) to obtain the plasmid vector (TERTp-Spike-IRES-mIL2).
  • ⁇ Plasmid for mIL2 alone> A DNA fragment encoding TERTp (including the first ATG) with EcoRI at the 5' end and BstXI at the 3' end was amplified by PCR using the chemically synthesized DNA/pEX plasmid as a template. The amplified DNA fragment was inserted into the EcoRI and BstXI cleavage sites of pIRES-mIL2 (see the procedure for constructing the 85A-mIL2 plasmid) to obtain a plasmid vector (mIL2-only plasmid).
  • C. Cell culture and transfection Colon26-Luc cells (JCRB1496) were purchased from the National Institutes of Biomedical Innovation, Health and Nutrition. Meth-A cells (TKG 0158) were purchased from the Cell Resource Center, Institute of Development, Aging and Cancer, Tohoku University. LLC-Luc cells (JCRB1716) were purchased from the National Institutes of Biomedical Innovation, Health and Nutrition. Renca cells (CRL-2947) were purchased from the American Type Culture Collection.
  • Cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone), 50 U/mL penicillin, and 50 ⁇ g/mL streptomycin (Invitrogen) at 37°C in a humidified atmosphere containing 5% CO2 .
  • Cell transfection was performed using Targefect-F1 (Nacalai Tesque) according to the manufacturer's protocol. After 48 hours of culture, the medium was replaced with fresh medium. After 48 hours, the cells were transferred to a 10 cm dish and cultured in medium containing 300 ⁇ g/mL hygromycin B (Thermo Fisher Scientific). All cell lines were tested for mycoplasma using a mycoplasma detection kit (Southern Biotech, Birmingham, AL).
  • Anti- ⁇ -actin (A5316, Sigma, 1:5000 dilution), anti-V5-tag (#R960-25, Invitrogen, 1:2000 dilution), anti-HLA A (EP1395Y, abcam, 1:2000 dilution), anti-HLA class I (W6/32, abcam, 1:100 dilution), anti- ⁇ 2-microglobulin (B1G6, BECKMAN COULTER, 1:100 dilution), goat anti-mouse IgG H&L (FITC) (ab6785, abcam, 1:2000 dilution), PE anti-mouse H-2Ld/H-2Db (28-14-8, BioLegend, 1:100 dilution), APC anti-mouse H-2Kb/H-2Db Antibody (28-8-6, BioLegend, 1:100 dilution), anti-mouse IgG, HRP-conjugated whole sheep antibody (NA931, Cytiva, 1:5000 dilution).
  • IVIS Imaging Mice were subcutaneously injected with 30 mg of luciferin (LK10000, OZ Biosciences), a luciferase substrate. Luciferin was dissolved in modified D-PBS (WAKOKKCl 0.02 w/v%, KH2PO4 0.02 w/v%, NaCl 0.8 w/v%, Na2HPO4 0.115 w/v%). Mice were then anesthetized with isoflurane and placed in a supine position on the imaging stage of the IVIS system. Images were collected 10 minutes after luciferin injection using an IVIS Imaging System (Caliper Life Sciences, PerkinElmer). Photons emitted from the tumor and its surrounding area were quantified using Living Image software (PerkinElmer).
  • Etoposide Treatment We examined how the proliferation rate of cancer cells changes with etoposide treatment. Specifically, we measured the proliferation rate (%) of cancer cells when Colon26-Luc cells were contacted with etoposide at 10 ⁇ M or 50 ⁇ M. The results are shown in Table 1.
  • BCG vaccine-derived 85A was selected as the antigen to be introduced into cancer cells to induce CTL in vivo, interleukin-2 (IL2) was linked via an Internal Ribosome Entry Site (IRES), and human telomerase reverse transcriptase (hTERT) was selected as the promoter to express them.
  • the plasmid (TERTp-85A-IRES-mIL2) was constructed according to (1-1) B above.
  • Balb/c mouse-derived colon cancer cells (Colon26-Luc cells) and C57BL/6 mouse-derived Lewis lung carcinoma cells (LLC-Luc cells), which stably express luciferase (Luc), were selected as cancer cells into which the antigen was introduced.
  • the antigen was introduced into the cancer cells by transfecting the cancer cells with the plasmid constructed as described above according to (1-1) (c) above.
  • the introduction of the antigen into the cancer cells was confirmed by the secretion of IL2 into the cell culture supernatant.
  • the amount of IL2 secreted was measured by ELISA according to (1-1) (e) above. As shown in Table 2, it was confirmed that Colon26-Luc cells expressing 85A and IL2 were established.
  • Cancer cells (85A-mIL2-expressing Colon26-Luc cells) transfected with antigen (85A) and cancer cells (Colon26-Luc cells) without antigen transfection were administered to CB6F1 mice, a hybrid of Balb/c and C57BL/6 mice, and the growth of the cancer cells was analyzed.
