WO2021126872A1 - Multi-specific t cell receptors - Google Patents

Multi-specific t cell receptors Download PDF

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Publication number
WO2021126872A1
WO2021126872A1 PCT/US2020/065147 US2020065147W WO2021126872A1 WO 2021126872 A1 WO2021126872 A1 WO 2021126872A1 US 2020065147 W US2020065147 W US 2020065147W WO 2021126872 A1 WO2021126872 A1 WO 2021126872A1
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Prior art keywords
mhc
tcr
antigen
recognizes
cancer
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PCT/US2020/065147
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English (en)
French (fr)
Inventor
Klaus Frueh
Louis Picker
Jonah SACHA
Scott Hansen
Benjamin BIMBER
Shaheed ABDULHAQQ
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Oregon Health and Science University
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Oregon Health and Science University
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Priority to CN202080087017.9A priority Critical patent/CN114828877A/zh
Priority to AU2020404933A priority patent/AU2020404933A1/en
Priority to EP20903063.4A priority patent/EP4076510A4/en
Priority to KR1020227021089A priority patent/KR20220116458A/ko
Priority to JP2022537047A priority patent/JP2023507142A/ja
Priority to CA3161826A priority patent/CA3161826A1/en
Priority to US17/786,186 priority patent/US20230272036A1/en
Publication of WO2021126872A1 publication Critical patent/WO2021126872A1/en
Anticipated expiration legal-status Critical
Priority to JP2025169565A priority patent/JP2026010019A/ja
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    • 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
    • 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
    • A61K39/12—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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • TCRs Conventionally restricted T cell receptors (TCRs) recognize a specific peptide, or epitope, within a given protein, or antigen, that is presented by a specific allele of major histocompatibility complex (MHC) class I or class II.
  • MHC major histocompatibility complex
  • the mouse T cell receptor OT-1 is specific to the murine MHC-I molecule Kb presenting the peptide SIINFEKL derived from the antigen ovalbumin.
  • TCR T cells are re-introduced into the patient for treatment (similar to T cells expressing a chimeric antigen receptor, or CAR).
  • the present invention relates to a method of generating CD8+ T cells comprising multi-specific TCRs, the method comprising: (a) administering to a subject a recombinant cytomegalovirus (CMV) vector comprising a nucleic acid sequence that encodes a first heterologous antigen, in an amount effective to generate a first set of CD8+ T cells that recognize a first MHC/heterologous antigen-derived peptide complex, wherein the CMV vector does not express an active UL128, UL130, UL146 and UL147 protein or orthologs thereof; (b) identifying a first CD8+ TCR from the first set of CD8+ T cells, wherein the first CD8+ TCR recognizes the first MHC/heterologous antigen- derived peptide complex; (c) administering to the subject a second heterologous antigen in an amount effective to generate a second set of CD8+ T cells that recognizes a second MHC/heterologous
  • the recombinant CMV vector does not express an active
  • the recombinant CMV vector expresses an active UL40 protein, or ortholog thereof, and an active US28 protein, or ortholog thereof.
  • the first MHC/heterologous antigen-derived peptide complex is a MHC-II/heterologous antigen-derived peptide complex, a MHC -E/heterologous antigen-derived peptide complex, or a MHC -I/heterologous antigen-derived peptide complex.
  • the second MHC/ heterologous antigen-derived peptide complex is a MHC-II/heterologous antigen-derived peptide complex or a MHC-E/ heterologous antigen-derived peptide complex.
  • the subject is a human or non-human primate.
  • the recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus macaque CMV vector.
  • the first and/or second heterologous antigen comprises a tumor antigen, pathogen-specific antigen, a tissue specific antigen, or a host-self antigen.
  • the tumor antigen is related to a cancer selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, ovarian cancer, colon cancer, renal cell carcinoma, and cervical cancer.
  • the pathogen-specific antigen is related to a pathogen selected from the group consisting of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, Epstein-barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Human T-lymphotropic virus type 1 (HTLV1), merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.
  • human immunodeficiency virus herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, Epstein-barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Human T-lymphotropic virus type 1 (HTLV1), merkel
  • the first CD8+ TCR recognizes specific MHC-II subtopes or supertopes. In one embodiment, the first CD8+ TCR recognizes specific MHC-E subtopes or supertopes. In one embodiment, wherein the first CD8+ TCR recognizes specific MHC-I subtopes or supertopes.
  • the first CD8+ TCR is identified by DNA or RNA sequencing. In another embodiment, the first CD8+ TCR is identified by single cell sequencing.
  • first heterologous antigen and second heterologous antigens are the same. In one embodiment, the first heterologous antigen and second heterologous antigen are different.
  • CD8+ T cells express CD69 and TNFa.
  • the second CD8+ TCR recognizes one or more specific
  • the second CD8+ TCR recognizes one or more specific MHC-E supertopes. In one embodiment, the second CD8+ TCR recognizes one or more specific MHC-I supertopes.
  • the second CD8+ TCR recognizes a MHC-II supertope and a
  • the second CD8+ TCR recognizes a MHC-I supertope and a MHC-E supertope. In one embodiment, the second CD8+ TCR recognizes a MHC-I supertope and a MHC-II supertope.
  • the second CD8+ TCR recognizes one or more specific
  • the second CD8+ TCR recognizes one or more specific MHC-E subtopes. In one embodiment, wherein the second CD8+ TCR recognizes one or more specific MHC-I subtopes.
  • the second CD8+ TCR recognizes a MHC-II subtope and a
  • the second CD8+ TCR recognizes a MHC-II subtope and a MHC-I subtope. In one embodiment, the second CD8+ TCR recognizes a MHC-E subtope and a MHC-I subtope.
  • the second CD8+ TCR recognizes a MHC-II subtope or supertope and a MHC-E subtope or supertope. In one embodiment, the second CD8+ TCR recognizes a MHC-II subtope or supertope and a MHC-I subtope or supertope. In one embodiment, the second CD8+ TCR recognizes a MHC-E subtope or supertope and a MHC-I subtope or supertope. [0021] In one embodiment, the second CD8+ TCR recognizes specific MHC-II supertopes and MHC-II subtopes.
  • the second CD8+ TCR recognizes specific MHC-E supertopes and MHC-E subtopes. In one embodiment, the second CD8+ TCR recognizes specific MHC-I supertopes and MHC-I subtopes.
  • the second CD8+ TCR recognizes more than one MHC-II supertope from the same antigen. In one embodiment, the second CD8+ TCR recognizes more than one MHC-E supertope from the same antigen. In one embodiment, the second CD8+ TCR recognizes more than one MHC-I supertope from the same antigen.
  • the second CD8+ TCR recognizes more than one MHC-II subtope from the same antigen. In one embodiment, the second CD8+ TCR recognizes more than one MHC-E subtope from the same antigen. In one embodiment, the second CD8+ TCR recognizes more than one MHC-I subtope from the same antigen.
  • the second CD8+ TCR recognizes one or more MHC-II supertopes and one or more MHC-II subtopes from the same antigen. In one embodiment, the second CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from the same antigen. In one embodiment, the second CD8+ TCR recognizes one or more MHC-I supertopes and one or more MHC-I subtopes from the same antigen.
  • the second CD8+ TCR recognizes more than one MHC-II supertope from more than one antigen. In one embodiment, the second CD8+ TCR recognizes more than one MHC-E supertope from more than one antigen. In one embodiment, the second CD8+ TCR recognizes more than one MHC-I supertope from more than one antigen.
  • the second CD8+ TCR recognizes more than one MHC-II subtope from more than one antigen. In one embodiment, the second CD8+ TCR recognizes more than one MHC-E subtope from more than one antigen. In one embodiment, the second CD8+ TCR recognizes more than one MHC-I subtope from more than one antigen.
  • the second CD8+ TCR recognizes one or more MHC-II supertopes and one or more MHC-II subtopes from different antigens. In one embodiment, the second CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from different antigens. In one embodiment, the second CD8+ TCR recognizes one or more MHC-I supertopes and one or more MHC-I subtopes from different antigens.
  • the third CD8+ TCR recognizes one or more specific MHC-II supertopes. In one embodiment, the third CD8+ TCR recognizes one or more specific MHC-E supertopes. In one embodiment, the third CD8+ TCR recognizes one or more specific MHC-I supertopes.
  • the third CD8+ TCR recognizes one or more specific MHC-II subtopes. In one embodiment, the third CD8+ TCR recognizes one or more specific MHC-E subtopes. In one embodiment, the third CD8+ TCR recognizes one or more specific MHC-I subtopes.
  • the third CD8+ TCR recognizes specific MHC-II supertopes and MHC-II subtopes. In one embodiment, the third CD8+ TCR recognizes specific MHC-E supertopes and MHC-E subtopes. In one embodiment, the third CD8+ TCR recognizes specific MHC-I supertopes and MHC-I subtopes.
  • the third CD8+ TCR recognizes more than one MHC-II supertope from one antigen. In one embodiment, the third CD8+ TCR recognizes more than one MHC-E supertope from one antigen. In one embodiment, the third CD8+ TCR recognizes more than one MHC-I supertope from one antigen.
  • the third CD8+ TCR recognizes more than one MHC-II subtope from one antigen. In one embodiment, the third CD8+ TCR recognizes more than one MHC-E subtope from one antigen. In one embodiment, wherein the third CD8+ TCR recognizes more than one MHC-I subtope from one antigen.
  • the third CD8+ TCR recognizes one or more MHC-II supertopes and one or more MHC-II subtopes from one antigen. In one embodiment, the third CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from one antigen. In one embodiment, the third CD8+ TCR recognizes one or more MHC-I supertopes and one or more MHC-I subtopes from one antigen.
