EP3463398A1 - Immun-checkpoint-inhibitoren und zytotoxische t-zellen zur behandlung von krebs - Google Patents

Immun-checkpoint-inhibitoren und zytotoxische t-zellen zur behandlung von krebs

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Publication number
EP3463398A1
EP3463398A1 EP17802270.3A EP17802270A EP3463398A1 EP 3463398 A1 EP3463398 A1 EP 3463398A1 EP 17802270 A EP17802270 A EP 17802270A EP 3463398 A1 EP3463398 A1 EP 3463398A1
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EP
European Patent Office
Prior art keywords
immune checkpoint
peptide
ctls
cells
subject
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Withdrawn
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EP17802270.3A
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English (en)
French (fr)
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EP3463398A4 (de
Inventor
Rajiv Khanna
Corey Smith
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QIMR Berghofer Medical Research Institute
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Queensland Institute of Medical Research QIMR
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Publication of EP3463398A1 publication Critical patent/EP3463398A1/de
Publication of EP3463398A4 publication Critical patent/EP3463398A4/de
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • C07K16/2818—Immunoglobulins [IGs], 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 CD28 or CD152
    • 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
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Nasopharyngeal carcinoma is a type of head and neck cancer that begins in the nasopharynx. Recently, there is emerging evidence to show that exposure to Epstein Barr virus (EBV) can contribute to the pathology of nasopharyngeal carcinoma. Epstein-Barr virus associated nasopharyngeal carcinoma (NPC) is endemic in regions of South-East Asia, with incidence as high as 25-50 cases per 100,000 people in southern China. While current standard therapy is often curative for a subset with stage I or II disease, a high proportion of patients relapse and many patients are still initially diagnosed with advanced stage HI or IV disease, where overall 5-year survival is significantly reduced. Thus, there exists a need to develop improved therapies for NPC.
  • EBV Epstein Barr virus
  • NPC Epstein-Barr virus associated nasopharyngeal carcinoma
  • provided herein are methods of treating cancer (e.g., NPC) in a subject by administering (e.g., conjointly) an immune checkpoint inhibitor and a
  • composition comprising cytotoxic T cells (CTLs) expressing a T cell receptor specific for a cancer-associated peptide presented on a class I MHC.
  • CTLs cytotoxic T cells
  • the immune checkpoint inhibitor is a protein or polypeptide (e.g., an antibody or antigen-binding fragment thereof) that binds to an immune checkpoint protein, such as CTLA4, PD-1, PD-L1 , PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG-3, ⁇ -3, IDO, TDO, and VISTA.
  • an immune checkpoint protein such as CTLA4, PD-1, PD-L1 , PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG-3, ⁇ -3, IDO, TDO, and VISTA.
  • the immune checkpoint protein is CTLA4, PD-1, PD-L1, ⁇ -3 or LAG-3.
  • the immune checkpoint inhibitor binds to the immune checkpoint protein such that it inhibits its activity.
  • the immune checkpoint inhibitor inhibits the interaction between the immune checkpoint protein and an associated receptor/ligand.
  • the immune checkpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI- Al 110, TSR-042, RG-7446, BMS-936559, BMS-936558, MK-3475, CT 01 1, MPDL3280A, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010.
  • the cytotoxic T cells in the composition can be specific for any cancer-associated peptide presented on a class I MHC (e.g., a cancer associated peptide expressed by a tumor and/or cancer cells in the subject).
  • the cancer- associated peptide is a viral peptide.
  • the viral peptide is an EBV peptide.
  • the EBV peptide comprises a LMP1 peptide, a LMP2A peptide, and/or an EBNA1 peptide.
  • the CTLs are allogeneic to the subject (e.g., obtained from a cell bank). In some embodiments, the CTLs are autologous to the subject. The CTLs and the immune checkpoint inhibitor may be co-administered or administered sequentially. In some embodiments, the method further comprises administering to the subject a chemotherapeutic agent.
  • provided herein are methods of treating cancer (e.g. nasopharyngeal carcinoma) in a subject, comprising generating peptide-specific CTLs by incubating a sample comprising CTLs and antigen-presenting cells (APCs) that present a CMV peptide, thereby inducing proliferation peptide-specific CTLs in the sample, and administering the peptide-specific CTLs to the subject in combination with an immune checkpoint inhibitor described herein.