  • the tumor volume of the cancer cells was measured by IVIS imaging as described above in (1-1) (K). Animal experiments involving subcutaneous administration to animals were performed as described above in (1-1) (A).
  • cancer cells expressing the antigen (85A) (85A-mIL2-expressing Colon26-Luc cells) were administered, and then cancer cells not expressing the antigen (Colon26-Luc cells) were re-transplanted into mice in which the cancer cells had disappeared (see (1-2)A above), and analysis was performed to determine whether sensitized CTLs against neoantigen-derived peptides against the cancer cells were induced.
  • CB6F1 mice administered with cancer cells (85A-mIL2-expressing Colon26-Luc cells) transfected with the antigen (85A) were generated according to the procedure described above in (1-2)A. Animal experiments involving subcutaneous administration of cancer cells (Colon26-Luc cells) not expressing the antigen (85A) to animals were performed according to the procedure described above in (1-1)A.
  • Table 8 shows the results of verifying the vaccine effect on etoposide-treated antigen-expressing cancer cells (85A-mIL2-expressing Colon26-Luc cells).
  • LLC-Luc cells were also used as cancer cells expressing the antigen (Spike). Furthermore, experiments in which etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing Colon26-Luc cells and Spike-mIL2-expressing LLC-Luc cells) were administered to experimental animals not only once, but also multiple times (see Figure 1).
  • Table 9 shows the results of administering cancer cells (Colon26-Luc cells) expressing the antigen (Spike) to experimental animals.
  • Table 10 shows the results of verifying the vaccine effect for cancer cells (Spike-mIL2-expressing Colon26-Luc cells) in which an antigen (Spike) was expressed.
  • Table 11 shows the results of administering cancer cells (LLC-Luc cells) expressing the antigen (Spike) to experimental animals.
  • Table 12 shows the results of testing the vaccine efficacy of cancer cells (Spike-mIL2-expressing LLC-Luc cells) expressing the antigen (Spike).
  • Table 13 shows the results of administering etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing Colon26-Luc cells) to experimental animals.
  • Table 14 shows the results of verifying the vaccine effect on etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing Colon26-Luc cells).
  • etoposide-treated antigen-expressing cancer cells 85A-mIL2-expressing Colon26-Luc cells
  • etoposide-treated antigen-expressing cancer cells also had a vaccine effect in inducing immunity against tumors.
  • Table 15 shows the results of administering etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing LLC-Luc cells) to experimental animals.
  • Table 16 shows the results of verifying the vaccine effect on etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing LLC-Luc cells).
  • etoposide-treated antigen-expressing cancer cells (Spike-mIL2-expressing LLC-Luc cells) also had a vaccine effect in inducing immunity against tumors.
  • cancer cells expressing only mIL2 (mIL2-expressing Renca cells) and wild-type cancer cells (Renca cells) that had not been transfected with mIL2 or antigen (Spike) were administered to CB6F1 mice, and the growth of the cancer cells was analyzed.
  • the transduction plasmid (mIL2 alone) was constructed according to (1-1) (i) above.
  • Table 18 shows the results of verifying the vaccine effect on cancer cells in which mIL2 was expressed (mIL2-expressing Renca cells).
  • mice were administered cancer cells expressing mIL2 (mIL2-expressing Renca cells) and the cancer cells disappeared, wild-type mouse cancer cells (Renca cells) were transplanted into the mice.
  • wild-type mouse cancer cells Renca cells
  • the remaining mice 50%
  • the cancer cells proliferated.
  • Table 20 shows the results of verifying the vaccine effect of cancer cells (Spike-mIL2-expressing Renca cells) expressing the antigen (Spike).
  • Example 2 Evaluation of the applicability of CTL-inducing antigen-expressing cancer cells to humans using an HLA expression system
  • an extrapolation experiment to humans was carried out on the antigen-expressing cancer cells prepared in Example 1 using HLA-expressing mouse cancer cells and a cancer-bearing mouse model of an HLA-expressing knock-in mouse, and efficacy in humans was verified by evaluating the antigen reactivity in a situation closer to that of humans.
  • pX458 (Cas9-2A-GFP) was used as the CRISPR-Cas9 vector (see Addgene: pSpCas9(BB)-2A-GFP(pX458) instructions).
  • a plasmid with the Exon1 or Exon2 target sequence inserted below the guide RNA sequence of pX458 (Cas9-2A-GFP) plasmid DNA was introduced into LLC-Luc cells by electroporation, and GFP (green fluorescent protein)-positive fractions (CRISPR-Cas9/Exon1, Exon2 plasmid-introduced cell population) were collected.