  • the third CD8+ TCR recognizes more than one MHC-II supertope from more than one antigen. In one embodiment, the third CD8+ TCR recognizes more than one MHC-E supertope from more than one antigen. In one embodiment, the third CD8+ TCR recognizes more than one MHC-E supertope from more than one antigen. [0035] In one embodiment, the third CD8+ TCR recognizes more than one MHC-II subtope from more than one antigen. In one embodiment, the third CD8+ TCR recognizes more than one MHC-E subtope from more than one antigen. In one embodiment, the third CD8+ TCR recognizes more than one MHC-I subtope from more than one antigen.
  • the third CD8+ TCR recognizes specific MHC-E subtopes or supertopes and MHC-II subtopes or supertopes. In one embodiment, the third CD8+
  • TCR recognizes specific MHC-E subtopes or supertopes and MHC-I subtopes or supertopes.
  • the third CD8+ TCR recognizes specific MHC-II subtopes or supertopes and MHC-I subtopes or supertopes.
  • the third CD8+ TCR recognizes more than one MHC-II subtope from the same antigen. In one embodiment, the third CD8+ TCR recognizes more than one MHC-E subtope from the same antigen. In one embodiment, the third CD8+ TCR recognizes more than one MHC-I subtope from the same antigen.
  • the third CD8+ TCR recognizes one or more MHC-II supertopes and one or more MHC-II subtopes from different antigens. In one embodiment, the third CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from different antigens. In one embodiment, the third CD8+ TCR recognizes one or more MHC-I supertopes and one or more MHC-I subtopes from different antigens.
  • the nucleic acid sequence encoding the third CD8+ TCR is identical to the nucleic acid sequence encoding the second CD8+ TCR.
  • one or more CD8+ T cells are isolated from a second subject and transfecting the one or more CD8+ T cells with a nucleic acid sequence encoding the selected third CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the third CD8+ TCR, thereby generating one or more CD8+ T cells that recognize the first MHC/heterologous antigen-derived peptide complex and the second MHC/heterologous antigen-derived peptide complex.
  • the first MHC-heterologous antigen-derived peptide complex is a MHC-Q/heterologous antigen-derived peptide complex, a MHC -E/heterologous antigen-derived peptide complex, or a MHC -I/heterologous antigen-derived peptide complex.
  • the second MHC-heterologous antigen-derived peptide complex is a MHC-II/heterologous antigen-derived peptide complex, a MHC- E/heterologous antigen-derived peptide complex, or a MHC-I/heterologous antigen- derived peptide complex.
  • the transfected CD8+ T cells are administered to the second subject to treat or prevent cancer.
  • the cancer is selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, ovarian cancer, colon cancer, renal cell carcinoma, and cervical cancer.
  • the transfected CD8+ T cells are administered to the second subject to treat a pathogenic-infection
  • the pathogenic infection is selected from the group consisting of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, Epstein-barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Human T-lymphotropic virus type 1 (HTLV1), merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.
  • the first subject is a nonhuman primate and the second subject is a human
  • the transfected CD8+ T cells comprises a chimeric nonhuman primate-human CD8+ TCR comprising the non-human primate CDR3a and CDR3P of the second CD8+ TCR.
  • the third CD8+ TCR comprises the non-human primate
  • the third CD8+ TCR comprises the CDRla, CDR2a, CDR3a, CDR 1 b, CDR2p, and CDR3p of the second CD8+ TCR.
  • the first subject is a nonhuman primate and the second subject is a human
  • the second CD8+ TCR is a chimeric nonhuman primate-human CD8+ TCR comprising the non-human primate CDR3a and CDR3P of the first CD8+ TCR.
  • the third CD8+ TCR is a chimeric CD8+ TCR.
  • administering the recombinant CMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration.
  • a CD8 + T cell comprising the multi-specific TCR is generated by the method.
  • the CD8+ T cell is administered to a subject in need thereof to treat or prevent cancer.
  • the cancer is selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, ovarian cancer, colon cancer, renal cell carcinoma, and cervical cancer.
  • the CD8+ T cell is administered to a subject in need thereof to treat a pathogenic infection.
  • the pathogenic infection is selected from the group consisting of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, Epstein-barr virus (EBV), Kaposi's sarcoma- associated herpesvirus (KSHV), Human T-lymphotropic virus type 1 (HTLV1), merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.
  • human immunodeficiency virus herpes simplex virus type 1
  • herpes simplex virus type 2 hepatitis B virus
  • hepatitis C virus papillomavirus
  • Plasmodium parasites Epstein-barr virus (EBV), Kaposi's sarcoma- associated herpesvirus (KSHV),
  • the recombinant CMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration.
  • the present invention also relates to a method of generating CD8+ T cells comprising a multi-specific T cell receptor (TCR) comprising: (a) administering to a subject a recombinant cytomegalovirus (CMV) vector comprising a nucleic acid sequence that encodes a first heterologous antigen, in an amount effective to generate a first set of CD8+ T cells that recognize a first MHC-E/ heterologous antigen-derived peptide complex, wherein the CMV vector does not express an active UL128, UL130, UL146 and UL147 protein or orthologs thereof, and wherein the recombinant CMV vector further comprises a microRNA recognition element (MRE); (b) identifying a first CD8+ TCR from the first set of CD8+ T cells, wherein the first CD8+ TCR recognizes the first MHC- E/heterologous antigen-derived peptide complex; (c) administering to the subject a second heterologous antigen in
  • the subject is a human or non-human primate.
  • the recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus macaque CMV vector.
  • the first heterologous antigen comprises a tumor antigen, pathogen-specific antigen, a tissue specific antigen, or a host-self antigen.
  • the tumor antigen is related to a cancer selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, ovarian cancer, colon cancer, renal cell carcinoma, and cervical cancer.
  • the pathogen-specific antigen is related to a pathogen selected from the group consisting of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, Epstein-barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Human T-lymphotropic virus type 1 (HTLV1), merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.
  • human immunodeficiency virus herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, Epstein-barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Human T-lymphotropic virus type 1 (HTLV1), merkel
  • the MRE contains target sites for microRNAs expressed in endothelial cells.
  • the MRE is specific for the miRNA selected from the group consisting of miR126, miR-126-3p, miR-130a, miR-210, miR-221/222, miR-378, miR-296, and miR-328.
  • the first CD8+ TCR recognizes specific MHC-E subtopes or supertopes.
  • the first CD8+ TCR is identified by DNA or RNA sequencing. In one embodiment, the first CD8+ TCR is identified by single cell sequencing.
  • the second heterologous antigen comprises a tumor antigen, pathogen-specific antigen, a tissue specific antigen, or a host-self antigen.
  • the tumor antigen is related to a cancer selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, ovarian cancer, colon cancer, renal cell carcinoma, and cervical cancer.
  • the pathogen-specific antigen is related to a pathogen selected from the group consisting of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, Epstein-barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Human T-lymphotropic virus type 1 (HTLV1), merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.
  • human immunodeficiency virus herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, Epstein-barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Human T-lymphotropic virus type 1 (HTLV1), merkel
  • first heterologous antigen and second heterologous antigens are the same. In one embodiment, the first heterologous antigen and second heterologous antigen are different.
  • CD8+ T cells express CD69 and TNFa.
  • the second CD8+ TCR is identified by DNA or RNA sequencing. In one embodiment, the second CD8+ TCR is identified by single cell sequencing.
  • the second CD8+ TCR recognizes one or more specific
  • the second CD8+ TCR recognizes one or more specific MHC-E subtopes. In one embodiment, the second CD8+ TCR recognizes specific MHC-E supertopes and MHC-E subtopes. [0065] In one embodiment, the second CD8+ TCR recognizes more than one MHC-E supertope from the same antigen. In one embodiment, the second CD8+ TCR recognizes more than one MHC-E subtope from the same antigen.
  • the second CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from the same antigen. In one embodiment, the second CD8+ TCR recognizes more than one MHC-E supertope from more than one antigen. In one embodiment, the second CD8+ TCR recognizes more than one MHC-E subtope from more than one antigen. In one embodiment, the second CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from different antigens.
  • the third CD8+ TCR recognizes one or more specific MHC-E supertopes. In one embodiment, the third CD8+ TCR recognizes one or more specific MHC-E subtopes. In one embodiment, the third CD8+ TCR recognizes specific MHC-E supertopes and MHC-E subtopes.
  • the third CD8+ TCR recognizes more than one MHC-E supertope from one antigen. In one embodiment, the third CD8+ TCR recognizes more than one MHC-E subtope from one antigen.
  • the third CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from one antigen. In one embodiment, the third CD8+ TCR recognizes more than one MHC-E supertope from more than one antigen.
  • the third CD8+ TCR recognizes more than one MHC-E subtope from more than one antigen. In one embodiment, the third CD8+ TCR recognizes specific MHC-E supertopes and MHC-E subtopes.
  • the third CD8+ TCR recognizes more than one MHC-E subtope from the same antigen. In one embodiment, the third CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from different antigens.
  • the nucleic acid sequence encoding the third CD8+ TCR is identical to the nucleic acid sequence encoding the second CD8+ TCR.
  • one or more CD8+ T cells are isolated from a second subject and transfecting the one or more CD8+ T cells with a nucleic acid sequence encoding the selected third CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the third CD8+ TCR, thereby generating one or more CD8+ T cells that recognize the first MHC -E/heterologous antigen-derived peptide complex and the second MHC-E/heterologous antigen-derived peptide complex.
  • the transfected CD8+ T cells are administered to the second subject to treat or prevent cancer.
  • the cancer is selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, ovarian cancer, colon cancer, renal cell carcinoma, and cervical cancer.
  • the transfected CD8+ T cells are administered to the second subject to treat a pathogenic-infection.
  • the pathogenic infection is selected from the group consisting of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, Epstein-barr virus (EBV), Kaposi's sarcoma- associated herpesvirus (KSHV), Human T-lymphotropic virus type 1 (HTLV1), merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.