  • cancer e.g. nasopharyngeal carcinoma
  • the APCs are made to present the EBV peptide by incubating them with a nucleic acid construct (e.g., AdEl-LMPpoly) encoding for the EBV peptide, thereby inducing the APCs to present the EBV peptide.
  • a nucleic acid construct e.g., AdEl-LMPpoly
  • the APCs may be B cells, antigen-presenting T cells, dendritic cells, or artificial antigen- presenting cells (e.g., a cell line expressing CD80, CD83, 41BB-L and/or CD86, such as aK562 cells).
  • the EBV peptide comprises a LMP1 peptide or a fragment thereof, a LMP2A peptide or fragment thereof, and/or an EBNA l peptide or fragment thereof.
  • the EBV peptide comprises a sequence listed in Table 1.
  • one or more immune checkpoint inhibitors are administered.
  • the immune checkpoint inhibitors may be administered by any technique known in the art.
  • the immune checkpoint inhibitor is administered intratumorally.
  • the sample comprises one or more cytokines or peripheral blood mononuclear cells (PBMCs). BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 has two panels showing the expression of immune checkpoint molecules ⁇ i.e., LAG-3, TIM-3, CTLA4, or PD-1).
  • Panel A depicts percentage of HLA-multimer positive CD8-positive lymphocytes that express PD-1, TIM-3, LAG-3 and CTLA-4.
  • Panel B depicts the percentage of PD-1 positive, TIM-3 positive, LAG-3 positive and CTLA-4 positive lymphocytes in the CTL immunotherapy administered in NPC patients with no/minimal residual disease (N/MRD) and active-recurrent/metastatic disease (ARMD) who either showed stable disease (SD) or progressive disease (PD) following adoptive T cell therapy.
  • N/MRD no/minimal residual disease
  • ARMD active-recurrent/metastatic disease
  • kits for treating cancer in a subject using a combination therapy that includes administration, e.g., conjoint administration, of one or more immune checkpoint inhibitors combined with cytotoxic T cell (CTL) immunotherapy.
  • the cancer is EBV-associated NPC and the CTLs administered to the subject express a T cell receptor that has binding specificity for a peptide expressing and EBV epitope presented on a class I MHC.
  • an element means one element or more than one element.
  • administering means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering.
  • an agent can contain, for example, peptide described herein, an antigen presenting cell provided herein and/or a CTL provided herein.
  • tissue sample each refers to a collection of cells obtained from a tissue of a subject.
  • the source of the tissue sample may be solid tissue, as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents, serum, blood; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid, urine, saliva, stool, tears; or cells from any time in gestation or development of the subject.
  • binding refers to an association, which may be a stable association, between two molecules, e.g., between a T cell receptor (TCR) and a T cell receptor (TCR) and a T cell receptor (TCR)
  • peptide/MHC due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions.
  • cancer includes, but is not limited to, solid tumors and blood borne tumors.
  • the term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels.
  • the term “cancer” further encompasses primary and metastatic cancers.
  • epitope means a protein determinant capable of specific binding to an antibody.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which a T cell receptor or antibody is capable of binding.
  • the phrase "pharmaceutically acceptable” refers to those agents, compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the phrase "pharmaceulically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydrox
  • a therapeutic that "prevents" a condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • subject means a human or non-human animal selected for treatment or therapy.
  • therapeuticatty-effective amount and “effective amount ' " as used herein means the amount of an agent which is effective for producing the desired therapeutic effect in at least a sub-population of cells in a subject at a reasonable benefit/risk ratio applicable to any medical treatment.
  • Treating" a disease in a subject or “treating” a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening.
  • the term "conjoint administration” means administration of two or more agents to a subject of interest as part of a single therapeutic regimen.
  • administration(s) can be either simultaneous or sequential, i.e., administering one agent followed by administering of a second (and/or a third one, etc.) at a later time, as long as the agents administered co-exist in the subject being treated, or at least one agent will have the opportunity to act upon the same target tissues of other agents while said target tissues are still under the influence of said other agents.
  • agents to be administered can be included in a single pharmaceutical composition and administered together.
  • the agents are administered simultaneously, including through separate routes, hi a certain embodiment, one or more agents are administered continuously, while other agents are administered only at predetermined intervals (such as a single large dosage, or twice a week at smaller dosages, etc.).
  • Immune Checkpoint Inhibitors in certain aspects, provided herein are methods related to treating cancer ⁇ e.g., nasopharyngeal carcinoma) in a subject by administering to the subject a combination therapy, the combination therapy comprising administering to the subject both an immune checkpoint inhibitor and a composition comprising cytotoxic T cells (CTLs) expressing a T cell receptor specific for a cancer-associated peptide presented on a class I MHC.
