Classifications

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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2827—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
  • A61K40/22—Immunosuppressive or immunotolerising
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/418—Antigens related to induction of tolerance to non-self
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
  • A61K40/421—Immunoglobulin superfamily
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
  • A61K40/421—Immunoglobulin superfamily
  • A61K40/4213—CD74, Ii, MHC class II invariant chain or MHC class II gamma chain
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/50—Cellular immunotherapy characterised by the use of allogeneic cells
• • 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
• • 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
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • 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
  • A61P37/08—Antiallergic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
  • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
• • 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
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • 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
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
  • C07K16/244—Interleukins [IL]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • 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
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]
  • C12N2501/2302—Interleukin-2 (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
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/50—Cell markers; Cell surface determinants
  • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)

Definitions

• Liver transplantation has been widely used as a definitive treatment for patients with end-stage liver failure. There are 20,000 cases overseas and more than 500 cases in Japan each year.
• Transplantation is one of the primary treatments of choice for end-stage renal, cardiac, hepatic, and pancreatic organ failure, and despite significant advances in the treatment of transplant rejection in recent years, the majority of transplants are ultimately rejected.
• Current immunosuppressive regimens which rely on continuous drug therapy, predispose organ transplant patients to increased susceptibility to infections and cancer, as even the drugs fail to inhibit responses specifically directed against the transplant.
• One technique for inducing immune tolerance is the induction of antigen-specific immune unresponsiveness (anergy) in T cells.
• One method involves co-culturing regulatory T cells (Treg) with donor cells in the presence of IL-2, and using the donor-specific Tregs that have been divided and proliferated for treatment, while another method involves administering a low dose of IL-2 to proliferate Tregs in vivo and using them for treatment.
• these methods have problems, such as the risk of IL-2 activating other T cells and the proliferation of polyclonal Tregs that are not donor-specific.
• antigen-specific induced suppressor T cells can be selectively induced by contacting cells with a specific factor. Furthermore, the inventors have discovered that regulating the production or function of IL-2 is important in inducing antigen-specific induced suppressor T cells, and have also discovered a new method for inducing antigen-specific induced suppressor T cells by regulating the production or function of IL-2.
• the present invention provides, for example: (Item 1) A method for selectively inducing antigen-specific induced inhibitory T cells from cells derived from a subject, the method comprising a step of mixing a factor that selectively induces antigen-specific induced inhibitory T cells, cells derived from the subject, and an antigen derived from the subject or an antigen not derived from the subject, or a material containing an antigen not derived from the subject. (Item 2) The method according to the above item, wherein the factor that selectively induces antigen-specific induced suppressor T cells is a regulatory factor capable of regulating the production of IL-2 or a regulatory factor capable of regulating the function of the produced IL-2.
• the factor that selectively induces antigen-specific induced suppressor T cells is an inhibitory factor capable of inhibiting the production of IL-2 or an inhibitory factor capable of inhibiting the function of the produced IL-2.
• the factor that selectively induces antigen-specific induced suppressor T cells is an inhibitory factor capable of inhibiting the interaction between IL-2 and IL-2 receptor (IL-2R) and/or an inhibitory factor capable of inhibiting the interaction between CD80 and/or CD86 and CD28.
• IL-2R IL-2 receptor
• the method according to any one of the above items, wherein the factor that selectively induces antigen-specific induced inhibitory T cells is an inhibitor capable of inhibiting the interaction between IL-2 and IL-2R, selected from an anti-IL-2 antibody, an anti-IL-2R antibody, an anti-IL-2R ⁇ (CD25) antibody, an anti-IL-2R ⁇ (CD122) antibody, or an antigen-binding fragment thereof.
• the factor that selectively induces antigen-specific induced inhibitory T cells is an inhibitor capable of inhibiting the interaction between CD80 and/or CD86 and CD28, selected from the group consisting of an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein.
• a method for producing induced suppressor T cells from cells derived from a subject comprising the step of mixing a regulatory factor capable of regulating the production of IL-2 or a regulatory factor capable of regulating the function of the produced IL-2, cells derived from the subject, and an antigen derived from the subject or an antigen not derived from the subject, or a substance containing the antigen.
• the regulatory factor capable of regulating the production of IL-2 is selected from an inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD28, and a small molecule compound that inhibits IL-2 production.
• the regulator capable of regulating the function of the produced IL-2 is selected from an inhibitor capable of inhibiting the interaction of IL-2 with IL-2 receptor (IL-2R) and a small molecule compound that inhibits the action of IL-2.
• the inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD28 is selected from the group consisting of an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein.
• compositions for selectively inducing antigen-specific induced suppressor T cells from cells derived from a subject comprising a regulatory factor capable of regulating the production of IL-2 or a regulatory factor capable of regulating the function of the produced IL-2, characterized in that the composition is contacted with cells derived from the subject and an antigen derived from the subject or an antigen not derived from the subject or a substance containing the antigen.
• a composition for treating a disease in a subject mediated by an immune response comprising a regulatory factor capable of regulating the production of IL-2 or a regulatory factor capable of regulating the function of the produced IL-2, and antigen-specific induced suppressor T cells selectively induced by mixing cells derived from the subject with an antigen derived from the subject or an antigen not derived from the subject or a substance containing the antigen.
• a method for treating a disease in a subject mediated by an immune response comprising the steps of mixing a regulatory factor capable of regulating the production of IL-2 or a regulatory factor capable of regulating the function of the produced IL-2, cells derived from the subject, and an antigen derived from the subject or an antigen not derived from the subject or a substance containing the antigen, and administering to the subject antigen-specific induced suppressor T cells selectively induced by the mixing.
• An antigen-specific induced suppressor T cell for treating a disease in a subject mediated by an immune response the antigen-specific induced suppressor T cell being selectively induced by mixing a regulatory factor capable of regulating the production of IL-2 or a regulatory factor capable of regulating the function of the produced IL-2, cells derived from the subject, and an antigen derived from the subject or an antigen not derived from the subject, or a substance containing the antigen.
• (Item 13C) Use of antigen-specific induced inhibitory T cells in the manufacture of a medicine for treating a disease in a subject mediated by an immune response, wherein the antigen-specific induced inhibitory T cells are selectively induced by mixing a regulatory factor capable of regulating the production of IL-2 or a regulatory factor capable of regulating the function of the produced IL-2, cells derived from the subject, and an antigen derived from the subject or an antigen not derived from the subject or a substance containing the antigen.
• transplant immune rejection allergy, autoimmune disease, graft-versus-host disease, and immune rejection caused by transplantation of iPS cells or ES cells, or cells, tissues, or organs differentiated therefrom.
• the subject is a human.
• the method disclosed herein makes it possible to selectively induce antigen-specific induced suppressor T cells by contacting cells with a specific factor.
• FIG. 1 shows an outline of the experiment in Example 1.
• Figure 2 shows the antigen-specific suppressive ability of whole cells and Tregs by culturing in the presence of anti-CD80/86 antibodies. The left figure shows the results of confirming Treg proliferation, and the right figure shows a graph of IFN- ⁇ production by stimulation with a stimulator.
• FIG. 3 shows an outline of the experiment in Example 1.
• Figure 4 shows the antigen-specific suppressive ability of whole cells and Tregs when IL-2 was further added to culture in the presence of anti-CD80/86 antibodies. The left figure shows the results of confirming Treg proliferation, and the right figure shows a graph of IFN- ⁇ production by stimulation with a stimulator.
• FIG. 5 shows an outline of the experiment in Example 2.
• Figure 6 shows the antigen-specific suppressive ability of whole cells and Tregs by culturing in the presence of anti-IL-2 antibody.
• the left figure shows the results of confirming Treg proliferation, and the right figure shows a graph of IFN- ⁇ production by stimulation with a stimulator.
• FIG. 7 shows a schematic diagram of acquisition of antigen-specific suppressive ability by anti-CD80/86 antibody.
• FIG. 8 shows a schematic diagram of the acquisition of antigen-specific suppressive ability by anti-IL-2 antibodies.
• FIG. 9 shows a graph of IL-2 production by anti-CD80/86 antibodies.
• FIG. 10 shows a table of IL-2 production by anti-CD80/86 antibodies.