  • human immunodeficiency virus herpes simplex virus type 1
  • herpes simplex virus type 2 hepatitis B virus
  • hepatitis C virus papillomavirus
  • Plasmodium parasites Epstein-barr virus (EBV), Kaposi's sarcoma- associated herpesvirus (KSH
  • the first subject is a nonhuman primate and the second subject is a human
  • the transfected CD8+ T cells comprises a chimeric nonhuman primate-human CD8+ TCR comprising the non-human primate CDR3a and CDR3P of the second CD8+ TCR.
  • the third CD8+ TCR comprises the non-human primate
  • the third CD8+ TCR comprises the CDRla, CDR2a, CDR3a, CDR1 b, CDR2p, and CDR3p of the second CD8+ TCR.
  • the first subject is a nonhuman primate and the second subject is a human
  • the second CD8+ TCR is a chimeric nonhuman primate-human CD8+ TCR comprising the non-human primate CDR3a and CDR3P of the first CD8+ TCR.
  • the third CD8+ TCR is a chimeric CD8+ TCR.
  • administering the recombinant CMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration.
  • a CD8 + T cell comprising the multi-specific TCR is generated by the method.
  • the CD8+ T cell is administered to a subject in need thereof to treat or prevent cancer.
  • the cancer is selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, ovarian cancer, colon cancer, renal cell carcinoma, and cervical cancer.
  • the CD8+ T cell is administered to a subject in need thereof to treat a pathogenic infection.
  • the pathogenic infection is selected from the group consisting of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, Epstein-barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Human T-lymphotropic virus type 1 (HTLV1), merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.
  • human immunodeficiency virus herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, Epstein-barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Human T
  • administering the recombinant CMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration.
  • FIGs. 1 A-B show an overview of the primary study cohort.
  • FIG. 1 A is a timeline showing the vaccination dates and sampling window used in this study.
  • FIG. IB shows the overlapping 15 SIVgag peptides recognized by rhesus macaques (RM) using intracellular cytokine staining (ICS) with each T cell-targeted peptide "box” colored based on their MHC restriction as determined by differentially blocking analysis.
  • Green MHC-E
  • red MHC- la
  • blue MHC -II
  • purple indeterminate.
  • the MHC-E and MHC-II restricted supertopes are labeled.
  • FIG. 2A-F show TCR clonotypic hierarchies of MHC-E supertope responses.
  • FIG. 2A shows peripheral blood mononuclear cells (PBMC) from RhCMV 68-1/SIVgag- vaccinated RM (Rh-1) that were stimulated with EK9 peptide in the presence of the secretion inhibitor Brefeldin A and intracellular cytokine (TNF-a v. IFN-g) analysis (ICS) was performed to identify EK9-specific CD8+ T cells (left).
  • PBMC peripheral blood mononuclear cells
  • Rh-1 RhCMV 68-1/SIVgag- vaccinated RM
  • ICS intracellular cytokine
  • STTS analysis right
  • FIG. 2B are bar plots illustrating the clonotypic hierarchies for each time point, based on CDR3 alpha and/or beta sequence.
  • a given TCR a/b pair clone was found in the responsive fraction after both EK9 and RL9 stimulation (asterisks).
  • FIG. 2F shows a representative ICS experiment, in which these transductants were cultured with BLCL pulsed with no peptide, a negative control peptide, Gag RL9, or Gag EK9.
  • FIGs. 3 A-3B show the SIVgag recognition by TCR transductants.
  • FIG. 3 A are the results of a flow cytometry experiment showing target cells that were generated by infecting purified SEB/CD3 -activated Rh-4 CD4+ T cells with SIVmac239 or transducing Rh-5 (Mamu-A*01+) BLCL with a retrovirus the expressing both SIV Gag and truncated NGFR, which provides a surface marker (NGFR-T2A-Gag).
  • FIG. 3B are ICS assays with the target cells and the indicated MHC-E-TCR CD8+ T cell transductants.
  • CD8+ T cell transductants expressing a Mamu-A*01 -restricted, CM9-specific TCR were used as a positive control.
  • Non-transduced CD8+ T cells or CD8+ T cell transductants expressing an (irrelevant) MR1 -restricted TCR were used as negative controls.
  • FIGs. 4A-4D show pie charts demonstrating the complete clonotypic hierarchies for SIV-infected recognition.
  • FIG. 5 shows the analysis of epitope cross-reactivity using TCR transductants.
  • ICS using CD8+ T cell transductants expressing Rh-1 MHC-E-TCR4 vs. TCR 6-1 were cultured with RM BLCL pulsed with SIVgag MHC-E optimal supertope and subtope peptides for Rh-1, as indicated. Responses were measured using IFN-g and TNF-a staining.
  • FIGs. 6A-6B show the response of CD8+ T cells expressing TCRs with MHC-E- presented SIVgag peptide recognition to MHC-II-presented SIVgag peptides and with peptides from an unrelated TB antigen.
  • PBMC from Rh-4 was stimulated with either of the MHC-II supertope peptides Gag2ii-222 (53) or Gag29o-3oi (73) (FIG. 6A) or with a pool of overlapping 15mer peptides from the TB protein Ag85B (FIG. 6B).
  • Activated cells were sorted based on sCD69 and stTNF-a, and TCRs were characterized by scRNAseq.
  • FIGs. 7A-7J show the cross-reaction of MHC-E-restricted TCRs with CMV IE peptides presented by MHC-Ia.
  • FIG. 7A is a flow cytometry experiment to analyze the response of the four RM to AN10 and VY9 tetramers.
  • FIG. 7B-7E are graphs showing the clonotypic hierarchies for each peptide-specific response identified by both approaches in each RM (note concordance of TCR identification by both approaches).
  • FIG. 7F shows ICS analysis of CD8+ transductants expressing TCR2 (top) or TCR4 (bottom) cultured with (Mamu-A*02+ and MHC-E+) BLCL pulsed with the indicated peptide.
  • FIGs. 7G-7J are pie charts showing the clonotypic hierarchies from SIV-infected cell recognition assays, identical to FIG. 4, except TCR clones are shaded based on whether they cross-react with AN10/VY9 or not.
  • FIGs. 8A-8B show validation of MHC-Ia restriction by VY9 blocking.
  • FIG. 8 A shows CD8+ transductants expressing TCR were cultured with Mamu-A*02+ and MHC- E+ BLCL pulsed with the indicated peptide (top row).
  • the BLCL were pre incubated with the strongly MHC -E-binding VL9 peptide prior to pulsing with the epitopic peptide to assess MHC-E restriction of the individual responses (bottom row).
  • FIG. 8 A shows CD8+ transductants expressing TCR were cultured with Mamu-A*02+ and MHC- E+ BLCL pulsed with the indicated peptide (top row).
  • the BLCL were pre incubated with the strongly MHC -E-binding VL9 peptide prior to pulsing with the epitopic peptide to assess MHC-E restriction of the individual responses (bottom row).
  • CM9 non- A* 02 binder
  • GY9 weak A* 02 binder
  • YY9 strong A*02 binder
  • FIG. 9 shows ICS demonstrating the specificity analysis of dual-TCR expressing clonotypes.
  • FIGs. 10A-10B show the functional avidity analysis of MHC-Ia- and MHC-E- restricted responses mediated by the same TCR.
  • Mamu-Al*002 BLCL were pulsed with ten-fold dilutions EK9 or VY9 peptides starting at 200mM.
  • BLCL were washed and incubated with TCR2 CD8+ T cell transductants in three separate experiments.
  • FIG. 10A shows representative flow cytometric data from one experiment.
  • FIG. 10B is a graph showing the results from all the experiments.
  • FIGs. 11 A-l 1G shows the transcriptomic response of MHC-E-restricted, SlVgag- reactive CD8+ T cells, with and without MHC-Ia-IE epitope cross-reactivity.
  • FIG. 11 A shows a tSNE plot of scRNA-seq of purified CD8+ T cells incubated with BLCL pulsed with EK9 and RL9. Cells were clustered based on transcriptional profile. Colors denote the results of unsupervised clustering. Dots indicate cells expressing previously identified TCR pairs previously identified as MHC-E-restricted (FIG. 4).
  • FIG. 1 IB is a heatmap of the scRNA-seq data.
  • FIG. 11C shows the activation score in the tSNE plot. The activation score was calculated based on the combined expression of nine canonical marker genes [IFNG, MIP-1B (CCL4), TNFRSF9,
  • FIG. 1 ID shows the gating of total CD69+ cells.
  • FIG. 1 IE shows the tSNE plot of scRNA-seq of purified CD8+ T cells incubated with BLCL pulsed with EK9 and RL9.
  • FIG. 1 IF shows the activation score in the tSNE plot.
  • FIG. 11G are graphs showing the activation score of the CD8+ T cells expressing each indicated TCR to each indicated antigen stimulus. Dotted blue line denotes the threshold at which cells are considered activated.
  • Antigen As used herein, the terms “antigen” or “immunogen” are used interchangeably to refer to a substance, typically a protein, which is capable of inducing an immune response in a subject. The term also refers to proteins that are immunologically active in the sense that once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector that encodes the protein) the protein is able to evoke an immune response of the humoral and/or cellular type directed against that protein.
  • Antigen-specific T cell A CD8 + or CD4 + lymphocyte that recognizes a particular antigen. Generally, antigen-specific T cells specifically bind to a particular antigen presented by MHC molecules, but not other antigens presented by the same MHC.
  • Administration means to provide or give a subject an agent, such as a composition comprising an effective amount of a CMV vector comprising an exogenous antigen by any effective route.
  • routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
  • Avidity refers to the strength of multiple affinities of individual non-covalent binding interactions such as antigen-antibody interactions. Avidity therefore gives a measure for the overall strength of an antigen- antibody complex.