  • CTLs cytotoxic T cells
  • the immune checkpoint inhibitor and CTL composition can be administered together or separately. They can be administered simultaneously or sequentially. When sequentially administered, in some embodiments the immune checkpoint inhibitor will be administered before the CTL composition (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before).
  • the CTL composition e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before.
  • the CTL composition will be administered before the immune checkpoint inhibitor (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before).
  • the immune checkpoint inhibitor e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before.
  • Immune checkpoint inhibition broadly refers to inhibiting the biological pathways that serve as checkpoints to prevent or downregulate an immune response. Such pathways are often used by cancer cells to avoid an anti-tumor immune response.
  • the method includes administering to the subject one or more immune checkpoint inhibitors that target immune checkpoint proteins.
  • Immune checkpoint proteins include, but are not limited to, CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG-3, ⁇ -3, IDO, TOO, and VISTA.
  • one or more immune checkpoint inhibitor may target one or more immune checkpoint proteins.
  • the immune checkpoint inhibitor is a protein, such as a soluble fusion protein.
  • a protein comprises a receptor/ligand binding domain (e.g., an extracellular domain) of CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7- H4, BTLA, KIR, LAG-3, ⁇ -3, IDO, TOO, or VISTA.
  • the receptor/ligand binding domain is fused to an immunoglobulin Fc domain.
  • a fusion protein can be produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different peptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger- ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, for example. Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety.
  • the immune checkpoint inhibitor is an antibody or antigen- binding fragment thereof that binds to and inhibits an immune checkpoint protein (e.g., CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KTR, LAG-3, ⁇ -3, IDO, TDO, or VISTA).
  • an immune checkpoint protein e.g., CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KTR, LAG-3, ⁇ -3, IDO, TDO, or VISTA.
  • an immune checkpoint protein e.g., CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KTR, LAG-3, ⁇ -3, IDO, TDO, or VISTA.
  • the term “antibody” may refer to both an intact antibody and an antigen binding fragment thereof.
  • An antigen-binding fragment of an antibody refers to one or more fragments of an antibody that retain the ability to bind to an antigen.
  • binding fragments include Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, camelid antibodies, isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody.
  • Such antibody fragments can be obtained using conventional recombinant and/or enzymatic techniques and can be screened for antigen-binding in the same manner as intact antibodies.
  • the immune checkpoint inhibitor is an inhibitory nucleic acid (e.g., an siRNA molecule, an shRNA molecule, an antisense RNA) that specifically binds to an mRNA that encodes an immune checkpoint inhibitor (e.g., CTLA4, PD-1, PD-L1 , PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG-3, ⁇ -3, IDO, TDO, or VISTA).
  • Inhibitory nucleic acid molecules can be prepared by chemical synthesis, in vitro transcription, or digestion of long dsRNA by Rnase III or Dicer.
  • Inhibitory nucleic acid molecules can be delivered in vitro to cells or in vivo, e.g., to tumors or hypoxic tissues of a mammal.
  • Typical delivery means known in the art can be used.
  • an interfering RNA can be delivered systemically using, for example, the methods and compositions described in PCT Application No: PCT/US09/036223, PCT/US09/061381 PCT/US09/063927,
  • the inhibitory nucleic acid is delivered locally.
  • delivery to a tumor can be accomplished by intratumoral injections, as described, for example, in Takahashi et al, Journal of Controlled Release 116:90-95 (2006) and Kim et al, Journal of Controlled Release 129: 107-116 (2008), each of which is incorporated by reference in its entirety.
  • the immune checkpoint inhibitor is a small organic molecule, e.g., a molecule having a molecular weight under about 5 kD, preferably less than about 2 kD, and typically exclude oligonucleotides and oligopeptides.
  • Small molecules include, for example, peptidomimeti.cs, oligosaccharides, steroids, etc.
  • Representative small- molecule checkpoint inhibitors are described in WO 2016/041511, WO 2015/034820, WO 2010/005958, WO 2014/159248, US Published Application 2011/0318373, and Weinmann, H., Chem. Med. Chem. 2016, 11, 450-466 (and in references cited therein).
  • the immune checkpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, BMS-936558, MK-3475, CT Oil, MPDL3280A, MEDI-4736, MSB-0020718C, AUR-012 and STI-AIOIO.