• FIG. 11 shows a table of the percentage of CD4 + T cells in cell populations cultured in the presence of anti-CD80/86 and/or anti-IL-2 antibodies.
• Figure 12 shows confirmation of the antigen-specific suppressive ability of whole cells and Tregs by culture in the presence of anti-CD80/86 antibodies and/or anti-IL-2 antibodies.
• FIG. 13 shows a table summarizing the results of FIG.
• immune tolerance refers to a state in which a specific immune response to a specific antigen is not shown or the specific immune response is suppressed.
• Immune tolerance may mean both or either of a state in which immune cells (especially T cells) do not show a specific immune response to a specific antigen or the specific immune response is suppressed, and a state in which a human does not show a specific immune response to a specific antigen or the specific immune response is suppressed.
• Immune tolerance has attracted attention because it makes it possible to treat immune rejection reactions and allergies by inducing immune tolerance.
• PBMCs or T cells in which immune tolerance has been induced
• anergy PBMCs or T cells
• subject includes domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
• domestic animals e.g., cows, sheep, cats, dogs, horses
• primates e.g., humans and non-human primates such as monkeys
• rabbits e.g., mice and rats
• rodents e.g., mice and rats.
• the subject is a human.
• the term "factor” may refer to any substance or other element (e.g., energy such as light, radioactivity, heat, electricity, etc.) as long as it can achieve the intended purpose.
• substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (e.g., hormones, ligands, signaling substances, other small organic compounds, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.)), and composite molecules thereof.
• proteins e.g., proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucle
• regulatory factor refers to any type of factor, such as a small molecule, protein, nucleic acid, lipid, or sugar, that positively or negatively regulates a given action (e.g., interaction, signal transduction, protein production, protein function, etc.).
• regulation includes modification or non-modification to any level, such as enhancement (increase), as well as reduction (inhibition, suppression), and disappearance, and as used herein, “regulatory factor” includes “inhibitory factor” and "enhancing factor”, etc.
• the proliferation of induced suppressive T cells specific to a particular antigen is promoted by regulating the production of IL-2 or the function of the produced IL-2.
• IL-2 also promotes the proliferation of induced suppressive T cells nonspecific to a particular antigen, it is possible to suppress the proliferation of nonspecific induced suppressive T cells by regulating the production of IL-2 or the function of the produced IL-2. Since complete inhibition of the production and function of IL-2 also suppresses the proliferation of antigen-specific induced suppressive T cells, it is preferable to have a certain amount of IL-2.
• the methods, factors, and compositions disclosed herein can be adjusted to achieve the presence of a predetermined amount of IL-2.
• the term "inhibitory factor” refers to any type of factor, such as a small molecule, protein, nucleic acid, lipid, or sugar, that can inhibit a certain action (e.g., interaction, signal transduction, protein production, protein function, etc.).
• a certain action e.g., interaction, signal transduction, protein production, protein function, etc.
• anergy is induced in T cells by blocking the interaction between CD80 and/or CD86 on the cell surface and CD28 and inhibiting the CD28 costimulatory signal.
• the inhibitor used to block the interaction between CD80 and/or CD86 and CD28 is selected from the group consisting of a small molecule, a protein, a nucleic acid, a lipid, a sugar, and a combination thereof.
• the protein is an antibody or a variant thereof, or a cell surface molecule or a variant thereof.
• the variant of the antibody is an antigen-binding fragment.
• the variant of the cell surface molecule is a fusion protein.
• the inhibitor is selected from the group consisting of an anti-CD80 antibody, an anti-CD86 antibody, a bispecific antibody against CD80 and CD86, an anti-CD28 antibody or an antigen-binding fragment thereof, a CTLA4-Ig fusion protein, and a CD28-Ig fusion protein.
• the CTLA4-Ig fusion protein is abatacept or belatacept. It is also envisioned that an inhibitor that indirectly inhibits the above interaction (e.g., an inhibitor of an upstream or downstream signal transduction) may also be used in combination.
• antibody a molecule or a group thereof that can specifically bind to a particular epitope on an antigen.
• a full-length antibody i.e., an antibody having an Fc portion
• an antibody lacking an Fc portion may be capable of binding to an antigen of interest, and examples of such an antibody include, but are not limited to, a Fab antibody, an F(ab') 2 antibody, an Fab' antibody, an Fv antibody, and an scFv antibody.
• the antibody may be any type of antibody, i.e., an immunoglobulin known in the art.
• the antibody is an antibody of the isotype IgA, IgD, IgE, IgG, or IgM class.
• the antibody described herein comprises one or more alpha, delta, epsilon, gamma, and/or mu heavy chains.
• the antibody described herein comprises one or more kappa or light chains.
• the antibody is an IgG antibody and is one of the four human subclasses: IgG1, IgG2, IgG3, and IgG4.
• Antibodies contemplated for use in the present disclosure also include camelid-derived antibodies (e.g., VHH antibodies), shark-derived antibodies (e.g., single-chain antibodies), peptibodies, nanobodies (single domain antibodies), minibodies, multispecific antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv), and the like, which are known in the art. See, e.g., Kortt et al., Biomol Eng. 2001 18:95-108, (2001) and Todorovska et al., J Immunol Methods. 248:47-66, (2001). The antibody also includes modified or unmodified antibodies.
• camelid-derived antibodies e.g., VHH antibodies
• shark-derived antibodies e.g., single-chain antibodies
• peptibodies e.g., single-chain antibodies
• nanobodies single domain antibodies
• minibodies
• the modified antibody may be bound to an antibody with various molecules such as polyethylene glycol.
• the modified antibody can be obtained by chemically modifying the antibody using a known method. For artificially produced antibodies and various methods of modifying/altering antibodies, see also Biochemistry (2016) 88:380-385.
• the term “antibody” in the narrow sense refers to an immunoglobulin or a group thereof that can specifically bind to a specific epitope on an antigen, and a variant thereof is referred to as a "variant of an antibody".
• the term “antibody” in the narrow sense may be a full-length antibody (i.e., an antibody having an Fc portion), and in the present specification, the term “variant of an antibody” may be a variant lacking the Fc portion of the antibody.
• an antibody in the narrow sense may also be referred to as a full-length antibody, and a variant of an antibody may also be referred to as a variant of a full-length antibody.
• a variant lacking an Fc portion may be capable of binding to an antigen of interest, and examples of such variants include, but are not limited to, Fab antibodies, F(ab') 2 antibodies, Fab' antibodies, Fv antibodies, and scFv antibodies.
• variants of an antibody include modified or unmodified antibodies.
• the modified antibody may be bound to an antibody with various molecules such as polyethylene glycol.
• the modified antibody can be obtained by chemically modifying an antibody using a known method.
• a "polyclonal antibody” can be produced, for example, by administering an immunogen containing an antigen of interest to a mammal (e.g., rat, mouse, rabbit, cow, monkey, etc.), bird, etc., to induce production of polyclonal antibodies specific to the antigen.
• the administration of the immunogen may be by injection of one or more immunizing agents and, if desired, an adjuvant.
• Adjuvants may be used to increase the immune response and may include Freund's adjuvant (complete or incomplete), mineral gels (e.g., aluminum hydroxide), or surfactants (e.g., lysolecithin), etc.
• Immunization protocols are known in the art and may be performed by any method that induces an immune response, tailored to the host organism of choice (Protein Experiment Handbook, Yodosha (2003): 86-91.).
• a "monoclonal antibody” includes a population in which the individual antibodies are substantially identical to a single epitope, except for antibodies with a small amount of naturally occurring mutations.
• the individual antibodies in the population may be substantially identical to a single epitope, except for antibodies with a small amount of naturally occurring mutations.
• Monoclonal antibodies are highly specific and differ from conventional polyclonal antibodies, which typically contain different antibodies that are directed to different epitopes and/or different antibodies that are directed to the same epitope. In addition to their specificity, monoclonal antibodies are useful in that they can be synthesized from hybridoma cultures that are uncontaminated by other immunoglobulins.
• monoclonal may indicate the characteristic of being obtained from a substantially homogeneous population of antibodies, but does not imply that the antibody must be produced by any particular method.
• monoclonal antibodies may be produced by methods similar to the hybridoma method described in "Kohler G, Milstein C., Nature. 1975 Aug 7; 256(5517): 495-497.”