  • Effective amount refers to an amount of an agent, such as a CMV vector comprising a heterologous antigen or a transfected CD8+ T cell that recognizes a MHC -E/heterologous antigen-derived peptide complex, a MHC-II/heterologous antigen-derived peptide complex, or a MHC- I/heterologous antigen-derived peptide complex, that is sufficient to generate a desired response, such as reduce or eliminate a sign or symptom of a condition or disease or induce an immune response to an antigen.
  • an agent such as a CMV vector comprising a heterologous antigen or a transfected CD8+ T cell that recognizes a MHC -E/heterologous antigen-derived peptide complex, a MHC-II/heterologous antigen-derived peptide complex, or a MHC- I/heterologous antigen-derived peptide complex
  • an "effective amount" is one that treats (including prophylaxis) one or more symptoms and/or underlying causes of any of a disorder or disease.
  • An effective amount may be a therapeutically effective amount, including an amount that prevents one or more signs or symptoms of a particular disease or condition from developing, such as one or more signs or symptoms associated with infectious disease or cancer.
  • Epitope refers to molecular structure which may completely make up a specific binding partner or be part of a specific binding partner to the binding domain or the T-cell receptor domain polypeptide of the present invention. Chemically, an epitope may either be composed of a carbohydrate, a peptide, a fatty acid, an organic, biochemical or inorganic substance or derivatives thereof and any combinations thereof. If an epitope is a polypeptide, it will usually include at least 3 amino acids, preferably 8 to 50 amino acids, and more preferably between about 10-20 amino acids in the peptide. There is no critical upper limit to the length of the peptide, which could comprise nearly the full length of a polypeptide sequence.
  • Epitopes can be either linear or conformational epitopes.
  • a linear epitope is comprised of a single segment of a primary sequence of a polypeptide chain.
  • Linear epitopes can be contiguous or overlapping.
  • Conformational epitopes are comprised of amino acids brought together by folding of the polypeptide to form a tertiary structure and the amino acids are not necessarily adjacent to one another in the linear sequence.
  • epitopes are at least part of diagnostically relevant molecules, i.e. the absence or presence of an epitope in a sample is qualitatively or quantitatively correlated to either a disease or to the health status of a patient or to a process status in manufacturing or to environmental and food status.
  • Epitopes may also be at least part of therapeutically relevant molecules, i.e. molecules which can be targeted by the specific binding domain which changes the course of the disease.
  • Heterologous antigen refers to any protein or fragment thereof that is not derived from CMV. Heterologous antigens may be pathogen-specific antigens, tumor virus antigens, tumor antigens, host self antigens, or any other antigen.
  • Immunogenic peptide A peptide which comprises an allele-specific motif or other sequence, such as an N-terminal repeat, such that the peptide will bind an MHC molecule and induce a cytotoxic T lymphocyte ("CTL”) response, or a B cell response (for example antibody production) against the antigen from which the immunogenic peptide is derived.
  • CTL cytotoxic T lymphocyte
  • B cell response for example antibody production
  • immunogenic peptides are identified using sequence motifs or other methods, such as neural net or polynomial determinations known in the art.
  • algorithms are used to determine the "binding threshold" of peptides to select those with scores that give them a high probability of binding at a certain affinity and will be immunogenic.
  • the algorithms are based either on the effects on MHC binding of a particular amino acid at a particular position, the effects on antibody binding of a particular amino acid at a particular position, or the effects on binding of a particular substitution in a motif-containing peptide.
  • a conserved residue is one which appears in a significantly higher frequency than would be expected by random distribution at a particular position in a peptide.
  • a conserved residue is one where the MHC structure may provide a contact point with the immunogenic peptide.
  • Mutation refers to any difference in a nucleic acid or polypeptide sequence from a normal, consensus, or "wild type" sequence.
  • a mutant is any protein or nucleic acid sequence comprising a mutation.
  • a cell or an organism with a mutation may also be referred to as a mutant.
  • Some types of coding sequence mutations include point mutations (differences in individual nucleotides or amino acids); silent mutations (differences in nucleotides that do not result in an amino acid changes); deletions (differences in which one or more nucleotides or amino acids are missing, up to and including a deletion of the entire coding sequence of a gene); frameshift mutations (differences in which deletion of a number of nucleotides indivisible by 3 results in an alteration of the amino acid sequence).
  • a mutation that results in a difference in an amino acid may also be called an amino acid substitution mutation.
  • Amino acid substitution mutations may be described by the amino acid change relative to wild type at a particular position in the amino acid sequence.
  • nucleotide sequences or nucleic acid sequences refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences, including, without limitation, messenger RNA (mRNA), DNA/RNA hybrids, or synthetic nucleic acids.
  • the nucleic acid may be single- stranded, or partially or completely double stranded (duplex).
  • Duplex nucleic acids may be homoduplex or heteroduplex.
  • Operably linked As the term "operably linked” is used herein, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in such a way that it has an effect upon the second nucleic acid sequence. Operably linked DNA sequences may be contiguous, or they may operate at a distance.
  • promoter may refer to any of a number of nucleic acid control sequences that directs transcription of a nucleic acid.
  • a eukaryotic promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element or any other specific DNA sequence that is recognized by one or more transcription factors. Expression by a promoter may be further modulated by enhancer or repressor elements. Numerous examples of promoters are available and well known to those of ordinary skill in the art.
  • a nucleic acid comprising a promoter operably linked to a nucleic acid sequence that codes for a particular polypeptide may be termed an expression vector.
  • Recombinant As used herein, the term "recombinant" with reference to a nucleic acid or polypeptide refers to one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence, for example a CMV vector comprising a heterologous antigen.
  • a recombinant polypeptide may also refer to a polypeptide that has been made using recombinant nucleic acids, including recombinant nucleic acids transferred to a host organism that is not the natural source of the polypeptide (for example, nucleic acids encoding polypeptides that form a CMV vector comprising a heterologous antigen).
  • compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein are conventional.
  • the nature of the carrier will depend on the particular mode of administration being employed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle.
  • non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Polynucleotide refers to a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • a polynucleotide is made up of four bases; adenine, cytosine, guanine, and thymine/uracil (uracil is used in RNA).
  • a coding sequence from a nucleic acid is indicative of the sequence of the protein encoded by the nucleic acid.
  • Polypeptide The terms "protein”, “peptide”, “polypeptide”, and “amino acid sequence” are used interchangeably herein to refer to polymers of amino acid residues of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
  • sequence identity counted over the full-length alignment with the amino acid sequence of specific protein using ALIGN set to default parameters. Proteins with even greater similarity to a reference sequence will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 98% sequence identity.
  • sequence identity can be compared over the full length of particular domains of the disclosed peptides.
  • Sequence identity/similarity As used herein, the identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity may be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity may be measured in terms of percentage identity or similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Polypeptides or protein domains thereof that have a significant amount of sequence identity and also function the same or similarly to one another (for example, proteins that serve the same functions in different species or mutant forms of a protein that do not change the function of the protein or the magnitude thereof) may be called "homologs.”
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, (1990) supra) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38 A, Room 8N805, Bethesda, MD 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information may be found at the NCBI web site.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
  • the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences.
  • 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2.
  • the length value will always be an integer.
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • Homologs are typically characterized by possession of at least 70% sequence identity counted over the full-length alignment with an amino acid sequence using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr database, swissprot database, and patented sequences database. Queries searched with the blastn program are filtered with DUST (Hancock & Armstrong, Comput Appl Biosci 10, 67-70 (1994.) Other programs use SEG. In addition, a manual alignment may be performed. Proteins with even greater similarity will show increasing percentage identities when assessed by this method, such as at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a protein.
  • nucleic acid sequences that do not show a high degree of identity may nevertheless encode identical or similar (conserved) amino acid sequences, due to the degeneracy of the genetic code. Changes in a nucleic acid sequence may be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.
  • homologous nucleic acid sequences can, for example, possess at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to a nucleic acid that encodes a protein.
  • binding refers to a binding reaction which is determinative of the cognate ligand of interest in a heterogeneous population of molecules.
  • the specified T-cell receptor domain polypeptide binds to its particular "target” and does, not bind in a significant amount to other molecules present in a sample.
  • Subject refers to a living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals.
  • Subtope refers to a subdominant epitope or peptide that is recognized by T cells.
  • Supertope As used herein, the term “supertope” or “supertope peptide” refers to a epitope or peptide that is recognized by T cells in greater than about 90% of the population regardless of MHC haplotype, /. e. , in the presence or absence of given MHC-I, MHC-II, or MHC-E alleles.
  • treatment refers to an intervention that ameliorates a sign or symptom of a disease or pathological condition.
  • treatment refers to any observable beneficial effect of the treatment.
  • the beneficial effect may be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of relapses of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease.
  • a prophylactic treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs, for the purpose of decreasing the risk of developing pathology.
  • a therapeutic treatment is a treatment administered to a subject after signs and symptoms of the disease have developed.
  • Vaccine An immunogenic composition that can be administered to a mammal, such as a human, to confer immunity, such as active immunity, to a disease or other pathological condition.
  • Vaccines can be used prophylactically or therapeutically.
  • vaccines can be used reduce the likelihood of developing a disease (such as a tumor or pathological infection) or to reduce the severity of symptoms of a disease or condition, limit the progression of the disease or condition (such as a tumor or a pathological infection), or limit the recurrence of a disease or condition (such as a tumor).
  • a vaccine is a replication-deficient CMV expressing a heterologous antigen, such as a tumor associated antigen derived from a tumor of the lung, prostate, ovary, breast, colon, cervix, liver, kidney, bone, or a melanoma.
  • a heterologous antigen such as a tumor associated antigen derived from a tumor of the lung, prostate, ovary, breast, colon, cervix, liver, kidney, bone, or a melanoma.
  • Vector Nucleic acid molecules of particular sequence can be incorporated into a vector that is then introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art, including promoter elements that direct nucleic acid expression.