  • the CTLs in the CTL composition administered to the subject express a T cell receptor that specifically binds to a peptide (e.g., a peptide comprising a cancer-associated epitope) presented on a class I MHC.
  • a peptide e.g., a peptide comprising a cancer-associated epitope
  • the class I MHC has an a chain polypeptide that is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-g, HLA-K or HLA-L.
  • the peptide is a peptide described herein.
  • the CTLs in the sample express a TCR specific for an Epstein-Barr Virus (EBV) peptide (e.g., a LMP1 peptide, a LMP2A peptide or an EBNA1 peptide) presented on a class I MHC.
  • EBV Epstein-Barr Virus
  • CTLs in the CTL compositions described herein may be generated by incubating a sample comprising CTLs with the antigen-presenting cells (APCs), thereby inducing the APCs.
  • APCs antigen-presenting cells
  • the APCs that present a peptide described herein e.g., a peptide comprising a LMPl, LMP2A, or EBNA1 epitope sequence.
  • the APCs are B cells, antigen-presenting T-cells, dendritic cells, or artificial antigen-presenting cells (e.g., aK562 cells).
  • Dendritic cells for use in the process may be prepared by taking peripheral blood mononuclear cells (PBMCs) from a patient sample and adhering them to plastic. Generally the monocyte population sticks and all other cells can be washed off. The adherent population is then differentiated with IL-4 and GM-CSF to produce monocyte derived dendritic cells. These cells may be matured by the addition of IL- ⁇ , IL-6, PGE-1 and TNF-a (which upregulates the important co-stimulatory molecules on the surface of the dendritic cell) and are then transduced with one or more of the peptides provided herein.
  • PBMCs peripheral blood mononuclear cells
  • APCs that present one or more peptides described herein may be generated by contacting an APC with a peptide comprising a CTL epitope and/or with a nucleic acid encoding a peptide comprising a CTL epitope. In some embodiments, the APCs are irradiated. In some embodiments, the APCs that present a peptide described herein (e.g., a peptide comprising a LMPl, LMP2A, or EBNA1 epitope sequence).
  • a cell presenting a peptide described herein can be produced by standard techniques known in the art. For example, a cell may be pulsed to encourage peptide uptake.
  • the cells are transfected with a nucleic acid encoding a peptide provided herein.
  • the methods provided herein include steps of generating, activating and/or inducing proliferation of T cells (e.g., CTLs) that recognize one or more of the CTL epitopes described herein.
  • a sample comprising CTLs i.e., a PBMC sample
  • an APC provided herein (e.g., an APC that presents a peptide comprising a CTL epitope (e.g., EBV epitope) on a class I MHC complex).
  • the APCs are autologous to the subject from whom the T cells were obtained. In some embodiments, the APCs are not autologous (i.e.
  • the sample containing T cells is incubated two or more times with APCs provided herein.
  • the T cells are incubated with the APCs in the presence of at least one cytokine.
  • the cytokine is IL-4, IL-7 and/or IL-15. Exemplary methods for inducing proliferation of T cells using APCs are provided, for example, in U.S. Pat. Pub. No. 2015/0017723, which is hereby incorporated by reference.
  • kits comprising the administration of samples, comprising immune checkpoint inhibitors and CTLs to a subject in order to treat and/or prevent cancer.
  • the method includes administering to the subject an effective amount of a composition comprising CTLs one or more immune checkpoint inhibitors, provided herein.
  • the composition includes a combination of multiple (e.g., two or more) CTLs and/ or immune checkpoint inhibitors provided herein.
  • the T cells are autologous to the subject.
  • the T cells are allogeneic to the subject.
  • the CTLs are stored in a cell bank before they are administered to the subject.
  • the methods provided herein include selecting allogeneic CTLs from a cell bank (e.g., a pre-generated third party donor derived bank of epitope specific CTLs) for adoptive immunotherapy by determining the level expression of a biomarker with in the CTL population. In some embodiments, the level of expression of two or more biomarkers is determined.
  • a cell bank e.g., a pre-generated third party donor derived bank of epitope specific CTLs
  • the method further includes selecting allogeneic CTLs because they express a TCR restricted to a class I MHC that is encoded by an HLA allele that is present in the subject, hi some embodiments, the CTLs are selected if the CTLs and subject share at least 2 (e.g., at least 3, at least 4, at least 5, at least 6) HLA alleles and the CTLs are restricted through a shared HLA allele.