• monoclonal antibodies may be produced by methods similar to the recombinant method described in U.S. Pat. No. 4,816,567.
• monoclonal antibodies may be isolated from phage antibody libraries using methods similar to the techniques described in "Clackson et al., Nature.
• monoclonal antibodies may be produced by methods described in "Protein Experiment Handbook, Yodosha (2003): 92-96.”
• a "chimeric antibody” is, for example, a combination of an antibody variable region and an antibody constant region between different organisms, and can be constructed by recombinant gene technology.
• a mouse-human chimeric antibody can be produced, for example, by the method described in "Roguska et al., Proc Natl Acad Sci USA. 1994Feb 1;91(3):969-973.”
• the basic method for producing a mouse-human chimeric antibody is, for example, to link a mouse leader sequence and a variable region sequence present in a cloned cDNA to a sequence encoding a human antibody constant region already present in an expression vector for mammalian cells.
• the mouse leader sequence and the variable region sequence present in the cloned cDNA may be linked to a sequence encoding a human antibody constant region, and then linked to a mammalian cell expression vector.
• the fragment of the human antibody constant region can be any human antibody H chain constant region or human antibody L chain constant region, for example, C ⁇ 1, C ⁇ 2, C ⁇ 3, or C ⁇ 4 for the human H chain, and C ⁇ or C ⁇ for the L chain.
• a "humanized antibody” is an antibody that has, for example, one or more CDRs from a non-human species, a framework region (FR) from a human immunoglobulin, and a constant region from a human immunoglobulin, and that binds to a desired antigen.
• Antibody humanization can be performed using various techniques known in the art (Almagro et al., Front Biosci. 2008 Jan 1;13:1619-1633.). For example, CDR grafting (Ozaki et al., Blood. 1999 Jun 1; 93(11): 3922-3930.), Re-surfacing (Roguska et al., Proc Natl Acad Sci USA.
• FR shuffling (Damschroder et al., Mol Immunol. 2007 Apr; 44(11): 3049-3060. Epub 2007 Jan 22.) can be used.
• amino acid residues in the human FR regions may be replaced with corresponding residues from the CDR donor antibody. This FR substitution can be performed by methods well known in the art (Riechmann et al., Nature. 1988 Mar 24;332(6162):323-327.).
• FR residues important for antigen binding can be identified by modeling the interactions of CDR and FR residues.
• unusual FR residues at specific positions can be identified by sequence comparison.
• a "human antibody” is, for example, an antibody in which the regions constituting the antibody, including the variable and constant regions of the heavy chain and the variable and constant regions of the light chain, are derived from genes encoding human immunoglobulin.
• Major production methods include the transgenic mouse method for producing human antibodies and the phage display method.
• the transgenic mouse method for producing human antibodies if a functional human Ig gene is introduced into a mouse in which endogenous Ig has been knocked out, human antibodies with diverse antigen-binding abilities are produced instead of mouse antibodies. Furthermore, if this mouse is immunized, human monoclonal antibodies can be obtained by the conventional hybridoma method.
• the phage display method is a system in which a foreign gene is expressed as a fusion protein on the N-terminus of the coat protein (g3p, g10p, etc.) of a filamentous phage, typically an E. coli virus such as M13 or T7, so that the phage does not lose its infectivity.
• a filamentous phage typically an E. coli virus such as M13 or T7, so that the phage does not lose its infectivity.
• it can be produced by the method described in "Vaughan et al., Nat Biotechnol. 1996 Mar;14(3):309-314.”
• antigen-binding fragment of an antibody refers to a fragment of any length that maintains the antibody's ability to bind to an antigen.
• antigen-binding fragments include, but are not limited to, Fab, F(ab') 2 , Fab', Fv, and scFv.
• fusion protein refers to a single protein in which two or more different proteins or fragments thereof are covalently linked, or in which the genes of these proteins are recombinantly expressed together.
• cells derived from a subject refers to cells obtained from a subject to which the composition, pharmaceutical, cell preparation, or cells disclosed herein is administered or which is the subject of the method disclosed herein, or cells derived from cells obtained from the subject.
• antigen derived from a subject refers to an antigen produced by the subject itself that elicits an immune response, for example, an antigen produced by the subject itself that causes an autoimmune disease in a subject with an autoimmune disease.
• antigen not derived from a subject refers to a foreign antigen that can elicit an immune response.
• a substance containing an antigen not derived from a subject refers to any substance or collection of substances that contains an antigen not derived from a subject, such as a cell, cell population, tissue, etc. that expresses an antigen not derived from a subject.
• transplant immune rejection refers to a condition in which the immune system of a subject who has received an organ, tissue, or cell transplant attacks and damages or destroys the transplanted organ, tissue, or cell.
• allergens not derived from the subject that cause allergies are also called allergens, and examples of such antigens include, but are not limited to, dust mite antigens, egg white antigens, milk antigens, wheat antigens, peanut antigens, soybean antigens, buckwheat antigens, sesame antigens, rice antigens, crustacean antigens, kiwi antigens, apple antigens, banana antigens, peach antigens, tomato antigens, tuna antigens, salmon antigens, mackerel antigens, beef antigens, chicken antigens, pork antigens, cat dander antigens, insect antigens, pollen antigens, dog dander antigens, fungal antigens, bacterial antigens, latex, haptens, and metals.
• autoimmune disease refers to any disease in which the immune system mounts an unwanted immune response against one's own cells, tissues, or organs.
• autoimmune diseases include rheumatoid arthritis, multiple sclerosis, type 1 diabetes, inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), systemic lupus erythematosus, psoriasis, scleroderma, autoimmune thyroid disease, alopecia areata, Graves' disease, Guillain-Barré syndrome, celiac disease, Sjögren's syndrome, rheumatic fever, gastritis, autoimmune atrophic gastritis, autoimmune hepatitis, insulitis, oophoritis, and sepsis.
• focal ulcers include, but are not limited to, focal ulcers, uveitis, phacogenic uveitis, myasthenia gravis, primary myxedema, pernicious anemia, autoimmune hemolytic anemia, Addison's disease, scleroderma, Goodpasture's syndrome, nephritis (e.g., glomerulonephritis), psoriasis, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, idiopathic thrombocytopenic purpura, idiopathic leukopenia, Wegener's granulomatosis, and poly/dermatomyositis.
• focal ulcers include focal ulcers, uveitis, phacogenic uveitis, myasthenia gravis, primary myxedema, pernicious anemia, autoimmune hemolytic anemia, Addison's disease, scleroderma, Goodpasture's
• graft-versus-host disease refers to an immune response in which a transplanted organ, tissue, or cell attacks, damages, or destroys the cells, tissues, or organs of the recipient.
• immune rejection caused by transplantation of iPS cells or ES cells, or cells, tissues, or organs differentiated from these cells refers to an immune rejection caused by an antigen present in iPS cells or ES cells, or an antigen present in cells, tissues, or organs differentiated from iPS cells or ES cells.
• regulatory T cells also referred to as Tregs
• regulatory T cells refer to T cells that control the immune response to an antigen. Regulatory T cells may be Foxp3 positive. Regulatory T cells may be CD4 positive or CD8 positive. Typically, regulatory T cells may be Foxp3 positive, CD25 positive, and CD4 positive.
• suppressor T cell refers to a T cell that suppresses the immune response to an antigen and/or that does not show or has a suppressed immune response to an antigen.
• antigen-specific suppressor T cells refers to T cells that specifically suppress immune responses to a specific antigen and/or T cells that do not show or are suppressed from showing a specific immune response to a specific antigen.
• induced suppressor T cells refers to suppressor T cells induced by stimulation with the methods, compositions, or factors disclosed herein, and refers to a cell population including regulatory T cells (e.g., FOXP3-positive CD4-positive CD25-positive T cells) and suppressor T cells (e.g., CD4-positive anergy T cells or CD8-positive anergy T cells).
• CD8-positive anergy cells and CD4-positive anergy cells contain many CD44-positive cells, and CD8-positive cells and/or CD4-positive cells may be CD44-positive.
• CD45RA/CD45RO can also be used to confirm the generation of anergy cells.
• CD8-positive anergy T cells and/or CD4-positive anergy T cells are CD45RA-negative and CD45RO-positive.