  • Vectors can be viral vectors, such as CMV vectors. Viral vectors may be constructed from wild type or attenuated virus, including replication deficient virus.
  • T-Cell Receptor refers to a heterodimeric molecule comprising an alpha polypeptide chain (alpha chain) and a beta polypeptide chain (beta chain), wherein the heterodimeric receptor is capable of binding to a peptide antigen presented by an HLA molecule.
  • Multiple-Specific T-Cell Receptor As used herein, the term "multiple-specific
  • T-cell receptor refers to a T-cell receptor that is capable of binding to multiple peptide antigens.
  • the peptide antigens may be from the same or different antigens.
  • the peptide antigens may be presented by the same or different HLA molecules.
  • TCRs Multi-specific T cell receptors
  • the present invention is directed to TCRs with multiple specificities to unrelated peptides. T cells bearing these TCRs can be used in patient treatments.
  • the present invention is also directed to a method of generating CD8+ T cells comprising a multi-specific T cell receptor (TCR), wherein the method comprises administering to a subject a recombinant CMV vector comprising a nucleic acid sequence that encodes a first heterologous antigen, in an amount effective to generate a first set of CD8+ T cells that recognize a first MHC/heterologous antigen-derived peptide complex, wherein the CMV vector does not express an active UL128, UL130, UL146 and UL147 protein or orthologs thereof; identifying a first CD8+ TCR from the first set of CD8+ T cells, wherein the first CD8+ TCR recognizes the first MHC/heterologous antigen- derived peptide complex; administering to the subject a second heterologous antigen in an amount effective to generate a second set of CD8+ T cells that recognizes a second MHC/heterologous antigen-derived peptide complex; iso
  • RhCMV Rhesus Cytomegalovirus
  • RhCMV homologues of human CMV UL128, UL130, UL146 and UL147 while expressing the homologs of UL40 and US28 efficiently elicit broadly targeted Mamu E- restricted CD8+ T cell responses in rhesus monkeys to virtually any protein expressed by this vector, including both RhCMV proteins and exogenous protein inserts, the latter including bacterial, viral and self-protein.
  • the subject is a human or non-human primate.
  • the recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus macaque CMV vector.
  • the recombinant CMV does not express an active UL128, UL130, UL146 and UL147 protein due to the presence of a mutation in the nucleic acid sequence encoding UL128, UL130, UL146 and UL147 or homologs thereof, or orthologs thereof (homologous genes of CMV that infect other species).
  • the recombinant CMV does not express an active UL128, UL130, UL146, UL147, and UL18 protein due to the presence of a mutation in the nucleic acid sequence encoding UL128, UL130, UL146, UL147, and UL18 or homologs thereof, or orthologs thereof (homologous genes of CMV that infect other species).
  • the mutation may be any mutation that results in a lack of expression of the active UL128, UL130, UL146, UL147 or US18 proteins.
  • Such mutations may include point mutations, frameshift mutations, deletions of less than all of the sequence that encodes the protein (truncation mutations), or deletions of all of the nucleic acid sequence that encodes the protein, or any other mutations.
  • Exemplary vectors are described in US Pat. Nos. 9,783,823 and 9,862,972, and US Appl. Pub. No. 2018/0298404 which are herein incorporated by reference.
  • the recombinant CMV vector does not express an active
  • the recombinant CMV vector does not express an active UL128, UL130, UL146, UL147, and UL18 protein, or homologs thereof, or orthologs thereof, and expresses an active UL40 and US28 protein, or homologs thereof, or orthologs thereof.
  • the first MHC/heterologous antigen-derived peptide complex is a MHC-II/heterologous antigen-derived peptide complex, a MHC- E/heterologous antigen-derived peptide complex, or a MHC-I/heterologous antigen- derived peptide complex.
  • the second MHC/ heterologous antigen- derived peptide complex is a MHC-IEheterologous antigen-derived peptide complex, a MHC-E/heterologous antigen-derived peptide complex, or a MHC -I/heterologous antigen-derived peptide complex.
  • Human or animal CMV vectors when used as expression vectors, are innately non-pathogenic in the selected subjects such as humans.
  • the CMV vectors have been modified to render them non-pathogenic (incapable of host-to-host spread) in the selected subjects.
  • a heterologous antigen can be any protein or fragment thereof that is not derived from CMV, including tumor antigens, pathogen-specific antigens, model antigens (such as lysozyme, keyhole-limpet hemocyanin (KLH), or ovalbumin), tissue-specific antigens, host self-antigens, or any other antigen.
  • model antigens such as lysozyme, keyhole-limpet hemocyanin (KLH), or ovalbumin
  • tissue-specific antigens such as lysozyme, keyhole-limpet hemocyanin (KLH), or ovalbumin
  • host self-antigens or any other antigen.
  • Pathogen specific antigens can be derived from any human or animal pathogen.
  • the pathogen may be a viral pathogen and the antigen may be a protein derived from the viral pathogen.
  • Viruses include, but are not limited to retroviruses, polyomaviruses, Adenovirus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, Herpes simplex, type 1, Herpes simplex, type 2, Varicella-zoster virus, Epstein-Barr virus, Kaposi's sarcoma herpesvirus, Human cytomegalovirus, Human herpesvirus, type 8, Hepatitis B virus, Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, Human immunodeficiency virus (HIV), Influenza virus, Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Human metapneumovirus, Human papillomavirus, Rabies virus, Rubella virus, Human bocavirus, human T-lymphotropic virus (HTLV
  • the pathogen may be a bacterial pathogen and the antigen may be a protein derived from the bacterial pathogen.
  • the pathogenic bacteria include, but are not limited to, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfr ingens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrog
  • the pathogen may be a parasite and the antigen may be a protein derived from the parasite pathogen.
  • the parasite may be a protozoan organism or a protozoan organism causing a disease such as, but not limited to, Acanthamoeba, Babesiosis, Balantidiasis, Blastocystosis, Coccidia, Dientamoebiasis, Amoebiasis, Giardia, Isosporiasis, Leishmaniasis, Primary amoebic meningoencephalitis (PAM), Malaria, Rhinosporidiosis, Toxoplasmosis— Parasitic pneumonia, Trichomoniasis, Sleeping sickness and Chagas disease.
  • a disease such as, but not limited to, Acanthamoeba, Babesiosis, Balantidiasis, Blastocystosis, Coccidia, Dientamoebiasis, Amoebiasis,
  • the parasite may be a helminth organism or worm or a disease caused by a helminth organism such as, but not limited to, Ancylostomiasis/Hookworm, Anisakiasis, Roundworm— Parasitic pneumonia, Roundworm— Baylisascariasis, Tapeworm— infection, Clonorchiasis, Dioctophyme renalis infection, Diphyllobothriasis— tapeworm, Guinea worm— Dracunculiasis, Echinococcosis— tapeworm, Pinworm— Enterobiasis, Liver fluke— Fasciolosis, Fasciolopsiasis— intestinal fluke, Gnathostomiasis, Hymenolepiasis, Loa filariasis, Calabar swellings, Mansonelliasis, Filariasis, Metagonimiasis— intestinal fluke, River blindness, Chinese Liver Fluke, Paragonimiasis, Lung Fluke, Schisto
  • the parasite may be an organism or disease caused by an organism such as, but not limited to, parasitic worm, Halzoun Syndrome, Myiasis, Chigoe flea, Human Botfly and Candiru.
  • the parasite may be an ectoparasite or disease caused by an ectoparasite such as, but not limited to, Bedbug, Head louse— Pediculosis, Body louse— Pediculosis, Crab louse— Pediculosis, Demodex— Demodicosis, Scabies, Screwworm and Cochliomyia.
  • the antigen may be a protein derived from cancer.
  • Tumor antigens are relatively restricted to tumor cells and can be any protein that induces an immune response. However, many tumor antigens are host (self) proteins and thus are typically not seen as antigenic by the host immune system. Tumor antigens can also be abnormally expressed by cancer cells. Tumor antigens can also be germline/testis antigens expressed in cancer cells, cell lineage differentiation antigens not expressed in adult tissue, or antigens overexpressed in cancer cells.
  • the cancers include, but are not limited to, Acute lymphoblastic leukemia; Acute myeloid leukemia; Adrenocortical carcinoma; AIDS- related cancers; AIDS-related lymphoma; Anal cancer; Appendix cancer; Astrocytoma, childhood cerebellar or cerebral; Basal cell carcinoma; Bile duct cancer, extrahepatic; Bladder cancer; Bone cancer, Osteosarcoma/Malignant fibrous histiocytoma; Brainstem glioma; Brain tumor; Brain tumor, cerebellar astrocytoma; Brain tumor, cerebral astrocytoma/malignant glioma; Brain tumor, ependymoma; Brain tumor, medulloblastoma; Brain tumor, supratentorial primitive neuroectodermal tumors; Brain tumor, visual pathway and hypothalamic glioma; Breast cancer; Bronchial adenomas/carcinoids; Burkitt lymphoma; Carcinoid tumor,
  • the first heterologous antigen and second heterologous antigens are the same. In some embodiments, the first heterologous antigen and second heterologous antigens are different.
  • the first CD8+ TCR recognizes specific MHC-II, MHC-E, or MHC-I subtopes or supertopes. In some embodiments, the first CD8+ TCR is identified by DNA or RNA sequencing. In some embodiments, the CD8+ TCR is identified by single cell sequencing.
  • the one or more isolated CD8+ T cells from the second set of CD8+ T cells express CD69 and TNFa.
  • the second CD8+ TCR recognizes one or more specific
  • the second CD8+ TCR recognizes a MHC-II supertope and a MHC-E supertope, a MHC- II supertope and a MHC-I supertope, or a MHC-I supertope and a MHC-E supertope.
  • the second CD8+ TCR recognizes one or more specific
  • second CD8+ TCR recognizes a MHC-II subtope and a MHC-E subtope, MHC-II subtope and a MHC-I subtope, or a MHC-I subtope and a MHC-E subtope.