  • the method comprises testing the TCR repertoire of the pre-generated third- party-donor-derived epitope-specific T cells (i.e., allogeneic T cells) with flow cytometry.
  • epitope-specific T cells are detected using a tetramer assay, an ELISA assay, a western blot assay, a fluorescent microscopy assay, an Edman degradation assay and/or a mass spectrometry assay (e.g., protein sequencing).
  • the TCR repertoire is analyzed using a nucleic acid probe, a nucleic acid amplification assay and/or a sequencing assay.
  • the methods and compositions provided herein relate to peptide specific CTLs.
  • the methods include the generation of such CTLs, for example, by incubating a sample comprising CTLs (i.e., a PBMC sample) with antigen-presenting cells (APCs) that present one or more of the CTL epitopes described herein (e.g., APCs that present a peptide described herein comprising a CTL epitope on a class I MHC complex).
  • APCs antigen-presenting cells
  • the peptides provided herein comprise a sequence of any EBV viral protein (e.g., a sequence of at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous amino acids of any EBV protein). In some embodiments, the peptides provided herein comprise no more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 contiguous amino acids of the EBV viral protein.
  • the peptides provided herein comprise a sequence of LMP1 (e.g., a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous amino acids of LMP1). In some embodiments, the peptides provided herein comprise no more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 contiguous amino acids of LMP1.
  • An exemplary LMPl amino acid sequence is provided below (SEQ ID NO: 1):
  • the peptides provided herein comprise a sequence of LMP2A (e.g., a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous amino acids of LMP2A). In some embodiments, the peptides provided herein comprise no more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 contiguous amino acids of LMP2A.
  • An exemplary LMP2A amino acid sequence is provided below (SEQ ID NO: 2):
  • the peptides provided herein comprise a sequence of EBNA1 (e.g., a sequence ofat least 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous amino acids of EBNA1). In some embodiments, the peptides provided herein comprise no more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 contiguous amino acids of EBNA1.
  • SEQ ID NO: 3 An exemplary EBNA1 amino acid sequence is provided below (SEQ ID NO: 3):
  • the peptide comprises the sequence of an epi tope listed in Table 1.
  • the peptides provided herein comprise two or more of the CTL epitopes (e.g., viral epitopes). In some embodiments, the peptides provided herein comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 CTL epitopes. For example, in some embodiments, the peptides provided herein comprise two or more of the CTL epitopes connected by linkers (e.g., polypeptide linkers).
  • linkers e.g., polypeptide linkers
  • the sequence of the peptides compri se a viral protein sequence except for 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) conservative sequence modifications.
  • conservative sequence modifications is intended to refer to amino acid modifications that do not significantly affect or alter the interaction between a T cell receptor (TCR) and a peptide containing the amino acid sequence presented on an MHC.
  • conservative modifications include amino acid substitutions, additions (e.g., additions of amino acids to the N or C terminus of the peptide) and deletions (e.g., deletions of amino acids from the N or C terminus of the peptide).
  • Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains
  • one or more amino acid residues of the peptides described herein can be replaced with o ther amino acid residues from the same side chain family and the altered peptide can be tested for retention of TCR binding using methods known in the art. Modifications can be introduced into an antibody by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • the peptides provided herein comprise a sequence that is at least 80%, 85%, 90%, 95% or 100% identical to a protein sequence (e.g., the sequence of a fragment of a viral protein).
  • a protein sequence e.g., the sequence of a fragment of a viral protein.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid sequence for optimal alignment and non- identical sequences can be disregarded for comparison purposes).
  • the amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the peptides provided herein can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques, and can be produced by recombinant DNA techniques, and/or can be chemically synthesized using standard peptide synthesis techniques.
  • the peptides described herein can be produced in prokaryotic or eukaryotic host cells by expression of nucleotides encoding a peptide(s) of the present invention. Alternatively, such peptides can be synthesized by chemical methods.
  • nucleic acid molecules encoding the peptides described herein.
  • the nucleic acid molecule is a vector.
  • the nucleic acid molecule is a viral vector, such as an adenovirus based expression vector, that comprises the nucleic acid molecules described herein.
  • the vector provided herein encodes a plurality of epitopes provided herein ⁇ e.g., as a polyepitope).
  • the vector provided herein encodes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 epitopes provided herein ⁇ e.g., epitopes provided in Table 1).
  • the vector is AdEl-LMPpoly.