• antigen-specific induced suppressor T cells refers to induced suppressor T cells that specifically suppress immune responses to a specific antigen and/or that do not show or are suppressed to show a specific immune response to a specific antigen.
• selectively inducing antigen-specific induced suppressive T cells does not necessarily require that the induced suppressive T cells be 100% antigen-specific, but refers to inducing an increase in the proportion of antigen-specific induced suppressive T cells when stimulated with the method, composition, or factor disclosed herein (for example, when cells are contacted with a factor that selectively induces antigen-specific induced suppressive T cells) compared to when not stimulated with the method, composition, or factor disclosed herein (for example, when cells are not contacted with a factor that selectively induces antigen-specific induced suppressive T cells).
• factor that selectively induces antigen-specific induced suppressor T cells refers to any factor that induces an increase in the proportion of antigen-specific induced suppressor T cells compared to when the factor is not in contact with cells, and does not necessarily require that the induced suppressor T cells be 100% antigen-specific.
• the inventors have found that in the induction of inducible suppressor T cells, regulating the production of the growth factor IL-2 or the function of the produced IL-2 induces antigen-specific suppressive ability in the inducible suppressor T cells. Furthermore, the inventors have also found a new culture method for inducing antigen specificity by adding a regulatory factor capable of regulating IL-2 production or a regulatory factor capable of regulating the function of the produced IL-2, with the aim of suppressing the antigen-nonspecific division and proliferation of inducible suppressor T cells.
• the present disclosure provides a method for selectively inducing antigen-specific induced suppressor T cells from cells derived from a subject, the method comprising the step of mixing a factor that selectively induces antigen-specific induced suppressor T cells, cells derived from the subject, and an antigen derived from the subject or an antigen not derived from the subject, or a substance containing the antigen.
• the substance containing the antigen may be a cell, which may be irradiated to prevent proliferation and activation of the cell.
• the present disclosure provides a method for producing induced suppressor T cells from cells derived from a subject, the method comprising the step of mixing an inhibitor capable of inhibiting the production of IL-2 or an inhibitor capable of inhibiting the function of the produced IL-2, cells derived from the subject, and an antigen derived from the subject or an antigen not derived from the subject, or a substance containing the antigen.
• the present disclosure provides a method for suppressing the proliferation of induced suppressive T cells non-specific to a specific antigen in the production of induced suppressive T cells from cells derived from a subject, the method comprising the step of mixing a factor that selectively induces antigen-specific induced suppressive T cells, cells derived from the subject, and an antigen derived from the subject or an antigen not derived from the subject, or a substance containing the antigen.
• the method of the present disclosure includes culturing a mixture containing the above-mentioned factor, cells derived from a subject, and antigens derived from the subject or antigens not derived from the subject or antigens contained therein.
• the induced suppressor T cells of the present disclosure can include CD4 positive anergy T cells, CD8 positive T cells, and regulatory T cells.
• the regulatory T cells exhibit cell surface markers such as Foxp3 + , preferably CD4 + , CD25 + , and Foxp3 + .
• the percentage of CD4 + T cells in the culture at the end of the culture using the disclosed methods, factors, and compositions can be about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more of the total cells. In a preferred embodiment, the percentage of CD4 + T cells in the culture can be about 35% or more of the total cells.
• the percentage of regulatory T cells (e.g., CD4 + Foxp3 + T cells) in the culture at the end of culture using the disclosed methods, factors, and compositions may be about 5% or more, about 10% or more, about 15% or more, about 20% or more, or about 25% or more of total cells. In a preferred embodiment, the percentage of regulatory T cells in the culture may be about 15% or more of total cells.
• the disclosed methods, factors, and compositions can increase the proportion of CD4 + regulatory T cells (e.g., CD4 + Foxp3 + T cells) among CD4 + T cells.
• the proportion of CD4 + regulatory T cells (e.g., CD4 + Foxp3 + T cells) among CD4 + T cells can be about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 80% or more, about 90% or more, or about 100%.
• the percentage of CD4 + regulatory T cells (e.g., CD4 + Foxp3 + T cells) among CD4 + T cells in cultures at the end of culture using the methods, factors, and compositions of the present disclosure may be about 50% or greater.
• the cells can be peripheral blood mononuclear cells (PBMCs), spleen cells, bone marrow cells, lymph node cells, or any combination thereof.
• PBMCs peripheral blood mononuclear cells
• spleen cells spleen cells
• bone marrow cells spleen cells
• lymph node cells or any combination thereof.
• the methods of the present disclosure are performed without the addition of interleukin-2 (IL-2).
• IL-2 interleukin-2
• the factor that selectively induces antigen-specific induced suppressor T cells may be a regulator capable of regulating the production of IL-2 or a regulator capable of regulating the function of the produced IL-2.
• the regulation may preferably be a negative regulation.
• IL-2 also promotes the proliferation of induced suppressor T cells that are non-specific to a particular antigen, so it is possible to suppress the proliferation of non-specific induced suppressor T cells by regulating the production of IL-2 or regulating the function of the produced IL-2. Since complete inhibition of the production and function of IL-2 would also suppress the proliferation of antigen-specific induced suppressor T cells, it is preferable that a certain amount of IL-2 is present.
• the predetermined amount of IL-2 can be about 100 pg/mL or less, about 90 pg/mL or less, about 80 pg/mL or less, about 70 pg/mL or less, about 60 pg/mL or less, about 50 pg/mL or less, about 40 pg/mL or less, about 30 pg/mL or less, about 20 pg/mL or less, about 19 pg/mL or less, about 18 pg/mL or less, about 17 pg/mL or less, about 16 pg/mL or less, about 15 pg/mL or less, about 14 pg/mL or less, about 13 pg/mL or less, about 12 pg/mL or less, about 11 pg/mL or less, about 10 pg/mL or less, about 9 pg/mL or less, about 8 pg/mL or less, about 7 pg/mL or
• the above-mentioned predetermined amount is the amount of IL-2 at a cell concentration of about 0.5 to 2 ⁇ 10 6 cells/mL, and the amount of IL-2 can be appropriately changed depending on the number of cells being cultured.
• the predetermined amount of IL-2 can also differ depending on the source.
• An appropriate predetermined amount of IL-2 can be appropriately determined based on Examples 3 and 6.
• IL-2 or an anti-IL-2 antibody may be added at the beginning or during the culture to achieve a predetermined amount of IL-2 during the culture.
• the suppressive ability of antigen-specific induced suppressive T cells or cell populations containing the same produced by the method or factor of the present disclosure against an antigen may be about 30% or more (i.e., an immune response of 70% or less relative to the standard), about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more, based on the immune response of T cells or cell populations cultured without using the method or factor of the present disclosure.
• the suppressive ability of antigen-specific induced suppressive T cells or cell populations containing the same produced by the method or factor of the present disclosure against an antigen may be about 50% or more.
• the factor that selectively induces antigen-specific induced suppressor T cells may be a regulator or inhibitor that can regulate or inhibit the production of IL-2, or a regulator or inhibitor that can regulate or inhibit the function of the produced IL-2, or a factor that has both functions.
• the factor that selectively induces antigen-specific induced suppressor T cells can be an inhibitory factor that inhibits the interaction between IL-2 and the IL-2 receptor (IL-2R) and/or an inhibitory factor that can inhibit the interaction between CD80 and/or CD86 and CD28.
• IL-2R IL-2 receptor
• inhibitors capable of inhibiting the interaction between IL-2 and IL-2 receptor include, but are not limited to, anti-IL-2 antibodies, anti-IL-2R antibodies, anti-IL-2R ⁇ (CD25) antibodies, anti-IL-2R ⁇ (CD122) antibodies, or antigen-binding fragments thereof.
• inhibitors that can inhibit IL-2 production include inhibitors that can inhibit the interaction between CD80 and/or CD86 and CD28, or small molecule compounds that inhibit IL-2 production.
• Regulatory or inhibitory factors capable of regulating or inhibiting the function of the produced IL-2 include, but are not limited to, inhibitory factors capable of inhibiting the interaction between IL-2 and the IL-2 receptor (IL-2R), small molecule compounds that inhibit the action of IL-2, etc.
• IL-2R IL-2 receptor
• small molecule compounds that inhibit the action of IL-2, etc.