  • the collected GFP-positive fractions (CRISPR-Cas9/Exon1, Exon2 plasmid-introduced cell population) were stained with mouse H-2D b -PE antibody (Monoclonal Antibody (28-14-8)-PE-labeled eBioscienceTM (Invitotogen/ThermoFisherSciene)), and the PE-negative fractions (M fractions) were collected and cultured. This procedure was repeated five times, and the cell populations that became almost negative fractions were designated as LLC-Luc cells in which the ⁇ 2M gene was disrupted ( ⁇ m ⁇ 2M LLC-Luc cells).
  • a DNA fragment containing the 5' leader sequence region of ⁇ 2M exon 1, which contains the CRISPR-Cas9 target sequence, to the 3' end of exon 2 was PCR amplified, and the resulting DNA was digested with restriction enzymes BamH1 and Sal1 and purified.
  • the purified DNA fragment was cut with EcoRV, and the DNA fragment containing exon 1 (BamHI-EcoRV ⁇ 300 bp) and exon 2 (SalI-EcoRV ⁇ 400 bp) was inserted into the pBluescriptSK(+) vector and transformed into E. coli. After insert checking, clones containing inserts were outsourced for DNA sequencing.
  • HLA-Expressing ⁇ m ⁇ 2M LLC-Luc cells were prepared by introducing an HLA expression unit into ⁇ m ⁇ 2M LLC-Luc cells.
  • HLA-A2 gene A0201
  • human HLA-A2 expression unit DNA (PGK promoter + human ⁇ 2M-cDNA - HLA ⁇ 1 ⁇ 2-gDNA + mouse H2Db ⁇ 3-gDNA) was introduced into the ⁇ m ⁇ 2M LLC-Luc cells prepared in (2-1) above, resulting in the expression of human HLA class I.
  • the detailed procedure for constructing the human HLA-A2 expression unit DNA is shown in Figure 5(A).
  • a schematic diagram of the chimeric MHC class I expressed by the human HLA-A2 expression unit and its behavior within the cells is shown in Figure 5(B).
  • HLA in the obtained cells was confirmed using flow cytometry and Western blotting. Specifically, this was confirmed using the following procedure.
  • Flow cytometry> The cultured cells were washed with PBS, then detached with EDTA-containing trypsin and collected. Primary antibodies diluted in PBS containing 2% FBS were added to the collected cells and incubated at 4°C for 1 hour. After centrifugation, the cells were washed three times with PBS. Fluorescently labeled secondary antibodies diluted in PBS containing 2% FBS were added and incubated at 4°C for 30 minutes. The cells were washed three times with PBS and analyzed using a FACSCalibur flow cytometer (BD Biosciences).
  • the primary antibody was diluted with Can Get Signal Solution 1 (TOYOBO) and incubated at room temperature for 1 hour.
  • the secondary antibody was diluted with Can Get Signal Solution 2 (TOYOBO) and incubated at room temperature for 30 minutes.
  • detection was performed using ECL (trademark) Select Western blot detection reagent.
  • HLA-A2-expressing ⁇ m ⁇ 2M LLC-Luc cells prepared in (2-2) above were transfected with a plasmid (TERTp-85A-IRES-mIL2) to prepare HLA-expressing cancer cells expressing BCG vaccine-derived 85A.
  • the plasmid was prepared and transfected according to the procedure in (1-2) above.
  • IL2 secretion levels were measured to confirm the establishment of HLA-expressing ⁇ m ⁇ 2M LLC-Luc cells expressing 85A and IL2.
  • HLA-A2-expressing ⁇ m ⁇ 2M LLC-Luc cells (85A-mIL2-expressing HLA-A2 cells) transfected with antigen (85A) and HLA-A2-expressing ⁇ m ⁇ 2M LLC-Luc cells (HLA-A2 cells) without antigen transfection were administered to HLA-A2-expressing knock-in mice (generated in accordance with WO 2015/056774), and cancer cell growth was analyzed. Cancer cell tumor volume was measured by IVIS imaging as described above in (1-1) (K). Animal experiments involving subcutaneous administration to animals were performed as described above in (1-1) (A).

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Abstract

Le but de la présente invention est de fournir une composition vaccinale contre le cancer efficace pour traiter ou prévenir le cancer. La présente invention concerne une cellule cancéreuse génétiquement modifiée comprenant : un acide nucléique codant pour un antigène induisant des lymphocytes T cytotoxiques et un acide nucléique codant pour IL-2. Cette cellule cancéreuse génétiquement modifiée peut être utilisée en tant que principe actif d'une composition vaccinale contre le cancer. L'antigène induisant des lymphocytes T cytotoxiques dans cette cellule cancéreuse génétiquement modifiée est de préférence un antigène dérivé d'un agent pathogène ou un antigène dérivé d'un virus.
PCT/JP2025/016891 2024-05-08 2025-05-08 Cellule cancéreuse modifiée et composition vaccinale contre le cancer la contenant Pending WO2025234450A1 (fr)

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