  • the AdEl-LMPpoly vector encodes a polyepitope of defined CTL epitopes from LMP1 and LMP2 fused to a Gly-Ala repeat-depleted EBNA1 sequence.
  • the AdEl-LMPpoly vector is described, for example, in Smith el al, Cancer Research 72: 1116 (2012); Duraiswamy et al, Cancer Research 64: 1483-9 (2004); and Smith et a!.., J. Immunol 117:4897-906, each of which is hereby incorporated by reference.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segm ents may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DN A segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced ⁇ e.g., bacterial vectors having a bacterial origin of replication, episomal mammalian vectors). Other vectors ⁇ e.g., non- episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby be replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).
  • nucleic acids operably linked to one or more regulatory sequences (e.g., a promotor) in an expression vector.
  • the cell transcribes the nucleic acid provided herein and thereby expresses a peptide described herein.
  • the nucleic acid molecule can be integrated into the genome of the cell or it can be extrachromasomal .
  • cells that contain a nucleic acid described herein e.g., a nucleic acid encoding a peptide described herein.
  • the cell can be, for example, prokaryotic, eukaryotic, mammalian, avian, murine and/or human.
  • the cell is a mammalian cell.
  • the cell is an APC (e.g. an antigen-presenting T cell, a dendritic cell, a B cell, or an aK562 cell).
  • a nucleic acid described herein can be administered to the cell, for example, as nucleic acid without delivery vehicle, in combination with a delivery reagent.
  • any nucleic acid delivery method known in the art can be used in the methods described herein.
  • Suitable delivery reagents include, but are not limited to, e.g., the Minis Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine), atelocollagen, nanoplexes, and liposomes.
  • liposomes are used to deliver a nucleic acid to a cell or subject.
  • Liposomes suitable for use in the methods described herein can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol.
  • lipids are generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream.
  • a variety of methods are known for preparing liposomes, for example, as described in Szoka el al.
  • the provided herein are methods of treating a cancer in a subject by administering to the subject a combination therapy described herein.
  • the methods provided herein can be used to treat any cancer.
  • the methods and CTLs described herein may be used to treat any cancerous or pre-cancerous tumor.
  • the cancer includes a solid tumor.
  • compositions provided herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma: carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma;
  • transitional cell carcinoma papillary transitional cell carcinoma; adenocarcinoma;
  • gastrinoma malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma;
  • nonencapsulating sclerosing carcinoma adrenal cortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous
  • adenocarcinoma adenocarcinoma
  • ceruminous adenocarcinoma adenocarcinoma
  • mucoepidermoid carcinoma adenocarcinoma
  • cystadenocarcinoma papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; mammary paget's disease; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; and malignant roblastoma; Sertoli cell carcinoma; malignant leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extra-mammary paraganglioma; pheochromocytoma;
  • glomangiosarcoma malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma;
  • myxosarcoma liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymoma; malignant brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma;
  • lymphangiosarcoma osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma;
  • ependymoma ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal;
  • cerebellar sarcoma cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; bas
  • the methods provided herein are used to treat EBV associated cancer.
  • the EBV-associated cancer is EBV-associated NPC.
  • the EBV associated cancer is post-transplant lymphoproliferative
  • PTLD tumor necrosis virus
  • the combination therapy further comprises a
  • chemotherapeutic agent e.g., alkylating agents or agents with an alkylating action, such as cyclophosphamide (CTX; e.g., CYTOXANcp), chlorambucil (CHL; e.g., LEUKERAN®), cisplatin (Cis P; e.g., PLATINOL®) busulfan (e.g., MYLERAN®), melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine (TEM), mitomycin C, and the like; antimetabolites, such as methotrexate (MTX), etoposide (VP 16; e.g., VEPESID®), 6- mercaptopurine (6MP), 6-thioguanine (6TG), cytarabine (Ara-C), 5-fluorouracil (5-FU), capecitabine (e.g.
  • CX cyclophosphamide
  • CHL chlorambuci
  • antibiotics such as actinomycin D, doxorubicin (DXR; e.g., ADRIAMYCIN®), daunoriibicin (daiinomycin), bleomycin, mithramycin and the like; alkaloids, such as vinca alkaloids such as vincristine (VCR), vinblastine, and the like; and other antitumor agents, such as paclitaxel (e.g., TAXOL®) and pactitaxel derivatives, the cytostatic agents, glucocorticoids such as dexamethasone (DEX; e.g., DECADRON®) and corticosteroids such as prednisone, nucleoside enzyme inhibitors such as hydroxyurea, amino acid depleting enzymes such as asparaginase, leucovorin and other folic acid derivatives, and similar, diverse antitumor agents.