• Small molecule compounds that inhibit IL-2 production include, but are not limited to, calcineurin inhibitors, steroidal anti-inflammatory drugs, and the like.
• Calcineurin inhibitors include tacrolimus, cyclosporine, and the like. Calcineurin dephosphorylates NFAT (nuclear factor of activated T cells), which allows NFAT to enter the nucleus and bind to interleukin-2 promoters. Blocking this process inhibits IL-2 production.
• Steroidal anti-inflammatory drugs include, but are not limited to, mometasone furoate, clobetasol propionate, loteprednol etabonate, difluprednate, dexamethasone, amcinonide, flurandrenolide, prednisolone, fluocinolone acetonide, desonide, triamcinolone acetonide, budesonide, fludrocortisone acetate, fluocinonide, methylprednisolone, betamethasone, desoximethasone, halcinonide, fluorometholone, beclomethasone dipropionate, and dutasteride.
• Small molecule compounds that inhibit the action of IL-2 include rapamycin (sirolimus) and SDZ RAD. Rapamycin suppresses intracellular signaling and cell proliferation, inhibiting the response of lymphocytes to IL-2 and suppressing the activation of T lymphocytes.
• Inhibitory factors capable of inhibiting the interaction of CD80 and/or CD86 with CD28 include, but are not limited to, for example, anti-CD80 antibodies, anti-CD86 antibodies, bispecific antibodies against CD80 and CD86, anti-CD28 antibodies or antigen-binding fragments thereof, CTLA4-Ig fusion proteins, and CD28-Ig fusion proteins.
• the CTLA4-Ig fusion protein may be abatacept or belatacept.
• CD80 and/or CD86 are expressed by antigen-presenting cells
• CD28 is expressed by T cells
• the inhibitor capable of inhibiting the interaction of CD80 and/or CD86 with CD28 may be an anti-CD80 antibody and/or an anti-CD86 antibody or a CTLA4-Ig fusion protein.
• Inhibitors contemplated for use in the present disclosure include CTLA-4 Ig fusion proteins as described above. CTLA-4 Ig fusion proteins function to compete with CD28, a costimulatory receptor on T cells, for binding to CD80/CD86 on antigen-presenting cells, thereby inhibiting T cell activation.
• the CTLA-4 Ig fusion protein is contemplated to be abatacept (Orencia®), belatacept, or Maxy-4.
• Belatacept contains two amino acid substitutions (L104E and A29Y) that significantly increase the binding avidity for CD80 and CD86 (see Davies JK et al., Cell Transplant. (2012); 21(9): 2047-61; Adams AB et al., J Immunol. (2016) 197(6): 2045-50).
• an inhibitor that is expected to have the same effect as the CTLA4-Ig fusion protein is the CD28-Ig fusion protein (Peach RJ et al., J Exp Med. (1994) 180(6): 2049-2058).
• the inhibitors of the present disclosure may also be used in the form of nucleic acids.
• a nucleic acid encoding a CTLA4-Ig fusion protein can be introduced into a cell via an adenovirus vector or the like and expressed. See, for example, Jin YZ et al., Transplant Proc. (2003); 35(8): 3156-9.
• a composition for selectively inducing antigen-specific inducible suppressor T cells from cells derived from a subject comprising a regulator capable of regulating the production of IL-2 or a regulator capable of regulating the function of the produced IL-2, the composition being contacted with cells derived from the subject and an antigen derived from the subject or an antigen not derived from the subject or a substance containing the antigen.
• the preliminary confirmation can be performed as follows.
• the various values, reagents, procedures, etc. exemplified below are representative examples, and those skilled in the art can appropriately modify them to perform the preliminary confirmation manufacturing.
• Infectious disease screening tests are conducted on donors and patients, and donors are confirmed to be negative for HBs antigen, HCV antibody, HIV-1/2, and HTLV-1 antibody.
• donor lymphocytes can be separated as follows: The various values, reagents, procedures, etc. exemplified below are representative examples, and those skilled in the art can make appropriate modifications to separate donor lymphocytes.
• - Donor lymphocytes are collected into a collection bag by apheresis, and the collection bag is then irradiated.
• the irradiated peripheral blood mononuclear cells are placed in a centrifuge tube containing an appropriate amount of Ficoll-Paque PREMIUM (GE Healthcare #17-5442-02) or Lymphocyte Separation Solution (Nacalai Tesque #20828) or the like (e.g., 20 mL), and centrifuged at 860 G for 20 minutes at 22°C. Discard the supernatant and transfer the cell suspension containing the lymphocyte layer to another centrifuge tube (for example, two 50 mL centrifuge tubes).
• Ficoll-Paque PREMIUM GE Healthcare #17-5442-02
• Lymphocyte Separation Solution Nacalai Tesque #20828
• saline to the centrifuge tube containing the cell suspension (for example, an appropriate amount until the total volume is 50 mL), and mix well by repeatedly aspirating and dispensing with a syringe (for example, a 50 mL syringe with an 18G needle attached) or a pipette. Centrifuge at 500G for 10 minutes at 22°C (you may set the centrifuge accelerator to fast and the brake to fast). Discard the supernatant, add saline again (for example, an appropriate amount until the total volume is 50 mL), and mix the cell pellet well by repeatedly aspirating and dispensing the cell pellet with a pipette.
• the cryopreservation of donor lymphocytes can be performed as follows.
• the various values, reagents, procedures, etc. exemplified below are representative examples, and those skilled in the art can modify them as appropriate to cryopreserve donor lymphocytes.
• a freezing bag for example, Frozebag F-050 25 mL freezing bag, Nipro 89-101
• a syringe e.g., a 30 mL syringe fitted with an 18G needle
• ACD liquid (Terumo TP-A05ACD, for example, 2 mL per 15 mL of cell suspension) to the freezing bag containing the cell suspension, sandwich it between ice packs cooled to 4°C, and chill for about 10 minutes.
• a syringe e.g., a 20 mL syringe with an 18G needle
• CP-1 Kinyokuto Pharmaceutical Co., Ltd. 551-27202-4 Cell Cryoprotectant CP-1
• CP-1 Kinyokuto Pharmaceutical Co., Ltd. 551-27202-4 Cell Cryoprotectant CP-1
• a syringe remove all air from the freezing bag and its ports. Seal the freezing bag using a tube sealer and first chill at 4°C for approximately 5-10 minutes, then store in a -80°C freezer.
• thawing of donor lymphocytes can be performed as follows: The various values, reagents, procedures, etc. exemplified below are representative examples, and those skilled in the art can modify them as appropriate to thaw donor lymphocytes. Thaw the frozen bag of stored donor cells, for example, in a constant temperature bath at 37° C. The subsequent operations are preferably performed under aseptic conditions.
• a syringe e.g., a 50 mL syringe with an 18G needle attached
• withdraw the cell suspension from the thawed freezing bag and transfer it to a centrifuge tube e.g., 12.5 mL each into two 50 mL centrifuge tubes.
• a centrifuge tube e.g. 12.5 mL each into two 50 mL centrifuge tubes.
• albumin solution Nahon Pharmaceutical Co., Ltd. 123146364 Blood Donation Albumin 5% Intravenous Injection 12.5 g/250 mL
• Intravenous Injection 12.5 g/250 mL
• Centrifuge for example, at 600G for 10 minutes at 22°C (for example, preferably with the centrifuge accelerator set to fast and the brake set to slow). Carefully discard the supernatant, and add an appropriate liquid such as albumin-added saline for washing (for example, made from 25 mL of 5% albumin solution and 19 mL of saline) to the cell pellet to suspend it.
• Centrifuge for example, at 600G for 10 minutes at 22°C (for example, preferably with the centrifuge accelerator set to fast and the brake to slow).
• ALyS505N culture medium for example, 10 mL for a 50 mL centrifuge tube
• -Anti-human CD80 antibody e.g., m2D10.4; Cat. No. 16-0809-85, eBioscience
• anti-human CD86 antibody e.g., IT2.2; Cat. No. 16-0869-85, eBioscience
• a culture bag containing ALyS505N-0 culture medium or a liquid equivalent thereto e.g., Nipro 87598 Nipro Medium ALyS505NB10
• a final concentration of, for example, 10 ⁇ g/mL or an inhibitor such as a CTLA4-Ig fusion protein (e.g., belatacept) is added
• the above cell suspension is added to this culture bag by injection with a syringe (e.g., a 20 mL syringe with an 18G needle).