  • the second CD8+ TCR recognizes a MHC-II subtope or supertope and a MHC-E subtope or supertope, a MHC-II subtope or supertope and a MHC-I subtope or supertope, or a MHC-I subtope or supertope and a MHC-E subtope or supertope.
  • the second CD8+ TCR recognizes specific MHC-II supertopes and MHC-II subtopes, MHC-E supertopes and MHC-E subtopes, or MHC-I supertopes and MHC-I subtopes. In some embodiments, the second CD8+ TCR recognizes more than one MHC-II supertope from the same antigen, more than one MHC-E supertope from the same antigen, or more than one MHC-I supertope from the same antigen.
  • the second CD8+ TCR recognizes more than one MHC-II subtope from the same antigen, more than one MHC-E subtope from the same antigen, or more than one MHC-I subtope from the same antigen.
  • the second CD8+ TCR recognizes one or more MHC-II supertopes and one or more MHC-II subtopes from the same antigen, one or more MHC- E supertopes and one or more MHC-E subtopes from the same antigen, or one or more MHC-I supertopes and one or more MHC-I subtopes from the same antigen. In some embodiments, second CD8+ TCR recognizes more than one MHC-II supertope from more than one antigen, more than one MHC-E supertope from more than one antigen, or more than one MHC-I supertope from more than one antigen.
  • the second CD8+ TCR recognizes more than one MHC-II subtope from more than one antigen, more than one MHC-E subtope from more than one antigen, or more than one MHC-I subtope from more than one antigen.
  • the second CD8+ TCR recognizes one or more MHC-II supertopes and one or more MHC-II subtopes from different antigens, one or more MHC- E supertopes and one or more MHC-E subtopes from different antigens, or one or more MHC-I supertopes and one or more MHC-I subtopes from different antigens.
  • the third CD8+ TCR recognizes one or more specific
  • the third CD8+ TCR recognizes one or more specific MHC-II subtopes, MHC-E subtopes, or MHC-I subtopes. In some embodiments, the third CD8+ TCR recognizes specific MHC-II supertopes and MHC-II subtopes, specific MHC-E supertopes and MHC-E subtopes, or specific MHC-I supertopes and MHC-I subtopes.
  • the third CD8+ TCR recognizes more than one MHC-II supertope from one antigen, more than one MHC-E supertope from one antigen, or more than one MHC-I supertope from one antigen. In some embodiments, the third CD8+
  • TCR recognizes more than one MHC-II subtope from one antigen, more than one MHC-E subtope from one antigen, or more than one MHC-I subtope from one antigen.
  • third CD8+ TCR recognizes one or more MHC-II supertopes and one or more MHC-II subtopes from one antigen, one or more MHC-E supertopes and one or more MHC-E subtopes from one antigen, or one or more MHC-I supertopes and one or more MHC-I subtopes from one antigen.
  • the third CD8+ TCR recognizes specific MHC-E subtopes or supertopes and MHC-II subtopes or supertopes, specific MHC-E subtopes or supertopes and MHC-I subtopes or supertopes, or specific MHC-II subtopes or supertopes and MHC-I subtopes or supertopes.
  • third CD8+ TCR recognizes third CD8+ TCR recognizes more than one MHC-II subtope from the same antigen, third CD8+ TCR recognizes more than one MHC-E subtope from the same antigen, or third CD8+ TCR recognizes more than one MHC-I subtope from the same antigen.
  • the third CD8+ TCR recognizes one or more MHC-II supertopes and one or more MHC-II subtopes from different antigens, one or more MHC-E supertopes and one or more MHC-E subtopes from different antigens, or one or more MHC-I supertopes and one or more MHC-I subtopes from different antigens.
  • the nucleic acid sequence encoding the third CD8+ TCR is identical to the nucleic acid sequence encoding the second CD8+ TCR.
  • the method comprises isolating one or more CD8+ T cells from a second subject and transfecting the one or more CD8+ T cells with a nucleic acid sequence encoding the selected third CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the third CD8+ TCR, thereby generating one or more CD8+ T cells that recognize the first MHC/heterologous antigen-derived peptide complex and the second MHC/heterologous antigen-derived peptide complex.
  • the first MHC -heterologous antigen-derived peptide complex is a MHC-II/heterologous antigen-derived peptide complex, a MHC- E/heterologous antigen-derived peptide complex, or a MHC-I/heterologous antigen- derived peptide complex.
  • the second MHC-heterologous antigen- derived peptide complex is a MHC-II/heterologous antigen-derived peptide complex, a MHC-E/heterologous antigen-derived peptide complex, or a MHC -I/heterologous antigen-derived peptide complex.
  • CD8+ T cells comprising the multi-specific TCRs can be used for prevention or treatment of disease.
  • the route of administration of the population of T cells and the amount to be administered to the human patient can be determined based on the condition of the human patient and the knowledge of the physician.
  • the route of administration is intravenous, intramuscular, intraperitoneal, or oral administration.
  • the administration is intravenous.
  • the CD8+ T cell is administered to treat or prevent cancer.
  • the cancer is prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, ovarian cancer, colon cancer, renal cell carcinoma, or cervical cancer.
  • the CD8+ T cell is administered to treat or prevent a pathogenic infection.
  • the pathogenic infection is human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, Epstein-barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Human T-lymphotropic virus type 1 (HTLV1), merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.
  • the administering is by infusion of the population of
  • the infusion is bolus intravenous infusion.
  • the administering comprises administering at least about lxlO 5 T cells of the population of CD8+ T cells per kg per dose per week to the human patient. In certain embodiments, the administering comprises administering at least about lxlO 6 T cells of the population of CD8+ T cells per kg per dose per week to the human patient.
  • the treatment methods comprise administering at least 2 doses of the population of CD8+ T cells to the human patient. In specific embodiments, the treatment methods comprise administering 2, 3, 4, 5, or 6 doses of the population of T cells to the human patient.
  • the transfected CD8+ T cells comprises a chimeric nonhuman primate-human CD8+ TCR comprising the non-human primate CDR3a and CDR3P of the second CD8+ TCR.
  • the third CD8+ TCR comprises the non-human primate CDRla, CDR2a, CDR3a, CDR 1 b, CDR2P, and CDR3P of the second CD8+ TCR.
  • the third CD8+ TCR comprises the CDRla, CDR2a, CDR3a, CDR 1 b, CDR2P, and CDR3P of the second CD8+ TCR.
  • the first subject is a nonhuman primate and the second subject is a human
  • the second CD8+ TCR is a chimeric nonhuman primate-human CD8+ TCR comprising the non-human primate CDR3a and CDR3P of the first CD8+ TCR.
  • the third CD8+ TCR is a chimeric CD8+ TCR.
  • a method of generating CD8+ T cells comprising a multi specific T cell receptor (TCR), wherein the method comprises administering to a subject a recombinant CMV vector comprising a nucleic acid sequence that encodes a first heterologous antigen, in an amount effective to generate a first set of CD8+ T cells that recognize a first MHC -E/heterologous antigen-derived peptide complex, wherein the CMV vector does not express an active UL128, UL130, UL146 and UL147 protein or orthologs thereof and wherein the recombinant CMV vector further comprises a microRNA recognition element (MRE); identifying a first CD8+ TCR from the first set of CD8+ T cells, wherein the first CD8+ TCR recognizes the first MHC -E/heterologous antigen-derived peptide complex; administering to the subject a second heterologous antigen in an amount effective to generate a second set of CD8+ T cells that recognize
  • MRE microRNA
  • the first heterologous antigen and second heterologous antigens are the same. In some embodiments, the first heterologous antigen and second heterologous antigens are different. In some embodiments, the subject is a human or non human primate. In some embodiments, the recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus macaque CMV vector.
  • the first heterologous antigen comprises a tumor antigen, pathogen-specific antigen, a tissue specific antigen, or a host-self antigen.
  • the tumor antigen is related to a cancer selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, ovarian cancer, colon cancer, renal cell carcinoma, and cervical cancer.
  • pathogen-specific antigen is related to a pathogen selected from the group consisting of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, Epstein-barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Human T-lymphotropic virus type 1 (HTLV1), merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.
  • the MRE contains target sites for microRNAs expressed in endothelial cells.
  • the MRE is specific for the miRNA selected from the group consisting of miR126, miR-126-3p, miR-130a, miR-210, miR-221/222, miR-378, miR-296, and miR-328.
  • the first CD8+ TCR recognizes specific MHC-E subtopes or supertopes. In some embodiments, the first CD8+ TCR is identified by DNA or RNA sequencing. In some embodiments, the CD8+ TCR is identified by single cell sequencing.
  • the one or more isolated CD8+ T cells from the second set of CD8+ T cells express CD69 and TNFa.
  • the second CD8+ TCR recognizes one or more specific
  • the second CD8+ TCR recognizes one or more specific MHC-E subtopes. In some embodiments, the second CD8+ TCR recognizes specific MHC-E supertopes and MHC-E subtopes.
  • the second CD8+ TCR recognizes more than one MHC-E supertope from the same antigen. In some embodiments, the second CD8+ TCR recognizes more than one MHC-E subtope from the same antigen.
  • the second CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from the same antigen. In some embodiments, the second CD8+ TCR recognizes more than one MHC-E subtope from more than one antigen.
  • the second CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from different antigens.
  • the third CD8+ TCR recognizes one or more specific
  • the third CD8+ TCR recognizes one or more specific MHC-E subtopes. In some embodiments, the third CD8+ TCR recognizes specific MHC-E supertopes and MHC-E subtopes.
  • the third CD8+ TCR recognizes more than one MHC-E supertope from one antigen. In some embodiments, the third CD8+ TCR recognizes more than one MHC-E subtope from one antigen.
  • third CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from one antigen. [0181] In some embodiments, third CD8+ TCR recognizes third CD8+ TCR recognizes more than one MHC-E subtope from the same antigen. In some embodiments, the third CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from different antigens.