  • antibiotics such as actinomycin D, doxorubicin (DXR;
  • cyclophosphamide lomustine (CCNU), doxorubicin lipo (e.g., DOXIL®), gemcitabine (e.g., GEMZAR®), daunoriibicin lipo (e.g., DAUNOXOME®), procarbazine, mitomycin, docetaxel (e.g., TAXOTERE®), aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan), 10-hydroxy 7-ethyl-camptothecin (SN38), fioxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon beta, interferon alpha, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pi
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions provided herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • the CTLs and the immune checkpoint inhibitors described herein may be co-administered or administered sequentially.
  • the immune checkpoint inhibitors may be administered by any technique known in the art.
  • the immune checkpoint inhibitor is administered intratumorally.
  • the immune checkpoint inhibitor is administered intravenously.
  • the immune checkpoint inhibitor is administered parenterally.
  • the subject has been exposed to a virus (e.g. EBV) such that virus particles are detectable in the subject's blood.
  • the method further comprises measuring viral load in the subject (e.g., before or after administering the peptide specific CTLs to the subject). Determining viral load in a subject may be a good prognostic marker for immunotherapy effectiveness.
  • selecting CTLs further comprises determining the number of viral DNA copies in the subject (e.g. in a tissue or blood sample). In some embodiments, viral load is measured two or more times.
  • the method further includes selecting allogeneic CTLs for combination therapy because they express a TCR restricted to a class I MHC that is encoded by an HLA allele that is present in the subject.
  • the CTLs are selected if the CTLs and subject share at least 2 (e.g., at least 3, at least 4, at least 5, at least 6) HLA alleles and the CTLs are restricted through a shared HLA allele.
  • the method comprises testing the TCR repertoire of the pre-generated mird-party-donor-derived epitope-specific T cells (i.e., allogeneic T cells) with flow cytometry.
  • epitope-specific T cells are detected using a tetramer assay, an ELISA assay, a western blot assay, a fluorescent microscopy assay, an Edman degradation assay and/or a mass spectrometry assay (e.g., protein sequencing).
  • the TCR repertoire is analyzed using a nucleic acid probe, a nucleic acid amplification assay and/or a sequencing assay.
  • the allogeneic CTLs are obtained from a cell bank.
  • PBMC peripheral blood mononuclear cells
  • the AdEl- LMPpoly vector was then used to infect 30% of the PBMC (MOT of 10: 1) that were then irradiated and co-cultured with the remaining PBMC for two weeks. Cultures were supplemented with fresh growth medium and 120lU/mL of recombinant IL-2 even' 3-4 days ( Komtur Pharmaceuticals, Frieburg, Germany). Cultured T cells were tested for antigen specificity using intracellular cytokine analysis and microbial contamination prior to release for infusion.
  • FACS profiling was performed to characterize the expression of immune checkpoint proteins by the T cells administered to the subjects.
  • MHC tetramers were generated in house. T cells were incubated for 20 minutes at 4°C with APC-labelled MHC class I tetramers specific for the HLA A1 1 -restricted epitope SSCSSCPLSKI (LMP2A), the HLA A24 restricted epitope TYGPVFMCL (LMP2A) and the HLA Cw03 restricted epitope
  • FVYGGSKTSL EBNA1
  • EBNA1 FVYGGSKTSL
  • Figure 1 shows the expression of immune checkpoint molecules (i.e., LAG-3, ⁇ -3, CTLA4, or PD-1).
  • Panel A depicts percentage of HLA-multimer positive CD8-positive lymphocytes that express PD-1, ⁇ -3, LAG-3 and CTLA-4.
  • Panel B depicts the percentage of PD-1 positive, TIM-3 positive, LAG-3 positive and CTLA-4 positive lymphocytes in the CTL immunotherapy administered in NPC patients with no/minimal residual disease (N/MRD) and active-recurrent/metastatic disease (ARMD) who either showed stable disease (SD) or progressive disease (PD) following adoptive T cell therapy.