• the total volume of the liquid in the culture bag is about 840 mL.
• the patient's lymphocytes can be separated as follows.
• the various values, reagents, procedures, etc. exemplified below are representative examples, and those skilled in the art can modify them as appropriate to separate the patient's lymphocytes.
• Plasma collected from the patient is inactivated by heating it in a constant temperature bath, for example, at 56°C for 30 minutes. If it is not used immediately, it is stored frozen.
• Peripheral blood collected from a patient is placed in a centrifuge tube containing an appropriate amount of a suitable medium, such as Ficoll-Paque (e.g., 20 mL), and centrifuged, for example, at 860 G for 20 minutes at 22°C (e.g., preferably with the accelerator and brake of the centrifuge set to slow). Discard the supernatant and transfer the cell suspension containing the lymphocyte layer to another centrifuge tube (for example, two 50 mL centrifuge tubes). Add saline to the centrifuge tube containing the cell suspension (for example, an appropriate amount until the total volume is 50 mL), and mix well by repeatedly aspirating and dispensing with a pipette.
• a suitable medium such as Ficoll-Paque (e.g., 20 mL)
• centrifuge at 500 G for 10 minutes at 22° C. the accelerator and brake of the centrifuge may be set to fast and fast, respectively.
• Discard the supernatant add saline again (for example, an appropriate amount until the total volume is 50 mL), and mix the cell pellet well by repeatedly aspirating and dispensing the cell pellet with a pipette.
• centrifuge at 500 G for 5 minutes at 22° C. the accelerator and brake of the centrifuge may be set to fast and fast, respectively).
• the cell pellet is suspended in, for example, ALyS505N-0 culture medium (for example, 10 mL) to prepare a cell suspension (for example, add ALyS505N-0 culture medium until the total volume becomes 20 mL). At this point, about 0.5 mL of the cell suspension is withdrawn, and the cell count, viable cell count, and surface antigen expression are confirmed.
• - Inactivated plasma derived from the patient is added to the culture bag containing the donor cells in the ALyS505N-0 culture medium prepared in "3. Thawing of donor lymphocytes" and inhibitors such as antibodies.
• the patient-derived cell suspension is added to the culture bag by injection using a syringe (e.g., a 20 mL syringe with an 18G needle) and the culture bag is sealed using a tube sealer.
• a syringe e.g., a 20 mL syringe with an 18G needle
• the total volume of the culture bag is about 1000 mL.
• Medium exchange (e.g., after 1 week, preferably under sterile conditions)
• the medium exchange can be performed as follows.
• the various values, reagents, procedures, etc. exemplified below are representative examples, and a person skilled in the art can appropriately modify them to exchange the medium.
• centrifuge tubes e.g., four 225 mL centrifuge tubes. Centrifuge, for example, at 600G for 10 minutes at 22°C (for example, preferably with the centrifuge accelerator set to fast and the brake set to fast).
• the cell pellet is suspended in, for example, ALyS505N-0 culture medium to prepare a cell suspension (for example, add ALyS505N-0 culture medium until the total volume becomes 20 mL). At this point, about 0.3 mL of the cell suspension is removed and the cell count and viable cell count are confirmed.
• the cell suspension is added to a culture bag containing ALyS505N-0 culture medium by injection using a syringe (for example, a 20 mL syringe with an 18G needle).
• a diluted solution of anti-human CD80 antibody e.g., 2D10.4
• an diluted solution of anti-human CD86 antibody e.g., IT2.2
• a final concentration of 10 ⁇ g/mL or an inhibitor such as a CTLA4-Ig fusion protein (e.g., belatacept)
• a syringe e.g., a 20 mL syringe attached with an 18G needle.
• thawing of donor lymphocytes, antigen restimulation, and initiation of secondary culture can be performed as follows.
• the various numerical values, reagents, procedures, etc. exemplified below are representative examples, and a person skilled in the art can appropriately modify the thawing of donor lymphocytes, antigen restimulation, and initiation of secondary culture.
• the frozen bag of stored donor cells and the inactivated plasma from the patient are thawed, for example, in a thermostatic bath at 37° C.
• the subsequent operations are preferably performed under aseptic conditions.
• a syringe e.g., a 50 mL syringe with an 18G needle
• withdraw the donor cell suspension from the thawed cryobag and transfer it to a centrifuge tube e.g., two 50 mL centrifuge tubes.
• 5% albumin solution for example, about 50 mL in total for two 50 mL centrifuge tubes
• centrifuge tube containing the donor cell suspension mix well, and then leave to stand for about 5 minutes. For example, centrifuge at 600 G for 10 minutes at 22° C. (set the centrifuge's accelerator to fast and the brake to slow).
• albumin-containing saline for washing prepared, for example, from 25 mL of 5% albumin solution and 19 mL of saline
• albumin-containing saline for washing prepared, for example, from 25 mL of 5% albumin solution and 19 mL of saline
• centrifuge at 600 G for 10 minutes at 22° C. set the centrifuge's accelerator to fast and the brake to slow.
• ALyS505N-0 culture medium for example, 10 mL for a 50 mL centrifuge tube.
• the thawed inactivated plasma from the patient e.g., 10 mL
• a syringe e.g., a 20 mL syringe with an 18 G needle
• the above-mentioned cell suspension is added by injection with a syringe (e.g., a 20 mL syringe with an 18 G needle) to this culture bag.
• the total volume of the liquid in the culture bag is about 1000 mL.
• Culture in a 37°C incubator for, for example, one week.
• test during the secondary culture can be performed as follows.
• the various values, reagents, procedures, etc. exemplified below are representative examples, and a person skilled in the art can appropriately modify them to perform the test during the secondary culture.
• a small amount of culture medium is withdrawn from the culture bag and tested for mycoplasma contamination, etc.
• Collection and filling of cultured lymphocytes (performed under sterile conditions)
• the collection and filling of cultured lymphocytes can be performed as follows.
• the various values, reagents, procedures, etc. exemplified below are representative examples, and those skilled in the art can modify them as appropriate to collect and fill cultured lymphocytes.
• centrifuge tubes for example, four 225 mL centrifuge tubes.
• centrifuge (set the centrifuge accelerator to slow and the brake to slow).
• a centrifuge tube e.g., a 50 mL centrifuge tube.
• Add saline to the centrifuge tube containing the cell suspension for example, an appropriate amount until the total volume is 50 mL
• a syringe for example, a 50 mL syringe with an 18G needle attached.
• centrifuge at 500 G for 10 minutes at 22° C. (the accelerator and brake of the centrifuge may be set to fast and fast, respectively).
• Secondary Packaging can be performed as follows: The various numerical values, reagents, procedures, etc. exemplified below are representative examples, and those skilled in the art can appropriately modify them to perform secondary packaging. Typically, enter and print the subject ID, serial number, and expiration date on a label based on the appropriate standard (typically, NUHCPC-M-12-ATREG) and attach the label to the container. - Issue "Dosage and administration, indications or effects, and precautions for use or handling" based on appropriate standards (typically, NUHCPC-PMF-ATREG14). - Place the test item and "usage, dosage, efficacy or effects, and precautions for use or handling" in a plastic bag with a zipper. - Store in a transport container in the monitoring unit until shipment.
• the cell preparation can be produced as follows: The various numerical values exemplified below are representative examples, and a person skilled in the art can appropriately modify them to produce the cell preparation. 1) Approximately 19 days prior to administration, apheresis will be performed on the donor at a medical institution, and the donor apheresis product will be irradiated with 30 Gy of radiation to eliminate cell proliferation ability, and then shipped to a cell processing facility where cell processing will be performed. 2) After receiving the donor apheresis product, the donor mononuclear cells will be separated and collected by density gradient centrifugation at the cell culture processing facility, then divided into two aliquots, frozen, and stored at -80 ⁇ 10°C.
• apheresis is performed on the recipient at a medical institution, and the recipient apheresis product is shipped to a cell culture processing facility where cell processing will be performed.
• the recipient mononuclear cells are separated and collected by density gradient centrifugation and co-cultured with thawed donor mononuclear cells and inhibitors such as anti-CD80 and anti-CD86 antibodies or CTLA4-Ig fusion protein.