  • the nucleic acid sequence encoding the third CD8+ TCR is identical to the nucleic acid sequence encoding the second CD8+ TCR.
  • the method comprises isolating one or more CD8+ T cells from a second subject and transfecting the one or more CD8+ T cells with a nucleic acid sequence encoding the selected third CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the third CD8+ TCR, thereby generating one or more CD8+ T cells that recognize the first MHC -E/heterologous antigen-derived peptide complex and the second MHC -E/heterologous antigen-derived peptide complex.
  • CD8+ T cells comprising the multi-specific TCRs can be used for prevention or treatment of disease.
  • the route of administration of the population of T cells and the amount to be administered to the human patient can be determined based on the condition of the human patient and the knowledge of the physician.
  • the route of administration is intravenous, intramuscular, intraperitoneal, or oral administration.
  • the administration is intravenous.
  • the CD8+ T cell is administered to treat or prevent cancer.
  • the cancer is prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, ovarian cancer, colon cancer, renal cell carcinoma, or cervical cancer.
  • the CD8+ T cell is administered to treat or prevent a pathogenic infection.
  • the pathogenic infection is human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, Epstein-barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Human T-lymphotropic virus type 1 (HTLV1), merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.
  • the administering is by infusion of the population of
  • the infusion is bolus intravenous infusion.
  • the administering comprises administering at least about lxlO 5 T cells of the population of CD8+ T cells per kg per dose per week to the human patient. In certain embodiments, the administering comprises administering at least about lxlO 6 T cells of the population of CD8+ T cells per kg per dose per week to the human patient.
  • the treatment methods comprise administering at least 2 doses of the population of CD8+ T cells to the human patient. In specific embodiments, the treatment methods comprise administering 2, 3, 4, 5, or 6 doses of the population of T cells to the human patient.
  • the transfected CD8+ T cells comprises a chimeric nonhuman primate-human CD8+ TCR comprising the non-human primate CDR3a and CDR3P of the second CD8+ TCR.
  • the third CD8+ TCR comprises the non-human primate CDRla, CDR2a, CDR3a, CDR 1 b, CDR2P, and CDR3P of the second CD8+ TCR.
  • the third CD8+ TCR comprises the CDRla, CDR2a, CDR3a, CDR 1 b, CDR2P, and CDR3P of the second CD8+ TCR.
  • the first subject is a nonhuman primate and the second subject is a human
  • the second CD8+ TCR is a chimeric nonhuman primate-human CD8+ TCR comprising the non-human primate CDR3a and CDR3P of the first CD8+ TCR.
  • the third CD8+ TCR is a chimeric CD8+ TCR.
  • the multi-specific TCRs disclosed herein may be used in methods of inducing an immunological response in a subject comprising administering to the subject a composition comprising a CD8+ T cell comprising the multi-specific TCR and a pharmaceutically acceptable carrier or diluent.
  • the term "subject” includes all animals, including non-human primates and humans, while “animal” includes all vertebrate species, except humans; and “vertebrate” includes all vertebrates, including animals (as "animal” is used herein) and humans. And, of course, a subset of "animal” is "mammal”, which for purposes of this specification includes all mammals, except humans.
  • tumor antigens For a list of antigens used in vaccine formulations; such antigens or epitopes of interest from those antigens may be used.
  • tumor antigens one skilled in the art may select a tumor antigen and the coding DNA therefor from the knowledge of the amino acid and corresponding DNA sequences of the peptide or polypeptide, as well as from the nature of particular amino acids (e.g., size, charge, etc.) and the codon dictionary, without undue experimentation.
  • TCR of the invention including but not limited to mammalian cells (animal cells) plant: cells, bacteria (e.g. Bacillus subtilis, Escherichia coli ), insect cells, and yeast (e.g. Pichia pastoris, Saccharomyces cerevisiae).
  • mammalian cells animal cells
  • bacteria e.g. Bacillus subtilis, Escherichia coli
  • yeast e.g. Pichia pastoris, Saccharomyces cerevisiae
  • a variety of cell lines that may find use in the present invention are described in the ATCC cell line catalog, available from the American Type Culture Collection.
  • plants and animals may be used as hosts for the expression of the T-cell receptor according to the present invention. The expression as well as the transfection vectors or cassettes may be selected according to the host used.
  • MHC major histocompatibility complex
  • MHC-E is highly conserved within and between mammalian species.
  • pharmaceutical and other compositions containing the disclosed multi-specific TCRs may be formulated so as to be used in any administration procedure known in the art.
  • Such pharmaceutical compositions may be via a parenteral route (intradermal, intramuscular, subcutaneous, intravenous, or others).
  • the administration may also be via a mucosal route, e.g., oral, nasal, genital, etc.
  • compositions may be prepared in accordance with standard techniques well known to those skilled in the pharmaceutical arts. Such compositions may be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the breed or species, age, sex, weight, and condition of the particular patient, and the route of administration. The compositions may be administered alone, or may be co-administered or sequentially administered with other with other immunological, antigenic or therapeutic compositions.
  • the disclosed CMV vectors may be administered in vivo , for example where the aim is to produce an immunogenic response, including a CD8+ immune response, including an immune response characterized by a high percentage of the CD8+ T cell response being restricted by MHC-E, MHC-II, or MHC-I (or a homolog or ortholog thereof).
  • an immunogenic response including a CD8+ immune response, including an immune response characterized by a high percentage of the CD8+ T cell response being restricted by MHC-E, MHC-II, or MHC-I (or a homolog or ortholog thereof).
  • a laboratory animal such as rhesus macaques for preclinical testing of immunogenic compositions and vaccines using RhCMV.
  • the disclosed CMV vectors are administered as a component of an immunogenic composition further comprising a pharmaceutically acceptable carrier.
  • the immunogenic compositions of the disclosure are useful to stimulate an immune response against the heterologous antigen, including a tumor antigen, a tumor virus antigen, or a host self-antigen and may be used as one or more components of a prophylactic or therapeutic vaccine against tumor antigens, tumor virus antigens, or host self-antigens for the prevention, amelioration or treatment of cancer.
  • the nucleic acids and vectors of the disclosure are particularly useful for providing genetic vaccines, i.e., vaccines for delivering the nucleic acids encoding the antigens of the disclosure to a subject, such as a human, such that the antigens are then expressed in the subject to elicit an immune response.
  • genetic vaccines i.e., vaccines for delivering the nucleic acids encoding the antigens of the disclosure to a subject, such as a human, such that the antigens are then expressed in the subject to elicit an immune response.
  • Immunization schedules are well known for animals (including humans) and may be readily determined for the particular subject and immunogenic composition. Hence, the immunogens may be administered one or more times to the subject. Preferably, there is a set time interval between separate administrations of the immunogenic composition. While this interval varies for every subject, typically it ranges from 10 days to several weeks, [and is often 2, 4, 6 or 8 weeks. For humans, the interval is typically from 2 to 6 weeks.
  • the interval is longer, advantageously about 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 32 weeks, 34 weeks, 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 58 weeks, 60 weeks, 62 weeks, 64 weeks, 66 weeks, 68 weeks or 70 weeks.
  • the immunization regimes typically have from
  • administrations of the immunogenic composition may have as few as one or two or four.
  • the methods of inducing an immune response may also include administration of an adjuvant with the immunogens.
  • annual, biannual or other long interval (5-10 years) booster immunization may supplement the initial immunization protocol.
  • the present methods also include a variety of prime-boost regimens. In these methods, one or more priming immunizations are followed by one or more boosting immunizations.
  • the actual immunogenic composition may be the same or different for each immunization and the type of immunogenic composition (e.g., containing protein or expression vector), the route, and formulation of the immunogens may also be varied.
  • an expression vector may either be of the same or different type (e.g., DNA or bacterial or viral expression vector).
  • One useful prime-boost regimen provides for two priming immunizations, four weeks apart, followed by two boosting immunizations at 4 and 8 weeks after the last priming immunization. It should also be readily apparent to one of skill in the art that there are several permutations and combinations that are encompassed using the DNA, bacterial and viral expression vectors of the disclosure to provide priming and boosting regimens. CMV vectors may be used repeatedly while expressing different antigens derived from different pathogens.
  • STTS was used longitudinally over a three-year period to sort EK9- and RL9-specific T cells (stTNF +/sCD69+) for each study RM. Sorted cells were analyzed by bulk- and/or single-cell RNAseq, allowing identification of their complete TCR a/b hierarchies.
  • EXAMPLE 2 SOME MHC-E RESTRICTED CD8+ TCRS RECOGNIZE SEQUENCE- UNRELATED SUPERTOPES AND ENDOGENOUSLY PROCESSED ANTIGEN
  • each of the major TCR alpha/beta chain pairs from all four RM were cloned for specificity analysis using transduction of primary control (SIV Ag naive) RM CD8+ T cells.
  • SIV Ag naive RM CD8+ T cells As shown in FIG. 2F, each scRNAseq-identified TCR mediated a specific response to Gag 27 6 -284 RL9, Gag 482-499 EK9 or both, confirming the specificities revealed by scRNAseq and unequivocally demonstrating that individual TCRs can have dual specificity to these 2 MHC-E-restricted supertopes.
  • TCRs can specifically recognize SIVmac239- infected CD4+ T cells and B lymphoblastoid cell lines (BLCL) transfected with SIVgag (FIGs. 3 A-3B), demonstrating that SIVgag epitopic peptides can be effectively processed and surface expressed in the context of MHC-E in non-RhCMV-infected cells.
  • EXAMPLE 4 THE BROAD EPITOPE SPECIFICITY OF THE MHC-E RESTRICTED CD8+ T CELL RESPONSE IS MEDIATED BY A SMALL NUMBER OF TCRS
  • RM recognize a minimum of 9-16 different MHC-E-restricted epitopes and 23-27 MHC- II-restricted epitopes.