  • N/MRD no/minimal residual disease
  • ARMD active-recurrent/metastatic disease
  • Example 2 Adoptive transfer o/EBV-CTLs and Checkpoint Inhibitor Therapy in NPC Patients Patients with platinum resistant or recurrent EBV-associated nasopharyngeal carcinoma (NPC) are treated with adoptive transfer of allogeneic Epstein-Barr virus cytotoxic T lymphocytes (EBV-CTLs) in combination with a checkpoint inhibitor
  • the study has two parts: Cohort 1 is enrolled as the Phase IB portion of the study to determine the Phase 2 dose; Cohort 2 is enrolled as the Phase 2 portion of the study to examine the clinical benefits of combined adoptiv e cellular and checkpoint inhibitor therapies for NPC.
  • the protocol will enroll 48 subjects in total.
  • Phase IB (Cohort 1) will enroll 12 subjects whose disease progressed despite prior PD1 inhibitor therapy, and Phase 2 (Cohort 2) will enroll 36 subjects naive to PD1 inhibitor therapy.
  • Allogeneic third-party EBV-CTLs are selected for each subject from the bank of available EBV-CTLs based on matching > 2 HLA alleles, at least one of which is a restricting HLA allele, shared between the EBV-CTLS source material (donor) and the subject.
  • High resolution HLA typing will be performed during screening to facilitate selection of the EBV-CTLS cell product.
  • Historical HLA typing will be acceptable if performed at high resolution (DNA based versus serologic assessment).
  • EBV-CTLs are administered at doses ranging from 500,000 to 200,000,000 T-cells per infusion intravenously on Day 1, Day 8, and Day 15 of a 21 Day Cycle to six subjects with advanced NPC.
  • the subject population is selected based on previous Phase 1 safety and efficacy data which showed adequate EBV CTL expansion and antitumor activity in patients with advanced NPC.
  • pembrolizumab is administered to Cohort 1 subjects at a dose of 200 mg IV Q3 weeks to adults (adults are greater than or equal to 18 years old) and at 2mg/kg IV Q3 weeks to pediatric subjects (less than 18 years old).
  • Phase IB Cohort 1 subjects experience dose limiting toxicity in the first 21 days, dose reduction of EBV-CTLS occurs, and the subsequent 6 subjects are treated with the combination of EBV-CTLS and pembrolizumab at the recommended dose level. Screening will begin up to 28 days prior to dosing (Cycle 1 Day 1 ). Subjects will be treated with EBV-CTLS in combination with pembrolizumab until disease progression or an unacceptable toxicity is observed.
  • reiractory/resistant defined as having at least one prior platinum-based
  • Phase IB (Cohort 1): patients who have received prior treatment with pembrolizumab anti-PDl, who have not received prior treatment with anti- PD-L1, anti-PD-L2, anti-CD137, anti-OX-40 or anti-CTLA-4 antibodies b.
  • Phase 2 (Cohort 2): patients who have not received prior treatment with
  • irradiated area are considered measurable if progression has been demonstrated in such lesions
  • Phase IB Patients in Phase IB must have a biopsy at baseline and on treatment of a metastatic lesion that can be biopsied with acceptable clinical risk (as judged by the investigator), and must agree to undergo biopsy.
  • Exclusion criteria A patient will not be eligible to participate in the study if any of the following criteria are met:
  • Pregnancy or breastfeeding females of childbearing potential must have a negative urine or serum pregnancy test. If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test will be required. The serum pregnancy must be confirmed negative within 72 hours of first dose for the patient to be eligible.
  • Exceptions include basal cell carcinoma of the skin, sq uamous cell carcinoma of the skin that has undergone potentially curative therapy or in situ cervical cancer.
  • Chemotherapy targeted small molecule therapy, hormonal therapy, or radiation therapy within 2 weeks of Cycle 1 Day 1 or who has not recovered (i.e., ⁇ Grade 1 or at baseline) from adverse events due to a previously administered agent.
  • Subjects with ⁇ Grade 2 neuropathy or ⁇ Grade 2 alopecia are an exception to this criterion and may qualify for the study.
  • metastases are treated and stable and the patient does not require systemic steroids
  • Subjects with previously treated brain metastases may participate provided they are stable (without evidence of progression by imaging (using the identical imaging modality for each assessment, either MR1 or CT scan) for at least four weeks prior to the first dose of trial treatment and any neurologic symptoms have returned to baseline), have no evidence of new or enlarging brain metastases, and are not using steroids for at least 7 days prior to trial treatment. This exception does not include carcinomatous meningitis which is excluded regardless of clinical stability.
  • Hepatitis B e.g. HBsAg reactive
  • Hepatitis C e.g. HCV RNA is detected.

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