• the medium is replaced about 7 days before administration.
• the intermediate product cultured for 7 days is collected and co-cultured with thawed donor mononuclear cells and inhibitors such as anti-CD80 and anti-CD86 antibodies or CTLA4-Ig fusion protein. 6) On the day of administration, the processed cells are collected by density gradient centrifugation, washed, and filled with physiological saline. 7) Ship it to a medical institution and administer it to the recipient at the medical institution.
• the disclosure provides a composition for treating a disease in a subject mediated by an immune response, the composition comprising an inhibitor that inhibits the interaction of IL-2 with the IL-2 receptor (IL-2R) and/or an inhibitor that can inhibit the interaction of CD80 and/or CD86 with CD28, and antigen-specific suppressor T cells selectively induced by mixing cells derived from the subject with an antigen derived from the subject or an antigen not derived from the subject or a substance containing the antigen.
• IL-2R IL-2 receptor
• the disease may be selected from the group consisting of transplant immune rejection, allergy, autoimmune disease, graft-versus-host disease, and immune rejection caused by transplantation of iPS cells or ES cells or cells, tissues, or organs differentiated therefrom.
• the transplant immune rejection is characterized as occurring following transplantation of kidney, liver, heart, skin, lung, pancreas, esophagus, stomach, small intestine, large intestine, nerves, blood, blood cells including immune system cells, bone, cartilage, blood vessels, cornea, eyeball, or bone marrow.
• anergy cells can be induced by mixing the inhibitor, recipient-derived cells, and donor-derived antigens or donor-derived antigen-containing material.
• the donor-derived antigen-containing material can be PBMCs, spleen cells, or cells derived from the organ to be transplanted.
• anergy cells can be induced by mixing the inhibitor, cells derived from the subject, and an antigen not derived from the subject that causes the allergy.
• anergy cells can be induced by mixing the inhibitor, cells derived from the subject, and an antigen derived from the subject that causes the autoimmune disease.
• anergy cells can be induced by mixing the inhibitor, PBMCs or spleen cells of the donor providing the graft, and a recipient-derived antigen or a substance containing the antigen.
• the recipient-derived antigen can be PBMCs, spleen cells, or cells in the vicinity of the site where the organ is transplanted or cells derived therefrom.
• anergy cells can be induced by mixing the above-mentioned inhibitor with cells derived from the subject and cells differentiated from iPS cells or ES cells to be used for transplantation.
• the present disclosure provides a method for treating or preventing allergies and/or autoimmune diseases using the pharmaceutical of the present disclosure, and a pharmaceutical, composition, and cell mixture for use therein.
• allergies and autoimmune diseases macrophages obtained from the peripheral blood of a patient are differentiated into dendritic cells (macrophage-derived dendritic cells) with high antigen-presenting ability by a conventional method, and these cells are made to present an antigen causing an overreaction in allergies or autoimmune diseases after irradiation (gamma rays), and co-cultured with a group of T cells obtained from the peripheral blood of the same patient in the presence of the above factors for 1 to 2 weeks to obtain anergy cells specific to the antigen causing the allergy or autoimmune disease.
• dendritic cells macrophage-derived dendritic cells
• gamma rays gamma rays
• anergy cells By administering these anergy cells to a patient, immune tolerance specific to the antigen causing the allergy or autoimmune disease is induced, and the anergy cells are used for the prevention and treatment of allergies and autoimmune diseases.
• the number of administrations may be multiple times depending on various conditions such as whether it is a preventive therapy or a treatment, and the strength of the symptoms.
• the present disclosure provides a method for treating or preventing graft-versus-host disease using the medicament of the present disclosure, and a medicament, composition, and cell mixture for use therein.
• cells that can cause graft-versus-host disease such as PBMCs or T cells of a donor providing a graft
• the reaction of the graft causing graft-versus-host disease to the host is suppressed (immune tolerance is induced), and graft-versus-host disease is prevented and treated.
• the number of administrations may be multiple, depending on various conditions, such as whether it is a preventive therapy or a treatment, the tissue to be transplanted, its size, and the severity of symptoms.
• the present disclosure provides a method for treating or preventing immune rejection or other side effects caused by transplantation of iPS cells or ES cells and cells, tissues or organs differentiated therefrom in prevention or therapy using iPS cells or ES cells using the pharmaceutical of the present disclosure, and a pharmaceutical, composition, and cell mixture used therein.
• immune rejection caused by transplantation of iPS cells or ES cells and cells, tissues or organs differentiated therefrom is typically exemplified as a treatment target.
• cells or dendritic cells to be used for transplantation that have been differentiated from iPS cells or ES cells are irradiated with radiation (gamma rays), and these cells are co-cultured with the PBMC or T cell group of the patient receiving the transplant in the presence of the above factors for 1-2 weeks to obtain anergy cells specific to the cells differentiated from iPS cells or ES cells.
• radiation gamma rays
• anergy cells specific to the cells differentiated from iPS cells or ES cells By administering these anergy cells to the host, specific immune tolerance is induced to the transplanted cells, tissues, and organs that have been differentiated from iPS cells or ES cells, and rejection reactions to them are prevented and treated.
• the number of administrations may be multiple depending on whether it is a preventive or therapeutic therapy, and on various conditions such as the tissue to be transplanted, its size, and the severity of symptoms.
• the present disclosure provides a method for treating or preventing allergy using the pharmaceutical of the present disclosure, and a pharmaceutical, composition, and cell mixture for use therein.
• the present inventors have demonstrated that a preparation containing cells in which anergy is induced antigen-specifically by the above-mentioned factor can induce immune tolerance to allergy. Therefore, in another aspect, the present disclosure provides a composition for treating or preventing allergy in a subject, the composition comprising cells in which immune tolerance is induced by mixing the above-mentioned factor, cells derived from the subject, and an antigen causing allergy or a substance containing the antigen.
• Allergens that cause allergies include foods, pollen, medicines, and metals, and more specifically include, but are not limited to, dust mite antigens, egg white antigens, milk antigens, wheat antigens, peanut antigens, soybean antigens, buckwheat antigens, sesame antigens, rice antigens, crustacean antigens, kiwi antigens, apple antigens, banana antigens, peach antigens, tomato antigens, tuna antigens, salmon antigens, mackerel antigens, beef antigens, chicken antigens, pork antigens, cat dander antigens, insect antigens, pollen antigens, dog dander antigens, fungal antigens, bacterial antigens, latex, haptens, and metals.
• the present disclosure provides a method for suppressing or preventing immune rejection caused by iPS cells, etc., and provides a medicine, composition, and cell mixture for the suppression or prevention.
• the present inventors have demonstrated that a preparation containing cells in which anergy is induced by the above-mentioned factor can induce immune tolerance to immune rejection caused by iPS cells, etc., or cells, tissues, or organs differentiated therefrom.
• the present disclosure provides a composition for suppressing or preventing immune rejection caused by iPS cells or ES cells, or cells, tissues, or organs differentiated therefrom, in a subject, the composition comprising cells in which immune tolerance has been induced by mixing the above-mentioned factor, cells derived from the subject, and an antigen differentiated from the iPS cells or ES cells, or a substance containing the antigen.
• Cells, tissues, or organs differentiated from iPS cells or ES cells include, but are not limited to, nerve cells or tissue, corneal cells or tissue, cardiac muscle cells or tissue, liver or tissue, cartilage cells or tissue, skin cells or tissue, kidney or tissue, etc.
• cells, tissues, or organs differentiated from iPS cells or ES cells include nerve cells or tissue, cardiac muscle cells or tissue, cartilage cells or tissue, and skin cells or tissue.
• Example 1 Effect of addition of IL-2 on allospecific suppression by Treg
• MLR mixed lymphocyte reaction
• Spleen cells were collected from Balb/c mice and irradiated with 30 Gy of X-rays, after which they were adjusted to a concentration of 4 x 106 cells/mL and mixed with hCD2-KI mouse spleen cells at a 1:1 ratio.
• Anti-CD80 mAb + anti-CD86 mAb (final concentration: 10 ⁇ g/mL each) and, if necessary, recombinant IL-2 (final concentration: 25 U/mL) were added, seeded on a 10 cm dish, and cultured at 37° C. in a 5% CO 2 incubator for 7 days.