  • CD8+ T cells from each study RM were stimulated with autologous SIV-infected CD4+ T cells, identified responding cells by STTS, sorted the responding cells on the basis of sCD69 and stTNF, and then analyzed the responding cells by scRNAseq, as shown in FIG. 2.
  • CD8+ T cells from each study RM were incubated with autologous SIV-infected CD4s. Activated cells were sorted based on sCD69 and s-tTNF-a staining, followed by single cell RNA-seq. Pie charts illustrate the relative frequency of each clone. Additionally, clones identified in >5% of responding cells in at least 2 separate supertope peptide stims, but present at ⁇ 5% in this experiment are also named. The clone name, alpha/beta CDR3 sequences, and V/J segment usage are shown in Tables 1-4.
  • TCR6-1/2, and TCR13-1/2; highlighted in grey) were identified. While no clonotypes were completely shared between RM, there is one alpha chain shared between Rh-3 and Rh-1 (FIGs. 4A and 4B, TCRl-1 and TCR4; red).
  • TCR hierarchies of the CD8+ T cells responding to SIV-infected cells were highly oligoclonal and comprised almost entirely (90%+) by TCRs previously identified by MHC-E-restricted supertope responsiveness (FIGs. 4A-4D). With the exception of one TCR alpha chain shared by Rh-3 and Rh-1, these TCRs were distinct in each RM. Of note, two clonotypes in Rh-3 and one in Rh-1 expressed two TCR alphas chains, resulting the in the potential for these cells to express two distinct TCR, in which the beta chain pairs with either alpha chain.
  • MHC-E-TCR CD8+ T cell transductants from the overall 4 RM study cohort were tested as shown in FIG. 5 against a panel of MHC-E-restricted optimal peptides that were recognized in any of the study RM.
  • the overall pattern of response for each TCR is shown in Tables 5-8 (note: not all targeted MHC-E- presented peptides trigger in all assays). ND: no data (analysis pending).
  • EXAMPLE 6 GENERATION OF MHC-E RESTRICTED TCRS RECOGNIZING MORE THAN ONE ANTIGEN
  • TCR9 Three of the five dominant clonotypes previously identified by their MHC-E restricted SIVgag reactivity also respond to one or both of the MHC-II-restricted SIVgag supertope peptides, and one of these TCR (TCR9) also responds to a TB Ag85B epitope (FIG. 6B).
  • RM vaccinated with 68-1 RhCMV vectors develop responses that are Ag-targeted by highly cross-reactive TCRs, with the cross-reactivity not only involving MHC-E-presented epitopes within a particular Ag insert, but also MHC-E-restricted epitopes within a heterologous insert expressed by a 68- 1 RhCMV-based vaccine that was administered at a different time.
  • EXAMPLE 7 SOME MHC-E RESTRICTED, SIVGAG- SPECIFIC TCRS ARE DERIVED FROM MHC-IA-RESTRICTED, RHCMV-IE1 SPECIFIC TCRS
  • RhCMV Immediate Early-1 (IE-1) protein a highly expressed viral protein that is frequently targeted by T cells.
  • the CD8+ T cells making up these responses were isolated by sorting on the basis of both Mamu-A*02/AN10/Mamu-A*02/VY9 tetramer binding and sCD69 and stTNF upregulation in response to peptide stimulation by STTS, and sorted cells were analyzed by scRNAseq, as described above. Strikingly and quite surprisingly, some the TCRs identified by this analysis turned out to be the same TCRs previously shown to be triggered by MHC-E-restricted SIVgag supertopes (FIG. 7B-7E).
  • TCRs recognizing AN10 and VY9 were distinct, but both also recognized with unconventionally restricted SIVgag supertopes/subtopes.
  • Functional analysis with TCR transductants confirmed the specific triggering of the relevant TCR by both one of the Mamu-A*02-restricted epitopes and one or both of the MHC-E-restricted SIVgag supertopes (FIG. 7F).
  • VL9 pre incubation blocks binding and TCR2 -mediated recognition of the SIVgag EK9 supertope, but does not block VY9 binding/recognition.
  • TCRs with both conventional IE- 1 -specific and unconventional SIVgag-specific reactivity comprised the majority (but not all) of TCRs involved in SIV-infected cell recognition in these four 68-1 RhCMV/SIVgag-vaccinated RM (FIG. 7G-7J).
  • EXAMPLE 8 DUAL-MHC- SPECIFICITY OF CD8+ T CELLS CAN RESULT FROM EXPRESSION OF TWO TCR SUBUNITS
  • both TCRs recognize Gag 482-499 EK9 and are broadly (but not identically) cross-reactive with multiple SIVgag subtopes (FIG. 5); however, only one of these pairs (TCR6.2) recognizes an Mamu-A*02 epitope (VY9) (FIG. 9).
  • VY9 Mamu-A*02 epitope
  • TCR2 was selected because it has been one of the most consistent and potent TCRs in terms of response to MHC-E supertope and also recognizes the IE-1 VY9 epitope. It was hypothesized that if triggering by the MHC-E supertope was compromised by either weak/unstable binding of the supertope peptide in the MHC-E peptide binding groove, or by low TCR avidity to the supertope-MHC-E complex, one would expect that supertope-mediated triggering would, at high epitope doses, start off similar to triggering by the conventional VY9 epitope (or possibly, less efficient than), but then would fall off fast with epitope dilution, such that demonstrable triggering by the conventional epitope would extend to much lower peptide doses than the unconventional epitope.
  • TCR transduction was performed on peripheral blood CD8+ T cells from control animals, and while the activation required for transduction converts all transductants to a memory phenotype, the origin of these cells is diverse, and thus there is likely heterogeneity in the epigenetic landscape of the transductants. Factors that dictate the ability of the cell to be triggered by MHC-E supertope remain, at this juncture of the project, to be determined. In addition, scRNA was used to determine whether MHC-E supertope non-responding transductants are in fact responding, but with a different activation response that does not include TNF-a or g-IFN production.
  • scRNAseq was used to analyze cells taken ex vivo from the study RM. Although the focus of our use of scRNAseq to this point has been single cell determination of TCR expression, the available data include whole transcriptomes. Ag-activated CD8+ T cells were predominantly sorted prior to scRNAseq analysis (stTNF+/sCD69+), this is primarily done to concentrate the Ag-responsive cells of interest to reduce costs, as the transcriptome of the Ag-responsive cells also provides clear evidence of TCR-mediated activation, easily recognizable by clustering of the responsive cells (with the relevant TCR) in a tSNE plot (FIG.
  • FIG. 11 A Isolated total CD69+ cells were studied, so as to generally enrich activated cells (FIG. 1 ID-1 IF, left panels) and then within this mostly activated subset, determine the activation score of CD8+ T cells expressing the relevant TCRs in this RM (See FIGs.
  • FIG. 1 ID-11G total CD69+ cells were sorted (FIG. 1 ID), which will enrich for activated cells, but contain background and scored cells for activation as above. Again, the cells expressing the cross-reactive TCR cluster with activation (FIGs. 1 IE-1 IF). The activation score for the response of CD8+ T cells expressing each indicated TCR to each indicated Ag stimulus was separately evaluated (FIG.
  • TCR4 and TCR6 have been shown to respond to AN10 or VY9, while TCR5 and TCR12 do not. Note that among the two TCRs reacting with MHC-E SIVgag supertopes and MHC-Ia epitopes, the activation distribution suggests more efficient activation (rightward shift) by the MHC-Ia epitopes, which at least for TCR4 is compensated for by the multiple MHC-E SIVgag epitopes presented by the SIV-infected cells.
  • TCR6+ cells express two TCRs sharing a common beta chain, and thus the response of these cells would likely reflect a composite of both TCRs.
  • TCR4 and TCR6 respond to IE- 1 VY9+AN10, whereas TCR5 and TCR12 are either not in the CD69+ gate at all, or if present, show a sub-zero activation score.
  • the TCR6 response to VY9/AN10 is robust and unimodal, despite the fact that only one of this clonotype's two TCRs responds to one of these peptides (VY9).
  • the TCR6 response to the supertope peptides and to SIV-infected cells is slightly, but discernably, left-shifted overall relative the IE-1 VY9 response and appears bimodal suggesting some cells with full activation and others with a lesser induction of the suite of activation genes.
  • the TCR4 response to IE peptide is slightly weaker than the TCR6 response, but the big difference with this TCR is its trimodal responses to MHC-E supertopes, including strong, weak and no response to these peptides, coupled with a robust response to SIV-infected cells.
  • TCR4 recognizes 4 MHC-E subtopes (FIG.
  • TCR5 is one of the supertope-only TCRs (e.g., no known recognized subtopes: FIG. 5) and the response of this TCR in its native cells to optimal supertope peptides is clearly stronger than to SIV-infected cells.
  • TCR12 has not yet been tested for subtope reactivity, but this TCR shows the same supertope > SIVinfected cell triggering pattern as TCR5, suggesting that it too might be less cross-reactive with subtopes.

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US20020127231A1 (en) * 1996-03-28 2002-09-12 Jonathan Schneck Soluble divalent and multivalent heterodimeric analogs of proteins
US20180133321A1 (en) * 2016-10-18 2018-05-17 Oregon Health & Science University Cytomegalovirus vectors eliciting t cells restricted by major histocompatibility complex e molecules
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US20020127231A1 (en) * 1996-03-28 2002-09-12 Jonathan Schneck Soluble divalent and multivalent heterodimeric analogs of proteins
US20180133321A1 (en) * 2016-10-18 2018-05-17 Oregon Health & Science University Cytomegalovirus vectors eliciting t cells restricted by major histocompatibility complex e molecules
US20190284577A1 (en) * 2018-03-19 2019-09-19 Boehringer Ingelheim Vetmedica Gmbh Ehv with inactivated ul18 and/or ul8

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