• B6 mouse spleen cells, LPS-stimulated Balb/c mouse spleen cells, or LPS-stimulated CBA/Nslc mouse spleen cells, and the cultured whole cells or isolated Tregs from 3. were prepared to the concentrations shown in Table 1 and seeded onto a U-bottom 96-well plate.
• the cells were cultured at 37°C in a 5% CO2 incubator for 3 days.
• Spleen cells were collected from Balb/c mice and irradiated with 30 Gy of X-rays, then adjusted to a concentration of 2 x 106 cells/mL and mixed with hCD2-KI mouse spleen cells at a 1:1 ratio.
• Anti-CD80 mAb + anti-CD86 mAb (final concentration 10 ⁇ g/mL each) or anti-IL-2 mAb (final concentration 10 ⁇ g/mL), and recombinant IL-2 (final concentration 25 U/mL) were added as required, and 200 ⁇ L each was seeded into a U-bottom 96-well plate and cultured at 37°C in a 5% CO2 incubator for 4 days.
• Example 2 Evaluation of allospecific suppressive ability of Treg in MLR with addition of anti-IL-2 antibody
• Example 2 Evaluation of allospecific suppressive ability of Treg in MLR with addition of anti-IL-2 antibody
• Spleen cells were collected from Balb/c mice and irradiated with 30 Gy of X-rays, after which they were adjusted to a concentration of 4 x 106 cells/mL and mixed with hCD2-KI mouse spleen cells at a 1:1 ratio.
• Anti-CD80 mAb + anti-CD86 mAb (final concentration 10 ⁇ g/mL each) or anti-IL-2 mAb (final concentration 10 ⁇ g/mL) was added, seeded on a U-bottom 96-well plate, and cultured at 37° C. in a 5% CO 2 incubator for 7 days.
• B6 mouse spleen cells, LPS-stimulated Balb/c mouse spleen cells, or LPS-stimulated CBA/Nslc mouse spleen cells, and the cultured whole cells or isolated Tregs from 3. were prepared to the concentrations shown in Table 2 and seeded onto a U-bottom 96-well plate.
• the cells were cultured at 37°C in a 5% CO2 incubator for 3 days.
• Example 3 Effect of IL-2 during culture
• the effect of IL-2 on the induction of antigen-specific inducible suppressor T cells using anti-CD80/86 antibody and/or anti-IL-2 antibody was confirmed.
• the method was the same as in Examples 1 and 2.
• the concentration of IL-2 in the culture medium on days 1 to 5 of culture was measured by ELISA.
• (result) 9 and 10 show that when cells were cultured in the presence of anti-CD80/86 antibody or anti-IL-2 antibody, the IL-2 concentration was 20 pg/mL or less on day 5. It is believed that the CD28 costimulatory signal was inhibited by anti-CD80/86 antibody, which reduced T cell activation and suppressed IL-2 production. On the other hand, when cells were cultured without anti-CD80/86 antibody or anti-IL-2 antibody, the IL-2 concentration exceeded 100 pg/mL already on day 2 of culture.
• Example 4 Change in the percentage of CD4 + T cells by anti-CD80/86 antibody or anti-IL-2 antibody
• changes in the proportion of CD4 + T cells due to anti-CD80/86 antibody and/or anti-IL-2 antibody were confirmed.
• the methods were similar to those in Examples 1 and 2.
• Example 5 Change in suppressive ability by anti-CD80/86 antibody or anti-IL-2 antibody
• changes in suppressive ability due to anti-CD80/86 antibody and/or anti-IL-2 antibody were confirmed.
• Tregs When IL-2 was completely blocked using both anti-CD80/86 antibody and anti-IL-2 antibody, isolated Tregs exhibited suppressive ability against Balb stimulation, but whole cells exhibited almost no suppressive ability. When cultured with only anti-CD80/86 antibody, stronger suppressive ability against Balb stimulation was observed. These results indicate that the suppressive ability of suppressive T cells is IL-2 dependent. Isolated Tregs also exhibited slight suppressive ability against CBA, but whole cells exhibited almost no suppressive ability (FIGS. 12 and 13). Therefore, the suppressive ability of whole cells has high antigen specificity and is also highly dependent on IL-2. Antigen non-specific suppression may be due to the influence of the suppressive cytokine TGF- ⁇ , etc.
• Example 6 Evaluation of inhibition of IL-2 production by anti-CD80/86 antibody or anti-IL-2 neutralizing antibody In this example, using human cells, it is confirmed that IL-2 production is suppressed by anti-CD80/86 antibody or anti-IL-2 neutralizing antibody.
• the culture supernatant is collected on days 1, 2, 3, and 5 of culture, and frozen and stored at -20°C until the day of measurement of IL-2 by ELISA. 5. Measure human IL-2 by ELISA.
• the IL-2 concentration in the culture supernatant is predicted to be lower than that in the control group.
• Example 7 Evaluation of the effect of adjusting the IL-2 concentration in the culture medium on the donor-specific suppressive ability of Treg.
• Method 1 Spleen cells are collected from Foxp3/hCD2-KI C57BL6 mice (hCD2-KI mice) and adjusted to 4 x 10 6 cells/mL. 2.
• Spleen cells from Balb/c mice are collected and irradiated with 30 Gy of X-rays, then adjusted to a concentration of 4 x 10 6 cells/mL and mixed with hCD2-KI mouse spleen cells at a 1:1 ratio.
• Anti-IL-2 mAb final concentration: 10 ⁇ g/mL
• the cells are seeded onto a 10 cm dish, and cultured at 37° C.
• B6 mouse spleen cells, LPS-stimulated Balb/c mouse spleen cells or LPS-stimulated CBA/Nslc mouse spleen cells, and the cultured whole cells or isolated Tregs from 5. are prepared to the concentrations shown in Table 1 and seeded on a U-bottom 96-well plate. 7. Culture in a 37°C, 5% CO2 incubator for 3 days.
• IL-2 Measurement Two types of commercially available human peripheral blood mononuclear cells (LONZA CC-2702) were used as donor and recipient cells.
• the donor cells (Batch#22TL290862) were suspended in 15mL of RPMI medium (FUJIFILM Wako Pure Chemical Industries, Ltd.) containing 10% FCS (SELBORNE), and then irradiated with 25Gy of radiation.
• anti-CD80 antibody (manufactured by JUNTEN BIO Co., Ltd.) was added to the donor cells and recipient cells (Batch#23TL043676) at a final concentration of 5 mg/L, respectively, and then allowed to stand on ice for 30 minutes.
• donor cells and recipient cells were collected by centrifugation, and CD86 antibody (manufactured by JUNTEN BIO Co., Ltd.) was added to the donor cells at a final concentration of 5 mg/L.
• donor cells and recipient cells were prepared to a final concentration of 5.0x10 ⁇ 7 cells/20mL in two T flasks (Thermo scientific, 25 cm2 ) to which 20mL of 10% FCS-containing RPMI medium had been added.
• Anti-CD80 antibody (manufactured by JUNTEN BIO Co., Ltd.) and anti-CD86 antibody (manufactured by JUNTEN BIO Co., Ltd.) were added to one T flask to a final concentration of 5mg/L, and the remaining one was used as a negative control without adding any antibody.
• the two T flasks were placed in a 5% CO2 incubator set at 37°C.
• the culture supernatant was collected before the start of the culture and on days 1, 2, 3, 4, and 7 after the start of the culture, and the IL-2 concentration was measured by the Cytometric Bead Array (CBA) method.
• CBA Cytometric Bead Array
• the cells cultured for 7 days were added to a co-culture system of donor cells (Batch #22 TL290862: 2.0x10 ⁇ 5 cells/well) and recipient cells (Batch #23 TL043676: 2.0x10 ⁇ 5 cells/well) irradiated with 25 Gy, and the concentrations of IFN- ⁇ and granzyme B in the culture supernatant 5 days after culturing in a 96-well plate (CORNING) were measured by the CBA method. The results are shown in Table 4. By adding cells cultured in the presence of anti-CD80 and anti-CD86 antibodies, the concentrations of IFN- ⁇ and granzyme B were reduced in a cell concentration-dependent manner, confirming that MLR was suppressed.

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